INFLUENCE OF HMGB1 ON ESTROGEN RESPONSIVE EXPRESSION AND STRUCTURE

Sachindra Raj Joshi

A Dissertation

Submitted to the Graduate College of Bowling Green State University in partial fulfillment of the requirements for the degree of

DOCTOR OF PHILOSOPHY

December 2009

Committee:

Dr. William M. Scovell, Advisor

Dr. Bruno Ullrich Graduate Faculty Representative

Dr. Carol Heckman

Dr. Carmen Fioravanti

Dr. George Bullerjahn

ii

Abstract

William M. Scovell, Advisor

High mobility group box-1 (HMGB1) increases the in vitro binding affinity of

estrogen (ER) to the various estrogen response elements (EREs) such as consensus

ERE (cERE), tandem cEREs, consensus half-site ERE (cHERE), tandem cHEREs, and variant spacer cEREn, n= 0-4bp), while decreasing the binding specificity. To test if this in vitro binding characteristic translates to the functional activity in the cell, firefly luciferase reporter vectors were constructed with different EREs noted above at 5’- to the TATA box upstream of the luciferase gene. Transient transfection of these constructs was performed in estrogen treated, ER negative human osteosarcoma cell lines, U2OS, along with the co-transfection of mammalian expression vectors pERα and pHMGB1. The in vitro binding activity of HMGB1 was correlated with the functional activity in cells, but there was no linear correlation between in vitro and in vivo. HMGB1 stimulated the transcriptional synergy for tandem cEREs and cHEREs which is reflected in its influence on in vitro binding co-operativity. cEREns with variant spacers, to which the HMGB1 had increased the ER binding affinity very strongly, did not produce a strong transcriptional activity even though it enhanced the activity. HMGB1 knock down experiments with siRNA in determining the influence of HMGB1 on estrogen (E2) responsive gene

expression clearly showed that HMGB1 is involved in E2 responsive .

ER binds strongly to cERE on free DNA but does not bind (KD >200nM) to cERE in

nucleosomal DNA. However, the presence of HMGB1 greatly facilitates ER binding

(KD~50nM) to cERE in the nucleosome. To determine the influence of HMGB1 on nucleosome

structure which could have facilitated enhanced binding of ER to cERE in nucleosomal DNA,

characteristic of the nucleosome were studied after treating it with increasing levels of HMGB1 iii

(up to 1600nM). The structure of the nucleosome is markedly altered by HMGB1 interaction,

with the population of remodeled increasing with increasing levels of HMGB1.

However, HMGB1 is not a stable component of the remodeled nucleosome. The HMGB1-

remodeling is ATP-independent. Two different forms of remodeled nucleosomes (N’ and N’’)

can be isolated after removal of HMGB1 which are stable in low salt buffer and low temperature.

The remodeled nucleosomes (N’/N’’) can be converted back to the canonical nucleosomes by

changing solution conditions. At high salt (100 mM NaCl), N’’ converts to N, while N’ remains

stable; increasing the temperature (37oC), converts N’’ to N’ and N’ remains stable. However, on

challenging the remodeled nucleosomes with excess “cold” competitor DNA (up to 1000 ng),

both N’ and N’’ reverts back to the canonical from, while at 100 ng of excess DNA, N’’ coverts

to N, and N’ remains stable. This suggests that the HMGB1-remodeled nucleosomes exhibit an altered structure stabilized at low salt buffer with electrostatic interactions. The formation of N’ and N’’ by possible interaction of tails with DNA was excluded as these two population can also be isolated by treating tailless nucleosomes with 1600 nM HMGB1. However, removal of histone tails enhance the activity of HMGB1 to distinctly reduce the EMSA mobility of nucleosome even at low concentrations ca. 400 nM, suggesting the HMGB1 needs to interact with histone tails prior to interact with DNA in the nucleosome. Furthermore, HMGB1 at 1600 nM can increase the DNase I sensitivity to alter the 10bp pattern on nucleosomes. Altogether, we find that HMGB1-remodeled nucleosomes exhibit many of the same characteristics of nucleosomes remodeled by ATP-dependent chromatin remodeling complexes. The presence of

HMGB1 appears to provide an alternate or complementary mechanism for nucleosome remodeling in which HMGB1 reduces interaction within the nucleosome to reorganize the nucleosome structure and/or increase its dynamic behavior in an ATP-independent manner. iv

This dissertation is dedicated to my parents Yogendra Raj Joshi and Sharada Joshi.

It is through their support, love and encouragement that made this endeavor possible.

Thank you very much for your never ending love and blessings.

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ACKNOWLEDGEMENTS

I would like to thank and acknowledge my advisor Dr. William M. Scovell for his constant support and enthusiasm in the development of this project and for my scientific and professional development. Dr. Scovell is an outstanding mentor, who has inspired me tremendously and has made an impact in my scientific career. I truly admire Dr. Scovell for his outstanding mentorship. I would like to thank him for playing an integral part in completion of this research.

Similarly, I would also like to thank my dissertation committee Dr. Carol Heckman, Dr.

Carmen F. Fioravanti, Dr. George S. Bullerjahn and Dr. Bruno Rene Ullrich for their teachings,

support and always being there with advice and help. Also, I would like to thank Drs. George S.

Bullerjahn and Mike McKay and Drs. Peter Lu, Yufan He for their contribution to this project by

letting us use the luminometer and atomic force microscopy, respectively. I would like to thank

Dr. Ila deSerna and Bridget for helping me with RT-PCR; Dr. D. Dignam for HPLC and Drs.

Sanchez and Sumudra for teaching me cell culture at University of Toledo, Health Science

Campus. I must thank Dr. Ron C. Peterson at Ohio Northern University for his guidance, support

and insight. With deep sincerity, I would like to thank you all.

My sincere appreciation goes to the Center of Biomolecular Science, for partial support

of this work and for a Fellowship (CBMS Pre-Doctoral Fellowship 2007-2008). Besides, I

would like to extend my gratitude to colleague, Yaw Sarpong, for wonderful years of research,

encouragement, friendship and support. Finally, I would like to thank the members of the

Biological Science and Chemistry Department faculty and staff for their support.

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TABLE OF CONTENTS

Page

CHAPTER I. INTRODUCTION ...... 1

High-mobility Group 1 Protein (HMGB1) ...... 1

Nomenclature and Structure ...... 1

Role of HMGB1 in Transcription ...... 4

HMGB1 Interaction with Nucleosome and Chromatin Remodeling ...... 7

Estrogen Receptor (ER) ...... 9

Progesterone Receptor (PR) ...... 14

Chromatin/Nucleosome ...... 16

Nucleosome Structure ...... 17

Chromatin Remodeling ...... 20

Objectives…… ...... 23

CHAPTER II. MATERIALS AND METHODS ...... 24

Plasmids and Cell Types ...... 24

Promoter Sequences for Regulating Extent of Activated Expression of

Luciferase Gene ...... 29

Transformation of E. coli Cells with Plasmids ...... 30

Construction of Luciferase Reporter Constructs...... 33

Isolation of Plasmid DNA ...... 36

Media Preparation for U2OS Cell Culture...... 38

Maintenance of U2OS Cells ...... 42

Transient Transfection of U2OS Cells ...... 45 vii

Hormone Treatment ...... 49

Cell Harvest ……...... 49

Dual Luciferase Assay ...... 49

Protein Assay ...... 52 siRNA Co-transfection...... 52

Isolation of Total RNA ...... 55 cDNA synthesis ...... 56

RT-PCR ………...... 57

Isolation and Purification of HMGB1 and HMGB2 ...... 57

DNA and Nucleosome ...... 61

Isolation of 161bp 2G2 DNA from pGEM-Q2 Plasmid ...... 62

End Labeling of DNA with γ-(32P)-ATP ...... 66

Measurement of Radioactivity by Scintillation Counter ...... 70

Preparation of Nucleosomes ...... 70

Preparation of Remodeled Nucleosomes ...... 72

Electrophoresis ...... 73

Agarose Gel Electrophoresis...... 73

PAGE……...... 74

Electrophoretic Shift Mobility Assay (EMSA)...... 75

DNase I Assay...... 78

SDS-PAGE ...... 81

Western Blot ………...... 84

Atomic Force Microscopy ...... 87 viii

CHAPTER III. RESULTS I ...... 89

Construction of Luciferase Reporter Constructs...... 89

Restriction Digestion of Luciferase Reporter Vector and Ligation of ERE

Sequences into the Vector ...... 89

Colony PCR ...... 91

DNA Sequencing of Constructs ...... 97

Determination of Specific Responsiveness of Luciferase Reporter Gene to

Estrogen and α ...... 108

Effect of Increasing Amounts of pCMVflag:hERα Expression Vector

on Luciferase Reporter Construct ...... 110

HMGB1 Overexpression in U2OS Cells ...... 110

Effect of Increasing Amounts of pHMGB1 Expression Vector on

Estrogen Responsive ERα Mediated Luciferase Reporter Activity ...... 112

Effect of Different ERE Inserts on Estrogen Responsive ERα Mediated

Luciferase Reporter Activity...... 113

Single or Tandem cEREs ...... 113

Single or Tandem cHEREs ...... 116

cEREs with Different Spacer Sizes...... 119

Effect of Overexpression of HMGB1 on Various ERE Constructs on Estrogen

Responsive ERα Mediated Luciferase Reporter Activity ...... 121

Single or Tandem cEREs ...... 121

Single or Tandem cHEREs ...... 122

cEREs with Different Spacer Sizes...... 123 ix

Effect of Silencing Endogenous HMGB1 Gene with siRNA on Estrogen

Responsive ERα Mediated Luciferase Reporter Activity ...... 127

CHAPTER IV. RESULTS II ...... 131

Isolation and Purification of HMGB Proteins from Calf Thymus ...... 131

Preparation and Isolation of Nucleosomes ...... 131

Influence of HMGB1 on Nucleosomes...... 133

Effect of Increasing Amounts of HMGB1 on Nucleosome Structure ...... 133

Determination of the Presence of HMGB1 as a Stable

Component of Nucleosomes ...... 136

DNase I 10bp Digestion Pattern for DNA, Nucleosomes, and

HMGB1-remodeled Nucleosomes ...... 139

Effect of Increasing Amounts of “Cold” Competitor DNA on

Nucleosomes Treated with 1600 nM HMGB1 ...... 139

Isolation of HMGB1-remodeled Nucleosomes ...... 141

Comparative Sedimentation Profile of Canonical Nucleosomes and

HMGB1-remodeled Nucleosomes ...... 143

Determination of the Presence of Core in

Nucleosomes, Nucleosomes Treated with 1600 nM HMGB1, and

HMGB1-remodeled Nucleosomes ...... 147

Determination of the Presence of HMGB1 as a Stable

Component of HMGB1-remodeled Nucleosomes ...... 147

Effect of Increasing Amounts of HMGB1 on of HMGB1-remodeled Nucleosomes 149

Characterization of HMGB1-remodeled Nucleosomes ...... 152 x

Effect of Different Buffers on HMGB1-remodeled Nucleosomes ...... 152

Effect of Different Components of ER Dilution Buffer on

HMGB1-remodeled Nucleosomes ...... 154

Effect of Increasing Salt Concentration on

HMGB1-remodeled Nucleosomes ...... 159

Effect of Temperature on HMGB1-remodeled Nucleosomes ...... 159

Effect of Increasing Amounts of “Cold” Competitor DNA on

HMGB1-remodeled Nucleosomes ...... 160

Effect of Increasing Amounts of Urea on

HMGB1-remodeled Nucleosomes ...... 164

Effect of Increasing Amounts of DTT on HMGB1-remodeled

Nucleosomes ...... 165

Atomic Force Microscopy of Canonical Nucleosomes and

HMGB1-remodeled Nucleosomes ...... 168

Effect of Increasing Amounts of HMGB1 on Tailless Nucleosomes ...... 168

Isolation of HMGB1-remodeled Tailless Nucleosomes ...... 170

Effect of HMGB1 on PR Binding to Free DNA ...... 171

Effect of HMGB1on PR Binding to Free DNA, Nucleosomal DNA, and

HMGB1-remodeled Nucleosomes ...... 175

CHAPTER V. DISCUSSION ...... 177

Role of Different EREs on In Vivo Transactivation and the

Influence of HMGB1 ...... 178

Single or Tandem cEREs ...... 181 xi

Single or Tandem cHEREs ...... 183

Variant Spacer cEREn (spacer size n=0-4bp) ...... 186

Role of HMGB1 on Estrogen Responsive ERα Mediated Gene Activity ..... 188

Role of HMGB1 on Nucleosome Remodeling ...... 190

Influence of HMGB1 on EMSA Mobility of Canonical Nucleosomes ...... 192

Influence of HMGB1 on DNase I 10bp Pattern of Canonical Nucleosomes 194

HMGB1-remodeled Nucleosomes ...... 196

Influence of HMGB1 on EMSA Mobility of Remodeled Nucleosomes ...... 201

Composition of Nucleosomes ...... 201

Characterization of HMGB1-remodeled Nucleosomes ...... 202

Role of Histone Tails on Nucleosomes ...... 205

Influence of HMGB1 on EMSA Mobility of Tailless Nucleosomes ...... 209

Binding Affinity of PR to PRE at Nucleosomal Level ...... 211

REFERENCES……………………………… ...... 213

APPENDIX I. HMGB1 Expression on Tissues/Cells/Tumors ...... 234

APPENDIX II. EREs Found in Estrogen Responsive ...... 236

APPENDIX III. Preparation of Oligonucleosomes from Chicken Erythrocytes ...... 254

APPENDIX IV. Preparation of Tailless Oligonucleosomes ...... 261

APPENDIX V. Media and Buffers ...... 263

APPENDIX VI. Sequences for pGL2-3cERE-TATA-Inr-Luc ...... 266

APPENDIX VII. Calculations of Nucleosome Reconstitution Buffer Compositions ...... 269

APPENDIX VII. Detail map of DNase I 10bp pattern on

nucleosome (N) + 1600 nM HMGB1...... 271 xii

LIST OF FIGURES Figure Page 1 Schematic diagram of HMGB1 protein showing different domains ...... 3 2 Schematic representation of the functional domain organization of nuclear receptors ……… ...... 10 3 Schematic diagram of human PR-B and PR-A ...... 15 4 Nucleosome core particle ...... 18 5 Schematic diagram of core histones showing tail domains ...... 19 6 A schematic diagram of 2G2 DNA ...... 25 7 Plasmid map for pGL2-3cERE-TATA-Inr-Luc...... 27 8 A schematic representation of DNA hybridization to produce Xho I and Bgl II overhangs ……… ...... 31 9 Schematic diagram for transient transfection of U2OS cells ...... 46 10 Schematic diagram of hemocytometer...... 47 11 Bioluminescent reactions catalyzed by firefly luciferase ...... 51 12 Bioluminescent reactions catalyzed by Renilla luciferase ...... 51 13 Agarose gel (1%) of restriction digests of pGL2-3cERE-TATA-Inr-Luc ...... 90 14 Agarose gel (1%) of restriction digestion and ligation to produce pGL2-2cERE-TATA-Inr-Luc………...... 92 15 Six % polyacrylamide gel of PCR products for colony PCR ...... 95 16 Dependence of 2cERE luciferase reporter expression on E2 and ER ...... 109 17 Effect of increasing amounts of pERα expression vector on p3cERE–Luc reporter vector...... 111 18 HMGB-1 overexpression in U2OS cells...... 114 19 Effect of increasing amounts of pHMGB1 on ER mediated transactivation of pGL2-3cERE-TATA-Inr-Luc reporter vector ...... 115 20 Effect of single or tandem cERE on ER mediated reporter activity ...... 117 21 Effect of single or tandem cHERE on ER mediated reporter activity ...... 118 22 Effect of ERE with different spacer size between two half sites on ER mediated reporter activity ……… ...... 120

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23 Effect of overexpression of pHMGB1 on ER mediated reporter activity of single or tandem cEREs ...... 124 24 Effect of overexpression of pHMGB1 on ER mediated reporter activity of single or tandem HEREs...... 125 25 Effect of overexpression of pHMGB1 on ER mediated reporter activity of cERE with different spacer size between two half sites on ER mediated reporter activity ...... 126 26 Effect of increasing amounts of siRNA for GAPDH on its down regulation ...... 129 27 Effect of HMGB1 silencing on ER mediated reporter activity...... 130 28 An 18% SDS-PAGE of HPLC purified calf thymus HMGB1 and HMGB2 stained with Coomassie blue ...... 132 29 Sedimentation and EMSA profile of canonical nucleosomes (N) ...... 134 30 Effect of HMGB1 on EMSA mobility of nucleosomes ……… ...... 135 31 Supershift assay to determine whether HMGB1 is a stable component of remodeled nucleosomes ...... 137 32 Reaction of glutaraldehyde with nucleosomes in the presence of 1600 nM HMGB1…...... 138 33 DNase I assay of nucleosomes in the presence of 1600 nM HMGB1 ...... 140 34 Effect of increasing levels of cold competitor DNA on nucleosomes treated with 1600 nM HMGB1……… ...... 142 35 Sedimentation and EMSA profile of HMGB1-remodeled nucleosomes (N’ & N”) ...... 144 36 Sedimentation and EMSA profiles of canonical nucleosomes (N), remodeled nucleosomes (N’ & N”)……...... 146 37 Supershift assay to determine the presence of core histones intact in nucleosomes, nucleosomes treated with HMGB1, and remodeled nucleosomes ...... 148 38 Supershift assay for the presence of HMGB1 as a stable component of the HMGB1- remodeled nucleosomes (N’/N’’) ……… ...... 150 39 Effect of increasing amounts of HMGB1 on remodeled nucleosomes...... 151 40 Effect of 1600 nM HMGB1 and ER buffer on nucleosomes and remodeled nucleosomes …...... 153 xiv

41 Effect of different reaction buffers on nucleosomes and remodeled nucleosomes ...... 155 42 EMSA to determine which components of ER dilution buffer destabilized the remodeled nucleosomes ...... 157 43 Effect of increasing concentration of NaCl on remodeled nucleosomes ...... 161 44 Effect of temperature on nucleosomes and remodeled nucleosomes ...... 162 45 Effect of increasing levels of cold competitor DNA on remodeled nucleosomes ..... 163 46 Effect of increasing concentration of urea on canonical and HMGB1-remodeled nucleosomes…...... 166 47 Effect of increasing concentration of DTT on canonical and HMGB1-remodeled nucleosomes …...... 167 48 Atomic force microscopy of canonical nucleosomes and HMGB1-remodeled nucleosomes…...... 169 49 Effect of increasing amounts of HMGB1 on tailless nucleosomes ...... 172 50 Sedimentation and EMSA profile of HMGB1-remodeled canonical and tailless nucleosomes … ...... 173 51 EMSA profile of PR binding to free DNA (2G2) in the presence and absence of 400nM HMGB1 ...... 174 52 EMSA profile of PR binding to free DNA (2G2), canonical nucleosomes (N), and HMGB1-remodeled nucleosomes (N’/N’’) in the presence and absence of 1600 nM HMGB1 ……… ...... 176 53 Energy landscape for HMGB1-remodeled nucleosomes ...... 206 54 Core histones in nucleosomes ...... 260 55 Detail map of DNase I 10bp pattern on nucleosome (N) + 1600 nM HMGB1 ...... 271

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LIST OF TABLES

Table Page 1 Nomenclature of HMG proteins ...... 2 2 Effect of HMGB1/2 on the superfamily ...... 6 3 Plasmids used in studies...... 26 4 Sequences for coding and non coding strands of single and tandem cEREs ...... 29 5 Sequences for coding and non coding strands of single and tandem cHEREs ...... 30 6 Sequences for coding and non coding strands of cEREn with different spacer sizes, when n=0,1,2,3,4 bps in between two half sites of the cERE ...... 30 7 Media composition for U2OS Maintenance Medium ...... 40 8 Media composition for U2OS Transfection Medium A ...... 41 9 Media composition for U2OS Transfection Medium B ...... 41 10 Media composition for Serum Free U2OS Transfection Medium B ...... 42 11 Sizes of PCR product for the colony PCR of the transformed E. coli (JM109) colonies with recombinant vectors containing various estrogen response elements ...... 94 12 Fold change in E2-responsive ERα-mediated luciferase reporter gene activity under the control of single cERE or tandem cEREs ...... 117 13 Fold change in E2-responsive ERα-mediated luciferase reporter gene activity under the control of single cHERE or tandem cHEREs ...... 118 14 Fold change in E2-responsive ERα-mediated luciferase reporter gene activity under the control of variant spacer cEREn (n=0-4) ...... 120 15 Effect of overexpression of HMGB1 on E2-responsive ERα-mediated luciferase reporter gene activity under the control of single cERE or tandem cEREs ...... 124 16 Fold change in E2-responsive ERα-mediated luciferase reporter gene activity under the control of single cERE or tandem cEREs when HMGB1 is overexpressed ...... 124 17 Effect of overexpression of HMGB1 on E2-responsive ERα-mediated luciferase reporter gene activity under the control of single cHERE or tandem cHEREs.… ... 125

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18 Fold change in E2-responsive ERα-mediated luciferase reporter gene activity under the control of single cHERE or tandem cHEREs when HMGB1 is overexpressed ...... 125 19 Effect of overexpression of HMGB1 on E2-responsive ERα-mediated luciferase reporter gene activity under the control of variant spacer cEREn (n=0-4)… ...... 126 20 Fold change in E2-responsive ERα-mediated luciferase reporter gene activity under the control variant spacer cEREn (n=0-4) when HMGB1 is overexpressed ...... 126 21 Final concentration of each component of the reaction buffer and the effect on HMGB1-remodeled nucleosomes (N’/N’’) ...... 158

22 KD values for in vitro ER binding to a single cERE and tandem cEREs ...... 179

23 KD values for in vitro ER binding to a single cHERE and tandem cHEREs ...... 179

24 KD values for in vitro ER binding to cEREn (spacer “n"=0-4bp) ...... 179 25 Comparison of in vitro binding affinity of ER to a single cERE and tandem cEREs to in vivo transcriptional activity ...... 184 26 Comparison of in vitro binding affinity of ER to a single cHERE and tandem cHEREs to in vivo transcriptional activity ...... 184 27 Comparison of in vitro binding affinity of ER to variant spacer cEREn (n=0-4bp) to in vivo transcriptional activity ...... 181 28 Binding affinities of various transcription factors (TFs) to DNA in the nucleosome compared to free DNA ...... 184 29 Levels of HMGB1 expression on tissues/tumor/cell lines ...... 234 30 Consensus ERE (cERE): 15bp inverted palindrome 5’-AGGTCAnnnTGACCT-3’...... 236 31 Minimal consensus ERE (cERE): 13bp inverted palindrome 5’-GGTCAnnnTGACC-3’ ...... 236 32 Imperfect EREs ...... 237 33 Multiple EREs ...... 244 34 cHEREs……………...... 251 35 Multiple HEREs ...... 251 36 Variant Spacer EREn ...... 253 xvii

37 Table for volumes and concentrations for MNase digestion ...... 255 38 Calculations of nucleosome reconstitution buffer compositions ...... 269 38 Final concentration of each component of the reaction buffer when remodeled nucleosomes were treated with different buffers ...... 270 xviii

LIST OF BOXES

Box Page 1 DNA sequence for 2G2 ...... 63 2 DNA sequences for p1cERE-TATA-Inr-Luc from Retrogen Inc using GL primer 1 and GL primer 2 ...... 98 3 DNA sequences for p2cERE-TATA-Inr-Luc from Retrogen Inc using GL primer 1 and GL primer 2 ...... 99 4 DNA sequences for p3cERE-TATA-Inr-Luc from Retrogen Inc using GL primer 1 and GL primer 2 ...... 100 5 DNA sequences for p1HERE-TATA-Inr-Luc from Retrogen Inc using GL primer 1 and GL primer 2 ...... 101 6 DNA sequences for p2HERE-TATA-Inr-Luc from Retrogen Inc using GL primer 1 and GL primer 2 ...... 102 7 DNA sequences for p3HERE-TATA-Inr-Luc from Retrogen Inc using GL primer 1 and GL primer 2 ...... 103 8 DNA sequences for pERE0-TATA-Inr-Luc from Retrogen Inc using GL primer 1 and GL primer 2 ...... 104 9 DNA sequences for pERE1-TATA-Inr-Luc from Retrogen Inc using GL primer 1 and GL primer 2 ...... 105 10 DNA sequences for pERE2-TATA-Inr-Luc from Retrogen Inc using GL primer 1 and GL primer 2 ...... 106 11 DNA sequences for pERE4-TATA-Inr-Luc from Retrogen Inc using GL primer 1 and GL primer 2 ...... 107

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ABBREVIATIONS

ACF ATP-utilizing chromatin assembly and remodeling factor AR AS Ammonium sulfate ATP Adenosine triphosphate BME Beta mercaptoethanol BSA Bovine serum albumin CD Charcoal dextran CDCS Charcoal dextran treated calf serum cERE Consensus estrogen response element cHERE Consensus half-site estrogen response element CHRAC Chromatin accessibility complex COUP-TF Chicken ovalbumin upstream promoter CPM Counts per minute CRC Chromatin remodeling complex CTE Carboxyl terminal extension DBD DNA binding domain DR Direct repeat DTT Dithiothreitol E2 Estrogen EDTA Ethylenediaminetetraacetic acid EMSA Electrophoretic mobility shift assay ER Human estrogen receptor ERE Estrogen response element EtOH Ethanol FACT Facilitates chromatin transcription GR Human GRE Glucocorticoid response element HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid HIFCS Heat inactivated fetal calf serum HMGB1 High mobility group box-1 protein HPLC High-performance liquid chromatography HRE Hormone response element Hsp Heat shock protein IDT Integrated DNA Technology Inc. KD Dissociation constant LB Luria-Bertani LBD Ligand binding domain Luc Luciferase MEM Minimum eagles medium xx

MOPS 3-(N-morpholino) propanesulfonic acid MR Mineralocorticoid receptor MWCO Molecular weight cut off NEAA Non essential amino acids NF-1 Nuclear factor-1 NPS Nucleosome positioning sequences NURF Nucleosome remodeling factor OD Optical density PBS Phosphate buffer saline PCI Phenol-chloroform-isopropanol PCR Polymerase chain reaction PMSF Phenylmethylsulphonylfluoride PR PRE Progesterone response element PXR RAR RSB Reticulocyte standard buffer RSC Chromatin structure remodeling RT-PCR Real time polymerase chain reaction RXR SDS Sodium dodecyl sulfate SDS-PAGE Sodium dodecyl sulfate- polyacrylamide gel electrophoresis SWI/SNF SWItch/Sucrose NonFermentable SXR Steroid X receptor TAE Tris acetate EDTA buffer TBE Tris borate EDTA buffer TBP TATA binding protein TC Tissue culture TCA Trichloroacetic acid TE Tris EDTA buffer TEMED N,N,N',N'-Tetramethylethylenediamine TF Transcription factor TR Thyroid receptor TRF1 Human telomeric-repeat binding factor Tris-HCl Tris hydroxy methyl amino methane hydrochloric acid U2OS Human osteosarcoma cells VDR

1

CHAPTER I: INTRODUCTION

1. High-mobility group 1 protein (HMGB1)

High-mobility group 1 protein (HMGB1) is a low molecular weight~25kDa, 215 amino

acid residues, non-histone chromosomal protein. These proteins are thought to be originated long

before the division of animal and plant kingdom. Only in 1973, it was first identified in

mammalian cells by Goodwin et al, and named according to its mobility in urea acid gels

(Goodwin et al., 1973). HMGB1 is highly conserved and ubiquitously expressed non-histone

protein in most eukaryotic cells. It is a highly abundant chromosomal protein with an average of

106 molecules per nucleus, which is only 10 times less than core histones (Bustin, 1999). It is

highly mobile with each individual nucleosomes visited in every 2 seconds with the residence

time of a fraction of a second (Agresti and Bianchi, 2003).These non-histone chromosomal proteins are considered as DNA architectural proteins (Wolffe, 1994), as they distort, bend or modify the structure of the DNA (Agresti and Bianchi, 2003). This bending feature has been implied in facilitation of transcription factor binding as well as in the stability of nucleosomes structure. HMGB1 enhances RAG1/2 cleavage in V(D)J recombination and DNA repair processes as well as RNA pol II transcription in a subset of genes, including many of those regulated by steroid hormone receptors. In 1979, HMGB1 was also reported to be found in cytoplasm (Bustin and Neihart, 1979). Its expression in intact organs and tissues can be developmentally regulated and/or respond to signals from the environment. Table 29, appendix I

shows the expression data for HMGB1 (reviewed by Bianchi, 2004).

1.1. Nomenclature and Structure

The HMGB1 belongs to the superfamily of HMG proteins, which according to

Bustin’s revised nomenclature for high mobility group proteins, is divided into three 2

subfamilies (Table 1). Each superfamily has a characteristic functional . The

functional motif of the HMGB family is called the “HMG-box”, that of the HMGN family is

called the “nucleosomal binding domain”, and that of the HMGA family called the “AT-

hook”. Proteins containing any of these functional motifs embedded in their sequence are

known as HMG-motif proteins. Previously known HMG-1 and HMG-2 are now known as

HMGB1 and HMGB2, respectively. Likewise, HMG-14 and HMB-17 are known as HMGN1 and HMGN2, respectively, and HMG-I and HMG-Y are known as HMGA1 and HMGA2, respectively.

Table 1: Nomenclature of HMG proteins (Bustin, 2001)

HMGB1 is extremely conserved and has 99% identity among all mammals. HMGB1 consists of two consecutive DNA binding domains, HMG-box A and HMG-box B and a 30 amino acid long acidic tail, connected by short linkers (figure 1). The A box (2-79) and B box (89-162) are highly basic domains, each containing residues with high percentage of arginines and lysines (Bustin et al., 1990; Weir et al., 1993). Therefore, HMGB1/2 have been shown to bind non-specifically to DNA (Bustin et al., 1990). The acidic tail, C-terminal is quite different. It is polyanionic, with 30 residues of aspartic acid and glutamic acid residues in a stretch, and therefore is completely negatively charged. 3

Figure 1. Schematic diagram of HMGB1 protein showing different domains: Red boxes represent HMG-box A and HMG-box B. Blue boxes represent hinge regions and Greenish blue represent C-terminal acidic tail domain (Bianchi and Manfredi, 2007).

4

1.2. Role of HMGB1 in Transcription

The process of transcription is highly regulated by the organization of genomic DNA into chromatin. Any changes in chromatin due to histone modification or chromatin remodeling can readily alter transcription (Felsenfeld and Groudine, 2003; Narlikar et al.,

2002). Therefore chromatin associated protein such as HMGB1 is likely to play an important role in transcription. It was shown in 1998 that that the HMGB protein stimulates transcription by RNA polymerase II and III (Tremethick and Molloy, 1988). Since a number of HMGB domain proteins are transcription factors (Landsman and Bustin, 1993), HMGB1 has been suspected of affecting transcription by preventing the binding of transcription factors to DNA.

In a number of studies HMGB1 was shown to promote the transcription by increasing the sequence-specific binding affinity of steroid hormone receptors to its hormone response elements (Boonyaratanakornkit et al., 1998; Melvin and Edwards, 1999; Verrier et al., 1997;

Verrijdt et al., 2002; Zhang et al., 1999). Binding of PR to its consensus response element was increased by addition of HMGB1, and increased further for imperfect non palindromic site. It also facilitated PR mediated transcriptional activity (Onate et al., 1994; Prendergast et al., 1994). Onate et al. also examined the effect of individual domains of HMGB1 protein, viz., HMG-box A and HMG-box B, expressed and purified in E. coli cells, on PR binding to the PRE. The HMGB1 B-box was nearly as effective as HMGB1 itself, whereas A-box only weakly enhanced PR binding to the PRE. HMGB1 facilitated binding of PR by inducing a structural change in the target DNA, and this activity was specific to HMG-box B. These experiments were consistent with the notion that HMGB1 is a nuclear “chaperone protein” which, perhaps by bending and unwinding the duplex, creates an enhanced site for 5

transcription factor binding and activation of gene expression (Ner and Travers, 1994).

HMGB1/2 has been reported to selectively influence the activity of steroid hormone

receptors (class I) but not other class II receptors (RXR, VDR, TR and RAR)

(Boonyaratanakornkit et al., 1998; Melvin and Edwards, 1999). The C-terminal extension

(CTE) of the nuclear DNA binding domain has been suggested to interact with HMGB1/2 proteins to facilitate the binding of nuclear hormone receptor to the DNA

(Melvin et al., 2004). The effect of HMGB proteins on nuclear receptor superfamily is shown in Table 2 (Melvin and Edwards, 1999).

HMGB1 dramatically enhanced the binding of purified, bacterially expressed DNA

binding domain (DBD) of ER to consensus vitellogenin A2 ERE in a dose-dependent manner

(Romine et al., 1998). HMGB1 enhances in vitro binding of estrogen receptor to estrogen

response elements (Das et al., 2004; El Marzouk et al., 2008; Ghattemani, 2004; Sarpong,

2006). Importantly, the binding affinity of ER to its half-site ERE was markedly enhanced in

the presence of HMGB1 (Das et al., 2004; El Marzouk et al., 2008). Moreover, HMGB1 has

been shown to stimulate the co-operative binding of ER to the tandem repeats of two cEREs

and direct repeats of two cHEREs (Das et al., 2004; Ghattemani, 2004). In addition, HMGB1

decreases binding specificity of ER to estrogen response element half-sites separated by

variant spacers (cEREn, n=0-4bp) while increasing its binding affinity (El Marzouk et al.,

2008).

HMGB1 is also reported to act as a general class II transcription factor in

transcriptional regulation (Singh and Dixon, 1990). The inhibition of transcription in HMG-

depleted nuclei was relieved by the addition of exogenous HMGB1 and it was also shown to

6

Table 2*: Effect of HMGB1/2 on the nuclear receptor superfamilya

Receptor In vitro DNA In vivo binding/element transactivation Steroid PRc 35-fold/GRE (4-12)-foldb AR 60-fold/GRE 10-fold GR (6-10)-fold/GRE 6-fold ER (3-4)-fold/ERE 3-fold ER +/ERE + Class II VDR -d/DR3 ND VDR-RXR -/DR3 (1.0-2.5)-fold RAR -/DR5 ND RAR-RXR -/DR5 - RXR -/DR1 ND TR -/DR4 ND TR -/TREpal ND *Table adapted from(Melvin and Edwards, 1999) aAdapted and updated from Boonyaratanakornkit et al. Mol Cell Biol 1998; 18:4471-87 bFold varies dependent on cell type and target promoter. cPR, progesterone receptor; AR, androgen receptor; GR, glucocorticoid receptor; ER, estrogen receptor; VDR, vitamin D3 receptor; RXR, retinioid X receptor; RAR, retinoic acid receptor; TR, . d-, no stimulation; +, stimulated, fold not reported; ND, not determined.

be co-purified along with TFIIB and therefore suggested that HMGB1 was a basal transcriptional factor (Singh and Dixon, 1990). HMGB1 and TBP interact in the presence of a TATA box containing oligonucleotide to form a specificHMGB1/TBP/promoter complex

(Ge and Roeder, 1994). Das and Scovell have demonstrated by using monoclonal antibody and deleted segment of the N-terminus of human TBP that the acidic C-terminal domain of

HMGB1 and the N-terminus of the human TBP are the domains that are essential for the formation of stable HMGB1/TBP/TATA complex (Das and Scovell, 2001). TFIIA can compete off HMGB1 from the HMGB1/TBP/TATA complex (HMGB1 is limiting), but 7

when there is excess HMGB1, an HMGB1/TFIIB/TBP/TATA complex is formed (Lu et al.,

2000). In contrast, it has been proposed that TFIIA can dissociate the HMGB1 from

HMGB1/TBP/TATA (Ge and Roeder, 1994). In this way increasing the level of TFIIA, but

not TFIIB, was shown to restore basal transcription in an in vitro assay (Ge and Roeder,

1994).

HMGB1 was reported to facilitate in vitro binding of several transcription factors

( proteins, Pou domain transactivators, TBP, , ) to their specific response

elements in DNA as well as influence the transcriptional activities in vivo (Jayaraman et al.,

1998; Stros et al., 2002; Verrier et al., 1997; Zappavigna et al., 1996; Zwilling et al., 1995).

Recently, HMGB1 was reported to up-regulate the human topoisomearse IIα gene (Stros et

al., 2009). HMGB1 deficient mice show a distinct phenotype and die shortly after birth due

to hypoglycemia and were deficient in the activation of GR responsive genes (Calogero et al.,

1999), suggesting that HMGB1 does not serve as an unspecific DNA binding protein but that

it contributes to gene specific transcriptional regulation.

1.3. HMGB1 Interaction with Nucleosome and Chromatin Remodeling

The distinct domains in HMGB1 have been reported to be involved in specific

intramolecular and nucleosomal interactions. The C-terminal acidic tail domain of the

HMGB1 interacts with the DNA-binding domain of HMGB1 (HMG-box A) in its native form and with core histone H3 in the nucleosome (Kawase et al., 2008). Photochemical

cross-linking studies have also suggested that HMGB1 and histone H3 in the nucleosomes interact with each other (Stros, 1987). These interactions have been indicated as an

important factor for modulation of the DNA and chromatin binding activities of HMGB1 as

well as biological functions of HMGB1. HMGB1 has been reported to interact with the 8

linker DNA in chromatin (Cato et al., 2008) and compete with linker histone H1 (Varga-

Weisz et al., 1994). Together with linker histone H1, HMGB1 was shown to silence proinflamatory genes during endotoxin tolerance by chromatin-specific remodeling (El

Gazzar et al., 2009).The C-terminal acidic tail of HMGB1 has been suggested to be necessary for its target binding to the linker DNA and stimulation of transcription (Ueda et al., 2004).

An HMGB1 homologue in Drosphila, HMG-D, interacts with the nucleosomal DNA and induces local changes in DNA accessibility. These changes involve the sites located on one face of the nucleosome and encompass the entry point of DNA as well as the dyad. In addition, it was found that the acidic C-terminal tail of HMG-D is required for this pattern of accessibility (Ragab and Travers, 2003).

Similarly, HMG-box containing proteins in yeast FACT, Nhp6a and Nhp6b also bind to the nucleosomes (Formosa et al., 2001). Binding of Nhp6 alone on the nucleosome altered the DNase I sensitivity of the nucleosomal DNA. However, when Spt16-Pob3 and the HMG box protein Nhp6 were both present (yFACT complex), the most dramatic changes in the nucleosome structure occurred (Rhoades et al., 2004). In addition, at high concentrations of

Nhp6, yFACT has been shown to increase in the accessibility of nucleosomal DNA to DNase

I (Ruone et al., 2003). Interestingly, the yFACT complex alters the nucleosome structure in an ATP-independent manner (Rhoades et al., 2004; Ruone et al., 2003). These reports suggest that the HMG-box containing protein Nhp6 is necessary for the enhanced activity of

ATP-independent chromatin remodeling complex, yFACT.

Interestingly, ATP-dependent chromatin remodeling complexes are also associated with a HMG box containing proteins, such as BAF57 in SWI/SNF complex, BAP111 in 9

Drosophila Brahma (BRM) complex. BAF57 subunit in the SWI/SNF complex has been

reported to be a critical regulator of ER function in breast cells (Garcia-Pedrero et al.,

2006). Likewise, the HMG-domain protein BAP111 was reported to be important for the

function of the BRM-complex in Drosophila (Papoulas et al., 2001).

Recently, HMGB1 was also reported to enhance the activity of chromatin remodeling

complex of SWI/SNF by~ 2-fold, while the acetylated HMGB1 increased by ~4-fold, along

with facilitating the binding of SWI/SNF to the nucleosomes (Ugrinova et al., 2009).

Altogether, these reports show that the HMG-box proteins can play a general role in

chromatin remodeling.

2. Estrogen Receptor

Estrogen receptor (ER) is a nuclear hormone receptor, a member of nuclear receptor superfamily that comprises about 60 different classical members of nuclear hormone receptor family (Mangelsdorf et al., 1995). It is activated by ligand (estrogen) to regulate target gene expression. Two subtypes of ER are expressed in mammalian cells, 1) ERα and 2) ERβ. In this study ER is referred to ERα.

A schematic structure of ER is shown in figure 2. It has six domains, A-F from N- to C-

terminus, encoded by 8–9 exons (Kuiper et al., 1998). The N-terminal domain (domains A and

B) controls transcription in a cell-specific as well as a gene specific manner through activation

function-1 (AF-1). The central DNA binding domain (DBD) is highly conserved and involves

the C domain. This DBD comprised of two functionally distinct zinc motifs, with one that ER

interacts directly with the DNA helix. The E domain is the ligand binding domain that contains

10

(b)

Figure 2. Schematic representation of the functional domain organization of nuclear

receptors. A) A/B, transactivation domain (AF1). C, DNA binding domain (DBD), D, hinge

regions, E, ligand binding domain (LBD), F, important domain for ERα function. B) The

percentages of identity with human ERα are given for the DBD and LBD of human ERβ, human PR (Ruff et al., 2000). 11

activation function-2 (AF-2). In ERα, the F domain has been shown to play a role in distinguishing estrogen agonists versus antagonists, perhaps through interaction with cell-specific factors (Montano et al., 1995).

On binding to the ligand, ER is activated, in which the conformation of ER is changed by dissociation of hsp90, hsp70 and other proteins (reviewed in (Klinge et al., 1997b)) and forms a ligand occupied ER dimer (Devin-Leclerc et al., 1998). The stimulation of gene expression in response to ligand (estrogen (E2) or other agonists) has been suggested to occur by either 1) “cis- acting” which is direct binding of activated ER to the specific sequences in the DNA called an estrogen response element (ERE) followed by protein-protein interaction with other co-activator proteins and components of the RNA polymerase II transcription initiation complex (Klinge,

2000), or 2) “trans-acting” which has ER interacting with other DNA bound transcription factor to stabilize the DNA binding of these transcription factor as well as recruiting co-activators to the complex. In the second type of interaction, ER does not bind to the DNA directly, but regulates gene expression by its interaction with other transcription factores, such as with Sp1 in conferring estrogen responsiveness on uteroglobin (Scholz et al., 1998), insulin-like growth factor binding protein-4 (Qin et al., 1999), TGFα (Vyhlidal et al., 2000). However, the focus of this study is to determine the regulation of a target gene by ER directly interacting with different

EREs.

Along with glucocorticoid, mineralocorticoid, progesterone and androgen receptors (GR,

MR, PR and AR, respectively), ERs are considered class I nuclear receptors (NR) that bind to

DNA as homodimers. ER binds to the common response element viz., the consensus ERE: 5'-

GGTCAnnnTGACC-3' (Klein-Hitpass et al., 1986). This ERE sequence is different from other response elements (Beato et al., 1989; Nelson et al., 1999). GR binds 12

with highest affinity to 5'-[GG (T/G) ACA (G/T) G (G/A)] GGTACAnnnTGTTCT-3'; AR binds with highest affinity to 5'-GGTAC (A/G) cgg TGTTCT-5'; and PR binds 5'- [(G/A) G (G/T) AC

(A/G)] tgg TGTTCT-3', where the nucleotides in the parenthesis indicates approximately equal preference for either nucleotide (Nelson et al., 1999).

These class I NRs are different from class II NRs (retinoic acid receptor (RAR), retinoid

X receptor (RXR), vitamin D receptor (VDR), thyroid receptor (TR) and peroxisome proliferator activated receptor (PPAR)). Class II NRs binds as a heterodimer with RXR to their response elements, which uses direct repeats of 5'-AGGTCA-3' with various spacers (Glass et al., 1997). The NR superfamily also includes ‘orphan receptors’. These receptors have their ligands either unknown e.g. chicken ovalbumin upstream promoter transcription factor (COUP-

TF) or have recently been identified, e.g. the pregnane X receptor/steroid X receptor

(PXR/SXR) that binds steroids and xenobiotics (Blumberg and Evans, 1998). The DBD of these nuclear receptor family is highly conserved and its ligand binding domain are variable (Ogawa et al., 1998). This accounts for the similarities in mechanisms of DNA binding and transcriptional activation among NR superfamily members.

In regards to the genes regulated by direct binding of ER to EREs, many in vitro binding studies have demonstrated that estrogen receptor recognizes and binds strongly with an extended 15bp consensus estrogen response element “5’-AGGTCAnnn TGACCT-3’” (Das et al., 2004; Driscoll et al., 1998; El Marzouk et al., 2008; Klinge et al., 1997a; Klinge et al., 1992;

Loven et al., 2001). Surprisingly, this high affinity 15bp inverted palindromic (perfect) sequence of cERE is not widely found in the genes of humans and other species (Tables 30-36, appendix II). Only a handful of 15bp cERE has been found in estrogen responsive genes, such as the vitellogenin A2 in a African wild frog (Klein-Hitpass et al., 1988), gene regulated by 13

estrogen in breast cancer protein (GREB1) (Ghosh et al., 2000) , sulfite oxidase (SUOX) gene

(Charpentier et al., 2000), and glyceraldehyde-3-phosphate dehydrogenase (GAPD) gene in humans (Revillion et al., 2000). Only a few genes have been screened to contain a minimal consensus sequence for estrogen response element, which is a 13bp perfect inverted palindrome

5’GGTCAnnnTGACC3’ (Table 31, appendix II). However, many EREs found in estrogen responsive genes are imperfect (Table 32, appendix II), which are non-palindromic since they deviate from the consensus sequence. Some of them have one of their consensus half sites retained while others have both the half sites that deviate from the consensus sequence. Also, multiple copies or tandem repeats of either consensus ERE or imperfect EREs have been found in estrogen responsive genes (Table 33, appendix II). Apart from perfect, imperfect and multiple copies of EREs, half site of consensus ERE (cHERE), multiple copies of cHEREs, or variant spacer EREs in which two of the half sites are separated by different number of nucleotides other than 3bps found in the consensus ERE have been extensively reported (Tables 34-36, appendix II). In addition, some genes regulated by estrogen on ER action have been reported to contain HEREs accompanied by Sp1 binding sites (O'Lone et al., 2004). In vitro binding assays with different EREs (single cERE, direct repeats of cERE, single cHERE, direct repeats of cHERE, and cERE half-sites separated by variant spacers “n”) it was observed that binding of

ER exhibits co-operativity for tandem repeats of cEREs and cHEREs (Das et al., 2004; El

Marzouk et al., 2008; Ghattemani, 2004). Also, it was observed that ER has a very weak binding affinity to a single cHERE and demonstrated the plasticity of ER-DNA complexes when it comes to a variant spacer EREs (El Marzouk et al., 2008)

A minimal core sequence of ERE, GGTCAnnnTGACC (Klein-Hitpass et al., 1986) was suggested to act as an enhancer element (Kato et al., 1992) because of its function in an 14

orientation and distance-independent manner. Since the identification of a canonical ERE,

several computational approaches have been undertaken to identify target genes based on the

presence of EREs within promoter proximal regions (Bajic et al., 2003; Bourdeau et al., 2004).

Bourdeau and co-workers screened for all EREs in the human and mouse genomes and

identified in excess of 70,000 EREs within the , over 17,000 of which were

within 15 kb of mRNA start sites. Likewise, genome-wide analysis of ER binding sites from

Brown’s lab reported only 4% of ER binding sites at 1kb promoter proximal regions (Carroll et

al., 2006). However, in a genomic study within a set of 58 direct E2 target genes based on ER-

dependent Pol II occupancy, one third of these direct E2 target genes contain promoter-proximal

ERα-binding sites (Kininis et al., 2007).

3. Progesterone receptor

The progesterone receptor (PR) also exists in two forms, PR-A and PR-B (Fig. 3). PR-A and PR-B are identical in amino acid sequence with the exception of an additional 164 aa on the

N-terminus of PR-B (Horwitz and Alexander, 1983; Kastner et al., 1990). Although they are similar, the two PR receptors exhibit different transcriptional activities. PR-B acts as a strong transcriptional activator, while PR-A acts as only a weak activator or in many cases, a repressor of transcription (Giangrande et al., 2000; Giangrande et al., 1997; Wen et al., 1994). In this study we have used only PR-B and referred to it as PR.

PR-A and B share similar structural features of the nuclear receptor superfamily, distinguished from other nuclear receptors by three additional features (Fig. 3). These are (1) an amino-terminal inhibitory domain (2) a B upstream sequence or “BUS”, which is only present in

PR-B, and (3) a small carboxy-terminal repressor region (Giangrande et al., 1997; Xu et al.,

1996). Although the inhibitory domain is present in both isoforms, it functions as a 15

Figure 3. Schematic diagram of human PR-B and PR-A. BUS: B upstream sequence for human PR-B. ID: inhibitory domain. AF1: activation function 1 domain. DBD: DNA binding domain. AF2: activation function 2 domain. HBD: hormone (ligand) binding domain

(Kimbrel, 2003).

16

repressor only within PR-A. If the inhibitory domain is removed, PR-A turns into a strong

transcriptional activator similar to PR-B (Giangrande et al., 1997). The repressive influence of

the inhibitory domain in full-length PR-B is overcomed by the BUS region. Therefore, the BUS

region allows PR-B to function as an activator of transcription, most likely by inducing

conformational changes that cause the inhibitory domain to be inactive (Xu et al., 1996). C-

terminal repressor region differs from that of other nuclear receptors, in which the co-repressor proteins are thought to bind to this region of the LBD when a ligand is not bound to it and are displaced when PR agonists binds to it. However, the binding of PR antagonist does not displace these co-repressors, and therefore they are able to contribute to the repression of the antagonist-

bound receptor (Wagner et al., 1999).

4. Chromatin/Nucleosome

Chromatin is a compaction of nucleosomes. The DNA in a nucleosome is wrapped

around an octamer of core-histones, composed of two copies of each of the four canonical

histones (H2A, H2B, H3, and H4) (Luger et al., 1997). The nucleosome core particle (~206kDa)

is basically composed of approximately 147 base pairs of DNA wrapped 1 and 3/4 times around

an octamer of core histone proteins (Arents et al., 1991; Luger et al., 1997; Van Holde, 1989).

Individual nucleosomes are linked together by short stretches of linker DNA of variable length

(10–90 base pairs), stabilized by linker histone H1, to form oligonucleosomal fibers that are

folded or coiled into a 30-nm diameter fiber of unknown configuration (Van Holde, 1989;

Wolffe, 1998; Woodcock and Dimitrov, 2001). The packaging of the nucleosome looks like

“beads on a string” and can be condensed to form the chromatin (Ehrenhofer-Murray, 2004).

These chromatin fibers are further coiled and compacted to from higher order chromatin structures (Belmont and Bruce, 1994) 17

4.1. Nucleosome Structure

The structure of the nucleosome resembles a disc of 11 nm diameter and 5.7 nm high

(Luger et al., 1997). About 75% of the core histone resides in the interior of the nucleosome including the “histone fold” domains. These domains are composed of structurally similar three-helix “handshake” motifs, which interact to form heterodimers of (H2A/H2B) and a heterotetramer of (H3/ H4) (Arents and Moudrianakis, 1993). In physiological salts, the

H2A/H2B dimer is a stable unit while the H3/H4 dimer forms a tetramer (Eickbush and

Moudrianakis, 1978; Karantza et al., 1995; Karantza et al., 1996). Within the nucleosome, two dimers and a tetramer are joined in an end-to-end fashion to form a tripartite protein helix, (H2A/H2B)–(H4/H3)–(H3/ H4)–(H2B/H2A), around which the nucleosome core DNA is wrapped (fig 4). The minor groove of the DNA helix makes contact with the core histones at ~14 points with > 120 direct atomic interactions within the nucleosome (Luger, 2003)).

The other ~25–30% of the mass of the core histones contains largely unstructured

“tail domains”, which are also evolutionarily highly conserved (Figure 5) (Zheng and Hayes,

2003). These domains are characterized by their sensitivity to proteases and are present in all four core histones at N-terminus while H2A contains a tail domain also in the C-terminus

(Figure 5). In the X-ray crystal structure, these tail domains are seen to follow the minor groove of the DNA to the exterior of the nucleosomes (Harp et al., 2000; Luger et al., 1997;

White et al., 2001). The tail domain of H2A and H4 pass through the minor grooves from above and below the top and bottom of superhelical turns whereas the tails of H3 and H2B pass through the minor grooves amid the adjoining DNA superhelical gyres (Figure 4). This exposes the tail domains to the exterior and is easily available for modification by the action

18

Figure 4. Nucleosome core particle: ribbon traces for 146bp DNA phosphodiester backbones (brown and turquoise) and eight histone protein main chains (blue: H3; green: H4; yellow: H2A; red: H2B. The views are down the DNA superhelix axis for the left particle and perpendicular to it for the right particle. For both particles, the pseudo- two fold axis is aligned vertically with the DNA centre at the top (Luger et al., 1997). 19

Figure 5. Schematic diagram of core histones showing tail domains. Helical regions found within the crystal structure of the nucleosome core are indicated as cylinders and the three helices comprising the histone fold domain in each protein are indicated by the gray box. The red T’s indicates the peptide bond closest to the histone fold domain susceptible to trypsin proteolysis in the nucleosome core. The vertical blue arrows indicate the approximate point where the tails exit either through (H2B and H3) or over/under (H2A/H4) the DNA superhelical gyres to the exterior of the nucleosome (Zheng and Hayes, 2003). 20

of enzymes. In addition, these domains facilitate internucleosomal interaction to from the

higher order chromatin structure.

4.2. Chromatin Remodeling

Chromatin remodeling implies the action of a complex containing an ATPase (or the

ATPase alone) that changes the structure of the nucleosome, in which the DNA-histone

interactions are weakened to alter the distribution of DNA. To a greater extent, disruption of

nucleosome such as displacement of histone dimers is also considered as chromatin

remodeling (Aalfs and Kingston, 2000). This remodeling of the chromatin is achieved by a

group of protein complexes called as chromatin remodeling complexes (CRCs). In eukaryotes, DNA is organized into chromatin, which makes segments of the DNA much less accessible to the proteins that act on the nucleosomal DNA. As a result, the cis-acting transcription factors may not easily gain access to the specific regions to transactivate the target gene. Therefore, CRCs alter the structure of nucleosomes to enhance the binding of cis-acting elements to the nucleosome to facilitate the downstream processes like transcription, replication and DNA repair and recombination (Clapier and Cairns, 2009).

One of the mechanisms to reorganize the nucleosome is by the action of ATP- dependent remodeling complexes. The ATP-dependent remodeling complexes: SWI/SNF,

NURF, RSC, CHRAC, and ACF have been reported to enhance the binding of transcription factor as well as to facilitate the restriction enzyme digestion of nucleosomal DNA (Cairns et al., 1994; Cairns et al., 1996; Cote et al., 1994; Ito et al., 1997; Kwon et al., 1994; Peterson and Herskowitz, 1992; Tsukiyama et al., 1994; Tsukiyama et al., 1995; Varga-Weisz et al.,

1997). 21

An ATP-dependent chromatin remodeling complex, SWI/SNF, is widely studied and

shown to remodel the nucleosome structure to facilitate transcription factor binding (Cote et

al., 1998; Cote et al., 1994; Imbalzano et al., 1994; Kwon et al., 1994; Utley et al., 1997).

Imbalzano reported that ATP-dependent hSWI/SNF complex alters the rotational phasing of

DNA in a nucleosome to facilitate the binding of TBP to the nucleosome as well as allows

DNase I to produce additional 15bp cuts to the 10bp DNase I pattern (Imbalzano et al.,

1994).

Furthermore, another SWI/SNF related chromatin remodeling complex, RSC, has

been shown to alter the nucleosome structure in an ATP-dependent manner (Lorch et al.,

1998; Saha et al., 2002). These chromatin remodeling complexes are associated with a HMG box containing proteins, such as BAF57 in SWI/SNF complex, BAP111 in Drosophila

Brahma. BAF57 subunit in the SWI/SNF complex has been reported to be a critical regulator of ER function in breast cancer cells (Garcia-Pedrero et al., 2006).

In yeast, facilitator of chromatin transactions (yFACT complex) promotes the reorganization of the nucleosome structure in an ATP-independent manner (Rhoades et al.,

2004; Ruone et al., 2003). This reorganization does not change the translational position of

DNA in the nucleosome (Rhoades et al., 2004). Human FACT, on the other hand, facilitates histone H2A/H2B dimer displacement in the nucleosome (Belotserkovskaya et al., 2003;

Reinberg and Sims, 2006) and has been shown to stimulate transcriptional elongation by

RNA polymerase II (Belotserkovskaya and Reinberg, 2004; Mason and Struhl, 2003). The human FACT complex comprises of Spt16/p140 and SSRP1 proteins (Orphanides et al.,

1999). A DNA-binding motif of the HMGB family is present in SSRP1 protein in human

FACT, whereas it is absent in its yeast homologue Pob3. Interestingly, yFACT complex is 22

associated with another protein, Nhp6, which contains a single ~70-residue high mobility group (HMG) box motif of the type found in HMGB family (Bustin, 2001). It was shown that multiple Nhp6 molecules facilitated the recruitment of Spt16-Pob3 to form the yFACT complex to remodel the nucleosome in an ATP-independent manner (Ruone et al., 2003).

Nhp6 subunit of yFACT has been shown to promote the formation of TBP-TFIIA-TATA complex (Eriksson et al., 2004), indicating its functional importance in transcriptional initiation and elongation. Likewise, an ATP-dependent remodeling complex SWI/SNF and

Gcn5 histone acetyltranferase are also reported to promote the formation of TBP-TFIIA-

TATA complex (Biswas et al., 2004). 23

Objectives

The main objectives of this dissertation are to determine:

1. The influence of HMGB1 to stimulate in vitro binding of ER to non-conventional EREs

translates into functional activity (gene expression) in cells.

2. The role of HMGB1 on estrogen responsive gene expression

3. The influence of HMGB1 on nucleosome structure.

24

CHAPTER II: MATERIALS AND METHODS

1. Plasmids and Cell Types

1.1. Plasmids

A firefly luciferase reporter vector “pGL2-3cERE-TATA-Inr-luc” was a gift from Dr.

D. P. McDonnell (Duke University) (Hall and McDonnell, 1999). This vector (Fig. 6)

consist of a “TATA-Inr” sequence at the 5’- region of the firefly luciferase gene in the pGL2

vector backbone (Promega), and three tandem cEREs between Xho I and Bgl II restriction

sites, upstream of the TATA sequence. Using this, other luciferase reporter vectors were

constructed with various EREs (a single cERE, tandem repeats of cEREs, a single cHERE,

tandem repeats of cHEREs, or cEREn with different spacer sizes “n” from n=0-4 ) upstream

of the firefly luciferase gene. The location of EREs with respect to TATA box has been

shown to influence the reporter gene activity mediated by ER on estrogen induction (Nardulli

et al., 1996). It has been reported that EREs placed at 3.6 helical turns away from TATA

sequence upstream of the reporter gene showed higher transcriptional activity of reporter

gene on exposure to estrogen (Nardulli et al., 1996). Therefore, all the reporter constructs

were designed and constructed by placing the nearest EREs (a single cERE, tandem repeats

of cEREs, a single cHERE, tandem repeats of cHEREs, or cEREn with different spacer sizes

“n” from n=0-4 ) 3.6 helical turns away from the TATA sequence. The distance was

measured from center of the TATA sequence to the center of the ERE.

The mammalian expression vector for (pCMVflag:hERα) was

provided by Dr. A. N. Nardulli (University of Illinois, Urbana-Champaign, IL) (Loven et al.,

2001; Reese and Katzenellenbogen, 1991) and Dr. D. P. Edwards (Baylor College of

Medicine, Houston, TX) provided the mammalian expression vector for HMGB1 (pHMGB1) 25

Figure 6. Plasmid map for pGL2-3cERE-TATA-Inr-Luc. A pGL2 basic vector with firefly luciferase gene, in which TATA-Inr sequence has been subcloned at Bgl II and Hind

III site, and three tandem cEREs subcloned at Xho I and Bgl II site upstream of the luciferase reporter gene. 26

and the empty vector pBluescript (Boonyaratanakornkit et al., 1998). A control reporter

vector with Renilla luciferase gene (pGL4.70 hRLuc) was purchased from Promega (Cat. #

E6881).

A plasmid “pGEM-Q2”containing 161bp DNA with a 15bp GRE (5’-

TGTACAggaTGTTCT-3’), corresponding to the -2509/-2495 segment of tyrosine aminotransferase gene (Jantzen et al., 1987; Li and Wrange, 1993), between EcoR I and

Hindi III was provided by Dr. Ronald Peterson (Ohio Northern University). The DNA was designed such that the GRE would be translationally positioned at the dyad axis within the nucleosome and rotationally phased such that the major groove of the GRE faces away from the core histones to have the optimum PR/GR binding in a nucleosome. The GRE is rotationally phased by using four nucleosome positioning sequences (NPS), two on each side of the GRE sequence (Fig. 7). This DNA fragment is referred to as 2G2.

Table 3: Plasmids used in studies

Antibiotic Vectors Protein Expressed Source Resistance pGL2-3cERETATA-Inr-Luc Firefly luciferase Ampicillin D. P. McDonnell pCMVflag:hERα Estrogen receptor α Ampicillin A. N. Nardulli pGL4.70(hRLuc) Renilla luciferase Ampicillin Promega pHMGB1 HMGB1 Ampicillin D.P. Edwards pBluescript None Ampicillin D. P. Edwards p2G2* None Ampicillin R. C. Peterson

27

Figure 7. A schematic diagram of 2G2 DNA: A 161bp DNA fragment containing 15bp

GRE “5’-TGTACAggaTGTTCT-3’” within a 30bp DNA segment flanked by two 20bp nucleosome positioning sequences on either side. This piece of DNA is named as 2G2, as a

GRE sequence is at the middle of the DNA surrounded by two NPSs on either side. The 5’ end and 3’ end are not blunt, but they have the EcoR I over hang at 5’ end and Hind III at 3’ end. 28

1.2. Cell Types

1.2.1. U2OS Cells

An estrogen receptor negative human osteosarcoma cell line (U2OS) was

provided by Dr. Ann Nardulli (University of Illinois, Urbana-Champaign, IL) (Schultz et

al., 2005). This cell line was used for the transient transfection studies with ERE driven

luciferase reporter vector. This cell line was chosen because it does not express ERα and

ERβ (Schultz et al., 2005), which makes an ideal cell line for the study where we can

examine the effect of estrogen receptor on luciferase reporter activity by exogenously

transfecting ER expression vector. This allows us to control the amount of estrogen

receptor and its effect on transactivation.

U2OS cell lines were received in dry ice in a 2 mL cryo freeze vials. The cells

were immediately transferred to the liquid nitrogen tank at Dr. Tami Stevenson’s lab

(Room # 406, Fourth floor Biology Department). These cells were later thawed in a 37oC

water bath and grown and maintained in the U2OS maintenance medium at 37oC in a

non-CO2 incubator by changing the media every two days, and splitting the confluent

cells. These cell lines were placed in liquid nitrogen (Dr. Tami Stevenson’s Lab Room #

406, Fourth floor Biology Department) for long term preservation.

1.2.2. Escherichia coli Cells

E. coli JM109 strain (Promega, Cat. # L1001) was used for transformation and

plasmid amplification. These cells were received in frozen state in a dry ice package. The

cells were stored in -80oC freezer. Luria-Bertani (LB) broth (appendix V) containing 100

µg/mL ampicillin was used to screen for cells that contained plasmids. LB agar

containing 100 µg/mL ampicillin was used to isolate colonies of the transformed cells. 29

2. Promoter Sequences for Regulating Extent of Activated Expression of Luciferase Gene

An array of consensus and non conventional estrogen response elements were used to

examine their regulatory effect of ER mediated transactivation of luciferase reporter gene. These

various estrogen response elements were cERE and its tandem repeats, cHERE and its tandem

repeats, and the spacer variants of cEREn (n= 0,1,2,3 & 4, where n is the number of nucleotides

in between two half sites of cERE-the so-called spacer). These ERE sequences (Tables 4, 5 and

6) were constructed synthetically as a single strand DNA (coding and non-coding strands) by

Integrated DNA Technologies Inc. They were designed in such a way that, when two single strands of each DNA insert are hybridized, they produce Xho I and Bgl II overhangs at 5’ and 3’ ends, respectively, as shown in figure 8. These regulatory promoter sequences were then cloned at Xho I and Bgl II restriction site upstream of TATA box in a pGL2 -TATA-Inr luciferase reporter vector.

2.1. cERE

Table 4: Sequences for coding and non coding strands of single and tandem cEREs

30

2.2. cHERE

Table 5: Sequences for coding and non coding strands of single and tandem

cHEREs

2.3. cEREs with Different Spacer Sizes

Table 6: Sequences for coding and non coding strands of cEREn with different spacer sizes, when n=0, 1, 2, 3, 4 bps in between two half sites of the cERE

3. Transformation of E. coli Cells with Plasmids

3.1. Preparation of Competent Cells

3.1.1. Cell Growth

A vial of frozen E. coli JM109 cells was thawed on ice and a loop of bacteria was

streaked on a LB agar plate. The plate was incubated overnight at 37oC in the incubator.

An isolated individual colony was picked up from the overnight culture and inoculated

into 10 mL LB broth and incubated at 37oC for 8 hrs with constant shaking at 250 rpm 31

Figure 8. A schematic representation of DNA hybridization to produce Xho I and Bgl II overhangs. In this figure, 2cERE insert for the pGL2-TATA-Inr-Luc is demonstrated. Coding and non-coding strands of the 2cERE insert were designed and synthetically constructed from

IDT Inc. with DNA sequences as shown. They were allowed to hybridize to get double stranded

DNA. As they were designed, they produce Xho I overhang at 5’ end and Bgl II overhang at 3’ end. The bottom DNA sequence shows the inserted DNA sequence in luciferase reporter gene with complete Xho I and Bgl II restriction sites. Likewise, all the other inserts were designed to hybridize accordingly. 32

3.1.2. Preparation of Competent Cells

A 5 mL aliquot of 8 hr culture of JM109 cells was inoculated into two 500 mL of

LB broth and incubated at 37oC for two hours with constant shaking at 250 rpm until

o O.D550=0.5-0.8. The cells were collected by spinning in Sorvall GSA rotor at 4 C for 10

min at 3000 x g. The cells were resuspended in 250 mL of ice cold sterile 0.1 M CaCl2, and then centrifuged at 3000 x g for 10min at 4oC in Sorvall GSA rotor and pellets were then gently resuspended in 250 mL of ice cold sterile 0.1 M CaCl2. The cell suspension

was incubated on ice in cold room for 8-10 hrs. The cells were then collected by spinning in Sorvall GSA rotor at 4oC for 10 min at 3000 g. The collected cells were gently

resuspended in 43 mL of ice cold 0.1 M sterile CaCl2 and 7 mL of 100% glycerol. The

cell suspension was aliquoted to 500 μL Eppendorf tubes and stored at -80oC.

3.1.3. Transfection of Plasmid into Competent Cells

The competent cells from -80oC were thawed on ice. At the same time, a 1.5 mL

Eppendorf tube was also chilled on ice. To 50 μL of the competent cells, 2 μL (5-10ng) of plasmid DNA was added in a pre-chilled Eppendorf tube and incubated on ice for 30 minutes. The DNA-competent cell mixture was incubated in 42oC water bath for 40

seconds for heat shock treatment and it was immediately placed back on ice for 2

minutes. A 250 μL of SOC medium (appendix V) at room temperature was added to it and incubated in a 37oC water bath for 1 hr. Following incubation, a 100 μL of the culture

was plated in LB agar plate containing 100 μg/mL ampicillin and incubated at 37oC for

16-18 hrs. The growth of transformed cells was observed as individual colonies.

33

4. Construction of Luciferase Reporter Constructs

4.1. Restriction Digestion of Luciferase Reporter Vector

A 3cERE-driven luciferase reporter vector has an Initiator sequence (Inr), a TATA

box and three tandem cEREs (3cERE) (fig. 7). This vector was constructed in a pGL2-Basic

vector backbone (Promega). The 3cERE sequence is located in between Xho I and Bgl II

restriction sites. Taking advantage of the presence of Xho I and Bgl II restriction sites,

restriction digestion on the pGL2-3cERE-TATA-Inr vector with Xho I and Bgl II restriction

enzymes was performed.

Ten microgram of the plasmid DNA, pGL2-3ERE-TATA-Inr-Luc was incubated

overnight with 40 units of Xho I in a 1X reaction buffer at 37oC. Following digestion, the

enzyme was heat inactivated and 40 units of Bgl II was added for the second digestion, and

incubated overnight at 37oC. The digest was run on a 1% agarose gel in 1X TAE buffer for

mapping the restriction fragment. With the restriction digestion confirmed (sizes 5683bp and

73bp), the total digest was run in a 6% polyacrylamide gel to isolate and purify the larger

fragment (5683bp). A gel slice corresponding to the DNA fragment was carefully cut out and

minced into small pieces The DNA fragment from the gel pieces was eluted overnight with

DNA elution buffer (0.5 M ammonium acetate, 10 mM magnesium acetate, 1 mM EDTA) at

37oC. The eluted DNA was concentrated by ethanol precipitation. To the eluted DNA, 1/10th

volume of 3 M sodium acetate and 2 volumes of ice cold absolute ethanol was added, mixed

well, and incubated at -80oC for at least 4 hrs to precipitate the DNA. The precipitate was

collected by centrifuging in a microcentrifuge at high speed for 10min at 4oC. The DNA

pellet was carefully rinsed with ice cold 70% ethanol, air dried and dissolved in TE buffer.

34

4.2. Ligation of Regulatory Promoter Inserts into the Luciferase Reporter Vector

A 3-fold molar excess of the dsERE inserts (Tables 4, 5 and 6, figure 8) were mixed

with 50ng of the linearized plasmid fragment (5683bp fragment of Xho I and Bgl II digest of

the pGL2-3cERE-TATA-Inr-Luc plasmid) in a 1X Quick ligation buffer. One µL of Quick

T4 DNA ligase (New England Bio Lab, Cat. # M2200S) was then added, mixed thoroughly

and the reaction was incubated for 5 minutes at 25oC (room temperature). After 5 minutes, the reaction mixture was chilled on ice and 2 µL of the reaction mixture was added to transform the competent JM109 E. coli cells.

4.3. Transformation of E. coli cells with Recombinant Luciferase Constructs

The competent cells from -80oC were thawed on ice. At the same time, a 1.5 mL

Eppendorf tube was also chilled on ice. To 50 μL of the competent cells, a 2 μL of the

ligation mixture was added to a pre-chilled Eppendorf tube and incubated on ice for 30

minutes. The competent cell-ligation mixture was incubated in 42oC water bath for 40

seconds for heat shock treatment. It was immediately placed back on ice for 2 minutes. A 250

μL of SOC medium was added to it and incubated at 37oC for 1 hr. Following incubation, a

100 μL of the culture was plated in LB agar plate containing 100 μg/mL ampicillin and

incubated at 37oC for 16-18 hrs. The growth of transformed cells was observed as individual

colonies. These transformed colonies may contain empty vector or the vector with ERE insert

subcloned. Using colony PCR, these colonies were screened for those containing the vector

with ERE inserts. The PCR products were then analyzed for the ERE inserts on a 6%

polyacrylamide gel.

35

4.4. PCR Screening of the Transformed Clones

Using a primer designing software – “web-primer” from

http://www.yeastgenome.org/cgi-bin/web-primer, both forward and reverse primers were

designed to amplify a segment of pGL2-TATA-Inr-Luc. The primers were designed such that

the amplified segment contained the subcloned insert of estrogen response element, the

TATA box and the Inr sequences flanked by the Xho I and Hind III site (see appendix VII for the segment of plasmid being amplified). After the primers were designed, the sequences were sent to Integrated DNA Technology Inc. for primer synthesis. A forward primer was named as pXHF and reverse primer was named XHR. The sequence of pXHF is:

5’-AGCTCTTACGCGTGCTAGCT-3’and the sequence of pXHR is:

5’-TTACCAACAGTACCGGAATGC-3’.

A 25 µL reaction mixture was set up with 12.5 µL of 10X Gotaq Green Master Mix

(Promega, Cat # M7122), 2.5 µL forward primer (10 µM), 2.5 µL reverse primer (10 µM) and 7.5 µL dH2O. An isolated individual colony from the transformed culture was touched

with the sterile pipette tip and the colony was transferred to the 25 µL PCR reaction mixture

and mixed thoroughly. The same colony was replica plated (in LB agar plate containing 100

µg/mL ampicillin) and the colony was marked “Cn”, where C stands for colony and n for the

number of colonies screened (n = 1, 2, 3…). A total of 7-9 colonies were screened for each

transformation. A PCR was performed in BioRad MJ Mini Personal Thermal Cycler at Dr.

Leontis lab, room 219 PSLB. The PCR was programmed for a cycle of: 30 seconds

denaturation at 95oC, 30 seconds annealing at 50oC, and 30 seconds extension at 72oC. This

cycle was repeated 30 times. The PCR product was analyzed in a 6% a polyacrylamide gel to

identify the transformed colony containing the recombinant vector with ERE insert. 36

4.5. DNA Sequencing of Constructs

From the polyacrylamide gel electrophoresis of PCR products, the transformed

colony containing the recombinant vector with ERE inserts were identified on the basis of the

sizes of PCR products as tabulated in table 11. Based on polyacrylamide gel analysis, the

transformed colony containing the ERE insert was grown in LB broth containing 100 µg/mL

ampicillin to isolate the recombinant plasmid. The recombinant plasmid was isolated and

purified using Qiagen HiSpeed Plasmid Maxi Kit (Qiagen). The isolated plasmid was then

sent for DNA sequencing to Retrogen Inc. for further verification of the cloned insert in the

recombinant vector. Universal Primers, GLprimer1 and GLprimer2 obtained from Retrogen

Inc. were used for DNA sequencing. Sequence for forward primer GLprimer1 is: 5' TGT

ATC TTA TGG TAC TGT AAC TG 3', and reverse primer GLprimer2 is: 5' CTT TAT GTT

TTT GGC GTC TTC C 3’. The correct sequence and alignment was verified for all the

inserts.

5. Isolation of Plasmid DNA

5.1. Cell Growth

A 1 mL of pure culture of transformed bacterial stock in a 1.5 mL Eppendorf tube

from -80oC was thawed on ice and inoculated in 5mL of LB broth containing 100 µg/mL

ampicillin, and incubated at 37oC with constant agitation at 250 rpm for 6-8 hrs. The 5 mL

(6-8 hrs) culture was added to 250 mL of LB broth containing 100µg/mL of ampicillin and

further incubated at 37oC for 12-16 hrs with constant agitation at 250 rpm.

5.2. Cell Harvesting

A pair of 250 mL centrifuge bottles was sterilized. The 250 mL (12-16 hr) culture of

bacterial suspension was then added to the sterile centrifuge bottle and spun down in a 37

Sorvall GSA rotor at 6000 g for 15 min at 4oC. The bacterial pellet harvested was

resuspended in a 10mL of resuspension buffer P1 (50 mM Tris-Cl, pH 8.0, 10 mM EDTA,

100 µg/mL RNaseA) provided with Qiagen HiSpeed Maxi Kit, Qiagen for cell lysis and plasmid isolation.

5.3. Cell Lysis and Isolation of Plasmid DNA (Procedure Supplied with Qiagen Hi Speed

Plasmid Maxi Kit)

The 10mL bacterial suspension in P1 buffer (50 mM Tris-Cl, pH 8.0, 10 mM EDTA, and 100 µg/mL RNaseA) was transferred to a sterile 50 mL Falcon tubes and 10 mL of lysis buffer P2 (200 mM NaOH, 1% SDS (w/v)) supplied with Qiagen Kit was added. Cell lysis was performed for no more than 5 minutes. After 5 minutes, 10 mL of ice cold neutralization buffer P3 (3 M potassium acetate, pH 5.5) was added. The precipitate was filtered through

Qiagen QIAfilter Cartridge equilibrated with equilibration buffer QBT (750 mM NaCl, 50 mM MOPS, pH7.0, 15% isopropanol (v/v), 0.15% Triton X-100 (v/v)). The filtrate was bound to the Qiagen-tip using only gravity flow. The tip was washed with 60mL of wash buffer QC (1 M NaCl, 50 mM MOPS, pH 7.0, 15% isopropanol (v/v)) through gravity flow.

The bound plasmid DNA was eluted with 15mL of elution buffer QF (1.25 M NaCl, 50 mM

Tris-HCl, pH 8.5, 15% isopropanol (v/v)). The eluted DNA was precipitated with 0.7 volumes (10.5 mL) of room-temperature isopropanol. The eluate/isopropanol mixture was incubated for 5 mins at room temperature. After incubation, the eluate/isopropanol mixture was filtered through QIAprecipitator using constant pressure. The precipitator was washed with 2 mL of 70% ethanol and dried by repeatedly applying pressure on the QIAprecipitator using 30 mL syringe. The QIAprecipitator was removed from the 30mL syringe and attached to the 5 mL syringe. The plunger was removed prior to the attachment of the 38

QIAprecipitator. The plasmid DNA was eluted to 1.5 mL collection tube by adding 1 mL of

TE buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA) through the QIAprecipitator using the

plunger. The DNA eluate was transferred to the 5 mL syringe again and eluted for the second

time to the same 1.5 mL tube for increased yield. The concentration of the plasmid was

determined using Nanodrop spectrophotometer, assuming A260=1 for 50 μg/mL of DNA.

6. Media Preparation for U2OS Cell Culture

6.1. Sterile Saline (0.2M)

A 4.4 g of NaCl was dissolved in about 400 mL dH2O, and the final volume was

brought to 500 mL with dH2O and mixed well. The prepared saline was autoclaved on liquid

cycle for 30 minutes and stored at ambient temperature, maintaining sterility.

6.2. Charcoal Dextran (CD)

A 50 g of charcoal (Sigma, Cat. # C-9157), a 5 g dextran (Sigma Cat. # C-4751) and a

4.4 g of sodium chloride was mixed in a 350mL of water. The mixture was autoclaved for 30

minutes on liquid cycle, cooled to room temperature and sterile water was added to 500 mL.

A cell culture bottle was used and all the preparation work was performed in a cell culture

hood.

Maintaining sterility, the CD mix was transferred to two 250 mL sterile centrifuge

bottles and centrifuged in IEC-CR6000 centrifuge at 4oC for 5 minutes at 2300 rpm. The

supernatant was decanted and discarded. In the cell culture hood, sterile water was added to

250 mL in each bottle and the CD pellet was mixed well and centrifuged again in IEC-

CR6000 centrifuge at 4oC for 5 minutes at 2300 rpm. This was repeated two times for a total

of three washes. 39

After the third wash, the CD mix was centrifuged in IEC-CR6000 centrifuge at 4oC

for 5 minutes at 2300 rpm and supernatant discarded. In the cell culture hood, sterile saline

was added to 250 mL bottles, mixed well and transferred to a sterile 1000 mL tissue culture

bottle. The mixture was shaken and swirled well enough to ensure homogenous mixture. The

homogenous CD mixture was then aliquot into 25 mL portions, shaking and swirling

between aliquots. The aliquots were labeled with “CD”, dated, and initialed, and stored at

4oC.

6.3. Charcoal-Dextran and Heat Treatment of Calf Serum

One the day prior to treatment, a 500mL bottle of the calf serum (Gibco, Cat. #

16010159) was placed in the refrigerator overnight to thaw. (NOTE: the treatment must be

completed in one day, with minimal interruptions in the procedure). The water bath was set

at 56oC and ensured that the water level was sufficient to meet serum height when the bottles

were standing in the bath. The serum was pre-warmed in a 37oC water bath.

A 25 mL portion of CD was mixed thoroughly by swirling. It was then centrifuged in

IEC-CR6000 centrifuge at 4oC for 5 minutes at 2300 rpm. The saline was aspirated in the

tissue culture (TC) hood and the 30 mL of serum was added to the CD and mixed well by

thoroughly agitating by hand and then with pipette. It was then added to the serum bottle,

mixed well and placed in 56oC water bath. The timer was set for 45 minutes and 15 minutes

for the serum to reach 56oC and 30 minutes for the incubation at 56oC. During incubation, at

every 6 minutes the CD-serum was mixed well by inverting the bottle and swirling gently.

The bottom of the bottle was observed to ensure that CD was not adhering.

In the TC hood, the contents of the bottle was transferred to two 250 mL centrifuge

bottles, and spun in Sorvall GSA rotor at 3000 rpm for 30 minutes at 4oC. The serum was 40

gently decanted into two new 250 mL centrifuge bottles and spun again in Sorvall GSA rotor at 3000 rpm for 30 minutes at 4oC. The serum was gently decanted and vacuum filtered in

TC hood. The filtered CD-calf serum (CDCS) was aliquoted into 50 mL falcon tubes. The aliquots were labeled “CDCS”, dated, and initialed, and stored at -20oC.

6.4. U2OS Medium

All the bottles of the ingredients (Minimum Eagles Medium (MEM), Non Essential

Amino Acids (NEAA), HEPES buffer, Penicillin/Streptomycin, Gentamycin, NaOH, and

Heat Inactivated Fetal Calf Serum (HIFCS)) or CDCS for the media preparation were wiped with 70% ethanol and placed in cell culture hood. Penicillin/Streptomycin and HIFCS (from the freezer) were allowed to thaw at room temperature. Using the sterile serological pipette, all the above mentioned ingredients were added into the 500 mL MEM media bottle. The compositions of all the ingredients for various U2OS media are tabulated below:

6.4.1. U2OS Maintenance Medium

Table 7: Media Composition for U2OS Maintenance Medium

Initial Final Ingredients Catalog # Volume Concentration Concentration MEM Gibco 11575 1X 500 mL 1X NEAA Gibco 11140 100X 5 mL 1X HEPES Gibco 15630 1 M 5 mL 1X Pen/Strep Gibco 11575 10,000 units/mL 5 mL 100 units/mL Gentamycin Gibco 15750 50 mg/mL 250 μL 25 µg/mL

NaOH - 1 M 1.5 mL 3 mM HIFCS Gibco 10082147 100% 75 mL 15%

41

6.4.2. U2OS Transfection Medium A

Table 8: Media Composition for U2OS Transfection Medium A

Initial Final Ingredients Catalog # Volume Concentration Concentration MEM Gibco 11575 1X 500 mL 1X NEAA Gibco 11140 100X 5 mL 1X HEPES Gibco 15630 1 M 5 mL 1X Pen/Strep Gibco 11575 10,000 units/mL 5 mL 100 units/mL Gentamycin Gibco 15750 50 mg/mL 250 μL 25 µg/mL

NaOH - 1 M 500 μL 1 mM CDCS Gibco 16010159 100% 25 mL 5%

6.4.3. U2OS Transfection Medium B

Table 9: Media Composition for U2OS Transfection Medium B

Initial Final Ingredients Catalog # Volume Concentration Concentration MEM Gibco 51200 1X 500 mL 1X NEAA Gibco 11140 100X 5 mL 1X Glutamax Gibco 35050 100X 5 mL 1X HEPES Gibco 15630 100X 5 mL 1X Pen/Strep Gibco 11575 10,000 units/mL 5 mL 100 units/mL Gentamycin Gibco 15750 50 mg/mL 250 μL 25 µg/mL

NaOH - 1 M 500 μL 1 mM CDCS Gibco 16010159 100% 25 mL 5%

42

6.4.4. Serum Free U2OS Transfection Medium B

Table 10: Media Composition for Serum Free U2OS Transfection Medium B

Initial Final Ingredients Catalog # Volume Concentration Concentration MEM Gibco 51200 1X 500 mL 1X NEAA Gibco 11140 100X 5 mL 1X Glutamax Gibco 35050 100X 5 mL 1X HEPES Gibco 15630 100X 5 mL 1X Pen/Strep Gibco 11575 10,000 units/mL 5 mL 100 units/mL Gentamycin Gibco 15750 50 mg/mL 250 μL 25 µg/mL

NaOH - 1 M 500 μL 1 mM

6.5. Quality Control

A 100 µL of the prepared media was inoculated in LB agar plates and incubated at

37oC overnight to monitor a possible bacterial contamination.

7. Maintenance of U2OS Cells

7.1. Cell Growth

A 2 mL (~6 x 105 cells) vial of frozen U2OS cell suspension in maintenance medium

with 10% glycerol was taken out from liquid nitrogen tank and thawed at 37oC water bath.

During thawing, 5mL of fresh maintenance medium was added to a T-25 flask. After the cell

suspension was thawed, it was aseptically transferred to the 5 mL of maintenance medium in

a T-25 flask. The cells were uniformly distributed by gently rocking the T-25 flask in east-

west and north-south direction for four to five times. The distribution of cells were verified

under the microscope (Nikon TMS Inverted Phase Contrast Microscope) and incubated at 43

o 37 C in a non-CO2 incubator. The next day, the media was aspirated off and the cells were

washed twice with sterile 1X PBS buffer. After washing, a 5 mL of fresh maintenance

o medium was added to it and incubated at 37 C in a non-CO2 incubator. Every two days, the

old medium was aspirated off and the cells were washed with sterile 1X PBS buffer and a

o new maintenance medium was added and incubated further at 37 C in a non-CO2 incubator

until confluent. Confluent cells were splitted and maintained in 10mL of maintenance

medium in a T-75 flask.

7.2. Cell Splitting

The cell culture in a T-25 flask was first observed under the microscope to determine

the state of confluence and observe the cell morphology. The maintenance medium stored at

4oC and 1X (0.05%) Trypsin-EDTA (Hyclone, Cat # SH 30236.01) stored at -20oC was thawed in a 37oC water bath. The working surface of the cell culture hood was sterilized with

70% ethanol. Also, gloved hands were wiped with 70% ethanol. A T-75 flask was taken and its surface was wiped with 70% ethanol. After thawing the media and 1X Trypsin-EDTA, the water on the surface of its containers was wiped thoroughly. The surface of the media bottle and Trypsin-EDTA bottle were also wiped with 70% ethanol. Sterile Pasteur pipette was attached to the vacuum suction and were flamed over the Bunsen burner to assure sterility. It was important to ensure that all the necessary items were there in the TC hood, and all of them were properly sterilized. The caps of the new T-75 flask were unscrewed and kept upright on the surface of the safety hood. The openings of the new T-75 flask were flamed to further decrease possible contamination. A 9mL of fresh maintenance media was added to the new T-75 flask. The cap(s) of the T-25 flask with the cell culture was unscrewed and its opening was also flamed. The flask with the cell culture was gently tilted to collect the old 44

media, and with the Pasteur pipette in vacuum suction, the old media was drawn off. It was

drawn from the other surface, opposite to the surface on which the cells were adhered. The

cells were washed with sterile 1X PBS buffer twice and a 500 µL of 1X Trypsin-EDTA was added to the T-25 flask and the flask was gently rocked in east-west north-south direction for three to four times and incubated at 37oC for 30 seconds to trypsinize the cells. Trypsin was

removed very carefully using the vacuum suction. A 4 mL of fresh maintenance medium was

added to T-25 flask and the cells were uniformly suspended to the media using the

serological pipette. One mL of the cell suspension was added to 9 mL fresh maintenance

medium in a T-75 flask and distributed uniformly by rocking in east-west north-south

direction for three to four times. The caps and the openings of the T-75 flasks were flamed

over the Bunsen burner and the caps were screwed loosely. It was then incubated at 37oC in a

non-CO2 incubator. Every two days, the old medium was aspirated off and the cells were

washed with sterile 1X PBS buffer. A fresh maintenance medium was then added and

o incubated further at 37 C in a non-CO2 incubator until confluent.

7.3. Cell Preservation

The cells were observed under the microscope to examine if they reached the

subconfluency level (70-80% confluent). Once the cells reached subconfluency, old media

was aspirated off and the cells were washed two times with sterile 1X PBS buffer and

subjected to trypsinization. The cells were detached and a 5 mL of fresh maintenance

medium containing 10% glycerol was added to the T-75 flask. The cells were suspended well

and 1 mL of it was aliquoted in cryo vial. The vials were labeled “U2OS cells”, “Passage #”,

dated, and initialized. The labeled vials were kept in Nalgene Cryo Freezing Container and

stored at -80oC for overnight to allow slow and synchronized freezing. The next day, the 45

vials from -80oC were transferred to liquid nitrogen at Dr. Tami Stevenson’s Lab in Biology,

Fourth Floor Room # 406.

8. Transient Transfection of U2OS Cells (Figure 9 shows a schematic diagram for transient

transfection of U2OS cells)

8.1. Cell Growth

The U2OS cells were maintained in T-75 flasks until confluent by changing the

medium every two days. The old medium was aspirated off with the sterile Pasteur pipette

attached to the vacuum suction and the cells were washed twice with sterile 1X PBS buffer

and a 10mL of fresh maintenance medium was added. Once 100% confluent, cells were

o splitted at 1:1 ratio into T-75 flasks and incubated at 37 C in a non-CO2 incubator. The next

day, the old maintenance medium was aspirated off and the cells were washed twice with 10

mL of sterile 1X PBS buffer. A 10 mL of transfection medium A was then added to it and

o further incubated at 37 C in a non-CO2 incubator. The next day, the medium was aspirated

off and the cells were washed twice with 10mL of sterile 1X PBS buffer. A 10 mL of

o transfection medium B was then added to it and further incubated at 37 C in a non-CO2

incubator. The next day, the medium was aspirated off and the cells were washed twice with

10 mL of sterile 1X PBS buffer. The cells were trypsinized with 1 mL of 1X Trypsin-EDTA

and resuspended in the transfection medium B for cell counting and then plating for

transfection.

8.2. Cell Counting

All the surfaces of a hemocytometer were cleaned with 70% ethanol. Cover slip was

also cleaned with 70% ethanol and placed at the center of the hemocytometer. A 500 µL

Eppendorf tube was taken and labeled. A 25 µL of the 0.4% (w/v) of trypan blue (Gibco, Cat 46

Figure 9. Schematic diagram for transient transfection of U2OS cells 47

# 25-900-CL) stain was added to the tube and a 225 µL of cell suspension was then homogenously mixed with the dye in the tube. A 10 µL of the homogenous mixture of the dye/cell suspension was loaded into the loading groove of the hemocytometer. Injecting bubbles into the chamber was avoided and the chambers were not overfilled. The cell suspension was allowed to settle for at least 10 seconds. All the cells in each of the four

1mm3 corner squares labeled A through D as shown in the figure given below was counted.

The cells touching the TOP and the LEFT borders were COUNTED while cells touching the

BOTTOM and the RIGHT borders were NOT COUNTED.

Figure 10. Schematic diagram of hemocytomer showing the four corners labled A, B,

C and D.

Calculation of the Total Number of Cells:

Number of cells = n X volume of sample loaded X dilution factor 0.9 Where, n = the average cell count per square of the four corners counted.

Total number of cells = cells/μL X total volume 48

8.3. Cell Plating

The transfection medium B was removed from T-75 flask and the cells were washed

twice with 10 mL of sterile 1X PBS buffer. After washing, the cells were detached from the

T-75 flasks by trypsinizing. One mL of 1X Trypsin-EDTA was added and gently swirled

around the flask, and incubated at 37oC for 30 seconds. After incubation, the trypsin was carefully aspirated and the cells were shaken off the sides of the flask by tapping, and resuspended in a 10 mL of transfection medium B. Total number of cells were counted using a hemocytometer, and the cells were diluted with transfection medium B to make the final concentration of the cell suspension ~4X105 cells per mL (total volume = 25 mL). Then,

1mL of the cell suspension was dispensed into each well of 24-well plate and incubated at

o 37 C in a CO2 incubator for the next day. A 5% CO2 concentration was maintained in a CO2

incubator.

8.4. Preparation of DNA/Lipofectamine LTX/PLUS Reagent Mixture

All the plasmids- reporter vectors pERE-Luc and pGL4.70, expression vectors pERα

and pHMGB1or pBluescript were diluted in 1X PBS buffer. In the experiments conducted

without pHMGB1, an equal amount (µg) of empty vector (pBluescript) was transfected. A 2

µL of PLUS reagent (Invitrogen, Cat # 15338-100) were added per 2 µg of total DNA to be

transfected per well, mixed gently and incubated at room temperature for 5 minutes. Then, a

5 µL of Lipofectamine LTX (Invitrogen, Cat # 15338-100) was added per 2 µg of total DNA

to be transfected per well in a 24-well plate, mixed thoroughly and incubated at room

temperature for 30 minutes.

49

8.5. Transient Transfection

The transfection medium B was removed from the 24-well plate and the cells were

washed with 500 µL of serum free transfection medium B. A 500 µL of serum free

transfection media B was then added to each well of the 24-well plate. A

DNA/Lipofectamine LTX/PLUS reagent mixture was added such that each well received 1

µg of pERE-Luc, 1 ng of pGL4.70hRluc, 1 µg of pHMGB1 or 1 µg of pBluescript, and 5 ng

o of pERα. The transfected cells were incubated at 37 C for 6.5 hrs +/- 30 minutes in a CO2

incubator.

9. Hormone Treatment

After 6.5 hrs to 7 hrs of transfection, the media from the wells were removed, and 1 mL

transfection medium B containing 10 nM 17β-estradiol (E2) or absolute ethanol (EtOH) was added for E2 treatment or without E2 treatment, respectively. The cells were then incubated at

o 37 C for 24 hrs in a CO2 incubator.

10. Cell Harvest

After 23 hrs +/- 1 hr, the media from the wells in the 24-well plate were removed and

washed once with 500 µL of 1X PBS buffer. A 100µL of 1X Passive Lysis Buffer (PLB) (Dual-

Luciferase® Reporter Assay Kit, Promega, Cat # E1910) was then added to each well of the 24-

well plate and incubated at room temperature for 15 minutes, constantly rocking at 50 rpm. The

lysates were collected and stored at -80oC until ready to measure the luciferase activity.

11. Dual Luciferase Assay

A Dual-Luciferase® Reporter (DLR) Assay System (Promega, Cat # E1910) was used to

measure the transcriptional activity of firefly luciferase gene under the control of various

estrogen response elements (a single cERE, tandem repeats of cEREs, a single cHERE, tandem 50

repeats of cHEREs, or cEREn with different spacer sizes “n” from n=0-4 ). Two different luciferase, Firefly (Photinus pyralis) and Renilla (Renilla reniformis, also known as sea pansy) were used in this assay. Because of their distinct evolutionary origins, they have dissimilar enzyme structures and substrate requirements. These differences make it possible to selectively discriminate between their respective bioluminescent reactions. Therefore, using the DLR. Assay

System, the luminescence from the firefly luciferase reaction is quenched by using Stop and

Glow Reaction buffer, while simultaneously activating the luminescent reaction of Renilla luciferase.

The firefly luciferase reporter is measured first by adding Luciferase Assay Reagent II

(LAR II) to generate a stabilized luminescent signal. After quantifying the firefly luminescence, this reaction is quenched, and the Renilla luciferase reaction is simultaneously initiated by adding Stop & Glo® Reagent to the same tube. The Stop & Glo® Reagent also produces a stabilized signal from the Renilla luciferase, which decays slowly over the course of the measurement.

A luciferase reporter assay was used as opposed to the conventional chloramphenicol acetyl transferase (CAT) assay because the luciferase (Luc) reporter assay has been largely improved over conventional assay methods in both sensitivity and simplicity (de Wet et al.,

1985; Seliger and Mc, 1960; Wood, 1991; Wood et al., 1984). This system yields linear results over at least eight orders of magnitude and less than 10–20 moles of luciferase can be detected under the optimal conditions (Wood, 1991). A 100-fold greater sensitivity has been reported for

Luc reporter assay over the CAT assay (Alam and Cook, 1990). A final quantitative measure of promoter activity can be obtained within 25 hours following transfection of luciferase reporter vector, compared to 108–160 hours with CAT reporter vector. Light emission significantly above 51

background (four-fold) is detectable 3 h after induction in a direct assay of extracts from transfected cells (Williams et al., 1989).

Firefly luciferase is a 61kDa monomeric protein that does not require posttranslational processing for enzymatic activity (de Wet et al., 1985; Wood et al., 1984). Thus, it functions as a genetic reporter immediately upon translation. Photon emission is achieved through oxidation of

2+ beetle luciferin in a reaction that requires ATP, Mg and O2 (Figure 11).

Figure 11. Bioluminescent reactions catalyzed by firefly luciferase.

Renilla luciferase, a 36kDa monomeric protein, is composed of 3% carbohydrate when purified from its natural source, Renilla reniformis (Matthews et al., 1977). However, like firefly luciferase, post-translational modification is not required for its activity, and the enzyme may function as a genetic reporter immediately following translation. The luminescent reaction catalyzed by Renilla luciferase utilizes O2 and coelenterate-luciferin (coelenterazine; Figure 12).

Figure 12. Bioluminescent reactions catalyzed by Renilla luciferase.

Luciferase activity was measured using a FB14 Single Tube Luminometer (Zylux

Corporation, Cat. # FB14) in Biology at Dr. Mike McKay/ Dr. George Bullerjhan’s Lab. In a 12 x 75 cm test tube, a 100 µL of LAR II was added, followed by a 20 µL of the cell lysate. It was 52

quickly mixed and placed in the FB14 Single Tube Luminometer reading chamber. The luminometer was programmed to measure the luminance for 10 seconds with a delay time of 2 seconds. After the measurement of Firefly luciferase activity, a 100 µL of the Stop and Glow reagent was added and the Renilla luciferase activity was measured. The luciferase activity was normalized by taking the ratio of Firefly luciferase Relative Light Units (RLU) and Renilla luciferase RLU.

12. Protein Assay

Bio-Rad Protein Assay was used to quantify the total protein, using a BSA plot as a standard curve.

13. siRNA Co-transfection

13.1. Preparation of siRNA for Transfection

All siRNAs were purchased from Dharmacon Inc. Five nmole of siRNA for human

HMGB1: ON-TARGET plus SMART pool (Dharmacon Cat # L-0189810-00-0005), a

positive control siRNA for glyceraldehyde 3-phosphate dehydrogenase (GAPDH): ON-

TARGET plus GAPD control pool (Dharmacon Cat # D-001830-10-05), and a negative

control siRNA: ON-TARGET plus Non-targeting pool (Dharmacon Cat # D-001810-10-05)

were resuspended in 250 µL of RNase Free 1X PBS buffer (Ambion, Cat # AM9624) to

make a 20 µM final concentration of the siRNAs. The resuspended siRNAs were stored at

-20oC.

13.2. Cell Growth

The U2OS cells were maintained in the T-75 flasks until confluent by changing the

medium every two days. The old medium was aspirated off using the sterile Pasteur pipette 53

attached to the vacuum suction. The cells were washed twice with sterile 1X PBS buffer and

10 mL of fresh maintenance medium was added. Once the cells were 100% confluent, they

o were splitted at 1:1 ratio into T-75 flasks and incubated at 37 C in a non-CO2 incubator. The

next day, the old maintenance medium was aspirated off and the cells were washed twice

with 10 mL of sterile 1X PBS buffer. A 10 mL of transfection medium A was then added to

o it and further incubated at 37 C in a non-CO2 incubator. The next day, the medium was

aspirated off and the cells were washed twice with 10mL of sterile 1X PBS buffer. A 10 mL

o of transfection medium B was then added to it and further incubated at 37 C in a CO2

incubator. A 5% CO2 concentration was maintained. The next day, the medium was aspirated off and the cells were washed twice with 10 mL of sterile 1X PBS buffer. The cells were then treated with 1 mL of 1X Trypsin-EDTA for 30 seconds at 37oC to detach the cell from the T-

75 flask. The trypsin was carefully aspirated off and the detached cells were resuspended in

transfection medium B for cell counting and then plating for transfection.

13.3. Cell Plating

The transfection medium B was removed from the T-75 flask and the cells were

washed twice with 10 mL of sterile 1X PBS buffer. One mL 1X Trypsin-EDTA was added to

it, gently swirled around the flask, and incubated at 37oC for 30 seconds to detach the cells.

The cells were shaken off the sides of the flask by tapping, and the detached cells were

resuspended in 10 mL transfection medium B. Total number of cells were counted using a

hemocytometer, and the cells were diluted with transfection medium B to make the final

concentration of the cell suspension ~4X105 cells per mL. Then, 1 mL of cell suspension was

o dispensed into each well of 24-well plate and incubated at 37 C in a CO2 incubator for the

next day. 54

13.4. Preparation of Lipofectamine 2000/siRNA Mixture

In one set of 1.5 mL Eppendorf tubes, reporter vectors (pERE-luc, pGL4.70hRluc,

pHMGB1/pBluescript, and pERα) and siRNAs were diluted in the serum and antibiotic free transfection medium B. In another set of Eppendorf tubes, Lipofectamine™ 2000

(Invitrogen, Cat # 11668-027) was diluted in serum and antibiotic free transfection medium

B, and incubated for 5 minutes at room temperature. After 5 minutes of incubation, the

diluted Lipofectamine™ 2000 was added to the diluted DNA/siRNA mixture, mixed gently

and incubated for 20 minutes at room temperature to allow the formation of complex.

13.5. Co-transfection

The transfection medium B was removed from the 24-well plate and the cells were

washed with 500 µL of serum and antibiotic free transfection B media. Then, a 500 µL of the

serum and antibiotic free transfection medium B was added. A DNA/siRNA/

Lipofectamine™ 2000 reagent mixture was added, such that each well received 1 µg of

pERE-Luc, 1 ng of pGL4.70hRluc, 100, 200 or 400 nM siRNA (for HMGB1) and 5 ng of

o pERα. The transfected cells were incubated at 37 C for 6.5 hrs +/- 30 minutes in a CO2

incubator.

13.6. Hormone Treatment

After 6.5 hrs to 7 hrs of transfection, media from the wells in a 24-well plate were

removed, and 1 mL transfection medium B containing 10 nM 17β-estradiol (E2) or EtOH

was added for E2 treatment or without E2 treatment, respectively. The cells were incubated

o at 37 C for 24 hrs in a CO2 incubator.

55

13.7. Cell Harvest / Lysis

For luciferase reporter analysis, media from the wells were removed after 23 hrs +/- 1

hr, and washed once with 500 µL of 1X PBS buffer. A 100 µL of 1X Passive Lysis Buffer

(PLB) (Dual-Luciferase® Reporter Assay Kit, Promega, Cat # E1910) was added to each

well and incubated at room temperature for 15 minutes, constantly rocking at 50rpm. The

lysates were collected and stored at -80oC until ready to measure luciferase activity.

For RNA isolation, media from the wells were removed after 23 hrs +/- 1 hr, and

washed once with 500 µL of 1X PBS buffer and direct cell lysis was performed in the wells

by adding 350 µL of the RLT buffer (Qiagen RNeasy Mini Kit, Cat # 74104). The lysates

were collected and used for total RNA isolation using Qiagen RNeasy Mini Kit (Cat #

74104).

14. Isolation of Total RNA

14.1. Cell Harvest and Lysis

The media from the wells were removed after 23 hrs +/- 1 hr, and washed once with

500 µL of 1X PBS buffer and direct cell lysis was performed in the wells by adding 350 µL

of the RLT buffer (Qiagen RNeasy Mini Kit, Cat # 74104). The lysates were collected and

used for total RNA isolation using Qiagen RNeasy Mini Kit (Cat # 74104).

14.2. Isolation of Total RNA (Procedure Supplied with Qiagen RNeasy Mini Kit, Qiagen)

One volume of 70% ethanol was added to the 350 µL of the homogenized cell lysate,

and mixed well by pipetting. The final volume of 700 μL of the lysate, including any

precipitate that may have formed was transferred to an RNeasy spin column placed in a 2 ml

collection tube supplied with the kit. The lid was gently closed and centrifuged for 15

seconds at 8000 x g (10,000 rpm). The flow-through was discarded. The spin column was 56

washed by adding 700 μL of buffer RW1 to the RNeasy spin column and centrifuged for 15

seconds at 8000 x g (10,000 rpm). A 500 μL of buffer RPE was added to the RNeasy spin

column and centrifuged for 15 seconds at 8000 x g (10,000 rpm) to wash the spin column

membrane. The flow-through was discarded. Another 500 μL of buffer RPE was added to the

RNeasy spin column and centrifuged for 2 min at 8000 x g (10,000 rpm) to wash the spin

column membrane for the second time. The RNeasy spin column was transferred to a new 2

ml collection tube supplied with the kit and centrifuged at full speed for 1 min to dry out the

wash buffer. The RNeasy spin column was transferred to a new 1.5 ml collection tube

supplied with the kit. A 30–50 μL of RNase-free water was directly added to the spin column

membrane and the total RNA was eluted by centrifuging for 1 min at 8000 x g (10,000 rpm).

To increase the yield, the eluted volume of 1 mL was added to the RNeasy spin column and

centrifuged for 1 min at 8000 x g (10,000 rpm) for the second time, and collected in the same

tube

15. cDNA Synthesis

A 500 ng of total RNA isolated was used to make cDNA. In a PCR tube, a 20 µL of reaction mixture of cDNA synthesis was set up. A 4 µL of 5X iScript Reaction Mixture, an estimated volume of total RNA to make 500ng of total RNA in the reaction mixture, and 1 µL of the iScript Reverse Transcriptase enzyme was added to the tube. Final volume of 20 µL was obtained by adding water for a difference in volume. The reaction was performed in BioRad MJ

Mini Personal Thermal Cycler at Dr. Leontis’ lab, room 219 PSLB. The thermal cycler was programmed as, 25oC for 5 min, 42oC for 30 min, 85oC for 5 min, and 4oC for 5 min.

57

16. RT-PCR

Primers for HMGB1, forward: 5’-CTC TGA GTA TCG CCC AAA AA -3’ and reverse:

5’-TTT TCA GCC TTG ACA ACT CC-3’ (Real Time Primers, Cat # VHPS-1427) and primers for GAPDH, forward: 5’-GAG TCA ACG GAT TTG GTC GT-3’ and reverse: 5’-TTG ATT

TTG GAG GGA TCT CG-3’ (Real Time Primers, Cat # VHPS-234) were obtained from Real

Time Primers. Primers were diluted to 1µM concentration and stored at -20oC. A 25µL reaction

was set up in a MicroAmp Optical 96 well plate (Applied Biosystems, Cat # N801-0560).

MicroAmp Optical Adhesive Film Kit (Applied Biosystems, Cat # 4313663) was used to seal the

96-well plate. The RT-PCR was performed in Applied Biosystems 7000series RT-PCR unit at

University of Toledo, Health Science Campus). In a 25 µL reaction mixture, 12.5 µL of 2X

iQSYBR Green Super Mix (QuantiTect SYBR Green PCR Kit, Cat # 204141, Qiagen), 2.5 µL of

1 µM Primers, 8 µL of nuclease free water, and 2 µL of cDNA template from cDNA synthesis

reaction was mixed well. The 96-well plate was centrifuged briefly to collect the reaction

mixture at the bottom of the well. Then, the plate was fed to the Applied Biosystems quantitative

real time PCR unit. The unit was programmed for quantitative RT-PCR as, 50oC for 2 minutes,

95oC for 15 minutes, a 40 cycles of [95oC for 15 seconds, 60oC for 1 minute, and 72oC for 1

minute], followed by a melt curve analysis program of 15 seconds at 95oC, 1 minute at 60oC, and

15 seconds at 95oC.

17. Isolation and Purification of HMGB1 and HMGB2 Proteins

17.1. Isolation of Nuclei

A 150 gm of calf thymus (Bellville Market, Bowling Green, OH) stored at -80oC, was

slowly thawed in the cold room at 4oC. The calf thymus was then blended to homogeneity, in

approximately 150 mL of buffer A ( 20 mM Tris-HCl, 50 mM NaCl, 1 mM EDTA, 5 mM 58

BME, 10% glycerol, 0.5 mM PMSF, pH 8.8) for 2 minutes at full speed in a blender. The

thick homogenate was filtered through two layers of cheesecloth. The relatively thick filtrate

was transferred into 8 autoclaved 50 mL plastic centrifuge tubes and sediment at 14000 x g

(Sorvall super speed RC-2, SS-34 rotor) for 40 minutes. The supernatant was discarded and

the pellet was repeatedly washed with 50 mL buffer A until no floating lipid was observed on

the supernatant. This usually required 4 washes approximately.

17.2. Salt Extraction and Ammonium Sulfate Precipitation

The pellet was then resuspended in two pellet volumes (usually about 200 mL) of

buffer B ( 20 mM Tris-HCl, 5 mM BME, 0.5 mM PMSF, pH 7.2) taken in 500 ml of

Erlenmeyer flask. The suspension was stirred at moderate speed with magnetic stirrer and a

stir bar. A 0.1 volume of total suspension of 1.65 M ammonium sulfate (AS) (all AS

solutions were prepared in buffer B) was slowly added, over a few minutes with moderate

stirring to the suspended nuclei. The suspension was gently stirred for an additional 30

minutes at 4oC and then sedimented at 13000 x g for 20 minutes at 4oC in a Sorvall

centrifuge with SS-34 rotor. The supernatant containing HMGB1/2 protein was saved and the

pellet was extracted twice with buffer B containing 0.15 M AS. The supernatants were

pooled together and the final AS concentration was brought to 2.6 M by very slow addition

of solid AS (0.4 g of salt per mL), after which the solution was stirred gently for one hour

and sedimented at 100,000 x g (25,000 rpm) for 20 minutes at 4oC in the Beckmen L8-M

ultracentrifuge using the SW-28 rotor. The supernatant was then transferred to ¾ inch

dialysis tubing (12 kDa molecular weight cut off (MWCO)) and dialyzed exhaustively

against 4 L of buffer until the AS concentration was in the µM range. The dialysis was done

six times, each time in a fresh buffer to reduce the AS concentration to lower than 20 µM 59

range. The protein solution was then concentrated to ca. 10 mL by ultrafiltration (10 kDa

o MWCO, Diaflow by Amicon) using N2 pressure unit (Amicon) at 4 C. It was further concentrated to 4 mL by using Millipore centrifuge concentrator (10 kDa MWCO) at 1600 rpm in a 219 rotor, IECCR centrifuge) at 4oC.

17.3. HPLC Purification of HMGB Proteins from an Ion Exchange Column

An aliquot of 5 M NaCl was added slowly to the protein solution, with gentle

agitation, to make the final NaCl concentration 0.8 M. This was done to precipitate out the

extraneous proteins, which might precipitate in the Mono Q column during (linear salt

gradient) HPLC purification. The solution is gently stirred, set for an hour at 4oC and then

sediment at 12000 x g (Sorvall super speed RC-2, SS-34 rotor) for 20 minutes. The

supernatant was carefully transferred to another tube, leaving a faint white film of proteins in

the first centrifuge tube. The protein solution of ca. 10 mL was dialyzed once again against 2

L buffer B. This solution was further concentrated down to 4 mL using Millipore

concentrator (10 kDa MWCO, at 1600 rpm). The concentrated protein solution was filtered

using sterile Acrodisc filter (pore size 0.2 µm) and 250 µl aliquots of protein sample were

routinely injected into HPLC unit during each run.

All the solutions and water used in HPLC were similarly filtered before use. Before

injecting in the sample, the Mono Q column was eluted with the linear gradient until a flat

baseline was typically observed. HMGB1 and HMGB-2 were separated on a Pharmacia

Mono Q HR 5/5 anion exchange column using an elution program for 18 minute linear

gradient (Low salt buffer: 0.2 M NaCl, 20 mM Tris, 5 mM BME, pH 7.4 and High salt

buffer: 0.8 M NaCl, 20 mM Tris, 5 mM BME, pH 7.4). The computer program was set up to

run the gradient containing a 2 minute wash with low salt buffer, to remove proteins unbound 60

to column; an 18 minute linear salt gradient was run to elute and fractionate HMGB1 and

HMGB2. After HMGB1/2 elution, a 2 minute wash with high salt buffer and finally a 4 minute wash with low salt buffer were done. HMGB2 and HMGB1 eluted at 14.05 minutes and 13.03 minutes, respectively, in the linear gradient with a flow rate of 1 mL/min with the

chart speed as 1 cm/min.

Following the collection of samples, the HPLC unit and the column was washed with distilled water for 4 hrs at 1 mL/minute and then at 0.1 mL per minute overnight. This was required to remove residual salts since residual salts in the HPLC will corrode and damage the system. The Mono Q column was stored in 20% ethanol by running the solvent for about

10 minutes, then the system was shut off and the column stored by covering both the ends properly with suitable caps. This work was often performed in the lab of Dr. D. Dignam

(University of Toledo, Health Science Campus) with his assistance, which we gratefully acknowledge.

17.4. Characterization and Storage of HMGB1 and HMGB2 Proteins

The individual protein fractions (1.33 mg/mL) were dialyzed into HMGB working buffer (160 mM NaCl, 10 mM Na2HPO4, 1 mM DTT at pH 7.3) and stored in 100 μL

aliquots at -80oC. To determine purity, the HMGB1 and HMGB2 fractions were analyzed

using SDS-PAGE. One band corresponding to ca. 25kDa molecular weight was observed for

both HMGB1 and HMGB2. The concentration was determined using an extinction

coefficient of 20,500 M-1 cm-1 at 280 nM (Chow et al., 1995). The percent yield typically for

HMGB1 was 3.25 mg/150 g calf thymus and percent yield for HMGB2 was 3.8 mg/150 g

calf thymus. The concentration of HMGB1 and HMGB2 were maintained at ca. 1 µg/µL in

HMGB working buffer. 61

17.5. Cleaning of Pharmacia 5/5 Mono Q HPLC Column

The Mono Q column required cleaning after about every 10 injections or if a pressure

fault in either pumps was observed. For cleaning, the column was attached to HPLC in the

reverse orientation and washed by repeated injections of the following: 2 M NaCl, 2 M

NaOH and 75% AcOH. The cleaning was done as follows – 500 µL of 2 M NaCl solution

was injected, washed with dH2O until pH of the elution was neutral; 500 µL of 2 M NaOH

solution was then injected and repeatedly washed with water until pH 7.0; 500 µL of 75%

AcOH was then injected and repeatedly washed with water until pH 7.0. The syringe was

washed with plenty of water between different solutions. NaOH degrades glass so this was

especially important after NaOH injection. NaOH solutions were prepared in plastic

containers. All the solutions were filtered before using them for cleaning the column.

18. DNA and Nucleosome

The 15bp synthetic GRE 5’-TGTACAggaTGTTCT-3’, corresponding to the -2509/-

2495 segment of tyrosine aminotransferase gene (Jantzen et al., 1987; Li and Wrange, 1993) was

placed at the middle of the 161bp DNA segment in the plasmid pGEM-Q2. A 161bp 2G2 DNA fragment with GRE flanked by four nucleosome positioning sequences (NPS), two NPS at 5’ end and two NPS at 3’ end of the GRE (Fig. 7) was designed and constructed by Dr. Ron Peterson, at

Ohio Northern University. The 15bp GRE insert was cloned into the plasmid pGEM-Q2 having ampicillin resistance gene. Nucleosome positioning sequence (NPS) is a repeat of:

5’-TCGGTGTTAGAGCCTGTAAC -3’ (Li and Wrange, 1995).

18.1. Sequence of DNA and Restriction Sites

A sequence of DNA fragment from SP6 promoter primer to T7 promoter primer of a

p2G2-pGEM-Q2 plasmid is shown in the Box 1. This fragment contains a 161bp 2G2 DNA 62

within the EcoR I and Hind III restriction sites. The 15bp GRE is placed at the middle of the

161bp DNA such that it is translationally positioned by the two nucleosome positioning

sequences at the dyad axis in the nucleosome. However, the center of 15bp GRE does not

align perfectly with the dyad axis. The dyad axis is at the first nucleotide of the second half

site of the GRE (illustrated with the “*”in the Box 1), while the center of the GRE is the

second nucleotide of three spacer nucleotides (illustrated with the “·” in the Box 1). The

161bp 2G2 was designed in such a way that the GRE is rotationally phased by facing the

major groove of GRE away from the core histones to facilitate the optimum binding of

PR/GR.

19. Isolation of 161bp 2G2 DNA from p2G2-pGEM-Q2 Plasmid

19.1. Restriction Digestion of p2G2-pGEM-Q2 Plasmid with EcoR I and Hind III

The plasmid p2G2-pGEM-Q2 contains the 161bp 2G2 DNA between EcoR I and

Hind III site. This 161bp 2G2 DNA contains PRE/GRE at the centre, which is flanked by two

nucleosome positioning sequences on either side (Box 1, Fig 7). About 200 µg of plasmid

DNA was taken and a double digestion with EcoR I and Hind III in 1X NEbuffer 3 (final

concentration) was performed overnight at 37oC to generate 161bp 2G2 DNA. One percent

agarose gel was run to verify the complete digestion of the plasmid DNA. After the

verification of complete digestion, the enzyme was heat inactivated at 68oC for 20 minutes

and chilled rapidly on ice. 63

Box 1: DNA sequence for 2G2: The 2G2 DNA fragment has 2 nucleosome positioning sequences, a repeat of 5’-TCG GTG TTA GAG CCT GTA AC -3’ (Li and Wrange, 1995)

(starting at the 28th bp, counting from the cleavage site of EcoR I) on each side of a GRE “5’-

TGTACAggaTGTTCT-3’”. The pink shaded nucleotides represent AT-rich region with the minor groove toward the histones and the major groove directed the outside. Green shaded nucleotides are GC-rich region with the major groove toward the histone core. The symbol (•) depicts the center of progesterone / glucocorticoid response element and (*) depicts the center of the DNA fragment produced by EcoR I and Hind III double digest.

64

19.2. Dephosphorylation of 5’ Ends

The DNA was dephosphorylated with Antarctic phosphatase (New England Biolabs,

Cat. # M0289S) at 37oC for 30 minutes. The reaction was stopped by adding 7 µL 0.5 M

EDTA (final concentration of 15 mM), 5.5 µL 20 % SDS (final concentration 0.5 %). The mixture was then treated with 1 µL proteinase K (10 mg/mL) and incubated at 56oC for 30

minutes. A second aliquot of 1 µL proteinase K was added and incubated 56oC for 30

minutes.

19.3. Phenol Chloroform Extraction

Two volumes of phenol-chloroform-isopropanol (PCI) was added to the

dephosphorylated DNA-protein mixture and mixed well by inverting the tube several times.

The mixture was spun down in a microcentrifuge at maximum speed for 30 seconds. The

upper aqueous phase was carefully transferred to a new tube, leaving behind the white

precipitate between the organic phase and aqueous phase. The PCI extraction was repeated

once again, and then two volumes of chloroform was added to the extracted aqueous phase

and mixed well by inverting the tube several times. The mixture was centrifuged in a

microcentrifuge at maximum speed for 30 seconds. The upper aqueous phase was carefully

transferred to a new tube. It was repeated for the second time.

19.4. DNA Concentration

To the upper aqueous phase collected from the phenol chloroform extraction, one

tenth volume of 3 M sodium acetate pH 7 and two volumes of ice cold absolute ethanol was

added. It was incubated at -80oC for at least 4 hours. After incubation, the tubes were

centrifuged in a microcentrifuge for 10 minutes at 14,000 rpm in the cold room and the

supernatant removed. The pellet was washed gently with 100 µL of ice cold 70% ethanol, air 65

dried and dissolved in TE buffer (10 mM Tris, 1 mM EDTA). The amount of DNA was

quantified using Nanodrop spectrophotometer assuming A260=1 for 50 μg/mL of DNA.

19.5. Separation of 161bp DNA Fragment from EcoR I and Hind III Digested pG2-

pGEM-Q2 Plasmid

All the DNA that was double digested with EcoR I and Hind III was recovered after

dephosphorylation, phenol chloroform extraction, and DNA concentration. The recovered

DNA was then loaded into a 6% polyacrylamide gel and run for 40 minutes at 80 volts. The

gels were stained with methylene blue until bands are clearly visible. This was followed by

destaining in water. The DNA bands corresponding to the 161bp DNA were cut out of the

gel with a razor blade and then weighed to estimate the volumes (1 mg ≡ 1 µL).

19.6. Elution of 161bp DNA Fragment from Polyacrylamide Gel

The gel slice containing the 161bp DNA fragment was transferred to a 1.5 mL

Eppendorf tube, and finely minced with a 1000 µL sealed pipette tip. Two volumes (for

estimated volume, which is 1 µL for 1 mg of gel slice) of gel elution buffer (0.5 M

ammonium acetate, 10 mM Mg acetate, 1 mM EDTA) were added to the minced gel and

vortexed vigorously. It was then incubated at 55oC for one hour. The tube was then centrifuged at 10,000 rpm and the supernatant transferred into a new 1.5 mL Eppendorf tube.

An additional 0.5 volume of gel elution buffer was added to the minced gel, vortexed, microcentrifuged, and the supernatants combined. One tenth volume of 3M sodium acetate and two volumes ice cold absolute ethanol was added to precipitate DNA for at least 4 hrs at

-80oC. The pellet was carefully washed with 100µL of ice cold 70 % ethanol, air dried, and

dissolved in TE buffer. The amount of DNA was quantified using Nanodrop

spectrophotometer assuming A260=1 for 50 μg/mL of DNA. 66

20. End Labeling of DNA with γ-(32P)-ATP

20.1. Labeling at Both Ends of Double Stranded DNA

Four microliters of 161bp DNA fragment (400 ng, 100 µg/µL) was aliquoted in the 1.5

32 mL Eppendorf tube. A 17 µL of dH20, 2.5 µL of 10X Opti-kinase buffer, 2.5 µL γ-( P)-ATP

and 1 µL of Opti-kinase enzyme was added to it, mixed well by gently swirling with the pipette

tip, and incubated at 37oC water bath for 30 minutes. The reaction was stopped by the addition

of 2 µL of 0.5 M EDTA (pH 8.0). The labeled DNA was separated from γ-(32P)-ATP by gel

permeation chromatography using a spin column containing G-50 Sephadex swollen in dH2O.

20.1.1. Separation of End Labeled 161bp 2G2 DNA from Free γ-32P-ATP

A gel permeation chromatography in a 1 mL syringe containing G-50 Sephadex beads

was used to separate the labeled DNA from free γ-(32P)-ATP. An aliquot of 0.2 grams of G-50

Sephadex beads were swollen in 10mL of distilled water at room temperature for overnight to

obtain 4 mL of bead slurry. A spin column was prepared in a 1mL syringe by plugging the

bottom of the spin column with a piece of glass wool and filling the column with swollen

beads. The column was spun at 1600 x g (IEC centrifuge: 219 rotor at 2600 rpm) for 2.5

minutes. The syringe was filled with swollen beads until the column volume reached 1 mL.

The column was equilibrated by washing several times with 100 µL portions of TE buffer (10

mM Tris-HCl, 1 mM EDTA, pH 8.0). It was ensured that a 100 μL of TE buffer was eluted

from column under these conditions. The volume of the DNA reaction mixture was made to

100 µL with TE buffer, and loaded on to the column. The column was spun at 1600 x g (IEC

centrifuge: 219 rotor at 2600 rpm) for 2.5 minutes, and the 100 µL of the first flow through was

collected in a 1.5 mL microcentrifuge tube. The first eluent usually contained most of the end- 67

labeled DNA, which was used for the studies. The end-labeled 161bp 2G2 DNA was stored at -

20oC.

20.2. Labeling at a Single End of Double Stranded DNA

In order to label the 161bp 2G2 DNA at one single end with 32P, the plasmid p2G2-

pGEM-Q2 was first digested with EcoR I. Following EcoR I digestion, the 5’ phosphate was

removed from the linear plasmid DNA using Antarctic phosphatase, DNA purified from DNA-

protein mixture with phenol chloroform extraction, DNA concentrated with sodium

acetate/ethanol precipitation, and 32P labeled at EcoR I end. It was then subsequently digested

for the second time by Hind III to produce 161bp 2G2 DNA labeled at single end.

20.2.1. Restriction Digestion of p2G2-pGEM-Q2 Plasmid with EcoR I

About 200 µg of plasmid DNA (p2G2-pGEM-Q2) was treated with 200 units of

EcoR I in 1X EcoR I reaction buffer (final concentration) and incubated overnight in a

37oC water bath. One percent agarose gel was run to verify the complete digestion of the

plasmid DNA and formation of linear plasmid DNA. After the verification of complete

digestion, the enzyme was heat inactivated at 68oC for 20 minutes and chilled rapidly on

ice.

20.2.2. Dephosphorylation of 5’ End

The linear plasmid DNA produced by EcoR I digestion was dephosphorylated

with Antarctic phosphatase (New England Biolabs, Cat. # M0289S) at 37oC for 30

minutes. The reaction was stopped by adding 7 µL 0.5 M EDTA (final concentration of

15 mM), 5.5 µL 20 % SDS (final concentration 0.5 %). The mixture was then treated

with 1 µL proteinase K (10 mg/mL) and incubated at 56oC for 30 minutes. A second

aliquot of 1 µL proteinase K was added and incubated at 56oC for 30 minutes. 68

20.2.3. Phenol Chloroform Extraction

The phenol chloroform extraction was performed as outline previously (Section

19.3).

20.2.4. DNA Concentration

The DNA concentration was performed as outlined previously (Section 19.4).

20.2.5. End Labeling of Double Stranded DNA with 32P and Separation of Free γ-(32P)-

ATP from Labeled DNA

The linear plasmid DNA produced by EcoR I digestion was labeled at both ends using Opti-kinase enzyme as described above. The enzyme was heat inactivated at 68oC

for 20 minutes. The labeled DNA was separated from γ-(32P)-ATP by ethanol

precipitating the DNA. The labeled DNA was precipitated by adding 1/10th volume of 3

M sodium acetate pH 7 and two volumes of ice cold absolute ethanol; and then incubated

at -80oC for at least 4 hours. After incubation, the tubes were centrifuged in a

microcentrifuge for 10 minutes at 14,000 rpm in the cold room and the supernatant

removed. The pellet was washed with 100 µL of ice cold 70% ethanol, air dried and

dissolved in TE buffer (10 mM Tris-HCl, 1 mM EDTA).

20.2.6. Restriction Digestion of EcoR I Digested Plasmid DNA with Hind III

Following 32P end labeling of the EcoR I digested linear plasmid DNA and

ethanol precipitation it was digested for the second time with Hind III at 37oC overnight.

This restriction digestion produced 161bp 2G2 DNA labeled at only one end (EcoR I

end). The enzyme was heat inactivated at 68oC for 20 minutes. To the reaction mixture,

5X loading dye (50% glycerol, 100 mM EDTA, 0.25% bromophenol blue) was added to 69

1X final concentration. It was then run on a 6 % acrylamide gel to separate the 161bp

DNA from the larger fragment of plasmid DNA.

20.2.7. PAGE Separation of 32P Labeled 161bp 2G2 DNA

A 6 % polyacrylamide gel in 1X TAE buffer was prepared using a 1.5 mm thick

spacer so that all of the reaction can be loaded onto a single well. The 6% gel was made

with 37.5 mL of dH2O, 10 mL of 30% acrylamide, and 2.5 mL of 20X TAE and 500 µL

IGEPAL. The mixture was degassed for 15 minutes and poured into a BioRad Mini gel

cassettes. The gel was run for 40 minutes at 80 volts and stained with methylene blue until bands were clearly visible. This was followed by distaining in water. The DNA bands corresponding to the 161bp DNA were cut out of the gel with a razor blade and then weighed to estimate the volumes (1 mg ≡ 1 µL).

20.2.8. Elution of 32P Labeled 161bp DNA Fragment from Polyacrylamide Gel

The gel slice containing the 161bp DNA fragment labeled at single end was

transferred to a 1.5 mL Eppendorf tube, and finely minced with a sealed 1000 µL pipette

tip. Two volumes gel elution buffer (0.5 M ammonium acetate, 10 mM Mg acetate, 1

mM EDTA) was added to the minced gel and vortexed vigorously. It was then incubated

at 55oC for one hour. The tube was then centrifuged at 10,000 rpm and the supernatant

transferred into a new 1.5 mL Eppendorf tube. An additional 0.5 volume of gel elution buffer was added to the minced gel, vortexed, microcentrifuged, and the supernatants combined. One tenth volume of 3 M sodium acetate and two volumes ice cold absolute ethanol was added to precipitate DNA for at least 4 hrs at -80oC. The pellet was carefully

washed with 100 µL of ice cold 70 % ethanol, air dried, and dissolved in TE buffer.

Although the pellet was virtually invisible, essentially most of the radioactivity was 70

found in the pellet; determined from Geiger counter reading and DuPont BC2000

counter.

21. Measurement of Radioactivity by Scintillation Counter

One µL of the labeled DNA was transferred onto a Skatron filter consisting of a membrane and a roll of absorbing paper located beneath the membrane. An aliquot of 25 µL of ice cold 10% TCA was applied to the membrane to precipitate the labeled DNA. The precipitated fragment was blocked by the membrane and separated from the free γ-32P-ATP which penetrated through the membrane and was absorbed by the paper. The TCA precipitation was repeated three times, and then transferred to a 1.5 mL scintillation vial and 1 mL of liquid scintillation solution

(ECOLUME) was added to measure the radioactivity on Beckman-LS-133 Scintillation System.

Using the reading from the Beckman-LS-133 Scintillation System specific activity of the DNA was determined. The typical specific activity was ~7 x 105 counts per minute (CPM)/μg of DNA.

22. Preparation of Nucleosomes

22.1. Nucleosome Reconstitution

About 10 µL labeled 161bp DNA in TE buffer was transferred to a 1.5 mL Eppendorf

tube and lyophilized in a Speed-vac without the application of heat. This sample contained

about 100,000 CPM (counted with DuPont BC2000 counter). Eight µL of 5 M NaCl, 4 µL of

10X reconstitution buffer (RC) (150 mM Tris-HCl pH 7.5, 2 mM EDTA, 2 mM PMSF) and

28 µL of donor chromatin in a Sepharose column buffer (650 mM NaCl, 5 mM Tris-HCl pH

7.5) was added to it. The final volume of the reaction mixture was 40 µL and the final

concentration of the donor chromatin was about 80 µg/mL in terms of DNA and the final

buffer contains 2 M NaCl, 20 mM Tris-HCl, pH 7.5, 0.5 mM EDTA and 0.2 mM PMSF.

(Calculations are shown in Table 38, appendix VII). 71

The mixture was incubated for 30 minutes in a 37oC water bath, followed by stepwise

dilution with 1X RC buffer (15 mM Tris-HCl pH 7.5, 0.2 mM EDTA, 0.2 mM PMSF) for 3 hours. The stepwise dilution was achieved by adding 5 µL of 1X RC buffer to the sample six times at 10 minutes interval followed by addition of 10 µL of 1X buffer six times every

10 minutes. Finally six additions of 30 µL of 1X RC buffer were added every 10 minutes.

The final volume was about 310 µL, and the final salt concentration of the buffer was 200 mM NaCl (Calculations are shown in Table 38, appendix VII).

22.2. Isolation of Nucleosome

A 5%, 30% and 50% (w/v) sucrose solution was prepared in TE buffer. It was then autoclaved and stored at 4oC. Four to six hours before ultracentrifugation, a 2.5 mL of 5% sucrose and 2.5 mL of 30% sucrose was used to prepare 5mL of 5-30% sucrose linear gradient, and stored at 4oC. The reconstituted nucleosome was then separated from the unlabelled DNA, free labeled DNA, donor chromatin and free histones by linear gradient

sedimentation in 5-30% sucrose linear gradient containing 10 mM Tris-HCl pH 7.5, 1 mM

EDTA and 0.2 mM PMSF. This was spun in a SW55Ti rotor at 36,000 rpm for 16 hours at

4oC. Thirty four fractions of 6 drops (a drop ~ 25 µL), which was about 150 µL were

collected with the Density Gradient Fractionator by poking the bottom of the tube and

pushing a 50% sucrose solution from the bottom. The flow rate was set to 0.375 mL/min and

the samples collected into the series of 1.5 mL Eppendorf tubes on ice. The CPM for each fractions collected were determined using DuPont BC2000 counter, and 10 µL of the

samples were run on a 4% EMSA gel to monitor the distribution of free DNA and

nucleosome.

72

22.3. Storage of Nucleosome

The fractionated nucleosomes were stored at -20oC in 18% sucrose/TE buffer.

23. Preparation of Remodeled Nucleosomes

23.1. Nucleosome Remodeling

Once the nucleosomes were fractionated and band positions verified by

polyacrylamide gel electrophoresis, peak fractions were collected (252 µL of nucleosomes)

and transferred to a 1.5 mL Eppendorf tube. A 48 µL of 250 ng/µL of HMGB1 (12 μg) was

added to it such that the final concentration of HMGB1 was 1600 nM. It was mixed well by

gently swirling with pipette tip and incubated on ice for 1 hour.

23.2. Isolation of HMGB1-Remodeled Nucleosomes

To the 5 mL of 5-30% sucrose linear gradient prepared at least 4-6 hrs ahead and

stored at 4oC, the nucleosomes treated with 1600 nM HMGB1 for 1 hour on ice was loaded

onto it. It was then centrifuged in SW55Ti rotor at 36,000 rpm for 16 hours at 4oC. Thirty

four fractions of 6 drops (a drop ~ 25 µL), which was about 150 µL, were collected with the

Density Gradient Fractionator by poking the bottom of the tube and pushing a 50% sucrose

solution from the bottom. The flow rate was set to 0.375 mL/min and the samples were

collected into a series of 1.5 mL Eppendorf tubes on ice. The CPM for each fractions

collected were determined using DuPont BC2000 counter, and 10 µL of the samples were

run on a 4% EMSA gel to monitor the distribution of nucleosomes and remodeled

nucleosomes.

23.3. Storage of HMGB1-Remodeled Nucleosomes

The fractionated HMGB1-remodeled nucleosomes were stored at -20oC in 18%

sucrose/TE. 73

24. Electrophoresis

24.1. Agarose Gel Electrophoresis

One percent agarose gel electrophoresis was typically run to analyze restriction

digestion profile of plasmid DNA.

24.1.1. Gel Preparation

In a 250 mL Erlenmeyer flask, 0.7 g of electrophoresis grade agarose (Sigma, cat

# A6013) was added and dissolved in a 70 mL of 1X TAE buffer. The mixture was

heated in a microwave until all the agarose was completely dissolved. After it was

completely dissolved, it was cooled to about 40-50oC and 7 μL of the 10,000X GelRedTM

Nucleic Acid Gel Stain (Biotium, Cat. # 41003) was added to it, mixed well and poured

into the pre-assembled gel tray. The gel was allowed to cool down to room temperature

and the combs were taken out.

24.1.2. Sample Preparation

The molecular weight markers: 1kb DNA ladder from Fisher Scientific (Cat. #

BP2578-100), or 100bp DNA ladder from Fisher Scientific (Cat. # BP2573-100) or

100bp DNA ladder from New England Biolabs (Cat. # N3231S) were thawed at room

temperature. At the same time, a 10 µL of DNA samples were prepared with a final

concentration of 1X loading buffer (1.6 mM Tris-HCl, pH 7.6, 0.005% bromophenol

blue, 10 mM EDTA, and 10% glycerol).

24.1.3. Electrophoresis

One % agarose gel, prepared earlier, was placed in the horizontal gel

electrophoresis unit filled with 1X TAE buffer and electrodes were connected to the

power supply. A 10 µL of the prepared DNA samples and the molecular weight markers 74

were loaded into the wells and electrophoresed for 1 hour at 100V. After electrophoresis,

the gel was observed under the UV light, and a photograph of the gel was taken in 4th

floor Biology Department Room #517.

24.2. PAGE

A 6% polyacrylamide gel electrophoresis was typically run to analyze the PCR product from colony PCR as well as to separate and isolate 161bp 2G2 DNA.

24.2.1. Gel Preparation

A 6 % polyacrylamide gel was prepared in 1X TAE buffer using a 1.5bmm thick

spacer. In a 50 mL Erlenmeyer flask, 10 mL of 30% acrylamide, 37.5 mL of dH2O, 2.5

mL of 20X TAE buffer, and 500 µL IGEPAL was mixed well and degassed for 15

minutes. After degassing, 125 μL of 10% ammonium persulfate and 25 μL of TEMED

was added and mixed well. The mixture was carefully loaded in the pre-assembled glass

plates and allowed to polymerize.

24.2.2. Sample Preparation

The molecular weight markers: 100bp DNA ladder from Fisher Scientific (Cat. #

BP2573-100) or 100bp DNA ladder from New England Biolabs (Cat. # N3231S) were

thawed at room temperature. At the same time, DNA samples were prepared with a final

concentration of 1X loading buffer (1.6 mM Tris-HCl, pH 7.6, 0.005% bromophenol

blue, 10 mM EDTA, and 10% glycerol).

24.2.3. Electrophoresis

The electrophoresis unit (BioRad Mini Gel) with 6% polyacrylamide gel was

filled with 1X TAE buffer and electrodes were connected to the power supply. The

prepared DNA samples and the molecular weight markers (100bp DNA ladder from 75

Fisher Scientific (Cat. # BP2573-100) or 100bp DNA ladder from New England Biolabs

(Cat. # N3231S) were loaded into the wells. It was then electrophoresed for 1 hour at

100V.

24.2.4. Gel Staining

For analysis of PCR product of colony PCR of transformed JM109 cells, the gel

was stained in a final concentration of 1000X GelRedTM Nucleic Acid Gel Stain

(Biotium, Cat. # 41003) in 0.1X TAE buffer until DNA bands were clearly visible under

UV light. The gel was then observed under the UV light, and a photograph of the gel was

taken in 4th floor Biology Department Room #517.

For separation and isolation of 161bp 2G2 DNA, the gel was stained using

0.002% methylene blue in 0.1X TAE buffer until the DNA bands were clearly visible.

Following staining, the gel was briefly destained in water or 0.1X TAE buffer.

24.3. Electrophoretic Mobility Shift Assay (EMSA)

Non-denaturing polyacrylamide gels separate DNA and protein according to charge,

shape and size. EMSA is a technique based on the observation of altered mobility of DNA-

protein complex or altered structure of nucleosome due to DNA-protein interaction. The

stable protein-DNA complexes migrate slower through polyacrylamide gels than free DNA

fragments and hence the DNA-protein complex band shifts to the higher position in the gel.

The γ-32P-ATP labeled DNA molecules enable us to observe bands by autoradiography and / or phosphoimager screen. The latter allows the detection of the exact number of counts in a complex between protein and labeled DNA and therefore permits the determination of

32 equilibrium dissociation constant (KD). The γ- P-ATP labeled DNA in a nucleosome was

reacted with HMGB1, PR to form a complex. The reaction was incubated on ice for varying 76

length of time depending on the experiment. The reaction products were run on 5% native polyacrylamide gel for 2 hours in a 0.35X TBE buffer. The dried gels were exposed to photographic film at -80oC and the results were observed by autoradiography.

24.3.1. Gel Preparation

A 4% native polyacrylamide gel was used for the EMSA studies. To prepare a

4% polyacrylamide gel, a 7 mL aliquot of 5X TBE buffer (450 mM Tris-HCl, 450 mM

boric acid, and 10 mM EDTA), 13.4 mL 30% (w/v) acrylamide (29:1, acrylamide:

bisacrylamide), 78 mL distilled water and 1 mL of 5% IGEPAL were dispensed into a

500 mL flask and degassed for 15 minutes. After degassing, a 500 µL of 10% (w/v)

ammonium persulfate and a 100 µL of TEMED were added and mixed well. The

mixture was carefully poured into a pre-assembled glass plates and left to polymerize.

24.3.2. Sample Preparation

The reactions were performed in a 500 µL Eppendorf tube maintained at 4oC.

Typically the reactions contained diluted proteins, reaction buffer, and dilution buffer to

attain the final reaction volume. In a typical reaction of nucleosome with varying

concentration of HMGB1, a 10 µL reaction, 8 µL (~100 pM) of nucleosome in

TE/sucrose (18%) buffer was added to the 500 µL Eppendorf tube. To it, varying amount

of 250 ng/µL HMGB1 was added and final volume of 10 µL was attained by adding

dH2O. The reaction mixture was incubated on ice for 1 hour. After incubation, the sample

was loaded in the pre-electrophoresed 4% native polyacrylamide gel.

24.3.3. Electrophoresis

The 4% polyacrylamide gel was assembled in the electrophoresis apparatus

(Vertical Gel Electrophoresis System, Model V16, Cat. # 21070, Whatman, Inc). The 77

upper and lower chambers were filled with 0.35X TBE. The gels were pre-

electrophoresed at 100V for 1.5-2 hours or until the current dropped to half its starting

value. The voltage was increased to 200V and then the samples were loaded into the

wells and electrophoresed typically for 2 hours at 4oC.

24.3.4. Gel Drying

After the electrophoresis was complete, the gel cassette was removed from the

apparatus and the two glass plates were pried apart. The fragile gel tended to stick to one

side of the glass plate. A piece of 3 M Whatman paper cut to the dimensions of the gel

was placed on top of the gel, and the gel was removed from the remaining glass plate.

The gel/filter paper combination was covered with Saran wrap and dried in a gel drying unit using vacuum pump (Physical Science Building, Room # 216).

24.3.5. Autoradiogram

After the gel was completely dried, it was transferred to an X-Ray cassette

containing an intensifying screen. An X-ray film (Merry X-Ray Corporation) was placed

on the dried gel in the dark and the cassette cover was closed. The gel cassette was

incubated at -80oC for various lengths of time depending on the activity of the

radiolabeled oligonucleotide. The cassettes were removed from the -80oC freezer and

allowed to warm up to room temperature.

The film was developed in the dark by immersing and gently agitating the film for

5 minutes in developer solution (Kodak GBX developer, Cat. # 1900984). The film was

rinsed for 20-30 seconds in distilled water and finally it was placed in fixer solution

(Kodak GBX fixer, Cat. # 1902485) for 1-2 minutes. The film was then rinsed once more with the distilled water and then hung up to dry. 78

24.4. DNase I Assay

24.4.1. DNase I Digestion

In a 1.5 mL Eppendorf tube, 100.8 μL of nucleosomes (~600 CPM) from two

peak fractions of a 5-30% sucrose linear gradient was taken and treated with 19.2 µL of

250 ng/µL HMGB1 (final concentration, 1600 nM) at 4oC for 1 hour. In another two 1.5

mL Eppendorf tubes, 100.8 μL of nucleosomes (~600 CPM) and 100.8 μL of DNA (~600

CPM) were taken and 19.2 µL of dH20 was added and placed on ice for 1 hour.

Another two sets of 1.5 mL Eppendorf tube were taken for each reaction (free

DNA, untreated nucleosome, and nucleosomes treated with 1600 nM HMGB1) and

labeled 60 seconds and 90 seconds. To each of the tubes, 7.3 μL of 10X stop buffer (50

mM EDTA, 2% SDS, 100 ng/μL tRNA) was added (1X final concentration).

After one hour treatment of nucleosome with 1600 nM HMGB1 at 4oC, 13.3 μL

of 10X DNase I buffer (100 mM Tris-HCl, pH 7.5, 25 mM MgCl2, 5 mM CaCl2) was

added (1X final concentration). To this, a freshly diluted DNase I was added as 1

U/reaction (2 μL of 0.5 U/μL) and incubated at 37oC for 60 seconds and an aliquot (66

µL) was taken and added to the Eppendorf tube containing the 7.3 µL of 10X stop buffer

(50 mM EDTA, 2% SDS, 100 ng/µL tRNA). The reaction mixture was vortexed and

mixed well to stop the reaction. After 90 seconds, another aliquot (66 µL) was taken and

added to the Eppendorf tube containing the stop buffer. The reaction mix was vortexed

and mixed well to stop the reaction. Likewise, the untreated nucleosomes and free DNA

was also digested with DNase I with 1 U/reaction for 60 seconds and 90 seconds at 37oC

and the reaction stopped by adding the stop buffer. The reaction mix was vortexed and

mixed well to stop the reaction. 79

After all the reactions were stopped, one tenth volume of 3 M sodium acetate pH

7 and two volumes of ice cold absolute ethanol was added. It was incubated at -80oC for

1 hour for the precipitation of DNA. The tubes were spun on microcentrifuge for 10 minutes, and the supernatant was removed carefully. The counts in the supernatant and the pellet were checked using Geiger counter and DuPont BC2000 counter to ensure the yield of DNA. The pellet was washed gently with 100 µL of ice cold 70% ethanol, air dried and dissolved in TE buffer (10 mM Tris-HCl, 1 mM EDTA). The DNA was then lyophilized using the Speed-vac with heat. In order to load the equal counts on each well, the dry counts of the DNA was taken by using DuPont BC2000 counter. Since the tube with the lowest counts was the limiting one, its CPM per μL was calculated and then variable volumes of loading buffer (98% formamide, 1 mM EDTA, trace amount of bromophenol blue and xylene cyanol) was added to all tubes to obtain the equal number of counts. The samples were heated on a 90oC heat blocker for 5 minutes and

immediately quenched on ice. From each tube 5 μL of the sample was loaded in the gel.

24.4.2. G/A Ladder Preparation

DNA labeled at one end was chilled to 4oC and 10 µL was pipetted into an

Eppendorf tube. The volume was adjusted to 20 µL with chilled dH2O and treated with

50 µL of formic acid (Fisher, cat # 7732-185). The tube was then incubated at 20oC for 5

minutes. The reaction was stopped with the addition of 180 µL of HZ stop solution (0.3

M EDTA, 25 µg/mL tRNA and 0.3 M sodium acetate). The DNA was precipitated by the

addition of 750 µL of ice cold absolute ethanol and incubating at -80oC for 15 minutes.

The mixture was then centrifuged at maximum speed in the microcentrifuge for 10

minutes. A 250 µL of 0.3 M sodium acetate and 750 µL of ice cold absolute ethanol was 80

added and incubated at -80oC for 15-20 minutes. The mixture was then centrifuged at

10,000 rpm and the supernatant carefully removed. The pellet was then washed with 100

µL of ice cold 70 % ethanol and vacuum dried. The dried pellet was then treated with 100

µL of 1 M piperidine and incubated at 90oC for 30 minutes. After the incubation period,

the mixture was lyophilized in a Speed-vac. A 10 µL of water was added, mixed and

lyophilized again in a Speed-vac. A second round of 10 µL of water was added, mixed

and lyophilized again and the pellet dissolved in 10 µL of loading buffer (80 % (v/v)

formamide, 50 mM Tris-borate (pH 8.3), 1 mM EDTA, 0.1 % (w/v) xylene cyanol, 0.1 %

bromophenol blue). The mixture was then vortexed and heated on a heating block for 1

minute at 90oC and chilled on ice. This was used to produce a G/A ladder for a

sequencing gel and used as a nucleotide sequence marker in DNase I digestion studies of a rotationally phased and translationally positioned DNA on a nucleosome.

24.4.3. Gel Preparation

A denaturating 8% polyacrylamide sequencing gel was prepared in 1X TBE buffer. A 5 mL aliquot of 10X TBE buffer (900 mM Tris-HCl, 900 mM boric acid, and

20 mM EDTA), a 16 mL 25% (w/v) acrylamide (19:1, acrylamide: bisacrylamide) and a

21 g of urea were dispensed into a 50 mL beaker. The mixture was heated gently while stirring to dissolve the urea. The volume was then adjusted with dH2O to 50 mL and

filtered through 3 M Whatman filter paper using a water aspirator for a vacuum suction.

The mixture was then degassed for 15 minutes and cold on ice to slow down the

polymerization process. Two hundred and fifty microliters of 10% ammonium persulfate

and 50 µL of TEMED were then added to it, mixed well and the gel mixture poured into

the pre-assembled glass plates. The gels are pre-electrophoresed until the temperature 81

increased to 45oC and the current decreased to half of its initial value before loading the

samples.

24.4.4. Electrophoresis

A denaturating 8% polyacrylamide sequencing gel, prepared earlier, was

assembled in the vertical gel electrophoresis apparatus. The upper and lower chambers

were filled with 1X TBE. The gel was pre-electrophoresed at 1500 V until the

temperature increases to 45oC and the current dropped to half its starting value. The

power supply was turned off and samples loaded into the wells and electrophoresed

typically for 2-3 hours.

24.4.5. Gel Drying

After electrophoresis was complete, the gel cassette was removed from the

apparatus and the two glass plates were pried apart. The fragile gel will tend to stick to

stick to one side of the plate. A piece of 3 M Whatman paper cut to the dimensions of the

gel was placed on top of the gel, and the gel was removed from the remaining glass plate.

The gel/filter paper combination was covered with Saran wrap and dried in a gel drying

unit using a vacuum pump (Physical Science Building, Room # 216).

24.4.6. Autoradiogram

After the gel was completely dried, it was transferred to an X-Ray cassette

containing intensifying screen, an X-ray film exposed and developed as described earlier.

24.5. SDS-PAGE

24.5.1. Gel Preparation

The BioRad protein mini-gel apparatus was used for both casting and running

gels. An appropriate amount of acrylamide solution was used to obtain a desired final 82

percentage of acrylamide in the separating layer. Typically, a 10% polyacrylamide gel was used. A 15 mL of separating gel solution was prepared. In a 25 mL Erlenmeyer flask, a 5 mL of 30% (29:1, acrylamide: bisacrylamide), 3.75 mL of 1.5M Tris-Cl, pH 8.8, 150

µL of 10% SDS and 6.1 mL of dH2O was added and mixed well. This solution was

degassed for 10-15 minutes and 50 µL of 10% ammonium persulfate (APS) and 10 µL of

N, N,N’N,-tetramethylethylenediamine (TEMED) was added to it and mixed well. The polymerizing solution was immediately poured between two glass plated up to the level of 1 cm below where the comb in the stacking layer would be. A layer of water saturated isobutanol was poured on top of the gel while polymerization was completed (in about

25-45 minutes). A solution of 4% acrylamide was used to prepare the stacking layer. In a

25 mL Erlenmeyer flask, 650 µL of 30% (29:1 acrylamide: bisacrylamide), 1.25 mL of

0.5 M Tris-HCl, pH 6.8, 50 µL of 10% SDS and 3.05 mL of dH2O was mixed well. The

stacking gel mixture was degassed for 15 minutes and 25 µL of 10% APS and 5 µL of

TEMED were added to it and mixed well. The polymerizing layer was poured on top of

the already polymerized separating gel. The comb was immediately placed between the

two glass plated of the gel to form the sample wells.

24.5.2. Sample Preparation

A calculated volume of Blue juice (10X SDS loading buffer (250 mM Tris-HCl

pH 6.8, 10% SDS, 30% glycerol, 5% β-mercaptoethanol, 0.02% bromophenol blue) was added to the sample to attain 1X final concentration. The samples were mixed well and briefly centrifuged at high speed in a microcentrifuge. The samples were heated in a boiling water bath for 5-10 minutes and spun down. The sample was then loaded on the gel. 83

24.5.3. Electrophoresis

After polymerization was complete, the comb in the stacking layer was removed

and the wells were thoroughly washed with distilled water. The BioRad gel cassette was

assembled and then fitted onto the BioRad mini gel electrophoresis unit. Pre-

electrophoresis was performed at 150V for 20-30 minutes. Samples prepared in a final

concentration of 1X loading buffer (Blue juice) was loaded into the wells and the gel was

electrophoresed at 200V in electrophoresis buffer (25 mM Tris-HCl, 192 mM glycine,

0.1% (w/v) SDS). The blue dye gave an indication as to when the gel should be stopped.

Once the dye ran off the gel, the electrophoresis was stopped.

24.5.4. Gel Staining

After electrophoresis was complete the gel was removed from the glass plate sandwich and placed into a Coomassie blue staining solution (0.1 % (w/v) Coomassie blue R-250, 50% (v/v) methanol, 10% (v/v) glacial acetic acid). The container was gently shaken gently for a minimum of three hours at room temperature. The Coomassie blue interacted with the protein bands while the gel itself did not. After staining was completed the gel was then place in destaining solution (10% (v/v) methanol, 10% (v/v) glacial acetic acid) to remove the unbound dye. Again the gel was shaken gently for overnight or until the blue dye has disappeared from all of the gel except for the protein

bands.

24.5.5. Gel Preservation

The gel was placed in soaking solution (20% (v/v) ethanol and 10% (v/v)

glycerol) in order to avoid cracks in the gel when they were dried. The cellophane used to

dry the gel was submerged in distilled water for 15 minutes. After the soaking process 84

was complete, one sheet of cellophane paper was smoothed across a plexi-glass plate.

Then the gel was placed on top of the cellophane sheet making sure that there were no air

bubbles. Finally the other piece of cellophane was placed on top of the gel and smoothed

out so that there were no air bubbles. A small plexi-glass frame was placed over the last

piece of cellophane, and it was secured by binder clips. The gel was allowed to dry for

several days at room temperature which it was placed in the lab notebook or scanned.

25. Western Blot

25.1. Transfer of Gel

A 1X transfer buffer (200 mL Methanol, 100 mL 10X Running Buffer, 700mL dH2O)

was prepared. The PVDF membrane (Amersham Hybond-P, Cat # RPN303F) was cut to (5.5

X 9.5 cm2) and filter paper was cut to (8 X 11 cm2) dimensions. The membrane was soaked

with methanol first and then with 1X transfer buffer. At the same time the fiber pads and

filter paper were soaked in 1X transfer buffer for at least 20 minutes. The gel was transferred

with the help of filter paper (soaked with 1X transfer buffer) and equilibrated with 1X

transfer buffer. The surface of the gel was flooded with 1X transfer buffer and the membrane

was placed on top of it. The membrane was marked (for the arrangements of the samples)

near the pre-stained marker (BioRad Cat # 161-0374) using a pencil. With the help of a glass

rod or a test tube, all air bubbles formed between the gel the membrane were removed by

gently rolling the test tube or a glass rod over the membrane surface. The surface of the

membrane was flooded with 1X transfer buffer and covered it with another filter paper

(soaked with 1X transfer buffer). The sandwich was completed by placing a pre-soaked (in

1X transfer buffer) fiber pad on top of the filter paper (one on the top of the membrane and

another on the bottom of the gel). 85

25.2. Protein Transfer from SDS-PAGE Gel to PVDF Membrane

The sandwich was held firmly and placed securely into the cassette such that the gel is facing towards the gray panel of the cassette (cathode, -ve) side and the membrane is facing towards the clear panel of the cassette (anode, +ve side). The cassette holding gel/membrane sandwich was placed in a buffer tank so that the gray panel of the holder is facing the gray cathode of the electrode panel. The protein was transferred electrophoretically for 1hour at

100V in cold room at 4oC. A magnetic stirrer was placed in the transfer buffer tank to uniformly distribute the heat generated and cool down the system during the transfer.

25.3. Blocking Non-Specific Antibody Sites on the Membrane

A 25 mL of the blocking buffer (5% (w/v) Non Fat Dry Milk in 1X TBST, (50 mM

Tris-HCl, 150 mM NaCl, 0.1% Tween)) was added into small pipette tip box lid. After the transfer was completed, the PVDF membrane was removed from the transfer cassette using a pair of tweezers, and the membrane was washed with 1X TBST. The membrane was then added to the 25 mL of blocking buffer with the transferred protein side facing upwards and incubated at 4oC for overnight by gently shaking the buffer continuously.

25.4. Probing with Anti-HMGB1

The membrane was washed three times with 1X TBST (5 minutes each), and then incubated with primary antibody (2 µg/mL final concentration) (HMGB1 rabbit polyclonal antibody, Upstate, Cat # 07-584) in 12 mL of 1xTBST buffer for 1 hr at room temperature in a small pipette tip box lid. After one hour of incubation, the membrane was washed thrice with 1X TBST (5minutes each). Then, the membrane was incubated with secondary antibody

(200 ng/mL final concentration) (Anti Rabbit IgG, Horseradish Peroxidase conjugated,

Upstate, Cat # A132P) in 12 mL of 1X TBST for 1 hr at room temperature in a small pipette 86

tip box lid. After incubation, the membrane was washed three times with 1X TBST (10 minutes / 5 minutes / 5 minutes). The membrane was then developed by using chemilluminance detection system (Super Signal West Femto Maximum Sensitivity

Substrate Pierce, Cat. # 34095).

25.5. Enhanced Chemilluminance Detection System

A working solution of Super Signal West Substrate was prepared by mixing 2.5 mL of Supersignal West Femto Luminol/Enhancer solution and 2.5 mL of Supersignal West

Femto Stable Peroxide solution (1:1 ratio) (Super Signal West Femto Maximum Sensitivity

Substrate, Cat. # 34095, Pierce). After the incubation with secondary antibody and a series of washes, 5 mL of working solution of Super Signal West Substrate was uniformly layered over the PVDF membrane using a 5ml serological pipette. The blot was then incubated for 5 minutes at room temperature. After incubation, the excess reagent was drained off and the blot was covered with saran wrap, scotch taped in a cassette. It was then exposed to a CL-

XPosure Film 5 x 7 in (13 x 18 cm) (Pierce, Cat # 34092) for 30 seconds or longer, depending on the amount transferred.

25.6. Membrane Development

The film was developed in the dark by immersing and gently agitating the film for 5 minutes in developer solution (Kodak GBX developer, Cat. # 1900984). The film was rinsed for 20-30 seconds in distilled water and finally it was placed in fixer solution (Kodak GBX fixer, Cat. # 1902485) for 1-2 minutes. The film was then rinsed once more with the distilled water and then hung up to dry.

87

26. Atomic Force Microscopy

26.1. Sample Preparation

Thirty microliters (~200 CPM) of both canonical nucleosomes (N) and HMGB1-

remodeled nucleosomes (N’/N’’) from the 5-30% sucrose linear gradient fraction was

aliquoted into two separate Eppendorf tubes, respectively. To it, 20 µL of TE/sucrose (18%

sucrose) buffer was added to make the final volume of 50 µL. One µL of 25% glutaraldehyde

was added to make 0.5% final concentration, and incubated at 4oC for 6 hours. After the

incubation, 150 µL of TE/sucrose (18% sucrose) buffer was added to make the final volume

of 200 µL. This 200 µL of cross-linked nucleosomes were dialyzed immediately against 1

liter of TE buffer at 4oC for overnight. The next day, 2 µL of the dialyzed sample was diluted

to 500 µL of TE buffer (250 times dilution). Again, from this dilution, 2 µL of the sample

was further diluted to 500 µL of TE buffer (250 times dilution), with a total dilution of

62,500 times. A 10 µL of the diluted sample was then used for preparing the slides.

26.2. Slide Preparation

A layer of mica surface on a mica slide (provided by Dr. Peter Lu) was peeled off by

using a cello tape. The fresh mica surface was then coated with 10 µL of 1 mM spermidine.

The spermidine was uniformly coated by using a spin dryer at Dr. Peter Lu’s Lab (Overman

Hall, Room # 141). To the spermidine treated mica surface, 10 µL of diluted nucleosomes

were added and uniformly layered by using a spin dryer at Dr. Peter Lu’s Lab. The mica

surface was gently washed for unbound samples using the dH2O, and air dried by using the

spin dryer.

88

26.3. Atomic Force Microscopy

With the assistance of Dr. Yufan He at Dr. Peter Lu’s lab (Overman Hall, Room #

141), the prepared samples of both canonical nucleosomes and HMGB1-remodeled nucleosomes were observed under the atomic force microscopy. The silicon tip cantilever was carefully mounted to atomic force microscope and calibrated. The prepared slide was then loaded with care using the cell tape and the samples on the mica surface were scanned for optimum image generation. The image was saved in the disk. 89

CHAPTER III: RESULTS I

1. Construction of Luciferase Reporter Constructs

1.1. Restriction Digestion of Luciferase Reporter Vector and Ligation of ERE

Sequences into the Vector

The luciferase reporter vector (pGL2-3cERE-TATA-Inr-Luc, fig 6) containing three

tandem copies of vitellogenin EREs (3cERE) was obtained from Dr. D. P. McDonnell (Duke

University) (Hall and McDonnell, 1999). The 3cEREs insert in the pGL2-3cERE-TATA-Inr-

Luc reporter vector is located at Xho I and Bgl II site 5’ to TATA-Inr sequence upstream of

the luciferase reporter gene. Therefore, various constructs of luciferase reporter vector with

different ERE inserts (single cERE, tandem cEREs, single cHERE, tandem cHEREs and

variant spacer cEREs n=0, 1, 2, 3 and 4, where n is the number of nucleotides in between two

half sites of the cERE) (Tables 4, 5 and 6) were prepared by excising the 3cERE at Xho I and

Bgl II sites by restriction digestion and ligation with the ERE inserts. The plasmid DNA

(pGL2-3cERE-TATA-Inr-Luc) was first digested with Xho I and then with Bgl II as

described in materials and methods (M&M). The Xho I and Bgl II digest of the plasmid

DNA was verified on a 1% agarose gel as shown in figure 13. The plasmid DNA was

completely digested as seen in the gel (Figure 13, Lane 3), with the circular plasmid DNA

(5756bp) in lane 2 exhibiting a relative mobility of ca. 3500bp, while the linear Xho I and

Bgl II digested fragment of length 5683bp in lane 3 has a relative mobility corresponding to

ca. 6kb DNA marker.

After Xho I and Bgl II digestion, the linear plasmid DNA was purified and

concentrated as described in M&M. The 5683bp plasmid DNA having Xho I and Bgl II

overhangs was mixed with ERE DNA inserts (single cERE, tandem cEREs, single cHERE, 90

Figure 13. Agarose gel (1%) of restriction digests of pGL2-3cERE-TATA-Inr-Luc. Lane 1:

1kb DNA ladder. Lane 2: Undigested 5.7kb circular plasmid DNA (pGL2-3cERE-TATA-Inr-

Luc). Lane 3: Xho I and Bgl II digested 5685pb linear plasmid DNA (pGL2-TATA-Inr-Luc). 91

tandem cHEREs or variant spacer cEREs: n=0, 1, 2, 3, & 4, where n is the number of nucleotides in between two half site of cERE) having Xho I and Bgl II overhangs and ligated with T4 DNA ligase as described in M&M. The ligation mixture, undigested plasmid DNA, and Xho I and Bgl II digested DNA was analyzed on a 1% agarose gel to verify the ligation reaction as shown in figure 14. The ligated product in lane 4 has a relative mobility corresponding to the undigested plasmid in lane 2. Since the plasmid DNA of 5756bp is circular, it has a greater relative mobility corresponding to ca. 3.5kb DNA marker. The Xho I and Bgl II digested fragment is linear 5683bp (lane 1) and has a relative mobility corresponding to ca. 6kb DNA marker. A multiple chain ligation of linear plasmid among themselves was seen as an aggregation on the well (Figure 14, Lane 4).The ligated product was then transformed to E. coli (JM109) cells. As yet from these data, it is only confirmed that the ligation of the digested plasmid DNA was successful, where as there is no evidence of the small ERE inserts having been ligated to the plasmid. Therefore, the colony PCR was performed to screen the ERE inserts containing transformants.

1.2. Colony PCR

The ligated product was transformed to the JM109 strain of E. coli cells. The ligated product might have a mixture of two plasmid , viz. 1) recircularized plasmid DNA without the insert, and 2) recircularized plasmid DNA with the one or more ERE inserts.

Therefore, the transformed E. coli, colonies grown on the LB/ampicillin agar plate may carry either of the plasmid DNAs. In order to screen the transformed colony containing the ligated product of plasmid DNA with the ERE insert, colony PCR was performed using the primers designed to amplify the DNA sequence from 5’ Xho I to 3’ Hind III end as described in 92

Figure 14. Agarose gel (1%) of restriction digestion and ligation to produce pGL2-2cERE-

TATA-Inr-Luc. Lane 1: Xho I and Bgl II digested 5685pb linear plasmid DNA (pGL2-TATA-

Inr-Luc). Lane 2: Undigested 5.7kb plasmid DNA (pGL2-3cERE-TATA-Inr-Luc). Lane 3: 1kb

DNA ladder. Lane 4. Crude ligation mixture of Xho I and Bgl II digest of pGL2-3cERE-TATA-

Inr-Luc and 2cERE insert. 93

M&M. A random number of colonies were taken for PCR and each colony was replica plated

by transferring into another LB/ampicillin agar plate. The colonies were labeled C1, C2, C3

…Cn, where, n is the number of colony used for PCR. The colony PCR was performed as

described in M&M, and the PCR product was analyzed on a 6% polyacrylamide gel (Fig 15).

The PCR amplified sequence without any insert had a greater mobility with respect to the

PCR amplified sequence with various inserts as shown in figure 15. The sizes of the product

of the colony PCR amplified by primers “5’-AGC TCT TAC GCG TGC TAG CT-3’” and

“5’-TTA CCA ACA GTA CCG GAA TGC-3’” for various ERE inserts are tabulated in

Table 11.

Four JM109 colonies (C2, C4, C5 and C7) with recombinant luciferase reporter vector were found to have a 167bp PCR amplified DNA sequence containing 1cERE insert

(Figure 15, panel A). Commensurate with the seven colonies screened, three colonies (C1,

C3 and C6) were found to have a 146bp PCR amplified DNA sequence and were without the

1cERE insert (Figure 15, panel A). Colony C4 was selected for amplification of recombinant

luciferase reporter vector with 1cERE. The cells were grown, plasmid isolated, and the

isolated plasmid were sent to Retrogen Inc. for DNA sequencing.

Six JM109 colonies (C1, C4, C5, C6, C7, and C9) with recombinant luciferase

reporter vector were found to have a 164bp PCR amplified DNA sequence containing 2cERE

insert (Figure 15, panel B). Commensurate with that of nine colonies screened, three colonies (C2, C3 and C8) were found to have a 146bp PCR amplified DNA sequence without

2cERE insert (Figure 15, panel B). Colony C4 was selected for amplification of recombinant

luciferase reporter vector with 2cERE. The cells were grown, plasmid isolated, and the

isolated plasmid were sent to Retrogen Inc. for DNA sequencing. 94

Table 11: Sizes of PCR product for the colony PCR of the transformed E. coli

(JM109) colonies with recombinant vectors containing various estrogen response

elements.

ERE DNA Size of PCR Insert Product (bp) Empty Vector 146 1cERE 167 2cERE 190 3cERE 219 1cHERE 167 2cHERE 190 3cHERE 219 cEREn=1 164 cEREn=2 165 cEREn=3 166 cEREn=4 168

Four JM109 colonies (C1, C2, C3, and C4) with recombinant luciferase reporter vector were found to have a 168bp PCR amplified DNA sequence containing cERE4 insert

(Figure 15, panel C). Commensurate with that of nine colonies screened for a PCR, five colonies (C5, C6, C7, C8 and C9) were found to have a 146bp PCR amplified DNA sequence without cERE4 insert (Figure 15, panel C). Colony C1 was selected for amplification of recombinant luciferase reporter vector with cERE4. The cells were grown, plasmid isolated, and the isolated plasmid were sent to Retrogen Inc. for DNA sequencing.

Six JM109 colonies (C1, C2, C6, C7, C8, and C9) with recombinant luciferase reporter vector were found to have a 164bp PCR amplified DNA sequence containing cERE0 insert (Figure 15, panel D). Commensurate with that of nine colonies screened for a PCR, three colonies (C3, C4, and C5) were found to have a 146bp PCR amplified DNA sequence without cERE0 insert (Figure 15, panel D). Colony C2 was selected for amplification of 95

Figure 15. Six % polyacrylamide gel of PCR products for colony PCR: All DNAs were

from transformed JM109 colonies. The transformed colony marked with red was the ones

selected for amplification of recombinant luciferase reporter vector with the ERE insert. Panel A-

I, lane 1: 100bp DNA ladder. Panel A, lanes 2-8: PCR products of C1-C7 with 1cERE insert.

Panel B, lanes 2-10: PCR products of C1-C9 with 2cERE insert. Panel C, lanes 2-10: PCR of C1-

C9 with cEREn=4 insert. Panel D, lanes 2-10: PCR products of C1-C9 with cEREn=0 insert.

Panel E, lanes 2-10: PCR products of C1-C9 with cEREn=1 insert. Panel F, lanes 2-10: PCR products of C1-C9 with cEREn=2 insert. Panel G, lanes 2-9: PCR products of C1-C8 with

1cHERE insert. Panel H, lanes 2-10: PCR products of C1-C9 with 2cHERE insert. Panel I, lanes

2-9: PCR products of C1-C8 with 3cHERE insert. 96

recombinant luciferase reporter vector with cERE0. The cells were grown, plasmid isolated,

and the isolated plasmid were sent to Retrogen Inc. for DNA sequencing.

Seven JM109 colonies (C1, C2, C3, C4, C6, C7, and C8) with recombinant luciferase

reporter vector were found to have a 165bp PCR amplified DNA sequence containing cERE1

insert (Figure 15, panel E). Commensurate with that of nine colonies screened for a PCR, two

colonies (C5, and C9) were found to have a 146bp PCR amplified DNA sequence without

cERE1 insert (Figure 15, panel E). Colony C3 was selected for amplification of recombinant

luciferase reporter vector with cERE1. The cells were grown, plasmid isolated, and the

isolated plasmid were sent to Retrogen for DNA sequencing.

Eight JM109 colonies (C1, C2, C3, C4, C6, C7, C8 and C9) with recombinant

luciferase reporter vector were found to have a 166bp PCR amplified DNA sequence

containing cERE2 insert (Figure 15, panel F). Commensurate with that of nine colonies screened for a PCR, one colony (C5) was found to have a 146bp PCR amplified DNA sequence without cERE2 insert (Figure 15, panel F). Colony C2 was selected for amplification of recombinant luciferase reporter vector with cEREn=2. The cells were grown, plasmid isolated, and the isolated plasmid were sent to Retrogen Inc. for DNA sequencing.

Four JM109 colonies (C2, C3, C4, and C6) with recombinant luciferase reporter vector were found to have a 167bp PCR amplified DNA sequence containing 1cHERE insert

(Figure 15, panel G). Commensurate with that of eight colonies screened for a PCR, four colonies (C1, C5, C7 and C8) were found to have a 146bp PCR amplified DNA sequence without 1cHERE insert (Figure 15, panel G). Colony C4 was selected for amplification of 97

recombinant luciferase reporter vector with 1cHERE. The cells were grown, plasmid

isolated, and the isolated plasmid were sent to Retrogen Inc. for DNA sequencing.

Five JM109 colonies (C1, C3, C4, C5, and C7) with recombinant luciferase reporter vector were found to have a 190bp PCR amplified DNA sequence containing 2cHERE insert

(Figure 15, panel H). Commensurate with that of nine colonies screened for a PCR, four colonies (C2, C6, C8, and C9) were found to have a 146bp PCR amplified DNA sequence without 2cHERE insert (Figure 15, panel H). Colony C3 was selected for amplification of recombinant luciferase reporter vector with 2cHERE. The cells were grown, plasmid isolated, and the isolated plasmid were sent to Retrogen Inc. for DNA sequencing.

Five JM109 colonies (C1, C5, C6, C7, and C8) with recombinant luciferase reporter vector were found to have a 219bp PCR amplified DNA sequence containing 3cHERE insert

(Figure 15, panel I). Commensurate with that of eight colonies screened for a PCR, three colonies (C2, C3, and C4) were found to have a 146bp PCR amplified DNA sequence without 3cHERE insert (Figure 15, panel I). Colony C5 was selected for amplification of recombinant luciferase reporter vector with 3cHERE. The cells were grown, plasmid isolated, and the isolated plasmid were sent to Retrogen Inc. for DNA sequencing.

1.3. DNA Sequencing of Constructs

After identification of the transformed JM109 cells with the recombinant luciferase vector with various estrogen response elements subcloned (single cERE, tandem cEREs, single cHERE, tandem cHEREs or variant spacer cEREs: n=0, 1, 2, 3, & 4, where n is the number of nucleotides in between two half site of cERE), the plasmid from the JM109 cells were isolated. The isolated plasmid DNA was sent to Retrogen Inc for DNA sequencing, and the DNA sequences for respective inserts subcloned were determined as shown in boxes 2- 98

Sequences for Series of Tandem Consensus EREs (cEREs ≡ ERE3)

1. p1cERE

P1cERE-TATA-INR-LUC GLPRIMER-1

NNNNNNNNNNNNNGNNNNAGCTCTTACGCGTGCTAGCTCGAGAGGTCACTGTGACCTAGATCTGATATCATC GATGAATTCGGGCTATAAAAGGGGGTGGGGGGAGCTCGGCCCTCATTCTGGAGACGGATCCTCTAGAGTCGA CCTGCAGGCATGCAAGCTTGGCATTCCGGTACTGTTGGTAAAATGGAAGACGCCAAAAACATAAAGAAAGGC CCGGCGCCATTCTATCCTCTAGAGGATGGAACCGCTGGAGAGCAACTGCATAAGGCTATGAAGAGATACGCC CTGGTTCCTGGAACAATTGCTTTTACAGATGCACATATCGAGGTGAACATCACGTACGCGGAATACTTCGAA ATGTCCGTTCGGTTGGCAGAAGCTATGAAACGATATGGGCTGAATACAAATCACAGAATCGTCGTATGCAGT GAAAACTCTCTTCAATTCTTTATGCCGGTGTTGGGCGCGTTATTTATCGGAGTTGCAGTTGCGCCCGCGAAC GACATTTATAATGAACGTGAATTGCTCAACAGTATGAACATTTCGCAGCCTACCGTAGTGTTTGTTTCCAAA AAGGGGTTGCAAAAAATTTTGAACGTGCAAAAAAAATTACCAATAATCCAGAAAATTATTATCATGGATTCT AAAACGGATTACCAGGGATTTCAGTCGATGTACACGTTCGTCACATCTCATCTACCTCCCGGTTTTAATGAA TACGATTTTGTACCAGAGTCCTTTGATCGTGACAAAACAATTGCACTGATAATGAATTCCTCTGGATCTACT GGGTTACCTAAGGGTGTGGCCCTTCCGCATAGAACTGCCTGCGTCAGANTCTCGCATGCCAGANATCCTATT TTTGGCAATCAAATCATTCCGGATACTGNGATTTTAAGTGNTGTTCCATTCCATCACGGTTTTGGAATGTTT ACTACACTCGGANNNTTGATATGTGGATTTCGAGTCGTCTTNATGNATANATTTGANANANCTGTTTTACNA TCCCNTCNGNANTNNNAAATTCAAAGTGCGTTGNTANNACCNNNNN

P1cERE-TATA-INR-LUC GLPRIMER-1

ANNNNNNNCGGNNGCCAGCTTGCATGCCTGCAGGTCGACTCTAGAGGATCCGTCTCCAGAATGAGGGCCGAG CTCCCCCCACCCCCTTTTATAGCCCGAATTCATCGATGATATCAGATCTAGGTCACAGTGACCTCTCGAGCT AGCACGCGTAAGAGCTCGGTACCTCCCGGGTTATGTTAGCTCAGTTACAGTACCATAAGATACATTGATGAG TTTGGACAAACCACAACTAGAATGCAGTGAAAAAAATGCTTTATTTGTGAAATTTGTGATGCTATTGCTTTA TTTGTAACCATTATAAGCTGCAATAAACAAGTTAACAACAACAATTGCATTCATTTTATGTTTCAGGTTCAG GGGGAGGTGTGGGAGGTTTTTTAAAGCAAGTAAAACCTCCACGTACCTTAATATTACTTACTTATCATGGTA GCTTGGGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCG AATGGCAAATTGTAAGCGTTAATATTTTGTTAAAATTCGCGTTAAATTTTTGTTAAATCAGCTCATTTTTTA ACCAATAGGCCGAAATCGGCAAAATCCCTTATAAATCAAAAGAATAGACCGAGATAGGGTTGAGTGTTGTTC CAGTTTGGAACAAGAGTCCACTATTAAAGAACGTGGACTCCAACGTCAAAGGGCGAAAAACCGTCTATCAGG GCGATGGCCCACTACGTGAACCATCACCCTAATCAAGTTTTTTGGGGTCGAGGTGCCGTAAAGCACTAAATC GGAACCCTAAAGGGAGCCCCCGATTTAGAGCTTGACGGGGAAAGCCGGCGAACGTGGCGAGAAAGGAAGGGA AGAAAGCGAAAGGAGCGGGCGCTAGGGCGCTGGCANNTGTANCGGTCACGCTGCGCGTAACCACCACACCCG CCGCGCTTAATGCGCCGCTACAGGGCGCGTCAGGNGGCACTTTTCGGGGAAATGTGCNCGGAACCCCNANTT GTTNATTTTTCTAATACNTTCAATNTGTATCCGCTCANGANNNNATNACCCTGANNNATGCTTCNNN

Box 2. DNA sequences for p1cERE-TATA-Inr-Luc from Retrogen Inc using GL

primer 1 and GL primer 2: Upper panel shows the plus strand DNA 5’-3’ direction

with yellow highlight showing the insert subcloned and blue sequences for ERE and pink

sequences for restriction sites. Lower panel shows the minus strand DNA 5’-3’ direction

with yellow highlight showing the insert subcloned and blue sequences for ERE and pink

sequences for restriction sites. 99

2. p2cERE

P2cERE-TATA-INR-LUC GLPRIMER-1

NNNNNNNNNNGNNNGNNCNGAGCTCTTACGCGTGCTAGCTCGAGAGGTCACTGTGACCTAGATCCGCAGGTC ACTGTGACCTAGATCTGATATCATCGATGAATTCGGGCTATAAAAGGGGGTGGGGGGAGCTCGGCCCTCATT CTGGAGACGGATCCTCTAGAGTCGACCTGCAGGCATGCAAGCTTGGCATTCCGGTACTGTTGGTAAAATGGA AGACGCCAAAAACATAAAGAAAGGCCCGGCGCCATTCTATCCTCTAGAGGATGGAACCGCTGGAGAGCAACT GCATAAGGCTATGAAGAGATACGCCCTGGTTCCTGGAACAATTGCTTTTACAGATGCACATATCGAGGTGAA CATCACGTACGCGGAATACTTCGAAATGTCCGTTCGGTTGGCAGAAGCTATGAAACGATATGGGCTGAATAC AAATCACAGAATCGTCGTATGCAGTGAAAACTCTCTTCAATTCTTTATGCCGGTGTTGGGCGCGTTATTTAT CGGAGTTGCAGTTGCGCCCGCGAACGACATTTATAATGAACGTGAATTGCTCAACAGTATGAACATTTCGCA GCCTACCGTAGTGTTTGTTTCCAAAAAGGGGTTGCAAAAAATTTTGAACGTGCAAAAAAAATTACCAATAAT CCAGAAAATTATTATCATGGATTCTAAAACGGATTACCAGGGATTTCAGTCGATGTACACGTTCGTCACATC TCATCTACCTCCCGGTTTTAATGAATACGATTTTGTACCAGAGTCCTTTGATCGTGACAAAACAATTGCACT GATAATGAATTCCTCTGGATCTACTGGGTTACCTAAGGGTGTGGCCCTTCCGCATAGAACTGCCTGCGTCAG ANTCTCGCATGCCAGANATCCTATTTTTGGCAATCNAATCATTCCGGATACTGCGATTTTAAGTGTTGTTCC ATTCCATCACGGTTTTGGAATGTTTACTACACTCGGATATTTNATATGNGGATTTCGAGTCGTCTTAATGNA TAGATTTNAAGAANANCTNNTTTTACNATCCCTTCNGGATNNNAAANTCNAAGNGCNTNCTAGNNCNNCNNN NTTTNNTNNNNNNAAAGCNNNCTGATNNNNAANNCNANTNNNNTAATTNNNNNAANTGCTNNNN

P2cERE-TATA-INR-LUC GLPRIMER-2

NNNNNNNNNNNCCGGNNNGCCAGCTTGCATGCCTGCAGGTCGANNNNAGAGGATCCGTCTCCAGAATGAGGG CCNAGCTCCCCCCACCCCCTTTTATAGCCCGAATTCATCGATGATATCAGATCTAGGTCACAGTGACCTGCG GATCTAGGTCACAGTGACCTCTCGAGCTAGCACGCGTAAGAGCTCGGTACCTCCCGGGTTATGTTAGCTCAG TTACAGTACCATAAGATACATTGATGAGTTTGGACAAACCACAACTAGAATGCAGTGAAAAAAATGCTTTAT TTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTATAAGCTGCAATAAACAAGTTAACAACAACAA TTGCATTCATTTTATGTTTCAGGTTCAGGGGGAGGTGTGGGAGGTTTTTTAAAGCAAGTAAAACCTCCACGT ACCTTAATATTACTTACTTATCATGGTAGCTTGGGCTGGCGTAATAGCGAANAGGCCCGCACCGATCGCCCT TCCCAACAGTTGCNCANCCTGAATGGCGAATGGCAAATTGTAAGCGTTAATATTTTGTTAAAATTCGCGTTA AATTTTTGTTAAATCANCTCATTTTTTAACCNATANGCCGAAATCNGCNAAATCCCTTATAAATCAAAAGAA TANACCNAGATAGGGTTGAGTGTTGTTCCNGTTTGGAACAAGAGTCCACTATTAAAGAACGTGGACTCCAAC GTCNAAGGGCGAAAAANCGTCTATCANGGCGATGGCCCACTACNNGAACCATCACCCTNATCNAGTTTTTTG GGGTCNAGGTGCCGTANAGCACTAAATCGGAACCCTNAAGGGAGCCCCCGATTTNNAGCTTGACNGGGAAAG CCNGCNAACNTGNCGAGAAANGANNGNAANANAGCNAAAGGAGCGGGCGCTANGGCGCTGGNAAGTGTANCG GNCACGCTGCNCGTANCNNNNCACCCGCCNCGCTTAANGCGCCGCTNCNGGGCGCGTCNGNNGNACTTTCNG GNAATGTGCNCGGNACCCCNATNNNNNNTTNNNANTNCNTNNAATATGTNNCNNNNNNNNNNNATNANCCNN NNNNTGNNTNATNNNNGAANNNNANANNNTNNNNNNNNNCNTTNNNNNN Box 3. DNA sequences for p2cERE-TATA-Inr-Luc from Retrogen Inc using GL

primer 1 and GL primer 2: Upper panel shows the plus strand DNA 5’-3’ direction

with yellow highlight showing the insert subcloned and blue sequences for ERE and pink

sequences for restriction sites. Lower panel shows the minus strand DNA 5’-3’ direction

with yellow highlight showing the insert subcloned and blue sequences for ERE and pink

sequences for restriction sites. 100

3. p3cERE

P3cERE-TATA-INR-LUC GLPRIMER-1

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNCGTGCNANCTCGAGATCTAGGTCACAGTGACCTGCGGATCCG CAGGTCACTGTGACCTAGATCCGCAGGTCACTGTGACCTAGATCTGATATCATCGANGNNNNCGGGCTATAA AAGGGGGNGGGGGGAGCTCGGCCCTCATTCTGGAGACGGATCCTCTAGAGTCGACCTGCAGGCATGCAAGCT TGGCATTCCGGTACTGTTGGTAAAATGGAAGACGCCAAAAACATAAAGAAAGGCCCGGCGCCATTCTATCCT CTAGAGGATGGAACCGCTGGAGAGCAACTGCATAAGGCTATGAAGAGATACGCCCTGGTTCCTGGAACAATT GCTTTTACAGATGCACATATCGAGGTGAACATCACGTACGCGGAATACTTCGAAATGTCCGTTCGGTTGGCA GAAGCTATGAAACGATATGGGCTGAATACAAATCACAGAATCGTCGTATGCAGTGAAAACTCTCTTCAATTC TTTATGCCGGTGTTGGGCGCGTTATTTATCGGAGTTGCAGTTGCGCCCGCGAACGACATTTATAATGAACGT GAATTGCTCAACAGTATGAACATTTCGCAGCCTACCGTAGTGTTTGTTTCCAAAAAGGGGTTGCAAAAAATT TTGAACGTGCAAAAAAAATTACCAATAATCCAGAAAATTATTATCATGGATTCTAAAACGGATTACCAGGGA TTTCAGTCGATGTACACGTTCGTCACATCTCATCTACCTCCCGGTTTTAATGAATACGATTTTGTACCAGAG TCCTTTGATCGTGACAAAACAATTGCACTGATAATGAATTCCTCTGGATCTACTGGGTTANCTNAAGGNTGT GGCCCTTCCGCATAGAACTGNCTGCGTCANANTCTCGCATGCCANANATCCTATTTTTGGCAATCAAATCAT TCCNGANACTGCGATTTTAAGTGTTGTTCCATTCCATCACGGTTTTGGAATGNTTACTACNCTCGNANNNTT NANNATGNNGNTTTCNAGTCNNCNTNATGNNNNNNNTTNAANANNNN

P3cERE-TATA-INR-LUC GLPRIMER-2

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNGNNNNNNNNNNNNNGCCGAG NNNNNNCCACCCCCNNNNNNAGCCCGAATTNATCGNNNNNNNCAGATCTAGGTCACAGTGACCTGCGGATCT AGGTCACAGTGACCTGCGGATCCGCAGGTCACTGTGACCTAGATCTCGAGCTAGCACGCGTANNNNCNCGGT ACCTCCCGGGTTATGTTAGCTCAGTTACAGTACCATAAGATACATTGATGAGTTTGGACAAACCACAACTAG AATGCAGTGAAAAAAATGCTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTATAAGCTG CAATAAACAAGTTAACAACAACAATTGCATTCATTTTATGTTTCAGGTTCAGGGNNANNNTGGGAGGTTTTT TAAAGCAAGTAAAACCTCCACGTACCTTAATATTACTTACTTATCATGGTAGCTTGGGCTGGCGTAATAGCG AAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCANCCTGAATGGCGAATGGCAAATTGTAAGCGTTA ATATTTTGTTAAAATTCGCGTTAAATTTTTGTTAAATCANCTCATTTTTTAACCAATAGGCCGAAATCGGCA AAATCCCTTATAAATCAAAAGAATANACCNAGATAGGGTTGAGTGTTGTTCCAGTTTGGAACAAGAGTCCAC TATTAAAGAACGTGGACTCCAACGTCAAAGGGCGAAAAANCGTCTATCNNGGCGATGGCCCACTACGTGAAC CATCACCCTAATCNAGTTTTTTGGGGTCGANGTGCCGTAAAGCACTAAATCGGAACCCTAAAGGGANCCCCC GANTTNNAGCTTGACGGGGAAAGCCGGCGAACGTGGCGAGAAANGAANGNNANAAAGCNAAAGGAGCGNCNN TANGCGCTGGNNNNTGTANCGNCACGCTGCNNGTANNCNCANCGCCNCNCTTNANGNNCNNTNCAGGCNCGT CNGNGNACTTTNNNAANNNN

Box 4. DNA sequences for p3cERE-TATA-Inr-Luc from Retrogen Inc using GL

primer 1 and GL primer 2: Upper panel shows the plus strand DNA 5’-3’ direction

with yellow highlight showing the insert subcloned and blue sequences for ERE and pink

sequences for restriction sites. Lower panel shows the minus strand DNA 5’-3’ direction

with yellow highlight showing the insert subcloned and blue sequences for ERE and pink

sequences for restriction sites. 101

Sequences for Series of Tandem ERE Half Sites (HEREs)

1. pHERE

P1HERE-TATA-INR-LUC GLPRIMER-1

NNNNNNNNNNNNNNNNGTNCCGAGCTCTTACGCGTGCTAGCTCGAGAGGTCACTGGTTGGGAGATCTGATAT CATCGATGAATTCGGGCTATAAAAGGGGGTGGGGGGAGCTCGGCCCTCATTCTGGAGACGGATCCTCTAGAG TCGACCTGCAGGCATGCAAGCTTGGCATTCCGGTACTGTTGGTAAAATGGAAGACGCCAAAAACATAAAGAA AGGCCCGGCGCCATTCTATCCTCTAGAGGATGGAACCGCTGGAGAGCAACTGCATAAGGCTATGAAGAGATA CGCCCTGGTTCCTGGAACAATTGCTTTTACAGATGCACATATCGAGGTGAACATCACGTACGCGGAATACTT CGAAATGTCCGTTCGGTTGGCAGAAGCTATGAAACGATATGGGCTGAATACAAATCACAGAATCGTCGTATG CAGTGAAAACTCTCTTCAATTCTTTATGCCGGTGTTGGGCGCGTTATTTATCGGAGTTGCAGTTGCGCCCGC GAACGACATTTATAATGAACGTGAATTGCTCAACAGTATGAACATTTCGCAGCCTACCGTAGTGTTTGTTTC CAAAAAGGGGTTGCAAAAAATTTTGAACGTGCAAAAAAAATTACCAATAATCCAGAAAATTATTATCATGGA TTCTAAAACGGATTACCAGGGATTTCAGTCGATGTACACGTTCGTCACATCTCATCTACCTCCCGGTTTTAA TGAATACGATTTTGTACCAGAGTCCTTTGATCGTGACAAAACAATTGCACTGATAATGAATTCCTCTGGATC TACTGGGTTACCTAAGGGTGTGGCCCTTCCGCATAGAACTGCCTGCGTCAGANTCTCGCATGCCAGAGATCC TATTTTTGGCAATCAAATCATTCCGGANACTGCGATTTTAAGTGTTGTTCCATTCCATCACGGTTTTGGAAT GTTTACTACACTCGGANATTTGATATGTGGATTTCGAGTCGTCTTAATGNATANATTTGAANAANAGCTGNT TTTACNATCCCTTCNNNNTACNAAATTCAAAGTGCGNNN

P1HERE-TATA-INR-LUC GLPRIMER-2

NNNNNNNNNNNCGNNANNGCCAGCTTGCATGCCTGCANGNNNANTCCTAGAGGANCCGTCTCCAGAATGAGG GCCNNNNTCCCCCCACCCCCTTTTATAGCCCGAATTCATCGATGATATCAGATCTCCCAACCAGTGACCTCT CGAGCTAGCACGCGTAAGAGCTCGGTACCTCCCGGGTTATGTTAGCTCAGTTACAGTACCATAAGATACATT GATGAGTTTGGACAAACCACAACTAGAATGCAGTGAAAAAAATGCTTTATTTGTGAAATTTGTGATGCTATT GCTTTATTTGTAACCATTATAAGCTGCAATAAACAAGTTAACAACAACAATTGCATTCATTTTATGTTTCAG GTTCAGGGGGAGGTGTGGGAGGTTTTTTAAAGCAAGTAAAACCTCCACGTACCTTAATATTACTTACTTATC ATGGTAGCTTGGGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGA ATGGCGAATGGCAAATTGTAAGCGTTAATATTTTGTTAAAATTCGCGTTAAATTTTTGTTAAATCAGCTCAT TTTTTAACCAATAGGCCGAAATCGGCAAAATCCCTTATAAATCAAAAGAATAGACCGAGATAGGGTTGAGTG TTGTTCCAGTTTGGAACAAGAGTCCACTATTAAAGAACGTGGACTCCAACGTCAAAGGGCGAAAAACCGTCT ATCAGGGCGATGGCCCACTACGTGAACCATCACCCTAATCAAGTTTTTTGGGGTCGAGGTGCCGTAAAGCAC TAAATCGGAACCCTAAAGGGAGCCCCCGANTTAGAGCTTGACGGGGAAAGCCGGCGAACGTGGCGAGAAAGG AAGGGAAGAAAGCGAAAGGAGCGGGCGCTAGGGCGCTGGCNNNTGTANCGGTCACGCTGCGCGTAACCACCA CACCCGCCGCGCTTAANGCGCCGCTACAGGGCGCGTCNGNTGGCACTTTTCGGGGAAATGTGCNCGGAACCC CTATTTGTTTATTTTTCTAATNCNTTCAANNATGTATCCGCNNNN

Box 5. DNA sequences for p1HERE-TATA-Inr-Luc from Retrogen Inc using GL

primer 1 and GL primer 2: Upper panel shows the plus strand DNA 5’-3’ direction with

yellow highlight showing the insert subcloned and blue sequences for ERE and pink

sequences for restriction sites. Lower panel shows the minus strand DNA 5’-3’ direction

with yellow highlight showing the insert subcloned and blue sequences for ERE and pink

sequences for restriction sites.

102

2. p2HERE

P2HERE-TATA-INR-LUC GLPRIMER-1

NNNNNNNNNNNNNNGNNNNAGCTCTTACGCGTGCTAGCTCGAGAGGTCACTGGTTGGGAGATCCGCAGGTCA CTGGTTGGGAGATCTGATATCATCGATGAATTCGGGCTATAAAAGGGGGTGGGGGGAGCTCGGCCCTCATTC TGGAGACGGATCCTCTAGAGTCGACCTGCAGGCATGCAAGCTTGGCATTCCGGTACTGTTGGTAAAATGGAA GACGCCAAAAACATAAAGAAAGGCCCGGCGCCATTCTATCCTCTAGAGGATGGAACCGCTGGAGAGCAACTG CATAAGGCTATGAAGAGATACGCCCTGGTTCCTGGAACAATTGCTTTTACAGATGCACATATCGAGGTGAAC ATCACGTACGCGGAATACTTCGAAATGTCCGTTCGGTTGGCAGAAGCTATGAAACGATATGGGCTGAATACA AATCACAGAATCGTCGTATGCAGTGAAAACTCTCTTCAATTCTTTATGCCGGTGTTGGGCGCGTTATTTATC GGAGTTGCAGTTGCGCCCGCGAACGACATTTATAATGAACGTGAATTGCTCAACAGTATGAACATTTCGCAG CCTACCGTAGTGTTTGTTTCCAAAAAGGGGTTGCAAAAAATTTTGAACGTGCAAAAAAAATTACCAATAATC CAGAAAATTATTATCATGGATTCTAAAACGGATTACCAGGGATTTCAGTCGATGTACACGTTCGTCACATCT CATCTACCTCCCGGTTTTAATGAATACGATTTTGTACCAGAGTCCTTTGATCGTGACAAAACAATTGCACTG ATAATGAATTCCTCTGGATCTACTGGGTTACCTAAGGGTGTGGCCCTTCCGCATAGAACTGCCTGCGTCAGA TTCTCGCATGCCAGANATCCTATTTTTGGCAATCAAATCATTCCGGATACTGCGATTTTAAGTGTTGTTCCA TTCCATCACGGTTTTGGAATGTTTACTACACTCGGATATTTGATATGTGGATTTCGAGTCGTCTTAATGNAT AGATTTGAAGANANCTGTTTTACNATCCCNTCNNNTNNNAANTCNAANTGCNTNCTAGTNCNNCCTNTTTNN TNCNTNNCAAAGNNNNNTGATGACNAATNCNANTNNNNTNNTTNNNNNAANNTGNTNN

P2HERE-TATA-INR-LUC GLPRIMER-2

NNNNNNNNNNNNGNNGCCAGCTTGCATGCCTGCAGGTCGACTCTAGAGGANCCGTCTCCAGAATGAGGGCCG AGCTCCCCCCACCCCCTTTTATAGCCCGAATTCATCGATGATATCAGATCTCCCAACCAGTGACCTGCGGAT CTCCCAACCAGTGACCTCTCGAGCTAGCACGCGTAAGAGCTCGGTACCTCCCGGGTTATGTTAGCTCAGTTA CAGTACCATAAGATACATTGATGAGTTTGGACAAACCACAACTAGAATGCAGTGAAAAAAATGCTTTATTTG TGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTATAAGCTGCAATAAACAAGTTAACAACAACAATTG CATTCATTTTATGTTTCAGGTTCAGGGGGAGGTGTGGGAGGTTTTTTAAAGCAAGTAAAACCTCCACGTACC TTAATATTACTTACTTATCATGGTAGCTTGGGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCC CAACAGTTGCGCANCCTGAATGGCGAATGGCAAATTGTAAGCGTTAATATTTTGTTAAAATTCGCGTTAAAT TTTTGTTAAATCANCTCATTTTTTAACCAATAGGCCGAAATCGGCAAAATCCCTTATAAATCAAAAGAATAN ACCGAGATAGGGTTGAGTGTTGTTCCAGTTTGGAACAAGAGTCCACTATTAAAGAACGTGGACTCCNACGTC AAAGGGCGAAAAANCGTCTATCANGGCGATGGCCCACTACNTGAACCATCACCCTNATCNAGTTTTTTGGGG TCGAGGTGCCGTAAAGCACTAAATCGNAACCCTAAAGGGAGCCCCCGATTTANAGCTTGACGGGGAAAGCCG GCGAACGTGGCGAGAAAGGANNGNAAGAAAGCGAAAGGAGCGGGCGCTANGGCGCTGGCNAGTGTAGCGGTC ACGCTGCNCGTAACCACCACACCCGCCNNNNTNNNGNGCCGCTNNNNNCGCGTCNNTGGNACTTTNNGGAAN TNNGCNCNNANCCTANTNGNNNNTTNCNNAATNNNTTCAANATNNNNCNNNNNTNANACNNNNNCCNGNNNN NNNNNNNNANNTNAANNNNANANTNTNANNN

Box 6. DNA sequences for p2HERE-TATA-Inr-Luc from Retrogen Inc using GL

primer 1 and GL primer 2: Upper panel shows the plus strand DNA 5’-3’ direction

with yellow highlight showing the insert subcloned and blue sequences for ERE and pink

sequences for restriction sites. Lower panel shows the minus strand DNA 5’-3’ direction

with yellow highlight showing the insert subcloned and blue sequences for ERE and pink

sequences for restriction sites. 103

3. p3HERE

P3HERE-TATA-INR-LUC GLPRIMER-1

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNCGTGCNANCTCGAGAGGTCACAGGTTGGGGCGGATCCGCAGG TCACTGGTTGGGAGATCCGCAGGTCACTGGTTGGGAGATCTGATATCATCGANGNNNNCGGGCTATAAAAGG GGGNGGGGGGAGCTCGGCCCTCATTCTGGAGACGGATCCTCTAGAGTCGACCTGCAGGCATGCAAGCTTGGC ATTCCGGTACTGTTGGTAAAATGGAAGACGCCAAAAACATAAAGAAAGGCCCGGCGCCATTCTATCCTCTAG AGGATGGAACCGCTGGAGAGCAACTGCATAAGGCTATGAAGAGATACGCCCTGGTTCCTGGAACAATTGCTT TTACAGATGCACATATCGAGGTGAACATCACGTACGCGGAATACTTCGAAATGTCCGTTCGGTTGGCAGAAG CTATGAAACGATATGGGCTGAATACAAATCACAGAATCGTCGTATGCAGTGAAAACTCTCTTCAATTCTTTA TGCCGGTGTTGGGCGCGTTATTTATCGGAGTTGCAGTTGCGCCCGCGAACGACATTTATAATGAACGTGAAT TGCTCAACAGTATGAACATTTCGCAGCCTACCGTAGTGTTTGTTTCCAAAAAGGGGTTGCAAAAAATTTTGA ACGTGCAAAAAAAATTACCAATAATCCAGAAAATTATTATCATGGATTCTAAAACGGATTACCAGGGATTTC AGTCGATGTACACGTTCGTCACATCTCATCTACCTCCCGGTTTTAATGAATACGATTTTGTACCAGAGTCCT TTGATCGTGACAAAACAATTGCACTGATAATGAATTCCTCTGGATCTACTGGGTTANCTNAAGGNTGTGGCC CTTCCGCATAGAACTGNCTGCGTCANANTCTCGCATGCCANANATCCTATTTTTGGCAATCAAATCATTCCN GANACTGCGATTTTAAGTGTTGTTCCATTCCATCACGGTTTTGGAATGNTTACTACNCTCGNANNNTTNANN ATGNNGNTTTCNAGTCNNCNTNATGNNNNNNNTTNAANANNNN

P3HERE-TATA-INR-LUC GLPRIMER-2

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNGNNNNNNNNNNNNNGCCGAG NNNNNNCCACCCCCNNNNNNAGCCCGAATTNATCGNNNNNNNCAGATCTCCCAACCAGTGACCTGCGGATCT CCCAACCAGTGACCTGCGGATCCGCCCCAACCTGTGACCTCTCGAGCTAGCACGCGTANNNNCNCGGTACCT CCCGGGTTATGTTAGCTCAGTTACAGTACCATAAGATACATTGATGAGTTTGGACAAACCACAACTAGAATG CAGTGAAAAAAATGCTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTATAAGCTGCAAT AAACAAGTTAACAACAACAATTGCATTCATTTTATGTTTCAGGTTCAGGGNNANNNTGGGAGGTTTTTTAAA GCAAGTAAAACCTCCACGTACCTTAATATTACTTACTTATCATGGTAGCTTGGGCTGGCGTAATAGCGAAGA GGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCANCCTGAATGGCGAATGGCAAATTGTAAGCGTTAATAT TTTGTTAAAATTCGCGTTAAATTTTTGTTAAATCANCTCATTTTTTAACCAATAGGCCGAAATCGGCAAAAT CCCTTATAAATCAAAAGAATANACCNAGATAGGGTTGAGTGTTGTTCCAGTTTGGAACAAGAGTCCACTATT AAAGAACGTGGACTCCAACGTCAAAGGGCGAAAAANCGTCTATCNNGGCGATGGCCCACTACGTGAACCATC ACCCTAATCNAGTTTTTTGGGGTCGANGTGCCGTAAAGCACTAAATCGGAACCCTAAAGGGANCCCCCGANT TNNAGCTTGACGGGGAAAGCCGGCGAACGTGGCGAGAAANGAANGNNANAAAGCNAAAGGAGCGNCNNTANG CGCTGGNNNNTGTANCGNCACGCTGCNNGTANNCNCANCGCCNCNCTTNANGNNCNNTNCAGGCNCGTCNGN GNACTTTNNNAANNNN

Box 7. DNA sequences for p3HERE-TATA-Inr-Luc from Retrogen Inc using GL

primer 1 and GL primer 2: Upper panel shows the plus strand DNA 5’-3’ direction

with yellow highlight showing the insert subcloned and blue sequences for ERE and pink

sequences for restriction sites. Lower panel shows the minus strand DNA 5’-3’ direction

with yellow highlight showing the insert subcloned and blue sequences for ERE and pink

sequences for restriction sites. 104

Sequences for Series of cERE with variable spacers (0-4) EREn (n=spacer size)

1. pERE0

PERE0-TATA-INR-LUC GLPRIMER-1

NNNNNNNNNNNNNNNNNNAGCTCTTACGCGTGCTAGCTCGAGAGGTCATGACCTAGATCTGATATCATCGAT GAATTCGGGCTATAAAAGGGGGTGGGGGGAGCTCGGCCCTCATTCTGGAGACGGATCCTCTAGAGTCGACCT GCAGGCATGCAAGCTTGGCATTCCGGTACTGTTGGTAAAATGGAAGACGCCAAAAACATAAAGAAAGGCCCG GCGCCATTCTATCCTCTAGAGGATGGAACCGCTGGAGAGCAACTGCATAAGGCTATGAAGAGATACGCCCTG GTTCCTGGAACAATTGCTTTTACAGATGCACATATCGAGGTGAACATCACGTACGCGGAATACTTCGAAATG TCCGTTCGGTTGGCAGAAGCTATGAAACGATATGGGCTGAATACAAATCACAGAATCGTCGTATGCAGTGAA AACTCTCTTCAATTCTTTATGCCGGTGTTGGGCGCGTTATTTATCGGAGTTGCAGTTGCGCCCGCGAACGAC ATTTATAATGAACGTGAATTGCTCAACAGTATGAACATTTCGCAGCCTACCGTAGTGTTTGTTTCCAAAAAG GGGTTGCAAAAAATTTTGAACGTGCAAAAAAAATTACCAATAATCCAGAAAATTATTATCATGGATTCTAAA ACGGATTACCAGGGATTTCAGTCGATGTACACGTTCGTCACATCTCATCTACCTCCCGGTTTTAATGAATAC GATTTTGTACCAGAGTCCTTTGATCGTGACAAAACAATTGCACTGATAATGAATTCCTCTGGATCTACTGGG TTACCTAANGGTGTGGCCCTTCCGCATAGAACTGCCTGCGTCANATTCTCGCATGCCANAGATCCTATTTTT GGCAATCAAATCATTCCGGANN

PERE0-TATA-INR-LUC GLPRIMER-2

NNNNNNNNNNNNGNNGCCAGCTTGCATGCCTGCAGGTCGACTCTAGAGGATCCGTCTCCAGAATGAGGGCCG AGCTCCCCCCACCCCCTTTTATAGCCCGAATTCATCGATGATATCAGATCTAGGTCATGACCTCTCGAGCTA GCACGCGTAAGAGCTCGGTACCTCCCGGGTTATGTTAGCTCAGTTACAGTACCATAAGATACATTGATGAGT TTGGACAAACCACAACTAGAATGCAGTGAAAAAAATGCTTTATTTGTGAAATTTGTGATGCTATTGCTTTAT TTGTAACCATTATAAGCTGCAATAAACAAGTTAACAACAACAATTGCATTCATTTTATGTTTCAGGTTCAGG GGGAGGTGTGGGAGGTTTTTTAAAGCAAGTAAAACCTCCACGTACCTTAATATTACTTACTTATCATGGTAG CTTGGGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGA ATGGCAAATTGTAAGCGTTAATATTTTGTTAAAATTCGCGTTAAATTTTTGTTAAATCAGCTCATTTTTTAA CCAATAGGCCGAAATCGGCAAAATCCCTTATAAATCAAAAGAATAGACCGAGATAGGGTTGAGTGTTGTTCC AGTTTGGAACAAGAGTCCACTATTAAAGAACGTGGACTCCAACGTCAAAGGGCGAAAAACCGTCTATCAGGG CGATGGCCCACTACGTGAACCATCACCCTAATCAAGTTTTTTGGGGTCGAGGTGCCGTAAAGCACTAAATCG GAACCCTAAAGGGAGCCCCCGANTTAGAGCTTGACGGGGAAAGCCGGCGAACGTGGCGAGAAAGGAAGGGAA NAAAGCGAAAGGAGCGGGCGCTAGGGCGCTGGNNNTGNANCGGTCANNN

Box 8. DNA sequences for pERE0-TATA-Inr-Luc from Retrogen Inc using GL

primer 1 and GL primer 2: Upper panel shows the plus strand DNA 5’-3’ direction

with yellow highlight showing the insert subcloned and blue sequences for ERE and pink

sequences for restriction sites. Lower panel shows the minus strand DNA 5’-3’ direction

with yellow highlight showing the insert subcloned and blue sequences for ERE and pink

sequences for restriction sites. 105

2. pERE1

PERE1-TATA-INR-LUC GLPRIMER-1

NNNNNNNNNNNNNNNGTNCCGAGCTCTTACGCGTGCTAGCTCGAGAGGTCACTGACCTAGATCTGATATCAT CGATGAATTCGGGCTATAAAAGGGGGTGGGGGGAGCTCGGCCCTCATTCTGGAGACGGATCCTCTAGAGTCG ACCTGCAGGCATGCAAGCTTGGCATTCCGGTACTGTTGGTAAAATGGAAGACGCCAAAAACATAAAGAAAGG CCCGGCGCCATTCTATCCTCTAGAGGATGGAACCGCTGGAGAGCAACTGCATAAGGCTATGAAGAGATACGC CCTGGTTCCTGGAACAATTGCTTTTACAGATGCACATATCGAGGTGAACATCACGTACGCGGAATACTTCGA AATGTCCGTTCGGTTGGCAGAAGCTATGAAACGATATGGGCTGAATACAAATCACAGAATCGTCGTATGCAG TGAAAACTCTCTTCAATTCTTTATGCCGGTGTTGGGCGCGTTATTTATCGGAGTTGCAGTTGCGCCCGCGAA CGACATTTATAATGAACGTGAATTGCTCAACAGTATGAACATTTCGCAGCCTACCGTAGTGTTTGTTTCCAA AAAGGGGTTGCAAAAAATTTTGAACGTGCAAAAAAAATTACCAATAATCCAGAAAATTATTATCATGGATTC TAAAACGGATTACCAGGGATTTCAGTCGATGTACACGTTCGTCACATCTCATCTACCTCCCGGTTTTAATGA ATACGATTTTGTACCAGAGTCCTTTGATCGTGACAAAACAATTGCACTGATAATGAATTCCTCTGGATCTAC TGGGTTACCTAAGGGTGTGGCCCTTCCGCATAGAACTGCCTGCGTCANANTCTCGCATGCCAGANATCCTAT TTTTGGCAATCAAATCATTCCGGANACTGCGATTTTAAGTGNTGNTCCATTCCATCACGGNTTTGNANGNTT ACTNCN

PERE1-TATA-INR-LUC GLPRIMER-2

NNNNNNNNNNNGNNGCCAGCTTGCATGCCTGCAGGTCGACTCTAGAGGATCCGTCTCCAGAATGAGGNNNNA NCTCCCCCCACCCCCTTTTATAGCCCGAATTCATCGATGATATCAGATCTAGGTCAGTGACCTCTCGAGCTA GCACGCGTAAGAGCTCGGTACCTCCCGGGTTATGTTAGCTCAGTTACAGTACCATAAGATACATTGATGAGT TTGGACAAACCACAACTAGAATGCAGTGAAAAAAATGCTTTATTTGTGAAATTTGTGATGCTATTGCTTTAT TTGTAACCATTATAAGCTGCAATAAACAAGTTAACAACAACAATTGCATTCATTTTATGTTTCAGGTTCAGG GGGAGGTGTGGGAGGTTTTTTAAAGCAAGTAAAACCTCCACGTACCTTAATATTACTTACTTATCATGGTAG CTTGGGCTGGCGTAATANCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGA ATGGCAAATTGTAAGCGTTAATATTTTGTTAAAATTCGCGTTAAATTTTTGTTAAATCANCTCATTTTTTAA CCAATAGGCCGAAATCGGCAAAATCCCTTATAAATCAAAAGAATANACCGAGATAGGGNTGAGTGTTGTTCC AGTTTGGAACAAGAGTCCACTATTAAAGAACGTGGACTCCNACGTCAAAGGNCNAAAAANCGTCTATCANGG CGANGGCCCACTACGTGAACCATCNNCCTAATCAAGTTTTTTGGGGNCNAGGNGCCGTAAAGCACTANATCG GANCCTANAGGGAGCCCCCGANTTNNAGCTTGACGGGAAAGCCNNCNAACNTNGCGAGANANGAANGANNNN NNAAAGGANNNNNNCNNNGNACTGGNNNNNNNNNNNNNCTGNNCGNANNCANACNNNNNNGN

Box 9. DNA sequences for pERE1-TATA-Inr-Luc from Retrogen Inc using GL

primer 1 and GL primer 2: Upper panel shows the plus strand DNA 5’-3’ direction

with yellow highlight showing the insert subcloned and blue sequences for ERE and pink

sequences for restriction sites. Lower panel shows the minus strand DNA 5’-3’ direction

with yellow highlight showing the insert subcloned and blue sequences for ERE and pink

sequences for restriction sites. 106

3. pERE2

PERE2-TATA-INR-LUC GLPRIMER-1

NNNNNNNNCNNNNNNNNCCGAGCTCTTACGCGTGCTAGCTCGAGAGGTCACGTGACCTAGATCTGATATCAT CGATGAATTCGGGCTATAAAAGGGGGTGGGGGGAGCTCGGCCCTCATTCTGGAGACGGATCCTCTAGAGTCG ACCTGCAGGCATGCAAGCTTGGCATTCCGGTACTGTTGGTAAAATGGAAGACGCCAAAAACATAAAGAAAGG CCCGGCGCCATTCTATCCTCTAGAGGATGGAACCGCTGGAGAGCAACTGCATAAGGCTATGAAGAGATACGC CCTGGTTCCTGGAACAATTGCTTTTACAGATGCACATATCGAGGTGAACATCACGTACGCGGAATACTTCGA AATGTCCGTTCGGTTGGCAGAAGCTATGAAACGATATGGGCTGAATACAAATCACAGAATCGTCGTATGCAG TGAAAACTCTCTTCAATTCTTTATGCCGGTGTTGGGCGCGTTATTTATCGGAGTTGCAGTTGCGCCCGCGAA CGACATTTATAATGAACGTGAATTGCTCAACAGTATGAACATTTCGCAGCCTACCGTAGTGTTTGTTTCCAA AAAGGGGTTGCAAAAAATTTTGAACGTGCAAAAAAAATTACCAATAATCCAGAAAATTATTATCATGGATTC TAAAACGGATTACCAGGGATTTCAGTCGATGTACACGTTCGTCACATCTCATCTACCTCCCGGTTTTAATGA ATACGATTTTGTACCAGAGTCCTTTGATCGTGACAAAACAATTGCACTGATAATGAATTCCTCTGGATCTAC TGGGTTACCTAAGGGTGTGGCCCTTCCGCATAGAACTGCCTGCGTCAGATTCTCGCATGNCAGANATCCTAT TTTTGGCAATCAAATCATTCCGGATNCTGNGATTTTAAGTGNTGNTCCATTCCATCACGGTTTTGGAATGNT TACTACACTCNN

PERE2-TATA-INR-LUC GLPRIMER-2

AANNNNNNNGGNNGCCAGCTTGCATGCCTGCNGGTCGACTCTAGAGGATCCGTCTCCAGAATGAGGGCCGAG CTCCCCCCACCCCCTTTTATAGCCCGAATTCATCGATGATATCAGATCTAGGTCACGTGACCTCTCGAGCTA GCACGCGTAAGAGCTCGGTACCTCCCGGGTTATGTTAGCTCAGTTACAGTACCATAAGATACATTGATGAGT TTGGACAAACCACAACTAGAATGCAGTGAAAAAAATGCTTTATTTGTGAAATTTGTGATGCTATTGCTTTAT TTGTAACCATTATAAGCTGCAATAAACAAGTTAACAACAACAATTGCATTCATTTTATGTTTCAGGTTCAGG GGGAGGTGTGGGAGGTTTTTTAAAGCAAGTAAAACCTCCACGTACCTTANNATTACTTACTTATCATGGTAG CTTGGGCTGGCGTAATANCGAANAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCNCANCCTGAATGGCGA ATGGCAAATTGTAAGCGTTAATATTTTGTTAAAATTCGCGTTAAATTTTTGTTANATCANCTCATTTTTTNN CNNATAGGCCGAAATCGGCNNNNTCCCTTATAAATCAAAANAATNNNNNNAGATAGGGTTGANTGNNGTTCC NGTTTGGAACAAGAGTCCACTATTAAAGAACGTGGACTCCACGNCNANGNNNAAAAANNNCTATNNNGNNNA TGNNNANNANNTGAATCNTCNNCTNNNNNNNTTTTGGNNNNNNNN

Box 10. DNA sequences for pERE2-TATA-Inr-Luc from Retrogen Inc using GL

primer 1 and GL primer 2: Upper panel shows the plus strand DNA 5’-3’ direction

with yellow highlight showing the insert subcloned and blue sequences for ERE and pink

sequences for restriction sites. Lower panel shows the minus strand DNA 5’-3’ direction

with yellow highlight showing the insert subcloned and blue sequences for ERE and pink

sequences for restriction sites. 107

3. pERE4

PERE4-TATA-INR-LUC GLPRIMER-1

NNNNNNNNNNNGNNNGNACNGAGCTCTTACGCGTGCTAGCTCGAGAGGTCACTAGTGACCTAGATCTGATAT CATCGATGAATTCGGGCTATAAAAGGGGGTGGGGGGAGCTCGGCCCTCATTCTGGAGACGGATCCTCTAGAG TCGACCTGCAGGCATGCAAGCTTGGCATTCCGGTACTGTTGGTAAAATGGAAGACGCCAAAAACATAAAGAA AGGCCCGGCGCCATTCTATCCTCTAGAGGATGGAACCGCTGGAGAGCAACTGCATAAGGCTATGAAGAGATA CGCCCTGGTTCCTGGAACAATTGCTTTTACAGATGCACATATCGAGGTGAACATCACGTACGCGGAATACTT CGAAATGTCCGTTCGGTTGGCAGAAGCTATGAAACGATATGGGCTGAATACAAATCACAGAATCGTCGTATG CAGTGAAAACTCTCTTCAATTCTTTATGCCGGTGTTGGGCGCGTTATTTATCGGAGTTGCAGTTGCGCCCGC GAACGACATTTATAATGAACGTGAATTGCTCAACAGTATGAACATTTCGCAGCCTACCGTAGTGTTTGTTTC CAAAAAGGGGTTGCAAAAAATTTTGAACGTGCAAAAAAAATTACCAATAATCCAGAAAATTATTATCATGGA TTCTAAAACGGATTACCAGGGATTTCAGTCGATGTACACGTTCGTCACATCTCATCTACCTCCCGGTTTTAA TGAATACGATTTTGTACCAGAGTCCTTTGATCGTGACAAAACAATTGCACTGATAATGAATTCCTCTGGATC TACTGGGTTACCTAAGGGTGTGGNCCTTCCGCATAGAACTGCCTGCGTCANATTCTCGCATGCCAGANATCC TATTTTTGGCAATCAAATCATTCCGGATACTGCGATTTTAAGTGNTGTTCCATTCCATCACGGTTTTGGAAT GNTNNN

PERE4-TATA-INR-LUC GLPRIMER-2

NNNNNNNNNNGNNGNNNNNTTGCATGCCTGCAGGTCGACTCTAGAGGATCCGTCTCCAGAATGAGGGCCGAG CTCCCCCCACCCCCTTTTATAGCCCGAATTCATCGATGATATCAGATCTAGGTCACTAGTGACCTCTCGAGC TAGCACGCGTAAGAGCTCGGTACCTCCCGGGTTATGTTAGCTCAGTTACAGTACCATAAGATACATTGATGA GTTTGGACAAACCACAACTAGAATGCAGTGAAAAAAATGCTTTATTTGTGAAATTTGTGATGCTATTGCTTT ATTTGTAACCATTATAAGCTGCAATAAACAAGTTAACAACAACAATTGCATTCATTTTATGTTTCAGGTTCA GGGGGAGGTGTGGGAGGTTTTTTAAAGCAAGTAAAACCTCCACGTACCTTAATATTACTTACTTATCATGGT AGCTTGGGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGC GAATGGCAAATTGTAAGCGTTAATATTTTGTTAAAATTCGCGTTAAATTTTTGTTAAATCAGCTCATTTTTT AACCAATAGGCCGAAATCGGCAAAATCCCTTATAAATCAAAAGAATAGACCGAGATAGGGTTGAGTGTTGTT CCAGTTTGGAACAAGAGTCCACTATTAAAGAACGTGGACTCCAACGTCAAAGGGCGAAAAACCGTCTATCAG GGCGATGGCCCACTACGTGAACCATCACCCTAATCAAGTTTTTTGGGGTCNAGGTGCCGTAAAGCACTAAAT CGNAACCCTAAAGGGAGCCCCCGANTTANAGCTTGACGGGGAAAGCCGGCGAACGTGGCGAGAAAGGAANGG AANAAAGCGAAAGGAGCGGGCGCTANGGCNCTGGCAAGTGTANCGGTCACGCTGNNN

Box 11. DNA sequences for pERE4-TATA-Inr-Luc from Retrogen Inc using GL

primer 1 and GL primer 2: Upper panel shows the plus strand DNA 5’-3’ direction

with yellow highlight showing the insert subcloned and blue sequences for ERE and pink

sequences for restriction sites. Lower panel shows the minus strand DNA 5’-3’ direction

with yellow highlight showing the insert subcloned and blue sequences for ERE and pink

sequences for restriction sites. 108

11. This confirmed that ligation of the ERE inserts at Xho I and Bgl II sites 5’ upstream to

TATA box into the vector was successful.

2. Determination of Specific Responsiveness of Luciferase Reporter Gene to Estrogen and

Estrogen Receptor α

After the successful construction of the luciferase reporter vectors with various estrogen response element (single cERE, tandem cEREs, single cHERE, tandem cHEREs or variant spacer cEREs: n=0, 1, 2, 3, & 4, where n is the number of nucleotides in between two half site of cERE) at the promoter region of the luciferase gene, the preliminary aim was to determine if the prepared construct was indeed estrogen responsive, that is, specifically responsive to E2 and also specifically dependent on ER. In this trial, a luciferase reporter vector constructed with 2cERE

insert was used to determine E2-responsive ER mediated transactivation of luciferase gene.

One µg of p2cERE-TATA-Inr-Luc, 1 ng of pGL4.70 (hRLuc), and 5 ng of pERα or 5 ng

of pBluescript was co-transfected into 4X105 U2OS cells, and the cells were treated with either

estrogen (E2) or ethanol vehicle. The luciferase activity was measured as described in M&M. As

shown in figure 16, virtually no luciferase activity was observed in the absence of E2 treatment

or in the absence of ERα. However, on induction with E2, and in the presence of ERα, the

luciferase activity was increased by 16-fold (Fig. 16). From these observations it is evident that in an ER negative cell line (U2OS cells), ER is required and transcription must be induced by E2 to activate the expression of the luciferase gene. Thus, the recombinant construct was both an

ER-dependent and estrogen-responsive luciferase reporter gene.

109

Figure 16. Dependence of 2cERE luciferase reporter expression on E2 and ER: 5 ng of

pCMVflag:hERα and or 5 ng of pBluescript were transfected to 4X105 U2OS cells. 1µg of

p2cERE-Luc reporter vector and 1 ng of pGL4.70 hRLuc control reporter vector was

transfected. The cells were treated with 10 nM of 17-β estradiol (E2) or vehicle (EtOH), after

6 hrs of transfection. The cells were harvested 24 hrs after treatment and the cell extracts were assayed for luciferase activity. Data are displayed as the ratio of Relative Light Units

(RLU) of Firefly and Renilla Luciferase activity. These data are the mean ± s.d of three separate determinations.

110

2.1. Effect of Increasing Amounts of pCMVflag:hERα Expression Vector on Luciferase

Reporter Construct

In order to determine the maximum transcriptional responsiveness of ERα on

luciferase reporter gene, induced by E2, a preliminary experiment was carried out to

determine the effect of different levels of pERα expression vector on luciferase activity. In

this trial, increasing amounts (2.5, 5, 10, 25, 50, 100, and 200 ng) of pERα were co-

transfected with 1µg of pGL2-3cERE-TATA-Inr-Luc and 1 ng of pGL4.70 hRLuc into 4 X

105 cells, and cells were induced with E2. As shown in figure 17, it was observed that at 5-10

ng of pERα transfected, the luciferase reporter gene showed the maximum transcriptional

responsiveness to ERα. Beyond 10 ng, the luciferase reporter gene activity progressively

decreased. A similar pattern of ERα responsive reporter gene activity was observed by

another study (Tyulmenkov et al., 2000). At 10 ng of pERα transfected, the promoter may

have just saturated with the transcription machinery to give the highest activity and beyond

which the activity progressively decreased. Therefore, 5 ng of pERα was co-transfected in

the all the experiments.

3. HMGB1 Overexpression in U2OS Cells

HMGB1 is a non histone chromosomal protein that dramatically decreased the dissociation constant for ER binding to non-conventional estrogen responsive elements in vitro

EMSA studies (Das et al., 2004; El Marzouk et al., 2008). Thus, ER binds strongly to cEREs and more importantly to non-conventional EREs, so-called weak promoters, in the presence of 400 nM HMGB1 (Das et al., 2004; El Marzouk et al., 2008; Ghattemani, 2004). HMGB1 is ubiquitously expressed protein all eukaryotic cells (Wolffe, 1994). Therefore, to determine the effect of HMGB1 on E2-responsive ERα mediated luciferase reporter activity in U2OS cells, 111

Figure 17. Effect of increasing amounts of pERα expression vector on p3cERE –Luc

reporter vector: 2.5, 5, 10, 25, 50, 100, and 200 ng of pCMVflag:hERα transfected into

4X105 U2OS cells. 1µg of p3cERE-Luc reporter vector and 1 ng of pGL4.70 hRLuc control

reporter vector was transfected. The cells were treated with 10 nM of 17-β estradiol (E2), after 6 hrs of transfection. The cells were harvested 24 hrs after treatment and the cell extracts were assayed for luciferase activity. Data are displayed as the ratio of Relative Light

Units (RLU) of Firefly and Renilla Luciferase activity. These data are the mean ± s.d of three separate determinations. 112

HMGB1 was over-expressed by transfecting exogenous HMGB1 expression vector (pHMGB1)

to the U2OS cells.

First, the level of HMGB1 overexpression in the U2OS cells was determined, when 0, 10,

100 and 1000 ng of pHMGB1 were transfected in the U2OS cells. A western blot analysis was

carried out to examine the overexpression of HMGB1 followed by semi-quantification of protein bands in western blot using densitometric analysis with ImageJ software

(http://rsbweb.nih.gov/ij/). As shown in figure 18A, lane1 and lane 2, 10 ng of pHMGB1 transfected did not produce significant overexpression HMGB1 in comparison to endogenous level of HMGB1. Also from densitometric analysis, it is evident that 10 ng of exogenous

HMGB1 expression vector co-transfected was able to increase the expression of total HMGB1 by ~5% (Fig. 18B). But, 1000 ng (1 µg) of pHMGB1 co-transfected produced about a 40%

increase in overexpression of HMGB1 (Fig. 18B & 18A, lane 4).Therefore, to over-express

HMGB1 by at least 40%, 1 µg of exogenous HMGB1 expression vector has to be co-transfected.

3.1. Effect of Increasing Amounts of pHMGB1 Expression Vector on Estrogen

Responsive ERα Mediated Luciferase Reporter Activity

Based on the western blot (Fig 18), we co-transfected 10, 100, and 1000 ng of

pHMGB1 or pBluescript expression vector with 1 µg of pGL2-3cERE-TATA-Inr-Luc, 1 ng

of pGL4.70 hRLuc and 5 ng of pERα in 4 X 105 U2OS cells and E2 treated in order to

determine the maximum effect that overexpression of HMGB1 would exert to produce an

increase or decrease in transcription of estrogen responsive ER mediated luciferase reporter

gene. With reference to the influence of endogenous HMGB1 alone on the estrogen

responsive ER mediated luciferase reporter gene activity (Fig 19, purple bar), 10 ng and 100

ng of pHMGB1 co-transfected had no additional influence (Fig. 19, pink and yellow bars). 113

But, 1000 ng of pHMGB1 co-transfected showed a 3-fold increase in transcriptional activity

of the estrogen responsive ER mediated luciferase reporter gene (Fig. 19, blue bar). Similar

observations were made by another study (Zhang et al., 1999) where, 12.5 ng of exogenous

HMGB1 expression vector transfected in HeLa cells did not influenced the transactivation of

2ERE-TK-CAT mediated by ER upon E2 induction, but a 3.5-fold increase in CAT activity

was observed when 250 ng of pHMGB1 co-transfected. Also in the same study (Zhang et al.,

1999), 1000 ng of pHMGB1 co-transfected in MDA-MB-231 cell lines stimulated a 4-fold

increase in transactivation of 4ERE-Luc by ER upon E2 induction. These data suggested to

the authors that HMGB1 levels are limiting for ER action in intact cells. Therefore, to

generate the maximum effect of HMGB1 overexpression to produce at least a 3-fold increase

in transcriptional activity of E2 responsive ER mediated luciferase reporter gene, 1 µg of

pHMGB1 has to be co-transfected. Thus, to determine the influence of overexpressed

HMGB1 on E2 responsive ER mediated luciferase reporter gene activity, 1µg of pHMGB1

expression vector was co-transfected in all the experiments.

4. Effect of Different ERE Inserts on Estrogen Responsive ERα Mediated Luciferase

Reporter Activity

4.1. Single or Tandem cEREs

In order to study the influence of a single cERE or tandem copies of cEREs in

transactivation of E2-responsive ERα-mediated luciferase reporter gene, we co-transfected 1

µg of pGL2-1cERE-TATA-Inr-Luc, pGL-2cERE-TATA-Inr-Luc or pGL2-3cERE-TATA-

Inr-Luc with 5 ng of pCMVflag:hERα and pGL4.70hRluc in 4 X 105 U2OS cells, and the

cells treated with 10 nM estrogen. A 7-fold increase in luciferase activity was observed from

1cERE to 2cERE, and a 12-fold increase in activity was observed from 1cERE to 3cERE, 114

Figure 18. HMGB-1 overexpression in U2OS cells: A. Western Blot of HMGB1 overexpressed when, Lane 1: 0 ng, Lane 2: 10 ng, Lane 3: 100 ng and Lane 4: 1000 ng of pHMGB1 expression vector was transfected into 4 X 105 U2OS cells, along with 5 ng of pERα,

1 µg of p3cERE-Luc, and 1 ng pGL4.70 hRLuc. The cells were treated with 10 nM of 17-β estradiol (E2) after 6 hrs of transfection. After 24 hrs of hormone treatment the cells were harvested and protein concentration was quantified using the Bio-Rad Protein Assay, taking BSA as a standard protein and 20µg of total protein was loaded for Western Blot analysis B. The graph was plotted by quantifying band intensities of the western blot and calculating the percent increase in intensity from lane 1 to lane 2, 3 and 4 using ImageJ software from NIH. 115

Figure 19. Effect of increasing amounts of pHMGB1 on ER mediated transactivation of pGL2-3cERE-TATA-Inr-Luc reporter vector: 10, 50, 100,500, and 1000 ng of pHMGB1 expression vector transfected into 4X105 U2OS cells along with 5 ng of pERα, 1 µg of p3cERE-Luc, and 1 ng pGL4.70 hRLuc. The cells were treated with 10 nM of 17-β estradiol (E2) after 6 hrs of transfection. The cells were harvested 24 hrs after treatment and the cell extracts were assayed for luciferase activity.

These data are the mean ± s.d of three separate determinations.

116

while only 1.5-fold increase in luciferase activity was observed from 2cERE to 3cERE (Fig

20 and Table 12). This is a huge increase from 1cERE to 2cERE, but not from 2cERE to

3cERE. However, a functional synergy was observed in both cases viz., 1cERE to 2cERE

and 1cERE to 3cERE. This functional synergy parallels the results from the co-operative

binding of ER to tandem EREs, which is also observed with ER binding to vitellogenin B1

ERU that contains two imperfect EREs (ERE2/ERE1) separated by 20bp center to center

(Das et al., 2004). Thus, in comparison to transcription driven by single cERE, the tandem

copies of cEREs were found to act as a strong promoter in increasing the E2 responsive ER

mediated transcription of luciferase gene.

4.2. Single or Tandem cHEREs

In order to determine the influence of a single cHERE or tandem copies of cHEREs,

we co-transfected 1 µg of pGL2-1cHERE-TATA-Inr-Luc, pGL-2cHERE-TATA-Inr-Luc or

pGL2-3cHERE-TATA-Inr-Luc with 5 ng of pCMVflag:hERα and pGL4.70 hRLuc in 4 X

105 U2OS cells, and the cells were treated with 10 nM estrogen. More than 3-fold increase in luciferase activity was observed from 1cHERE to 2cHERE (Fig 21 and Table 13). In case of

1cHERE to 3cHERE, more than 8-fold increase in luciferase activity was observed and a 3- fold increase in activity was observed from 2cERE to 3cERE (Fig 20 and Table 12).

Although, it appears that going from 1cHERE to 2cHERE is slightly more than a linear increase in activation, a transcriptional synergy for 1cHERE to 3cHERE was observed.

Thus, in this experiment also, tandem copies of cHERE acted as a strong promoter than a single cHERE, where 3cHERE was found to be much stronger in eliciting the E2-responsive

ER-mediated transcription of luciferase gene. 117

Figure 20. Effect of single or tandem cERE on ER mediated reporter activity: 1 µg of

p1cERE, p2cERE and 3cERE was transfected into 4X105 U2OS cells along with 5 ng of

pERα and 1 ng of pGL4.70hRluc. The cells were treated with 10 nM of 17-β estradiol (E2)

or vehicle (EtOH), after 6 hrs of transfection, and the cell extracts were assayed for luciferase

activity. Data are displayed as the ratio of Relative Light Units (RLU) of Firefly and Renilla

Luciferase activity. These data are the mean ± s.d of four separate determinations.

Table 12:

Fold Change in E2-responsive ERα-mediated Luciferase Reporter Gene Activity under the control of single cERE or tandem cEREs From Increase in Activity 1cERE to 2cERE 7.5 fold 1cERE to 3cERE 12 fold 2cERE to 3cERE 1.5 fold

118

Figure 21. Effect of single or tandem cHERE on ER mediated reporter activity: 1 µg of

p1HERE, p2HERE and p3HERE was transfected into 4X105 U2OS cells along with 5 ng of

pERα and 1 ng of pGL4.70 hRLuc vector. The cells were harvested 24 hrs after E2 treatment

and the cell extracts were assayed for luciferase activity. Data are displayed as the ratio of

Relative Light Units (RLU) of Firefly and Renilla Luciferase activity. These data are the

mean ± s.d of four separate determinations.

Table 13:

Fold Change in E2-responsive ERα-mediated Luciferase Reporter Gene Activity under the control of single cHERE or tandem cHEREs From Increase in Activity 1cHERE to 2cHERE 3+ fold 1cHERE to 3cHERE 8+ fold 2cHERE to 3cHERE 3 fold

119

4.3. cEREs with Different Spacer Sizes

From our in vitro ER binding studies to cEREs with different spacer sizes in between

two half sites (cEREn, n=0-4bps), it was found that the binding affinity of ER decreased

when the spacer size was deviated from 3bp, except for n=0bp (El Marzouk et al., 2008). The

binding affinity of ER to cERE3 (KD=7.5 nM) and cERE0 (KD=10 nM) was observed to be comparable, while the binding affinity of ER to cERE1 (KD=80 nM) was found to be the weakest, and binding affinity of ER to cERE2 (KD=25 nM) and cERE4 (KD=25 nM) was the

same but weaker than consensus ERE3 (KD=7.5 nM) (El Marzouk et al., 2008). Therefore, in

order to examine if this in vitro binding has any functional relevance to transactivation in the

cell, various constructs of luciferase reporter vector containing variant spacer cEREn (where

n is the spacer sizes in bp between two half sites of the ERE) to the 5’ region of luciferase

reporter gene were co- transfected with pERα and pGL4.70 hRLuc into 4 x 105 U2OS cells,

and the cells treated with estrogen. A 4-fold decrease in estrogen responsive ER mediated luciferase activity was observed for cERE4 in comparison to cERE3 (Fig 22 and Table 14).

Similarly, with reference to cERE3, more than 3-fold decrease in transcriptional activity was

observed for cERE1 and a 2-fold decrease in transcriptional activity was observed for

cERE0. Interestingly, the transcriptional activity driven by cERE2 was increased by 1.4-fold

in comparison to cERE3 (Fig 22 and Table 14). In contrast, the transcriptional activity driven by cERE2, the transcriptional activity of estrogen responsive ER mediated luciferase gene driven by cERE0, cERE1, and cERE4 decreased in comparison to cERE3. Thus, this observation reflected the in vitro ER binding assays, where the binding affinity for ER was decreased when the spacer size was varied from 3 to 1, 2 and 4 (El Marzouk et al., 2008). 120

Figure 22. Effect of ERE with different spacer size between two half sites on ER

mediated reporter activity: 1 µg of pERE0, pERE1, pERE2, pERE3 and pERE4 was

transfected into 4X105 U2OS cells along with 5 ng of pERα and 1 ng of pGL4.70 hRLuc

vector. The cells were harvested 24 hrs after E2 treatment and the cell extracts were assayed

for luciferase activity. Data are displayed as the ratio of Relative Light Units (RLU) of

Firefly and Renilla Luciferase activity. These data are the mean ± s.d of four separate

determinations.

Table 14:

Fold Change in E2-responsive ERα-mediated Luciferase Reporter Gene Activity under the control of variant spacer cEREn (n=0-4) From Increase in Activity Decrease in Activity cERE3 to cERE4 4 fold cERE3 to cERE2 1.4 fold cERE3 to cERE1 3+ fold cERE3 to cERE0 2 fold

121

5. Effect of Overexpression of HMGB1 on Various ERE Constructs on Estrogen

Responsive ERα Mediated Luciferase Reporter Activity

5.1. Single or Tandem cEREs

In order to examine the influence of overexpression of HMGB1 on estrogen

responsive ER mediated transcriptional activity of luciferase reporter gene driven by a single

cERE or tandem copies of cEREs, 1 µg of pHMGB1 expression vector was co-transfected

with 1 µg of either pGL2-1cERE-TATA-Inr-Luc, pGL2-2cERE-TATA-Inr-Luc or pGL2-

3cERE-TATA-Inr-Luc, and 5ng of pERα and 1ng of pGL4.70 hRLuc into 4 X 105 cells, and

the cells treated with estrogen. In experiments conducted without HMGB1 overexpression 1

µg of empty vector (pBluescript) was co-transfected. A 5-fold increase in luciferase activity

was observed for 1cERE when HMGB1 was overexpressed (Fig 23 and Table 15). Similarly,

when HMGB1 was overexpressed, more than 3-fold increase in luciferase activity was

observed for both 2cERE and 3cERE (Fig 23 and Table 15). Also, in vitro binding studies

from our lab showed that HMGB1 enhanced the overall binding of ER to two tandem EREs

by almost 3 fold (Das et al., 2004). This illustrated that HMGB1 stimulates the binding co-

operativity in vitro as well as transcriptional synergy in vivo to comparable levels. From this

observation, it is evident that the influence of HMGB1 is greater for “so-called” weak

promoter, for example, 1cERE in this trial. In comparison to the influence of overexpression

of HMGB1 on a single cERE, the influence of overexpression of HMGB1 on estrogen

responsive ER mediated transcription of luciferase gene under the control of 2cEREs was

increased by more than 6-fold and 3cERE by more than 7-fold. But, only 1.2-fold increase in

luciferase activity was observed from 2cERE to 3cERE (Fig 23 and Table 16). This

suggested that HMGB1 overexpression increased the functional synergy in both the cases 122

viz., from 1cERE to 2cERE and 1cERE to 3cERE. Thus, overexpression of HMGB1

enhanced the estrogen-responsive ER-mediated transcription of luciferase gene for a single

cERE and tandem cEREs and the tandem cEREs produced transcriptional synergy.

5.2. Single or Tandem cHEREs

In order to examine the influence of overexpression of HMGB1 on estrogen-

responsive ER mediated transcriptional activity of luciferase reporter gene driven by a single

cERE or tandem copies of cEREs, 1 µg of pHMGB1 expression vector was co-transfected with 1 µg of either pGL2-1cHERE-TATA-Inr-Luc, pGL2-2cHERE-TATA-Inr-Luc or pGL2-

3cHERE-TATA-Inr-Luc, and 5 ng of pERα and 1 ng of pGL4.70 hRLuc into 4 X 105 cells,

and the cells treated with estrogen. In experiments conducted without HMGB1

overexpression 1 µg of empty vector (pBluescript) was co-transfected. On overexpression of

HMGB1, the estrogen responsive ER mediated transcriptional activity of the luciferase gene under the control of 1cHERE as well as 2cHEREs was increased by more than 3-fold (Fig 24 and Table 17). Likewise, the estrogen responsive ER mediated transcriptional activity of luciferase gene under the control of 3cHEREs was increased by more than 2-fold (Fig 24 and

Table 17). Again, from these observations it is evident that the influence of HMGB1 is greater for weaker promoters, this is for 1cHERE and 2cHERE as compared to 3cHERE in this case. In comparison to the influence of overexpression of HMGB1 on a single cHERE, the influence of overexpression of HMGB1 on reporter activity under the control of

2cHEREs was increased by more than 3-fold and 3cHERE by more than 5-fold. However, only a 2-fold increase in luciferase activity was observed from 2cHERE to 3cHERE (Fig 24 and Table 18). This suggested that HMGB1 overexpression increased the functional synergy in both the cases viz., from 1cHERE to 2cHERE and 1cHERE to 3cHERE. Also, in the 123

binding studies, a surprisingly strong binding affinity of ER to one of the half sites in the

direct repeats of two half sites (2cHEREs) was observed, while stimulation by HMGB1

exhibited a co-operative binding (KD=4 nM) to both the half sites (Ghattemani, 2004). This

reflected that in all cases, in vitro co-operativity was translated into functional synergy in

vivo study. Thus, overexpression of HMGB1 enhanced the estrogen- responsive ER-mediated transcription of luciferase gene for a single cHERE and tandem cHEREs as well as stimulated the transcriptional synergy for tandem cHEREs.

1.1. cEREs with Different Spacer Sizes

In order to examine the influence of overexpression of HMGB1 on estrogen responsive ER mediated transcriptional activity of luciferase reporter gene driven by a cEREn with different spacer (n) sizes in between two half sites, 1 µg of pHMGB1expression vector was co-transfected with 1 µg of either pGL2-cERE0-TATA-Inr-Luc, pGL2-cERE1-

TATA-Inr-Luc, pGL2-cERE2-TATA-Inr-Luc, pGL2-cERE3-TATA-Inr-Luc or pGL2- cERE4-TATA-Inr-Luc, and 5 ng of pERα and 1 ng of pGL4.70 hRLuc into 4 X 105 cells,

and the cells treated with estrogen. In experiments conducted without HMGB1

overexpression 1 µg of empty vector (pBluescript) was co-transfected. On overexpression of

HMGB1, the estrogen responsive ER mediated transcriptional activity of the luciferase gene under the control of cERE0, cERE3 as well as cERE4 was increased by more than 3-fold

(Fig 25 and Table 19). Likewise, the estrogen responsive ER mediated transcriptional activity of luciferase gene under the control of cERE2 was increased by more than 2-fold (Fig 25 and

Table 19). Surprisingly, the overexpression of HMGB1 decreased the estrogen responsive

ER mediated transcriptional activity of the luciferase gene under the control of cERE1 1.4- fold (Fig 25 and Table 19). Except for cERE1, the transcriptional activity of estrogen- 124

Figure 23. Effect of overexpression of pHMGB1 on ER mediated reporter activity of single or tandem cEREs: 1 µg of p1cERE, p2cERE and 3cERE was transfected into 4X105 U2OS cells along with 5 ng of pERα and 1 ng of pGL4.70 hRLuc with and without 1000 ng of pHMGB1 expression vector. The cells were harvested 24 hrs after treatment and the cell extracts were assayed for luciferase activity. Data are displayed as the ratio of Relative Light Units (RLU) of Firefly and Renilla Luciferase activity. These data are the mean ± s.d of four separate determinations. Table 15:

Effect of Overexpression of HMGB1 on E2-responsive ERα-mediated Luciferase Reporter Gene Activity under the control of single cERE or tandem cEREs Luciferase Activity (RLU) Regulatory Promoter Endogenous Overexpressed Increase in Activity HMGB1 HMGB1 1cERE 10 50 5 fold 2cERE 72 321 3+ fold 3cERE 117 367 3+ fold

Table 16:

Fold Change in E2-responsive ERα-mediated Luciferase Reporter Gene Activity under the control of single cERE or tandem cEREs when HMGB1 is overexpressed From Increase in Activity 1cERE to 2cERE 6+ fold 1cERE to 3cERE 7+ fold 2cERE to 3cERE 1.2 fold 125

Figure 24. Effect of overexpression of pHMGB1 on ER mediated reporter activity of single or tandem HEREs: 1 µg of p1HERE, p2HERE and 3HERE was transfected into 4X105 U2OS cells along with 5 ng of pERα and 1 ng of pGL4.70 hRLuc with and without 1000ng pHMGB1 expression vector. The cells were harvested 24 hrs after treatment and the cell extracts were assayed for luciferase activity. Data are displayed as the ratio of Relative Light Units (RLU) of Firefly and Renilla Luciferase activity. These data are the mean ± s.d of four separate determinations. Table 17:

Effect of Overexpression of HMGB1 on E2-responsive ERα-mediated Luciferase Reporter Gene Activity under the control of single cHERE or tandem cHEREs Luciferase Activity (RLU) Regulatory Promoter Endogenous Overexpressed Increase in Activity HMGB1 HMGB1 1cHERE 5 18 3+ fold 2cHERE 15 55 3+ fold 3cHERE 42 100 2+ fold

Table 18:

Fold Change in E2-responsive ERα-mediated Luciferase Reporter Gene Activity under the control of single cHERE or tandem cHEREs when HMGB1 is overexpressed From Increase in Activity 1cHERE to 2cHERE 3 fold 1cHERE to 3cHERE 5+ fold 2cHERE to 3cHERE 2fold

126

Figure 25. Effect of overexpression of pHMGB1 on ER mediated reporter activity of cERE with different spacer size between two half sites on ER mediated reporter activity: 1 µg of pERE0, pERE1, pERE2, pERE3 and pERE4 was transfected into 4X105 U2OS cells along with 5 ng of pERα and 1 ng of pGL4.70 hRLuc vector with and without 1000 ng of pHMGB1. The cells were harvested 24 hrs after treatment and the cell extracts were assayed for luciferase activity. Data are displayed as the ratio of Relative Light Units (RLU) of Firefly and Renilla Luciferase activity. These data are the mean ± s.d of four separate determinations. Table 19: Effect of Overexpression of HMGB1 on E2-responsive ERα-mediated Luciferase Reporter Gene Activity under the control of variant spacer cEREn (n=0-4) Luciferase Activity (RLU) Regulatory Promoter Endogenous Overexpressed Increase in Activity HMGB1 HMGB1 cERE0 14 46 3+ fold cERE1 7 5 1.5 fold (decrease) cERE2 38 85 2+ fold cERE3 27 98 3+ fold cERE4 7 23 3+ fold

Table 20: Fold Change in E2-responsive ERα-mediated Luciferase Reporter Gene Activity under the control of variant spacer cEREn (n=0-4) when HMGB1 is overexpressed From Increase in Activity Decrease in Activity cERE3 to cERE4 - 4+ fold cERE3 to cERE2 - 1.2 fold cERE3 to cERE1 - 20 fold cERE3 to cERE0 - 2 fold 127

responsive ER-mediated luciferase gene driven by cERE0, cERE2, cERE3 and cERE4 was

increased with overexpression of HMGB1. In comparison to the influence of overexpression

of HMGB1 on a cERE3, the influence of overexpression of HMGB1 on estrogen

responsiveER mediated transcription of luciferase gene under the control of cERE1 was

decreased by 20-fold and cERE4 by more than 4-fold. Similarly, a 2-fold decrease in activity

was observed for cERE0 and 1.2-fold decrease in activity was observed for cERE2 (Fig 25

and Table 20). Thus, it was observed that when the spacer size is deviated from the

consensus 3bps in between two half sites of ERE, the transcriptional activity was decreased.

2. Effect of Silencing Endogenous HMGB1 Gene with siRNA on Estrogen Responsive ERα

Mediated Luciferase Reporter Activity

Overexpression of HMGB1 does not reflect normal cellular conditions; instead it helps to understand the role of HMGB1 in cancer cells in which HMGB1 overexpression has been

reported (Brezniceanu et al., 2003). However, the role of HMGB1 in normal cellular functioning

can be investigated by knocking down the endogenous level of HMGB1 by using siRNA

technology. Therefore, to understand the role of HMGB1 in ER mediated regulation of estrogen

responsive gene, the endogenous level of HMGB1 was decreased by using siRNA specific to the

human HMGB1 gene and the effect of HMGB1 knock down (KD) on transcriptional regulation

of estrogen-responsive genes was determined.

In order to determine efficiency of gene silencing using siRNAs, preliminary experiments were carried out using siRNAs for human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene in which the relevant amount of siRNA (nM) required to significantly knock down GAPDH was determined. At 50 nM siRNA for GAPDH transfected, a 40% decrease in GAPDH mRNA 128

levels was observed by RT-PCR analysis (Fig 26). Likewise, when 100 to 400 nM siRNA for

GAPDH were transfected, GAPDH mRNA levels were decreased by 80-90% (Fig 26).

Therefore, to be certain to silence the HMGB1 gene, 200 nM and 400 nM siRNA for the human

HMGB1 was co-transfected with 1 µg of pGL2-3cERE-TATA-Inr-Luc, 5 ng of pERα and 1 ng of pGL4.70 hRLuc into 4 X 105 U2OS cells, and the cells were treated with estrogen. At 200 nM

siRNA of hHMGB1 co-transfected to knock down HMGB1 gene, more than 4-fold decrease in

estrogen responsive ER mediated transcription of luciferase reporter gene activity was observed

(Fig 27). Similarly, more than 5-fold decrease in luciferase reporter activity was observed when

400 nM of siRNA for hHMGB1 was transfected to knock down HMGB1 gene (Fig 27). This sharp decrease in luciferase activity when HMGB1 siRNA knocked down endogenous HMGB1 gene expression indicates that HMGB1 is required to facilitate estrogen responsive gene mediated by ER action. 129

Figure 26. Effect of increasing amounts of siRNA for GAPDH on its down regulation.

50 nM, 100 nM, 200 nM and 400 nM of siRNA cocktail for GAPDH (DHARMACON), 5 ng of pCMVflag:hERα, 1 µg of p3cERE-Luc reporter vector, and 1 ng of pGL4.70 hRLuc control reporter vector were co- transfected into 4X105 U2OS cells. The cells were treated

with 10 nM of 17-β estradiol (E2), after 6 hrs of transfection. The cells were harvested 24 hrs

after treatment and the total RNA was isolated using RNase Assay mini kit (Qiagen). The

RNA isolated was converted to cDNA using PCR (BioRad) and then RT-PCR (Applied

Biosystems) was performed to analyze the gene knock down. 130

Figure 27. Effect of HMGB1 silencing on ER mediated reporter activity. 200 nM and

400 nM of siRNA cocktail for human HMGB1 gene (DHARMACON), 5 ng of pCMVflag:hERα, 1 µg of p3cERE-Luc reporter vector, and 1 ng of pGL4.70 hRLuc control reporter vector were co- transfected into 4X105 U2OS cells. The cells were treated with 10 nM of 17-β estradiol (E2), after 6 hrs of transfection. The cells were harvested 24 hrs after treatment and the cell extracts were assayed for luciferase activity. Data are displayed as the ratio of Relative Light Units (RLU) of Firefly and Renilla Luciferase activity. These data are the mean ± s.d of four separate determinations. 131

CHAPTER IV: RESULTS II

1. Isolation and Purification of HMGB Proteins from Calf Thymus

HMGB1 and HMGB2 proteins were isolated from calf thymus as described in M&M.

The proteins were purified by HPLC using linear salt gradient elution (0.2 M NaCl to 0.8 M

NaCl) on a Mono Q 5/5 anion exchange column. The HPLC purification was performed at Dr.

D. Dignam’s Lab in University of Toledo, Health Science Campus. The purity of the fractionated

HMGB1 and HMGB2 proteins were verified using 18% SDS-PAGE (Fig. 28). HMGB2 fraction was eluted early at about 13.03 mins in the 18minute linear NaCl gradient, while HMGB1 was eluted following the HMGB2 at about 14.05 mins in the 18 minute linear salt gradient. A 215 amino acid containing HMGB1 isolated from calf thymus has a molecular weight of 25kDa as shown in fig 28 and a faster moving band corresponds to the HMGB2.

2. Preparation and Isolation of Nucleosomes

In an attempt to extend our ER binding studies at the DNA level to the nucleosomal level, an investigation was initiated to determine how, or if, HMGB1 alters or remodels nucleosome to facilitate ER binding. As such, nucleosomes were prepared in which the cERE or GRE were incorporated into a 161bp DNA at specific positions and rotational settings to produce a homogenous population of nucleosomes to determine the effect of translational positioning and rotational phasing has in ER binding. This study led to my specific study with PR binding to

GRE within a nucleosomal DNA and specifically, if HMGB1 alters or remodels nucleosome structure with GRE positioned at the dyad axis.

This 161bp DNA was first radiolabeled at 5’OH with γ-P32-ATP and incorporated in the nucleosome. The incorporation was done by reconstituting the P32 labeled 161bp DNA (2G2) (a

GRE positioned at the dyad axis) in the partial micrococcal nuclease digested chicken 132

Figure 28. An 18% SDS-PAGE of HPLC purified calf thymus HMGB1 and HMGB2 stained with Coomassie blue: Lane 1: Protein molecular weight marker, Lane 2: HMGB1, Lane

3: HMGB2.

133

erythrocytes chromatin prepared by Yaw Sarpong to produced oligonucleosomes. A step-wise salt dialysis (Li and Wrange, 1995) method was used to reconstitute the radiolabeled DNA into nucleosomes. The nucleosomes incorporated with P32 labeled 2G2 were separated from the

unincorporated DNA by 5-30% sucrose linear gradient sedimentation. A total of 40 fractions

were collected with 5 drops in each tube (each drop equivalent to 25µL). Early fractions 9-12

contained unincorporated DNA and later fractions 17-27 contained the 2G2 nucleosomes as

shown in figure 29 A. The fractions were then run in a 4% EMSA gel to verify the reconstituted

nucleosome. Fig 29 B lanes 1-4 shows free unincorporated DNA corresponding to the fractions

9-12 from the sucrose gradient, while lanes 5-15 shows reconstituted nucleosome corresponding

to the fractions 17-27.

3. Influence of HMGB1 on Nucleosomes

3.1. Effect of Increasing Amounts of HMGB1 on Nucleosome Structure

To determine the influence of HMGB1 on nucleosome structure, increasing amounts

of HMGB1 (400 nM, 800 nM, 1600 nM) were incubated with nucleosomes in a TE/sucrose

(18%) buffer (the gradient buffer) for 1 hr at 4oC. The HMGB1 treated nucleosomes were

analyzed on a 4% polyacrylamide EMSA gel for possible alteration, as reflected in EMSA

mobility. Figure 30 shows that on increasing the levels of HMGB1 from 400 nM to 1600 nM,

electrophoretic mobility of the nucleosome decreased. This suggested that the mobility

changed as a result of either 1) HMGB1 binding to the nucleosome and forming a stable

complex or 2) HMGB1 altered the nucleosome structure without being a stable component of

the altered structure.

134

A.

B.

Figure 29. Sedimentation and EMSA profile of canonical nucleosomes (N): (A)

Sedimentation profile of canonical nucleosomes from 5-30% sucrose linear gradient fraction collections as monitored by P32 counts per minute. The fraction were collected from the top by

injecting 50% sucrose from the bottom of the tube. (B) The EMSA profile of the fractions

showing electrophoretic mobility of DNA and canonical nucleosomes (N). A 10 µL of the

fractions collected were run in a 4% polyacrylamide EMSA gel. Lanes 1-4 correspond to the

fractions 9-12 and lanes 5-15 corresponds to fractions 15-27 from the 5-30% sucrose linear

gradient. 135

Figure 30. Effect of HMGB1 on EMSA mobility of nucleosomes: Lane 1: free DNA (2G2),

Lane2: Nucleosomes (n2G2), Lane3: Nucleosomes + 400 nM HMGB1, Lane4: Nucleosomes +

800 nM HMGB1, Lane 5: Nucleosomes + 1600 nM HMGB1. In 10 µL reaction volume, nucleosomes with and without increasing concentration of HMGB1 was incubated in

TE/sucrose(18%) buffer for 1 hour on ice and loaded onto a EMSA gel and run for 2 hours at

200V at 4oC

136

3.2. Determination of the Presence of HMGB1 as a Stable Component of Nucleosome

In order to examine if the reduced mobility of the nucleosome was due to a stably

bound HMGB1 to the nucleosome, an antibody supershift assay of the nucleosomes was

done. As shown in fig. 31, the α-HMGB1 did not supershift the remodeled nucleosomes for

all the three conditions corresponding to nucleosomes treated with 400 nM, 800 nM and

1600 nM HMGB1, respectively. This suggests that HMGB1 is not a stable component of the

nucleosome. HMGB1 appears to interact with the nucleosome to alter it from the canonical

state since the EMSA mobility differed from that of the canonical nucleosomes. This

behavior has been observed in many DNA complexes formed in the presence of HMGB1,

which led to the suggestion that HMGB1 interacts transiently in a “hit-&-run” mechanism

(Das et al., 2004; Hager et al., 2009).

In an attempt to covalently cross-link HMGB1 to the nucleosome, before it dissociates, the HMGB1/nucleosome mixture was reacted with 0.25% glutaraldehyde, which is widely used as a covalent cross-linking agent. As shown in figure 32, 0.25% (25 mM) glutaraldehyde was added to the 1600 nM HMGB1 treated nucleosome reaction mixture and incubated for one hour to potentially cross-link HMGB1 with nucleosome. The cross-linking reaction was stopped by adding glycine. We observed that the mobility of the band was reduced (in 4% EMSA gel) nearly reverting back to the position of the canonical nucleosome. We conclude that glutaraldehyde reacted extensively with the lysine residues in

HMGB1 and the nucleosomes. This reduced the positive charge on lysine and arginine and lowered the effectiveness of HMGB1 binding to nucleosomes. Thus, there would be little or no interaction of HMGB1 with nucleosomes and the nucleosomes could not be remodeled. 137

Figure 31. Supershift assay to determine whether HMGB1 is a stable component of

remodeled nucleosomes: Lane 1: free DNA (2G2), Lane2: Nucleosomes (n2G2), Lane3:

Nucleosomes + 400 nM HMGB1, Lane4: Nucleosomes + 800 nM HMGB1, Lane 5:

Nucleosomes + 1600 nM HMGB1, Lane 6: Nucleosomes + 400 nM HMGB1 + α-HMGB1

(2µg), Lane 7: Nucleosomes + 800 nM HMGB1 + α-HMGB1 (2µg), Lane 8: Nucleosomes +

1600 nM HMGB1 + α-HMGB1 (2 µg). In 20 µL reaction volume, nucleosomes with and

without increasing concentration of HMGB1 was incubated in TE/sucrose (18%) buffer for 1

hour on ice. After the 1 hour incubation, 2 µL of 1 mg/mL α-HMGB1 (Upstate) was added to the

lanes 6, 7 and 8 reaction mixture and incubated on ice for 20 mins and loaded onto a EMSA gel

and run for 2 hours at 200V at 4oC. 138

Figure 32. Reaction of glutaraldehyde with nucleosomes in the presence of 1600 nM

HMGB1: Lane 1: Nucleosomes (n2G2), Lane2: Nucleosomes + 1600 nM HMGB1, Lane 3:

Nucleosomes + 1600 nM HMGB1 + 33 mM glycine, Lane 4: Nucleosomes + 1600 nM HMGB1

+ α-HMGB1 (2µg) + 33 mM glycine, Lane 5: Nucleosomes + 1600 nM HMGB1 + 25 mM

glutaraldehyde + 33 mM glycine, Lane 6: Nucleosomes + 1600 nM HMGB1 + 25 mM

glutaraldehyde (0.25%) + 33 mM glycine (0.25%) + α-HMGB1 (2µg). In 10 µL reaction

volume, nucleosomes with and without 1600 nM HMGB1 was incubated in TE/sucrose (18%)

buffer for 1 hour on ice. After the 1 hour incubation, glutaraldehyde was added to 25 mM final

concentration and incubated for 1 hr on ice. Glycine was added to the reaction mixture to make

33 mM final concentration and 2µL of 1 mg/mL α-HMGB1 (Upstate) was added to it and

incubated for 20 minutes and loaded onto a EMSA gel and run for 2 hours at 200V at 4oC.

139

This made it clearer that under these conditions, HMGB1 is persistently required to remodel

the nucleosome or to maintain the remodeled sate of nucleosome.

3.3. DNase I 10bp Digestion Pattern for DNA, Nucleosomes and HMGB1-remodeled

Nucleosomes

To further understand how HMGB1 might alter the interactions in the nucleosomes

when treated with 1600 nM HMGB1, a DNase I digestion profile of nucleosomes treated

with 1600 nM HMGB1, free DNA, and canonical nucleosome was examined. Fig. 33 shows

DNase I profile of free DNA, the canonical nucleosome, and the nucleosome in the presence of 1600 nM HMGB1. The 10bp periodicity observed in the canonical nucleosome ( ) was

disrupted in the HMGB1-remodeled nucleosomes as evident by at least 17 additional bands

observed ( ) (Fig 33), whereas at 400 nM HMGB1 (done by Yaw Sarpong), there was no

change in 10bp pattern. This altered pattern was comparable with the DNase I digestion

profile of free DNA, which is an indicative of the remodeled and or dynamic nature of the

DNA-histone complex in the presence of HMGB1.

3.4. Effect of Increasing Amounts of “Cold” Competitor DNA on Nucleosomes Treated

with 1600 nM HMGB1

Additionally, to determine if the DNA in the remodeled nucleosomes (in the presence

of 1600 nM HMGB1) is dynamic and weakly interacting with the core histones, an attempt to

dissociate nucleosomal DNA from the nucleosome by competing with excess unlabeled

DNA was done. If DNA-histone interactions are weakened significantly, the “hot”

nucleosomal DNA may dissociate from the nucleosomes. It might also be possible for the

unlabeled 2G2 DNA to interact with the core histones. If excess DNA competed for histone 140

Figure 33. DNase I assay of nucleosomes in the presence of 1600 nM HMGB1: Lanes 1 and 8: GA

ladder for 2G2, Lanes 2 and 3: Free DNA, Lanes 4 and 5: Nucleosomes, Lanes 6 and 7: Nucleosomes in the presence of 1600 nM HMGB1. Nucleosomes were incubated with 1600 nM HMGB1 for 1 hour on ice and DNase I digestion was performed for 60 and 90 seconds subsequently. Arrows ( ) on the left indicate DNase I 10bp pattern on nucleosomes, arrows ( ) on the right indicate additional DNase I cuts

on nucleosomes in the presence of 1600 nM HMGB1, which more resembles free DNA cuts. The bar on

the left represents GRE binding site and arrow ( • ) indicates dyad axis. NOTE: Detail map of DNase I 10

bp pattern on N+1600nM HMGB1 is shown in figure 55, appendix VIII. 141

octamers and facilitated DNA dissociation from the nucleosomes, we would expect to see

free DNA bands, suggesting that P32 labeled 2G2 DNA would dissociate from the

nucleosomes and unlabeled 2G2 DNA would interact with the core histones. However, on

addition of “cold” competing DNA in increasing amounts (12.5 ng, 25 ng, 50 ng, 100 ng and

1000 ng) to the nucleosomes treated with 1600 nM HMGB1 (Fig. 34), the low mobility band

associated with HMGB1-remodeled nucleosomes disappeared and two bands of greater

mobility appear at low levels of competitor DNA, while at higher level of competitor DNA

(1000 ng), a single band at the same electrophoretic mobility as the canonical nucleosomes

appeared. This suggests that the excess DNA successfully competed with the nucleosomes

for HMGB1 and as a result, led to much less HMGB1 interaction with nucleosomes. Thus,

under these conditions, a continuous presence of HMGB1 is required to interact with

nucleosomes to produce the altered forms of nucleosomes or maintain a dynamic nature of

nucleosomes as evident from DNase I profile.

4. Isolation of HMGB1-remodeled Nucleosomes

It was of interest to determine if the altered form of nucleosomes produced by 1600

nM HMGB1 could be further isolated and characterized. At this point, it was not clear if

there were multiple altered forms or a single form of nucleosome. Therefore, an attempt was

made to isolate these altered nucleosomes by 5-30% sucrose linear gradient sedimentation. In

this attempt to isolate HMGB1-remodeled nucleosomes, the canonical nucleosomes were

treated with 1600 nM HMGB1 in a TE/sucrose (18%) buffer for 1 hour at 4oC. A 300 µL of

nucleosome treated with 1600 nM HMGB1 was loaded on the top of a five mL 5-30%

sucrose linear gradient and centrifuged for 16 hrs at 35,000 rpm in SW55 Ti rotor at 4oC. A 142

Figure 34. Effect of increasing levels of cold competitor DNA on nucleosomes treated with

1600 nM HMGB1: Lane 1: Nucleosomes (n2G2), Lane 2: Nucleosomes + 1600 nM HMGB1,

Lane 3-7: Nucleosomes + 1600 nM HMGB1 with 12.5 ng, 25 ng, 50 ng, 100 ng, 1000 ng of cold

DNA (2G2) respectively. In 10 µL reaction volume, nucleosomes with and without 1600 nM

HMGB1 was incubated in TE/sucrose (18%) buffer for 1 hour on ice. After the 1 hour

incubation, increasing amounts of cold DNA was added to the reaction mixtures (lanes 3-7) and

incubated for additional 1 hr and loaded onto a EMSA gel and run for 2 hours at 200V at 4oC.

143

total of 40 fractions were collected with 5 drops in each fraction (each drop equivalent to

25µL).Fractions 29-37 contained the remodeled nucleosomes (figure 35 A). A 10 µL of the

fractions 29-37 along with the canonical nucleosome used for preparation of remodeled

nucleosome were then run in a 4% polyacrylamide EMSA gel to verify the remodeled

nucleosome. Fig 30 B lane 1 shows canonical nucleosome, and lanes 2-10 shows remodeled

nucleosomes corresponding to the fractions 29-37, respectively. Two different populations of

the HMGB1-remodeled nucleosomes (N’ and N’’) with reduced mobility were observed.

Although, two different (N’ and N’’) populations of HMGB1-remodeled were observed in a

4% EMSA gel, they exhibited the same sedimentation rate in a 5-30% sucrose linear gradient. These nucleosome structures (N’ and N’’) were produced by HMGB1 transient interaction, but now, under these conditions appear stable in the absence of HMGB1 which would have been fractionated differentially in the 5-30% sucrose linear gradient sedimentation.

4.1. Comparative Sedimentation Profile of Canonical Nucleosomes and HMGB1-

remodeled Nucleosomes

Interestingly, the sedimentation rates for canonical nucleosomes (N) and HMGB1- remodeled nucleosomes (N’/N’’) were observed to be different (Figures 29 A and 35 A).

N’/N’’ had a greater sedimentation rate than N. This higher sedimentation rate closely resembled “dinucleosomes” formed when nucleosomes were remodeled by SWI/SNF

(Schnitzler et al., 1998). On reflection of our results, it was apparent that there was a problem with our initial procedure. The isolation of nucleosome initially from a sucrose gradient led to canonical nucleosomes being fractionated in 18% sucrose/TE buffer. In the preparation of

N’/N’’, the nucleosomes were reacted with 1600 nM HMGB1 in the 18% sucrose/TE buffer. 144

A.

B.

Figure 35. Sedimentation and EMSA profile of HMGB1-remodeled nucleosomes (N’ &

N”): (A.) Sedimentation profile of HMGB1-remodeled nucleosomes from 5-30% sucrose linear gradient fraction collections as monitored by P32 counts per minute. The fractions were collected

from the top by injecting 50% sucrose from the bottom of the tube. (B.) The EMSA profile of the

fractions showing electrophoretic mobility of canonical nucleosomes (N) and two different bands

(N’ and N’’) for nucleosomes altered by reaction with 1600 nM HMGB1. Lanes 1: Canonical

nucleosomes (N) from which N’/N’’ were produced by reacting with 1600 nM HMGB1. Lane 2-

10: Fractions 29-37 from the 5-30% sucrose linear gradient. 145

This was then loaded on top of 5-30% sucrose linear gradient. Loading 18% sucrose on 5% does not permit it to reside at top of the gradient and as a result it produced an incorrect sedimentation rate. Thus, during the isolation of canonical nucleosomes (N) and HMGB1- remodeled nucleosomes (N’/N’’) in a 5-30% sucrose linear gradient sedimentation, N and

N’/N’’ did not start at the same point, which resulted in two different sedimentation profiles.

Therefore, in order to compare the relative sedimentation rate for canonical nucleosome (N) and HMGB1-remodeled nucleosome (N’/N’’), the isolated fraction of nucleosomes (N and N’/N’’) (~300 µL) from the 5-30% sucrose linear gradient were dialyzed against 1 L of TE buffer to decrease the sucrose concentration to 0.006%. This insured that the sample loaded on top of the 5-30% sucrose gradient initially rested at the top, and all the nucleosomes started at the same point during the centrifugation. The dialyzed nucleosomes (N and N’/N’’) were then loaded on to 5-30% sucrose linear gradient and centrifuged as described earlier. As shown in figure 36 A, both the canonical nucleosomes

(N) and the HMGB1-remodeled nucleosomes (N’/N’’), which were run in individual tube, have nearly the same sedimentation rate with N’/N’’ being detectably greater. This suggested that HMGB1-remodeled nucleosomes (N’/N’’) are remodeled mononucleosomes and clearly not “dinucleosomes”. Also, the peak fraction collected were analyzed on a 4% polyacrylamide EMSA gel to verify that the N’ and N’’ occurred and their relative mobility

(Fig 36 B). Therefore, N’ and N’’ are both altered mononucleosomes, but with two different mobility profile in a 4% EMSA gel, suggesting that there are two distinct forms of the remodeled nucleosomes and distinct from the canonical nucleosomes.

146

A.

B.

Figure 36. Sedimentation and EMSA profiles of canonical nucleosomes (N), remodeled

nucleosomes (N’ & N”): Canonical nucleosomes (blue) and HMGB1-treated nucleosomes (red) were sedimented individually. (A) Fractions of nucleosomes, either canonical (N, blue) or

HMGB1-remodeled (N’/N’’, red) from sedimentation in 5-30% sucrose linear gradient as monitored by P32 counts per minute. (B) EMSA of fractions 14-22 from 5-30% sucrose linear

gradient sedimentation study, with a single bands for canonical nucleosomes (N) and two

different (N’ and N’’) bands for HMGB1- remodeled nucleosomes. 147

4.2. Determination of the Presence of Core Histones in Nucleosomes, Nucleosomes

Treated with 1600 nM HMGB1, and HMGB1-remodeled Nucleosomes

Although canonical nucleosomes (N) and remodeled nucleosomes (N’/N’’) are both mononucleosomes, they have a different mobility in a 4% polyacrylamide EMSA gel.

Therefore, to investigate if the remodeled nucleosomes have all the four core histones intact,

nucleosomes, nucleosomes treated with 1600 nM HMGB1, and HMGB1-remodeled

nucleosomes (N’/N’’) were reacted with antibodies (αH2A, αH2B, αH3, and αH4) to

histones (H2A, H2B, H3, and H4), respectively. All the nucleosomes treated with antibodies

to histones were analyzed on a 4% polyacrylamide EMSA gel. As shown in figure 37, αH2B,

αH3, and αH4 supershifted nucleosomes, the nucleosomes treated with 1600 nM HMGB1,

and the HMGB1-remodeled nucleosomes (N’/N’’) indicating the presence of H2B, H3, and

H4. However, αH2A did not supershift any of the nucleosomes, including the canonical

nucleosomes which contained all four histones (H2A, H2B, H3 and H4). The core histones

from micrococcal nuclease (MNase) digested oligonucleosomes prepared by Yaw Sarpong,

which was used to prepare the nucleosomes initially is shown in appendix III, figure 54. This

suggested that antibody to H2A was not a valid antibody for supershift experiments. Based

on these observations, we concluded that canonical nucleosomes, 1600 nM HMGB1 treated

nucleosomes, and fractionated N’/N’’ nucleosomes clearly have the three core histones, and

probably all four, although the stoichiometry is uncertain.

4.3. Determination of the Presence of HMGB1 as a Stable Component of HMGB1-

remodeled Nucleosomes

The N’/N’’ are HMGB1-remodeled nucleosomes produced from canonical nucleosomes (N) by reacting with 1600 nM HMGB1. Therefore, we were interested to know 148

Figure 37. Supershift assay to determine the presence of core histones intact in

nucleosomes, nucleosomes treated with HMGB1, and remodeled nucleosomes: Lane 1:

Nucleosomes (n2G2) in TE/sucrose (18%) buffer, Lanes 2-5: Nucleosomes (n2G2) in

TE/sucrose (18%) buffer with α-H2A, α-H2B, α-H3, and α-H4, respectively. Lane 6:

Nucleosomes in TE/sucrose (18%) buffer treated with 1600 nM HMGB1, Lanes 7-10:

Nucleosomes in TE/sucrose (18%) buffer treated with 1600 nM HMGB1 with α-H2A, α-H2B,

α-H3, and α-H4, respectively. Lane 11: Remodeled nucleosomes in TE/sucrose (18%) buffer,

Lanes 12-15: Remodeled nucleosomes in TE/sucrose (18%) buffer with α-H2A, α-H2B, α-H3, and α-H4, respectively. In 10µL reaction volume, nucleosomes, nucleosomes treated with 1600 nM HMGB1 for 1 hour on ice or remodeled nucleosomes from the TE/sucrose gradient was incubated with 3 µL of anti-histone antibody (α-H2A, α-H2B, α-H3, and α-H4) as indicated and incubated for 10 minutes on ice. After the incubation, it was and loaded onto an EMSA gel and run for 2 hours at 200V at 4oC. 149

if HMGB1 was a stable component of the remodeled nucleosome (N’/N’’). This led us to

investigate remodeled nucleosomes (N’/N’’) for the presence of HMGB1 as a stable

component of the HMGB1-remodeled nucleosome. Isolated N’/N’’ were incubated with 1 µg

of α-HMGB1 for 20 mins and analyzed for the supershifted complex on a 4% EMSA gel. As

shown in Fig. 38, the HMGB1-remodeled nucleosomes (N’/N’’) did not produce a supershift

(seen as a reduced mobility band representing a complex of α-HMGB1 and HMGB1-

remodeled nucleosome (N’/N’’)). This makes clear that HMGB1 is not a stable component of

the HMGB-remodeled nucleosomes, N’/N’’. On fractionation, N’/N’’ have little or no

HMGB1 and the HMGB1-remodeled nucleosomes are stable in the absence of HMGB1.

5. Effect of Increasing Amounts of HMGB1 on HMGB1-remodeled Nucleosomes

From the earlier observations, it was evident that HMGB1 is not a stable component of

the HMGB1-remodeled nucleosome (N’/N’’). This led us to investigate if HMGB1 has any

influence on this HMGB1-remodeled nucleosome. In order to determine the effect of HMGB1

on the remodeled nucleosomes, an increasing amounts of HMGB1 (400 nM, 800 nM, and 1600

nM) were incubated with N’/N’’ for 1 hour at 4oC and then analyzed on a 4% polyacrylamide

EMSA gel. The EMSA band for N’/N’’ treated with HMGB1 shifted to more reduced mobility, which we called the N’’’ population (Fig. 39). However, a progressive effect of increasing amounts of HMGB1 on N’/N’’ was not obvious as that for canonical nucleosomes (N). There was not any significant difference between the effect produced by 400 or 1600 nM HMGB1, but the band has a more reduced mobility and suggests possibly further alterations.

150

Figure 38. Supershift assay for the presence of HMGB1 as a stable component of the

HMGB1-remodeled nucleosomes (N’/N’’): Lane 1: Free DNA (2G2), Lane2: Nucleosomes

(n2G2), Lane3: Remodeled nucleosomes, Lane 4: Remodeled nucleosomes + α-HMGB1 (2 µg).

In 20 µL reaction volume, HMGB1-remodeled nucleosomes (N’/N’’) in TE/sucrose (18%) buffer fractionated from the 5-30% sucrose linear gradient were incubated with 2 µL of 1 mg/mL

α-HMGB1 (Upstate) on ice for 20 mins and loaded onto a EMSA gel and run for 2 hours at

200V at 4oC.

151

Figure 39. Effect of increasing amounts of HMGB1 on remodeled nucleosomes: Lane 1:

Free DNA (2G2), Lane 2: Nucleosomes (n2G2), Lane3: Remodeled nucleosomes (from a 5-30%

sucrose linear gradient sedimentation fraction), Lane 3: Remodeled nucleosomes + 400 nM

HMGB1, Lane 4: Remodeled nucleosomes + 800 nM HMGB1, Lane 5: Remodeled nucleosomes

+ 1600 nM HMGB1. In 10 µL reaction volume, remodeled nucleosomes with and without increasing amounts of HMGB1 was incubated in TE/sucrose (18%) buffer for 1 hour on ice.

After the 1 hour incubation, it was loaded onto an EMSA gel and run for 2 hours at 200V at 4oC.

152

6. Characterization of HMGB1-remodeled Nucleosomes

6.1. Effect of Different Buffers on HMGB1-remodeled Nucleosomes

In an attempt to determine the in vitro binding affinity of ER to the HMGB1-

remodeled nucleosomes (N’/N’’), these remodeled nucleosomes (in 18% sucrose/TE buffer)

were added to the 1X ER dilution buffer (80 mM KCl, 10% glycerol, 15 mM Tris-HCl pH

8.0, 0.2 mM EDTA, 0.4 mM DTT, 100 ng/µL BSA, 2 ng/µL poly (dI-dC)) in a 1:1 volume

ratio. The final concentrations of the components in the reaction buffer is 40 mM KCl, 5%

glycerol, 10 mM Tris-HCl, 0.6 mM EDTA, 50 ng/µL BSA, 1 ng/µL poly (dI-dC) and 9%

sucrose. The position of the EMSA band indicated that HMGB1-remodeled nucleosomes

(N’/N’’) reverted to canonical nucleosomes (N) in these solution conditions as shown by the

change of mobility to coincide with that of canonical nucleosomes (Fig. 40). As shown in

Fig. 40, although the EMSA band position for canonical nucleosomes was unaffected (lane 1

and 3), the band corresponding to the HMGB1-remodeled nucleosome changed from two

distinct populations with reduced mobility for the remodeled nucleosomes (N’/N’’) (lane 2)

to a single band which has essentially the same mobility as that of canonical nucleosome (N)

(lane 4). This suggests that the HMGB1-remodeled nucleosomes (N’/N’’) are sensitive to salt

concentration or higher ionic strength.

Next, the effect of various other reaction buffers with relatively low salt

concentrations (low ionic strength) in the final reaction mixture such as 1X DNase I buffer

(10 mM Tris-HCl, pH 7.5, 2.5 mM MgCl2, 0.5 mM CaCl2), 1X Exo III buffer (33 mM Tris-

153

Figure 40. Effect of 1600 nM HMGB1 and ER buffer on nucleosomes and remodeled

nucleosomes. Lane 1: Nucleosomes, Lane 2: Remodeled nucleosomes, Lane 3: Nucleosomes in

ER buffer (pH 8), Lane 4: Remodeled nucleosomes in ER buffer. In 10 µL reaction volume, nucleosomes or remodeled nucleosomes in ER buffer was incubated for 1 hr on ice. After the

incubation, it was loaded onto an EMSA gel and run for 90 minutes at 200V at 4oC. 154

acetate, pH 7.8, 66 mM KAc, 10 mM MgAc, 0.5 mM DTT), 1X Exo III buffer from Fisher

Scientific (66 mM Tris-HCl, pH 8, 0.6 mM MgCl2), and TE/sucrose (18%) buffer (10 mM

Tris-HCl, pH 8.0, 1 mM EDTA, 18% sucrose) along with the 0.5X ER dilution buffer (40

mM KCl, 5% glycerol, 10 mM Tris-HCl, 0.6 mM EDTA, 0.2 mM EDTA, 50 ng/µL BSA, 1

ng/µL poly (dI-dC)) which was used in the initial ER binding studies was examined. The

final concentration of each component in a reaction mixture is tabulated in table 39, appendix

VII. As shown in figure 41, the canonical nucleosomes were unaffected by any of these

buffers and stable to these conditions. However, ER dilution buffer reverted N’/N’’ to N, and

Exo III buffers converted N’’ to N. But, DNase I buffer and TE/sucrose (18%) buffer did not

affect the HMGB1-remodeled nucleosomes N’/N’’. This further suggests that the stability of

N’/N’’ are very sensitive to solution conditions. This led us to investigate the critical

components of ER dilution buffer.

6.2. Effect of Different Components of ER Dilution Buffer on HMGB1-remodeled

Nucleosomes

In order to investigate the critical components of ER dilution buffer that destabilized both N’ and N’’ resulting it to revert back to N, one volume of HMGB1-remodeled nucleosomes (N’/N’’) were treated with one volume of 1X ER dilution buffer or 1X ER dilution buffer minus one of the components in the ER dilution buffer for 15 mins on ice. The various 1X ER dilution buffer ± one of the components are: ER dilution buffer minus BSA and poly (dI-dC) (80 mM KCl, 10% glycerol, 15 mM Tris-HCl pH 8, 0.2 mM EDTA, 0.4 mM DTT), ER dilution buffer minus poly (dI-dC) (80 mM KCl, 10% glycerol, 15 mM Tris-

HCl 155

Figure 41. Effect of different reaction buffers on nucleosomes and remodeled nucleosomes:

Lanes 1-6: Nucleosomes (n2G2) in TE/sucrose (18%) buffer, ER buffer, TE/sucrose (18%)

buffer, DNase I buffer, Exo III buffer, Exo III buffer (Fisher Scientific), respectively, Lanes 7-

12: Remodeled nucleosomes in TE/sucrose (18%) buffer, ER buffer, TE/sucrose (18%) buffer,

DNase I buffer, Exo III buffer, Exo III buffer (Fisher Scientific), respectively In 10 µL reaction

volume, nucleosomes or remodeled nucleosomes was incubated in different reaction buffers for

15 minutes on ice. After the incubation, it was and loaded onto an EMSA gel and run for 2 hours at 200V at 4oC. 156

pH 8, 0.2 mM EDTA, 0.4 mM DTT, 100 ng/µL BSA), ER dilution buffer minus BSA (80

mM KCl, 10% glycerol, 15 mM Tris-HCl pH 8, 0.2 mM EDTA, 0.4 mM DTT, 2 ng/µL poly

(dI-dC)), ER dilution buffer minus DTT, poly (dI-dC), and BSA (80 mM KCl, 10% glycerol,

15 mM Tris-HCl pH 8, 0.2 mM EDTA), and ER binding buffer minus KCl and poly (dI-dC)

(10% glycerol, 15 mM Tris-HCl pH 8, 0.2 mM EDTA, 0.4 mM DTT, 100 ng/µL BSA). The

treated HMGB1-remodeled nucleosomes (N’/N’’) were analyzed in a 4% polyacrylamide

EMSA gel. The final concentration of each of the components in the reaction mixture and the

effect on N’/N’’ is tabulated in table 21.

In figure 42, the EMSA bands corresponding to HMGB1-remodeled nucleosomes

(N’/N’’) in TE/sucrose (18%) buffer in lane 2 and to N’/N’’ bands in lane 8 were

comparable, suggesting that N’/N’’ are stable when they were treated with ER binding buffer

minus KCl and poly (dI-dC), signifying that N’/N’’ are stable in low ionic strength. The

EMSA bands in lane 3 (N’/N’’ treated with ER dilution buffer minus poly (dI-dC) and BSA),

lane 5 (N’/N’’ treated with ER dilution buffer minus poly (dI-dC)), and lane 7 (N’/N’’ treated with ER dilution buffer minus DTT, poly (dI-dC), and BSA) showed that N’’ reverted to the

nucleosomal (N) band while N’ was unaffected. This suggested that N’’ was destabilized by

40 mM KCl or 1 ng/µL poly (dI-dC) alone, while DTT has no effect on it. The EMSA bands

corresponding to HMGB1-remodeled nucleosomes (N’/N’’) in lane 4 (N’/N’’ treated with

ER dilution buffer), and lane 6 (N’/N’’ treated with ER dilution buffer minus BSA)

converted back to the bands corresponding to the canonical nucleosome (N). This suggested

that the both N’ and N’’ were destabilized when 40 mM KCl and 1 ng/µL poly (dI-dC) were

present and acted in concert. Thus, HMGB1-remodeled population (N’’) was energetically

157

Figure 42. EMSA to determine which components of ER dilution buffer destabilized the remodeled nucleosomes: Lane 1: Nucleosomes (n2G2) in TE/sucrose (18%) buffer, Lane 2:

Remodeled nucleosomes (N’/N’’) in TE/sucrose (18%) buffer, Lane 3: Remodeled nucleosomes

(N’/N’’) in ER dilution buffer minus BSA and poly (dI-dC), Lane 4: Remodeled nucleosomes

(N’/N’’) in ER dilution buffer, Lane 5: Remodeled nucleosomes (N’/N’’) in ER dilution buffer minus poly (dI-dC), Lane 6: Remodeled nucleosomes (N’/N’’) in ER dilution buffer minus BSA,

Lane 7: Remodeled nucleosomes (N’/N’’) in ER dilution buffer minus DTT, poly (dI-dC), and

BSA, Lane 8: Remodeled nucleosomes (N’/N’’) in ER binding buffer minus KCl and poly (dI- dC). In 10 µL reaction volume, 5 µL nucleosomes or remodeled nucleosomes were incubated in

5µL of TE/sucrose (18%) buffer or ER binding buffer with or without one of its components for

15 mins on ice. After the incubation, it was and loaded onto an EMSA gel and run for 2 hours at

200V at 4oC.

158

Table 21: Final concentration of each component of the reaction buffer and the effect on HMGB1-remodeled nucleosomes (N’/N’’) Buffer Components* Final Concen. Effect ER dilution buffer Tris-HCl 10 mM KCl 40 mM (80 mM KCl, 10% glycerol, 15 mM Tris- EDTA 0.6 mM HCl pH 8.0, 0.2 mM EDTA, 0.4 mM N’/N’ N DTT 0.2 mM DTT, 100 ng/µL BSA, 2 ng/µL poly (dI- BSA 50 ng/µL dC)) poly (dI-dC) 1 ng/µL ER dilution buffer minus BSA and Tris-HCl 10 mM poly (dI-dC) (80 mM KCl, 10% glycerol, 15 mM Tris- KCl 40 mM N’’N HCl pH 8.0, 0.2 mM EDTA, 0.4 mM EDTA 0.6 mM DTT) DTT 0.2 mM ER dilution buffer minus poly (dI-dC) Tris-HCl 10 mM KCl 40 mM (80 mM KCl, 10% glycerol, 15 mM Tris- EDTA 0.6 mM N’’N HCl pH 8, 0.2 mM EDTA, 0.4 mM DTT, DTT 0.2 mM 100 ng/µL BSA) BSA 50 ng/µL ER dilution buffer minus BSA Tris-HCl 10 mM KCl 40 mM (80 mM KCl, 10% glycerol, 15 mM Tris- EDTA 0.6 mM N’/N’’N HCl pH 8.0, 0.2 mM EDTA, 0.4 mM DTT 0.2 mM DTT, 2 ng/µL poly (dI-dC)) poly (dI-dC) 1 ng/µL ER dilution buffer minus DTT, BSA Tris-HCl 10 mM and poly (dI-dC) N’’N (80 mM KCl, 10% glycerol, 15 mM Tris- KCl 40 mM HCl pH 8.0, 0.2 mM EDTA) EDTA 0.6 mM ER dilution buffer minus KCl and Tris-HCl 10 mM poly (dI-dC) No change (10% glycerol, 15 mM Tris-HCl pH 8.0, EDTA 0.6 mM (N’/N’’) 0.2 mM EDTA, 0.4 mM DTT, 100 ng/µL DTT 0.2 mM BSA) BSA 50 ng/µL *Note: The reaction mixture was prepared by mixing one part ER dilution buffer and one part nucleosomes in 18% sucrose/TE buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA). Therefore, the final concentration of sucrose is 9% and glycerol is 5% in all the reactions.

159

more unstable than HMGB1-remodeled population (N’). This led to investigate the effect of

increasing concentration of salt to the stability of HMGB1-remodeled nucleosomes (N’/N’’).

6.3. Effect of Increasing Salt Concentration on HMGB1-remodeled Nucleosomes

In order to examine the stability of HMGB1-remodeled nucleosomes (N’/N’’) in different solutions of increasing salt concentration, N’/N’’ in 18% sucrose/TE buffer was treated with only increasing concentration of one electrolyte, NaCl, (6.25 mM, 12.5 mM, 25 mM, 50 mM, and 100 mM). Figure 43 shows that as HMGB1-remodeled nucleosomes

(N’/N’’) are treated with increasing salt concentration from 6.25 mM to 100 mM, the EMSA band corresponding to N’’ progressively decreased, while the level of the bands corresponding to canonical nucleosomes (N) increased. The EMSA band for N’ virtually remained the same. This suggests that N’’ is less stable than N’, with both N’ and N stable in

100 mM NaCl.

6.4. Effect of Temperature on HMGB1-remodeled Nucleosomes

To further characterize the HMGB1-remodeled nucleosomes (N’/N’’), the effect of temperature on the stability of N’/N’’ was examined. The HMGB1-remodeled nucleosomes

(N’/N’’) in 18% sucrose/TE buffer were incubated at 4oC, and 37oC for increasing time and

compared it to the effect of temperature on canonical nucleosomes (N) at 37oC for increasing

time. The EMSA bands corresponding to the canonical nucleosomes (N) did not show any

change when they were incubated at 37oC for up to 24 hours (overnight (O/N)) (Fig 44), suggesting that canonical nucleosomes are thermodynamically stable at 37oC. The EMSA

bands corresponding to HMGB1-remodeled nucleosomes (N’/N’’) also did not show any

change when they were incubated at 4oC for increasing time (0 min to O/N incubation) (Fig

44). However, when the (N’/N’’) population was incubated at 37oC for increasing time, the 160

EMSA band corresponding to N’’ reverted to bands corresponding to N’ with increasing time

(Fig 44). This suggested that the HMGB1-remodeled population N’’ was again less stable and required only a minimal energy for it to convert to thermodynamically stable HMGB1- remodeled population N’. However, N’ was stable and did not convert to N.

6.5. Effect of Increasing Amounts of “Cold” Competitor DNA on HMGB1-remodeled

Nucleosomes

From the experiments on the effect of salt and temperature on HMGB1-remodeled nucleosomes (N’/N”), it was understood that the HMGB-remodeled population N’’ is sensitive to salt and temperature (Figures 43 and 44). Also, from the experiment to determine the critical component of ER dilution buffer, both N’ and N’’ were destabilized in the ER buffer containing both salt and poly (dI-dC) such that both N’ and N’’ converted to canonical nucleosomes (N) (Fig 42, lane 4 and 6). This instability of N’’ to both temperature and high ionic strength solution suggests the DNA-histone interaction in the remodeled population are different and weaker than in canonical nucleosomes (N). Therefore, to determine the extent to which the DNA interaction with the core histones in the HMGB1-remodeled nucleosomes

(N’/N’’) have been weakened, an attempt was made to dissociate nucleosomal DNA from the nucleosome by competing with excess unlabeled DNA. If the interactions were weak, the

“hot” nucleosomal DNA may dissociate from the nucleosomes. It might also be possible for the unlabeled 2G2 DNA to interact with the core histones. If this would occur, we would expect to see the free DNA band, suggesting that the P32 labeled 2G2 DNA would dissociate

from the nucleosomes and unlabeled 2G2 DNA would interact with the core histones.

However, as shown in figure 45, addition of “cold” competing DNA in increasing amounts 161

(12.5 ng, 25 ng, 50 ng, and 100 ng) to the remodeled nucleosomes (N’/N’’), the EMSA bands

corresponding to the remodeled nucleosome population N’’ progressively converted back to

Figure 43. Effect of increasing concentration of NaCl on remodeled nucleosomes: Lane 1:

Nucleosomes (n2G2) in TE/sucrose (18%) buffer, Lane 2: Remodeled nucleosomes in

TE/sucrose (18%) buffer, Lanes 3-7: Remodeled nucleosomes in TE/sucrose (18%) buffer with

6.25 mM, 12.5 mM, 25 mM, 50 mM, and 100 mM NaCl, respectively. In 10 µL reaction volume, remodeled nucleosomes were incubated with increasing concentration of NaCl as indicated for

15 minutes on ice. After incubation, the salt concentration in the reaction mixture was diluted to

50 mM or less and immediately loaded onto an EMSA gel and run for 2 hours at 200V at 4oC. 162

Figure 44. Effect of temperature on nucleosomes and remodeled nucleosomes: Lanes 1-5:

Nucleosomes (n2G2) in TE/sucrose (18%) buffer at 37oC for 0 min, 30 min, 60 min, 120 min

and overnight (O/N), respectively. Lanes 6-10: Remodeled nucleosomes in TE/sucrose (18%) buffer at 4oC for 0 min, 30 min, 60 min, 120 min and overnight (O/N), respectively. Lanes 11-

15: Remodeled nucleosomes in TE/sucrose (18%) buffer at 37oC for 0 min, 30 min, 60 min, 120 min and overnight (O/N), respectively. A 10 µL of nucleosomes or remodeled nucleosomes in

TE/sucrose (18%) buffer was incubated at 37oC or 4oC for 0 min, 30 min, 60 min, 120 min and overnight (O/N). After the incubation, it was and loaded onto an EMSA gel and run for 2 hours at 200V at 4oC. 163

Figure 45. Effect of increasing levels of cold competitor DNA on remodeled nucleosomes:

Lane 1: Nucleosomes (n2G2). Lane2: Remodeled nucleosomes (N’/N’’). Lane 3-7: Remodeled nucleosomes (N’/N’’) with 12.5 ng, 25 ng, 50 ng, 100 ng, 1000 ng of cold DNA (2G2), respectively. In 10 µL reaction volume, remodeled nucleosomes were treated with increasing amounts of cold DNA (lanes 3-7) and incubated for 1 hr and loaded onto an EMSA gel and run for 2 hours at 200V at 4oC. 164

the canonical nucleosomes (N) as was observed as a result of increasing salt concentration.

Interestingly, when the “cold” competitor DNA in very large excess (up to 1000 ng) was added, the bands corresponding to both the HMGB1- remodeled nucleosome populations, N’ and N’’, were converted back to the canonical nucleosome (N) (Fig 45, Lane 7). Besides reversion of the thermodynamically unstable forms of the remodeled nucleosomes (N’ and

N’’) to canonical nucleosome (N), there is no evidence for dissociation of nucleosomal DNA from the HMGB1-remodleled populations, i.e, no band for free “hot” DNA. Thus, successfully isolated forms of two different HMGB1-remodeled nucleosome populations N’ and N’’ were found to be stable at low ionic strength and unstable at higher ionic strength

(100 mM NaCl) and higher temperature (37oC). This observation also supports the contention that all histones reside in N’ and N’’.

6.6. Effect of Increasing Amounts of Urea on HMGB1-remodeled Nucleosomes

Additionally, to further characterize the stability of HMGB1-remodeled nucleosomes (N’/N’’), an attempt was made to destabilize hydrogen bonding that might have helped (if any) to stabilize N’/N’’ in low ionic strength and low temperature. To examine this possibility, an increasing amounts of urea (0.06 M, 0.12 M, 0.25M, 0.5M, and 1.0M) was added to disrupt the hydrogen bonds in N’/N’’ in TE/sucrose (18%) buffer. The EMSA bands corresponding to both the canonical nucleosomes (N) and HMGB1-remodeled nucleosomes

(N’/N’’) did not show any change in the presence of increasing amounts of urea (Fig 46).

This suggests that altered forms of nucleosomes are stable in low ionic strength and low temperature because of the electrostatic interactions as evident by the instability of N’ and

N’’ in the presence of high salt concentration (high ionic strength) and in the presence of excess “cold” competitor DNA. In the presence of excess “cold” competitor DNA, the 165

negative charges of the excess DNA might have disrupted the electrostatic interaction.

However, although urea disrupts hydrogen boding, increasing levels of urea had no

observable effect on the EMSA profile for N’/N’’.

6.7. Effect of Increasing Amounts of DTT on HMGB1-remodeled Nucleosomes

Initially, in an attempt to isolate the HMGB1-remodeled nucleosomes, the

sedimentation rate for the canonical nucleosomes (N) and the HMGB1-remodeled

nucleosomes (N’/N’’) were found to be different, with the sedimentation profile of N’/N’’

comparable to that of altered nucleosomes formed by the action of SWI/SNF resembling the

sedimentation profile of dinucleosomes (Schnitzler et al., 1998)). Therefore, in an attempt to

examine if the two altered populations of nucleosomes N’ and N’’ were indeed a putative

“dinucleosome”, which could be formed by disulfide bonds between the 110 cysteine residue

of H3 of one nucleosome to another (Camerini-Otero and Felsenfeld, 1977), the altered

nucleosomes were treated with an increasing amount of DTT. The EMSA bands

corresponding to N’ and N’’ as well as canonical nucleosomes (N) did not show any change

when they were treated with increasing amounts of DTT (Fig 47). This suggested that DTT

has no effect on these altered forms of nucleosomes, ruling out that the altered form of

nucleosomes are were two mononucleosomes that were covalently linked by disulfide (S-S)

bonds to form dinucleosomes. This possibility became evident when the procedure for

running a 5-30% sucrose linear gradient sedimentation profile was corrected as mentioned

earlier, in which the HMGB1-remodeled nucleosomes and canonical nucleosome

demonstrated similar sedimentation rate with HMGB1-remodeled being detectable greater,

suggesting that HMGB1-remodeled nucleosomes (N’/N’’) are mononucleosomes. 166

Figure 46. Effect of increasing concentration of urea on canonical and HMGB1-remodeled nucleosomes: Lane 1-6: Canonical nucleosomes (n2G2) with increasing amounts of urea (0,

0.06, 0.12, 0.25, 0.5 and 1.0 M), respectively. Lane 7-12: HMGB1-remodeled nucleosomes

(N’/N’’) with increasing amounts of urea (0, 0.06, 0.12, 0.25, 0.5 and 1.0 M), respectively. In 10

µL reaction volume, either canonical nucleosomes or HMGB1-remodeled nucleosomes with increasing concentration of urea were incubated in TE/sucrose (18%) buffer for 1 hour on ice.

After the incubation it was loaded onto an EMSA gel and run for 2 hours at 200V at 4oC. 167

Figure 47. Effect of increasing concentration of DTT on canonical and HMGB1-remodeled nucleosomes: Lane 1-6: Canonical nucleosomes (n2G2) with increasing amounts of DTT (0,

0.1, 0.2, 0.4, 0.8 and 1.6 mM), respectively. Lane 7-12: HMGB1-remodeled nucleosomes

(N’/N’’) with increasing amounts of DTT (0, 0.1, 0.2, 0.4, 0.8 and 1.6 mM), respectively. In 10

µL reaction volume, either canonical nucleosomes or HMGB1-remodeled nucleosomes with increasing concentration of DTT were incubated in TE/sucrose (18%) buffer for 1 hour on ice.

After the incubation it was loaded onto an EMSA gel and run for 2 hours at 200V at 4oC.

168

7. Atomic Force Microscopy of Canonical Nucleosomes and HMGB1-remodeled

Nucleosomes

In addition to the assay to determine if the remodeled nucleosomes were indeed

dinucleosomes, an attempt was made to see if any structures resembling dinucleosomes are

present by using atomic force microscopy with help of Dr. Yufan He at Dr. Peter Lu’s Lab,

Chemistry Department. Diluted samples of canonical nucleosomes (N) and HMGB1-remodeled nucleosomes (N’/N’’) from 5-30% sucrose linear gradient fractions were used to prepared slides for microscopy, as described in materials and methods. As shown in figure 48 A and B, both the canonical nucleosomes and HMGB1-remodeled nucleosomes demonstrated a structure resembling mononucleosomes. Therefore, we came to a conclusion that HMGB1-remodeled nucleosomes (N’/N’’) were altered form of mononucleosomes.

8. Effect of Increasing Amounts of HMGB1 on Tailless Nucleosomes

To understand if the two different HMGB1-remodeled nucleosome populations N’ and

N’’ were influenced by the dynamic behavior of histone tails (that could have been enhanced by the influence of HMGB1), tailless nucleosomes were prepared by removing the tails by trypsin digestions (Zheng and Hayes, 2003). The oligonucleosomes prepared by micrococcal nuclease

(MNase) digestion of chicken erythrocyte chromatin were treated with trypsin to cut off the N- terminal tails (prepared by Yaw Sarpong). These tailless oligonucleosomes were used to make nucleosomes with P32 labeled 161bp 2G2 DNA as described earlier. The nucleosomes were

isolated using 5-30% sucrose linear gradient sedimentation. The isolated fraction of tailless nucleosomes was then treated with increasing amounts of HMGB1 (400 nM, 800 nM, 1600 nM) to determine if HMGB1 had any effect on this population of nucleosomes. As shown in figure 169

A B

Figure 48. Atomic force microscopy of canonical nucleosomes and HMGB1-remodeled nucleosomes: Samples were fixed, deposited, and imaged with silicon tip as described in materials and methods. (A) Canonical mononucleosomes (N) on spermidine-treated mica.

DNA tails where visible are indicated by arrows. (B) HMGB1-remodeled nucleosomes

(N’/N’’) seen as mononucleosomes on spermidine-treated mica. 170

49, on increasing the levels of HMGB1 from 400 nM to 1600 nM, the mobility of the nucleosome was progressively reduced in the presence of increasing amounts of HMGB1. On comparing the effect of increasing amounts of HMGB1 on canonical nucleosomes to tailless nucleosomes, it appears that at higher levels of HMGB1 (800 or 1600 nM), there was no obvious difference with normal nucleosomes. However, when tailless nucleosomes were treated with 400 nM HMGB1, the mobility of tailless nucleosomes was distinctly reduced as compared to the 400 nM HMGB1 treated canonical nucleosomes (Fig 25, Lane 3 and Fig 49, Lane 2). This suggests that in order to alter the structure of nucleosomes by HMGB1, it has to first interact with histone tails and in the absence of histone tails, even lower concentrations of HMGB1 were effective to alter the nucleosome structure, as evident by the EMSA bands of nucleosomes, both canonical and tailless, treated with 400 nM HMGB1.

9. Isolation of HMGB1-remodeled Tailless Nucleosomes

It was of interest to determine if tailless nucleosomes treated with 1600 nM HMGB1 when allowed to sediment in 5-30% sucrose linear gradient would form two different population of nucleosomes (N’ and N’’) as observed for canonical nucleosomes. Therefore, the tailless nucleosome from 5-30% sucrose linear gradient sedimentation fraction was incubated with 1600 nM HMGB1 in TE/sucrose (18%) buffer for 1 hour on ice. A 300 µL of tailless nucleosome treated with 1600 nM HMGB1 was loaded on the top of a 5mL 5-30% sucrose linear gradient and centrifuged for 16 hrs at 35,000 rpm in SW55 Ti rotor at 4oC. A total of 30 fractions were collected with 7 drops in each fraction (each drop equivalent to 25µL). Fractions 21-27 contained the remodeled tailless nucleosomes (figure 50 A, Red squares). The 1600 nM HMGB1 treated canonical nucleosomes were run parallel to the 1600 nM HMGB1 treated tailless nucleosomes.

Interestingly, sedimentation profile of HMGB1- remodeled canonical nucleosome (with tail) and 171

tailless nucleosomes were the same, with HMGB1-remodeled nucleosome (with tail) collected at

the similar factions (fraction 21-26) (figure 50 A, Blue triangles). A 10 µL of the fractions from

both the HMGB1-remodeled canonical nucleosomes and tailless nucleosomes were then run in a

4% polyacrylamide EMSA gel to verify the remodeled nucleosome. Fig 50 B lane 1 shows

canonical nucleosome (N), and lanes 2-9 shows remodeled nucleosomes corresponding to the fractions 20-27, respectively. Two different populations of the HMGB1-remodeled nucleosomes

(N’) and (N’’) with reduced mobility than that of canonical nucleosomes were observed. Fig 50

B lane 10 shows tailless nucleosome ((∆T) N), and lanes 11-18 shows remodeled tailless

nucleosomes corresponding to the fractions 21-28, respectively. Two different populations of the

HMGB1-remodeled tailless nucleosomes ((∆T) N’) and ((∆T) N’’) with reduced mobility than that of tailless nucleosomes were observed. Although, two different populations of the HMGB1- remodeled nucleosomes were observed for both the nucleosomes with and without tail, the mobility of tailless nucleosome was reduced as compared to the mobility of the canonical nucleosomes.

10. Effect of HMGB1 on PR Binding to Free DNA

It has been observed from earlier work in our lab (Yaw’s thesis, 2006), that PR binds strongly (KD=2.5 nM) to PRE/GRE at the DNA level. Therefore, in order to determine if

HMGB1 will have an influence in the binding of low concentrations PR to the PRE/GRE at

DNA level, PR at different concentration (0.02 nM, 0.08 nM, 0.16 nM, 0.63 nM, 1.25 nM, 2.5

nM, and 5 nM) were incubated with 161bp 2G2 for 20 minutes in the presence and absence of

400 nM HMGB1. In the figure 51, the EMSA profile of PR-PRE/GRE reaction showed that in

the presence of 400 nM HMGB1 PR is bound to PRE at very low concentration of 0.02 nM. This

172

Figure 49. Effect of increasing amounts of HMGB1 on tailless nucleosomes: Lane 1:

Tailless nucleosomes ((∆T) N), Lane2: Tailless nucleosomes + 400 nM HMGB1, Lane3:

Tailless nucleosomes + 800 nM HMGB1, Lane 4: Tailless nucleosomes + 1600 nM HMGB1.

In 10 µL reaction volume, tailless nucleosomes with and without increasing concentration of

HMGB1 were incubated in TE/sucrose (18%) buffer for 1 hour on ice and loaded onto a

EMSA gel and run for 2 hours at 200V at 4oC 173

A.

B.

Figure 50. Sedimentation and EMSA profile of HMGB1-remodeled canonical and tailless nucleosomes: (A.) Sedimentation profile of HMGB1-remodeled nucleosomes with (Blue) and without (Red) tails from 5-30% sucrose linear gradient fraction collections as monitored by P32 counts per minute. The fractions were collected from the top by injecting 50% sucrose from the bottom of the tube. (B.) The EMSA profile of the fractions showing electrophoretic mobility of canonical nucleosomes (N) and two different bands (N’ and N’’) for nucleosomes altered by reaction with 1600 nM HMGB1, tailless nucleosomes ((∆T) N) and two different bands ((∆T) N’and (∆T) N’’) for nucleosomes altered by reaction with 1600 nM HMGB1. Lanes 1: Canonical Nucleosome (N) from which N’/N’’ were produced by reacting with 1600 nM HMGB1. Lane 2-9: Fractions 20-27 from the 5-30% sucrose linear gradient. Lanes 10: Nucleosome without tails ((∆T) N) from which (∆T) N’/ (∆T) N’’ were produced by reacting with 1600 nM HMGB1. Lane 11-18: Fractions 21-28 from the 5-30% sucrose linear gradient. 174

Figure 51. EMSA profile of PR binding to free DNA (2G2) in the presence and absence of

400 nM HMGB1: Lanes 1and 9, 0 nM PR, Lanes 2 and 10, 0.02 nM PR, Lanes 3 and 11, 0.08 nM PR, Lanes 4 and 12, 0.16 nM PR, Lanes 5 and 13, 0.63 nM PR, Lanes 6 and 14, 1.25 nM PR,

Lanes 7 and 15, 2.5 nM PR, and Lanes 6 and 16, 5 nM PR.

175

suggests the HMGB1 facilitates the binding of PR to PRE at DNA level even at very low

concentration of PR. However, we estimated the effect of HMGB1 is probably less than 2-

fold.

11. Effect of HMGB1 on PR Binding to Free DNA, Nucleosomal DNA, and HMGB1-

remodeled Nucleosomes.

Also from our previous studies (Yaw’s Thesis 2006), it was observed that PR did not

bind at the nucleosomal level even when PRE/GRE was rotationally phased such that the

major of groove of PRE is faced away from the core histones to allow optimal binding and at

the same time translationally positioned at dyad axis, 20bp away from the dyad axis or 40bp

away from the dyad axis. Further, in the presence of 400 nM HMGB1, PR did not show any

binding to PRE/GRE at the nucleosome level. Therefore, an attempt was made to examine if

PR could bind to PRE/GRE at the nucleosomal level in the presence of 1600 nM HMG1. In

addition, PR was allowed to interact with PRE/GRE in the HMGB1-remodeled nucleosomes

(N’/N’’) both in the presence and absence of 1600 nM HMGB1. Surprisingly, even in the

presence of 1600 nM HMGB1 and up to 200 nM PR, there was no evidence for a PR/PRE

complex (Fig 52, Lanes 5-10). Also, when PR was allowed to interact with PRE in the

HMGB1-remodeled complex (N’/N’’), the EMSA bands corresponding to PR/PRE complex

were also not observed (Fig 52, Lanes 11-20). This was probably due to the instability of

HMGB1-remodeled nucleosome (N’/N’’) in the ER dilution buffer as was observed earlier

(Fig 40, Lane 4, and Fig 41, Lane 8). 176

Figure 52. EMSA profile of PR binding to free DNA (2G2), canonical nucleosomes (N), and

HMGB1-remodeled nucleosomes (N’/N’’) in the presence and absence of 1600 nM

HMGB1: Lanes 1- 4 are PR interacting with free DNA (2G2), Lanes 5-10 are PR interacting with Nucleosomes (N), and lanes 11-20 are PR interacting with modified nucleosomes (N’/N’’).

Reactions were performed at increasing concentration of PR, 0-2 nM for free DNA, 0-200 nM for canonical nucleosomes (N) and remodeled nucleosomes (N’/N’’) with and without 1600 nM

HMGB1 in ER dilution buffer. 177

CHAPTER V: DISCUSSION

Understanding the role of HMGB1 in eukaryotic transcription has been the major interest of our lab for a number of years. Previous findings from our group have shown that HMGB1 enhances in vitro binding of estrogen receptor to estrogen response elements (Tables 22-24) (Das et al., 2004; El Marzouk et al., 2008; Ghattemani, 2004; Sarpong, 2006). Importantly, the binding affinity of ER to its half-site ERE was markedly enhanced in the presence of HMGB1

(Table 23) (Das et al., 2004; El Marzouk et al., 2008). Moreover, HMGB1 was shown to stimulate the co-operative binding of ER to the tandem repeats of two cEREs and direct repeats of two cHEREs (Das et al., 2004; Ghattemani, 2004). In addition, HMGB1 decreases binding specificity of ER to estrogen response element half-sites separated by variant spacers (cEREn, n=0-4bp) while increasing its binding affinity (Table 24) (El Marzouk et al., 2008). It is clear

from our previous findings that HMGB1 facilitates in vitro binding of ER to various non-

conventional estrogen response elements and relaxes the binding specificity. Therefore, our next

goal was to understand if the in vitro binding characteristics of ER to a spectrum of EREs (single

and tandem repeats of cERE, single and tandem repeats of cHEREs and for a series of cEREn,

for n=0, 1, 2, 3, and 4) translates to functional relevance within the cell.

As the aim of this study was to understand the role of various non-conventional EREs on

in vivo transactivation and examine the influence of HMGB1, ERE-driven luciferase reporter

constructs were co-transfected with an exogenous ER expression vector and HMGB1 expression

vector in U2OS cells, which are ER negative. A series of luciferase reporter vectors was

engineered with either a single cERE, tandem repeats (two and three) of cEREs, single cHERE,

tandem repeats (two and three) of cHEREs, or cEREn with different spacer sizes “n” between the 178

two half sites of cERE at the 5’ end of the luciferase gene to construct a luciferase reporter gene

under the control of various non-conventional EREs.

1. Role of Different EREs on In Vivo Transactivation and the Influence of HMGB1

Initially in the transient transfections, it was important to establish the levels of the

transfected DNAs to be used. Previous studies in similar systems (Loven et al., 2001; Nardulli et

al., 1996; Schultz et al., 2005; Tyulmenkov et al., 2000; Wood et al., 2001) suggested that 10 nM

of E2 and 1 µg of reporter vector luciferase in this case, was sufficient to determine reporter gene

activity. Figure 16 shows that with 2cERE 5’- to the promoter TATA box, the level of ER

dependent reporter activity of this construct on E2 induction was increased by 16-fold, which

without ER present had minimal or no luciferase activity. This confirmed the specific

responsiveness of the luciferase reporter gene to estrogen and ERα.

In an attempt to get the maximum transcriptional responsiveness to ERα on the luciferase

reporter construct, an increasing amount of exogenous ERα expression vector was initially co-

transfected along with the reporter vector. Interestingly, the profile of reporter gene activity

mediated by ER on increasing amounts of exogenous ERα expression vector transfected (Fig.

17) was similar to that observed by the other group (Tyulmenkov et al., 2000), where an increase in the amount of ERα expression vector beyond 10 ng, resulted in a progressive decrease in the transcriptional activity. It may be that the ERE promoter was saturated with the transcription machinery at 10 ng of transfected ERα expression vector to give the highest activity and beyond which the activity progressively decreased. Therefore, 5 ng of pERα was co-transfected in all the experiments. Furthermore, previous studies in similar systems (Loven et al., 2001; Nardulli et al.,

1996; Tyulmenkov et al., 2000; Wood et al., 2001) have shown that 5ng of exogenous ERα is sufficient to determine the transcriptional activity. In addition, it has been observed in a 179

Table 22: KD values for in vitro ER binding to a single cERE and tandem cEREs KD DNA [nM] Effect -HMGB1 + HMGB1 1cERE 7.4 5.1 1-2 2ERE* 11.0 5.1 2+

*KD was determined using vit B1 ERU, in which contains two imperfect EREs in tandem (Das et al., 2004)

Table 23: KD values for in vitro ER binding to a single cHERE and tandem cHEREs KD DNA [nM] Effect -HMGB1 + HMGB1 1cHERE 80 15 5-6 2cHERE (2DR) 7 4 1-2 (Ghattemani, 2004)

Table 24: KD values for in vitro ER binding to cEREn (spacer n=0-4bp) KD DNA [nM] Effect -HMGB1 + HMGB1 cERE0 10 5.5 2 cERE1 80 15 5-6 cERE2 25 7.3 3-4 cERE3 7.4 5.1 1-2 cERE4 25 12 2 (El Marzouk et al., 2008) 180

number of reports that high level of ER expression is extremely toxic to most mammalian cells

(Kushner et al., 1990; Webb et al., 1992). In another study conducted in rat osteosarcoma cells, overexpression of ERα by stably transfecting ERα expression vector decreased the viability of the cells (Harmston et al., 2005). However, it has also been reported that ER can be expressed

50-100 times more in COS cells than that found in normal estrogen target tissues such as rat uterus or pituitary, and human breast cancer cells that contain high levels of ER

(Katzenellenbogen et al., 1987; Reese and Katzenellenbogen, 1991; Toney and

Katzenellenbogen, 1986). Taken together, these suggest that the effects of expressing ERα are clearly cell type specific.

Using this level of ERα expression vector and the luciferase reporter construct (for each

ERE in the series of constructs), the effect of increasing amounts of HMGB1 mammalian expression vector, pHMGB1, on ER-dependent reporter activity was investigated to maximize the reporter activity. Activity increased to maximum level at 1 µg of transfected HMGB1 expression vector (Fig 19). Western blot of cellular levels of HMGB1 showed that HMGB1 was clearly overexpressed in comparison to the cells in which HMGB1 expression vector was not transfected (Fig. 18 A, lane 1, lane 4 and Fig. 18 B). Therefore, in all the experiments, 1 µg of

HMGB1 expression vector was transfected to determine the influence of HMGB1, whereas to compare the effect on transcriptional activity with and without HMGB1 overexpression, an equal amount of empty vector (pBluescript) was transfected in place of the HMGB1 expression vector transfected.

Altogether, 1 µg of luciferase reporter construct (for each ERE in the series of constructs), 5 ng of ERα expression vector and 1 µg of HMGB1 expression vectors were co- transfected into the U2OS cells. One ng of pGL4.70 hRLuc (Renilla luciferase) was transfected 181

as an internal control in all the experiments. Therefore, for all ERE in the series, the luciferase activity was then normalized by taking the ratio of relative light units (RLU) for firefly luciferase reporter construct to that for the Renilla luciferase. The control experiment in Fig 16 showed that the activity of the ERE-driven luciferase expression vector was dependent of both 10 nM E2 and the presence of ER. Therefore, the transcriptional activities for the luciferase reporter transactivation were directly dependent on E2 and ER, and the variation in the transactivation was dependent on the character of the EREs.

1.1. Single cERE and Tandem cEREs

In the first series of experiments with cERE and two or three tandem cEREs, the

activity progressively increased as the number of cEREs increased, from 1cERE to 3cERE

(Fig. 20). Compared to 1cERE, the activity was ~7-fold higher with 2cERE and ~12-fold

higher with 3cERE (Table 25). This tremendous increase in luciferase activity was more than

an additive effect of one additional cERE to the existing cERE, which is suggestive of

synergistic effect. A similar type of transcriptional synergy with ~4.5-fold functional synergy

from 1cERE to 2cERE, and ~6.5-fold from 1cERE to 3cERE was reported in transiently

transfected CHO cells (Tyulmenkov et al., 2000). Likewise, identical ERE pairs located

175bp further upstream to the TATA box were reported to act synergistically and in a

stereoalignment-dependent manner, whereas the pairs of perfect EREs proximal to the TATA

box act additively and in a spacing-independent manner (Ponglikitmongkol et al., 1990). The

transcriptional synergy from two or four tandem EREs has been reported to be cell specific

(Klinge, 2001). Functional synergism was reported in CHO cells and not in XL-10, HepG2

or CTC-2 cells (Mattick et al., 1997). 182

An increase in ~1.5-fold transcriptional activity from 2cERE to 3cERE (Table 12) may account for the saturation of all the available transcriptionary machinery including the co-activators at 5 ng of pERα co-transfected, as suggested by a similar study (Tyulmenkov et al., 2000). The distance of ERE from the promoter, and distance between each EREs in a tandem repeats of EREs also accounts for a significant influence in transcriptional activity

(Kato et al., 1995; Nardulli et al., 1996) . The reporter activity observed for 1cERE was relatively low as compared to that observed in other studies (Loven et al., 2001; Nardulli et al., 1996; Wood et al., 2001). The factors contributing to this difference may be due to the use of a different reporter gene (CAT reporter vector) and the amount (3-5 µg) used.

HMGB1 greatly influences the activity (5-fold) on a single cERE as compared to

2cERE (4+-fold) and 3cERE (3+-fold) (Fig. 23, Tables 15, 16 and summary Table 25). This indicated that HMGB1 has a greater influence on promoters with relatively weak transcriptional activity, in this case 1cERE, as opposed to 2cERE and 3cERE (Table 25).

Increase in expression of HMGB1 protein was demonstrated to stimulate a ~6-fold increase in transcriptional from 1cERE to 2cERE and ~7-fold increase in activity from 1cERE to

3cERE. The transcriptional synergy produced in this case clearly seems to be the translation of in vitro binding co-operativity induced by HMGB1 on ER binding to tandem copies of

EREs, in which HMGB1 stimulated increased binding of ER to ERE1 in vit B1 ERU when

ER was bound to ERE2, with a 4-fold increase in co-operativity (Das et al., 2004). Likewise, synergistic estrogen-dependent transcriptional activation conferred by the pair of hormone- responsive DNA elements of the vit B1 ERU was reported to be the result of cooperative binding of two estrogen receptor dimers to these two adjacent imperfect EREs (Martinez and

Wahli, 1989). 183

Although the in vitro binding co-operativity translated to functional synergy in vivo, the translation was not identical ( a 2-fold effect of HMGB1 on in vitro binding versus a 4+- fold effect on in vivo activity, Table 25). In a number of studies (Boonyaratanakornkit et al.,

1998; Melvin and Edwards, 1999; Stros et al., 2002; Stros et al., 2009; Verrijdt et al., 2002) it was reported that DNA-architectural protein, HMGB1, besides facilitating in vitro binding of

activator proteins to DNA, also influences the transcriptional activity by acting as a co-

activator protein.

1.2. Single cHERE and Tandem cHEREs

The second series of ER target sites in DNA involves the ERE half-sites, cHERE, in

which there was a single cHERE and two or three tandem cHEREs. The activity increased

progressively from 1cHERE to 3cHERE. The activity without HMGB1 was 3-fold higher with 2cHERE and more than 8-fold higher with 3cHERE as compared to 1cHERE (Table

26). This more than additive increase in luciferase activity suggests again a transcriptional synergy for the direct repeats of half-site EREs. These data also indicate that single cHERE has a weak transcriptional activity as compared to tandem copies of cHEREs. That is why in natural promoters like chicken ovalbumin promoter, multiple cHEREs in direct repeats are present, which are reported to act synergistically to produce an activity comparable to a full site ERE (Kato et al., 1992). There are a number of reports for the natural promoters containing multiple cHERE, some of which are human Na+/H+ exchange regulatory factor

(13 half-site EREs) (Ediger et al., 2002), human oxytocin (2cHEREs)(Richard and Zingg,

1990), human prothymosin α (2cHEREs) (Martini and Katzenellenbogen, 2001), and others

(Table 35,appendix II).

184

Table 25: Comparison of in vitro binding affinity of ER to a single cERE and tandem cEREs to in vivo transcriptional activity

KD Transcriptional Activity DNA [nM] Effect (Ratio of RLU) Effect -HMGB1 + HMGB1 -HMGB1 + HMGB1 1cERE 7.4 5.1 1-2 10 50 5 2cERE* 11.0 5.1 2+ 72 321 4+ 3cERE - - 117 367 3+

* KD was determined using vit B1 ERU, which contains two imperfect EREs in tandem(Das et al., 2004)

Table 26: Comparison of in vitro binding affinity of ER to a single cHERE and tandem cHEREs to in vivo transcriptional activity

KD Transcriptional Activity DNA [nM] Effect (Ratio of RLU) Effect -HMGB1 + HMGB1 -HMGB1 + HMGB1 1cHERE 80 15 5-6 5 18 3+ 2cHERE (2DR) 7 4 1-2 15 55 3+ 3cHERE (3DR) - - 42 100 2+

Table 27: Comparison of in vitro binding affinity of ER to variant spacer cEREn (n=0-4bp) to in vivo transcriptional activity

KD Transcriptional Activity DNA [nM] Effect (Ratio of RLU) Effect -HMGB1 + HMGB1 -HMGB1 + HMGB1 cERE0 10 5.5 2 14 46 3+ cERE1 80 15 5-6 7 5 1-2 cERE2 25 7.3 3-4 38 85 2+ cERE3 7.4 5.1 1-2 27 98 3+ cERE4 25 12 2 7 23 3+ 185

Furthermore, the genes containing cHEREs at the promoter region, such as human

TGFα (El-Ashry et al., 1996; Vyhlidal et al., 2000), human cathepsin D (Cavailles et al.,

1993; Krishnan et al., 1994; Wang et al., 1997), human RARα (Rishi et al., 1995), human (Petz and Nardulli, 2000), rat creatine kinase B (Wang et al., 2001;

Wu-Peng et al., 1992), are reported to be accompanied by the transcription factor Sp1 binding sites.

The degree of E2-dependent ER mediated transcriptional activity produced by direct repeats of two half-site EREs, 2cHEREs, was reported to be dependent on the distance between the two half-sties (Kato et al., 1995), in which two cHEREs separated by 15 - 20bp produced a comparable activity as produced by a single cERE. Similar to this report, in our case the 2cHEREs are separated by 17bp (Table 5) and demonstrated a comparable activity

(15 RLU) to a single cERE (10 RLU) (Table 25, and 26).

Overexpression of HMGB1 by transfecting exogenous HMGB1 expression vector increased the transcriptional activity for both 1cHERE and 2cHERE by more than 3-fold, as opposed to 2+-fold increase for 3cHERE (Table 26). This suggests that 1cHERE and

2cHERE have relatively weak transcriptional activity, which was highly influenced by

HMGB1 as observed for a single cERE compared to two and three tandem cEREs (Table 25

and Table 26). In addition to an increase in activity for each cHEREs (single or tandem

cHEREs), overexpression of HMGB1 also stimulated a 5-fold and a 3-fold transcriptional

synergy for 3cHERE and 2cHERE, respectively from 1cHERE (Table 26). This suggests that the influence of HMGB1 on in vitro binding of ER to direct repeats of half-site ERE studied previously (Ghattemani, 2004), in which HMGB1 stimulated the co-operative

binding of ER to the second half-site in the 2cHERE, translated to the functional synergy in 186

cell. Even if the in vitro binding co-operativity was translated to functional synergy in vivo,

there was no direct correlation between the binding activity and transcriptional activity

(Table 26). Similar to our observation, a number of studies (Reviewed in (Klinge, 2001)

report that there is no linear correlation between the binding affinity and transcriptional

activity.

1.3. Variant Spacer cEREn ( spacer size n = 0-4 bps)

In the third series of experiments with cEREn separated by variant spacers (n=0-4),

the activity without HMGB1 was decreased when the spacer between the two half-sites

deviated from the consensus 3bp to 0, 1 and 4bp (Table 27). The decrease in transcriptional

activity in these variant spacer EREs reflected the decrease in binding affinity without

HMGB1 found in in vitro EMSA studies (El Marzouk et al., 2008) although there are was

clearly no linear correlation (Table 27). The transcriptional activity for cERE1 was decreased

by ~4-fold, while the binding affinity was decreased by ~10-fold. Likewise, the

transcriptional activity for cERE4 was decreased by ~4-fold as compared to 3-fold decrease

in the binding affinity, and the transcriptional activity for cERE0 was decreased by 2-fold as

compared to 1.4-fold decrease in the binding affinity. Surprisingly, the activity for cERE

with the spacer size 2, cERE2, was increased by 1.4-fold (Table 27) whereas the in vitro binding affinity of ER to cERE2 was reported to decrease by a factor of 3-fold (Table 27).

This non-linear correlation of binding affinity and transcriptional activity was reported in number of cases (reviewed in (Klinge, 2001). This suggests that a myriad of other factors also play an important role in influencing the transcriptional activity in vivo. Thus, strong binding of ER to ERE is not sufficient to produce a strong functional transcriptional activity. 187

In the experiment with overexpression of HMGB1, the transcriptional activity for cERE0, cERE2, cERE3, and cERE4 was increased (Table 27), reflecting the in vitro binding characteristics, in which the presence of HMGB1 increased the binding affinity of ER to these EREs. However, there was no linear correlation between the effect of HMGB1 in the binding affinity and transcriptional activity (Table 27). Two fold increase in binding affinity in the presence of HMGB1 was translated to a greater than 3-fold increase in transcriptional activation for cERE0, a 3-fold increase in binding affinity in the presence of HMGB1 was translated to almost 3-fold increase in transcriptional activity for cERE2, a 2-fold increase in binding affinity in the presence of HMGB1 was translated to more than 3-fold increase in transcriptional activity for cERE3 as well as cERE4 when HMGB1 was overexpressed.

Interestingly, a decrease in transcriptional activity by 1.5-fold was observed for cERE1 when HMGB1 was overexpressed although HMGB1 enhanced the binding of ER to cERE1 in vitro by more than 5-fold in the presence of HMGB1. In similar findings, although

HMGB1/2 proteins was found to stimulate the binding of p73 to different p53-responsive elements, transient transfection studies with HMGB1 expression vector showed it significantly inhibited p73 or p53-dependent transactivation in p53-deficient SAOS-2 cells, whereas in p53-deficient H12999 cells, HMGB1/2 stimulated p73 or p53-dependent transactivation (Stros et al., 2002). In addition, HMGB1 has been proposed to inhibit the formation of pre-initiation complex, in a transcriptional regulation by RNA polymerase II, where it was demonstrated to act as a transcriptional repressor by binding with TBP to form

HMGB1/TBP/TATA complex on in vitro assay (Das and Scovell, 2001). Collectively, this suggests that ubiquitously expressed HMGB1/2 proteins have potential cell and promoter specificity to up or down regulate in vivo transcriptional activity. 188

Although overexpression of HMGB1 enhanced the transcriptional activity for the

cERE0, cERE2, cERE3, and cERE4 promoters, there was a decrease in promoter activity for

EREs when deviated from cERE3. When HMGB1 was overexpressed the cERE1 exhibited a

20-fold decrease in activity as compared to cERE3, cERE4 exhibited a 4-fold decrease,

cERE0 exhibited a 2-fold decrease, and cERE2 exhibited a1.2-fold decrease in activity

(Table 27). These observations may indicate why natural promoters containing these variant

spacer EREs also contain additional regulatory elements, such as in NHERF gene, where

cERE0 is accompanied by 13 multiple half-sites, cHEREs (Ediger et al., 2002); TGFα gene,

in which cERE4 is accompanied by an additional half-site ERE and Sp1-binding site (El-

Ashry et al., 1996; Vyhlidal et al., 2000); a promoter for human NMDA receptor 2D subunit, in which cERE2 is accompanied by additional seven cHEREs (Watanabe et al., 1999).

1.4. Role of HMGB1 on Estrogen Responsive ERα Mediated Gene Activity

The estrogen responsive ERα mediated reporter gene activity under the influence of

HMGB1 for different EREs (Tables 25- 27) discussed above was in the presence of

overexpressed HMGB1 rather than the endogenous level. Although the data in Tables 25-27

clearly suggest that the influence of HMGB1 in the in vitro binding of ERα to this series of

EREs translated into in vivo transcriptional activity, the overexpressed level of HMGB1 may

well reflect the conditions in a number of cancer cells.

An estrogen receptor positive breast cancer cell line, MCF, was reported to express

higher levels of HMGB1 (Brezniceanu et al., 2003). Taken together, it suggest that

overexpression of HMGB1 may play a significant role in cancer. Apart from breast cancer

cells, a number of studies have reported the expression of higher level of HMGB1 in other

cancer cell lines, which are human hepatocarcinoma (Kawahara et al., 1996), gastric mucosa 189

and colon (Xiang et al., 1997), smooth muscle tissue of gastro intestinal tract (Choi et al.,

2003), and mammary and pancreatic adenocarcinoma (Nestl et al., 2001). Interestingly, as a number of normal tissues are investigated, HMGB1 levels differ in a cell specific manner, such as in spleen, thymus, testis, endothelial cells, and (Bonaldi et al., 2003; Degryse et al., 2001; Gardella et al., 2002; Mosevitsky et al., 1989).

The elevated expression of HMGB1 in malignant cells as well as varying levels of

HMGB1 expression in normal tissue/cell explains the importance of HMGB1 in regulation of cellular activities. From our observation it is clear that overexpressed HMGB1 enhances the transcriptional activity of “so-called” weak promoter, in this study, a single cERE, a single cHERE and tandem cHERE and variant spacer ERE n ≠ 3. Moreover, overexpressed

HMGB1 adds another degree of regulation apart from the combination of these non- conventional EREs. This probably explains why expression of HMGB1 differs from cell to cell (Table 29, appendix I).

Irrespect of the levels of HMGB1 expression in cells, an interesting question is what effect HMGB1 exerts on transcriptional regulation? Interestingly, a transient transfection of

400 nM of HMGB1 specific siRNA to knock down endogenous level of HMGB1 to less than

20% of normal levels decreased estrogen responsive ER mediated transactivation of luciferase reporter gene (Fig. 27). A 5-fold decrease in luciferase activity, when cellular level of HMGB1 was knocked down, suggests that HMGB1 is clearly involved in E2-ER dependent activity of transcription. 190

2. Role of HMGB1 on Nucleosome Remodeling

Our lab has been investigating the role that HMGB1 exerts on eukaryotic

transcription by facilitating the binding of transcription factors. Studies done in our lab and

many others have shown that this DNA architectural protein facilitates the binding of steroid

hormone receptors to its hormone response elements at the DNA level (Boonyaratanakornkit

et al., 1998; Das et al., 2004; El Marzouk et al., 2008; Melvin and Edwards, 1999; Zhang et

al., 1999). In addition to facilitating the binding, we (in this study, Tables 25-27) and others

(Boonyaratanakornkit et al., 1998; Zhang et al., 1999) have shown that HMGB1 influences

steroid hormone receptor mediated transcriptional activity. A number of “HMGB1-sensitive”

activators have been reported, some of which are Oct, 1, 2, 4, 6 (Zwilling et al., 1995),

HOXD9 (Zappavigna et al., 1996), p53 (Jayaraman et al., 1998), p73 (Stros et al., 2002), and

Zebra (Ellwood et al., 2000). Recently, HMGB1 was reported to up-regulate the human

topoisomearse IIα gene (Stros et al., 2009).

However, DNA is not naked in eukaryotes. It is found wrapped around an octamer of

core histones, composed of two copies of each of the four core histones (H2A, H2B,

H3, and H4). This fundamental unit of chromatin is called the nucleosome. Therefore, our lab

was interested in understanding how nucleosome structure influences the ER accessibility

and binding affinity to cERE, that is, how ER binding affinity to cERE at the DNA level

translates to the nucleosomal level. A number of studies with other transcription factors have

been reported along these lines and the findings are tabulated in Table 28.

Our lab primarily wanted to test the position dependence of cERE on ER

accessibility, in which cERE was in a homogenous population of nucleosomes, and the major

groove (ER binding site) of the cERE was rotationally phased outward for optimum binding. 191

To achieve this, 161bp DNA fragments were prepared (Dr. Ron Perterson) that contained the

four nucleosome positioning sequences (NPS) and one cERE positioned at the dyad axis, at

20 or 40 bps off the dyad axis. It was found (Sarpong, 2006) that ER does not bind to cERE

in the

Table 28: Binding affinities of various transcription factors (TFs) to DNA in the nucleosome compared to free DNA TF binding site in Nucleosomes Relative Binding Position TF Affinity Rotationally References from ( Free DNA/Nucleosome) Phased dyad axis (Yes/No) (bp) ER 60-70 fold Yes 0, 40 (Sarpong, 2006) GR 1.9 fold Yes 0 9.7 fold Yes 3 >50 fold Yes 5 >50 fold Yes 7 (Li and Wrange, 1995) 1.2 fold Yes 40 4.5 fold Yes 43 7.8 fold Yes 45 >50 fold Yes 47 PR >200 fold Yes 0, 40 (Sarpong, 2006) TBP 105 fold Yes N/A (Imbalzano et al., 1994) NF1 100-300 fold Yes * (Blomquist et al., 1999) GAL4 10-100 fold** No N/A (Taylor et al., 1991) Sp1 10-20 fold No 27, 47 (Li et al., 1994) TRF1 6 fold No *** (Galati et al., 2006) 20 fold No 0  Calculated as the ratio of binding affinity for free DNA to nucleosome * NF1 site located near the end of the DNA and away from the dyad axis ** Depending on multiple or single GAL4 sites *** TRF1 sites are located away from the dyad axis N/A: Not available

nucleosomal DNA (KD>250 nM) and this was independent of translational position.

However, in the presence of 400 nM HMGB1, ER was bound to cERE at the nucleosomal

level with a KD of 60 nM (Sarpong, 2006). This suggested that HMGB1 either made the 192

cERE within the nucleosome much more accessible for ER binding or that HMGB1

influenced the character of ER itself to facilitate the stronger binding. The C-terminal extension (CTE) of ER has been suggested to play an important role in interacting with

HMGB1 to facilitate the binding of ER to cERE at DNA level (Melvin et al., 2002). Our studies do not show a significant change in KD at DNA level with the presence or absence of

HMGB1 (less than 2-fold effect of HMGB1) (Sarpong, 2006). Therefore, from our study it appears that HMGB1 might alter the nucleosome structure to facilitate ER binding to cERE at the nucleosomal level. Therefore, our next goal was to determine if HMGB1 has an influence in changing the nucleosome structure.

Since our aim was now directed to the determination of the role of HMGB1 on altering or remodeling the nucleosome structure, we addressed this by constructing a 161bp

DNA and incorporating it into a nucleosome. In this case, we used GRE, which was rotationally phased and translationally positioned at the dyad axis. Canonical nucleosomes were prepared by histone exchange and salt dialysis technique using the histones from oligonucleosomes obtained from limited micrococcal nuclease digestion of chicken erythrocytes. The mononucleosomes formed were isolated from 5-30% sucrose linear gradient sedimentation (Fig. 29) and the nucleosomes were stored in TE/sucrose (18%) buffer at -20oC.

2.1. Influence of HMGB1 on EMSA Mobility of Canonical Nucleosomes

To examine nucleosome structure as a function of HMGB1 (Fig 30), the homogenous

populations of nucleosomes were treated with increasing amounts of HMGB1 (400, 800, and

1600 nM), in which we monitored any difference in EMSA mobility as a function of

HMGB1. Note that at 400 nM, there is little change in mobility. However, the mobility is 193

further reduced at 800 nM and 1600 nM HMGB1 (Fig 30). This altered EMSA mobility

could result from HMGB1 binding to the nucleosomes forming a stable (HMGB1/N)

complex, or alternatively, a form of altered nucleosome without HMGB1. If HMGB1 formed

a stable complex with the nucleosome, reaction with α-HMGB1 should produce a supershift

in EMSA. At 2 µg of α-HMGB1, we could not detect any supershift of the complex (Fig.

31). HMGB1 is a highly mobile protein in cells with very low resident time (Scaffidi et al.,

2002), and it has been proposed that HMGB1 interacts with DNA with “hit and run”

mechanism (Das et al., 2004; Hager et al., 2009). Therefore, the supershift experiment

suggests that HMGB1-nucleosome interaction is a transient interaction and that HMGB1 is

not a stable component of the HMGB1-treated nucleosome with reduced mobility in the

EMSA.

In an attempt to “lock” the highly dynamic and transient interaction of HMGB1 to the

nucleosome, we tried to covalently cross-link the HMGB1 to the nucleosomes by using

glutaraldehyde, a widely used cross-linking agent. On addition of glutaraldehyde to the reaction system (N+1600 nM HMGB1), the reduced mobility band of the remodeled nucleosome nearly reverted back to the position of the canonical nucleosome (Fig. 32). We believe that this may be the result of inactivating HMGB1 molecules by glutaraldehyde extensively reacting with the lysine and arginine residues in HMGB1, reducing its positive charge and lowering the effectiveness of HMGB1 to interact with nucleosomes. This further suggests that HMGB1 is not a stable component of the complex and no cross-linking was observed between HMGB1 and nucleosomes.

HMGB1 has been shown to compete with H1 linker histones to bind at the linker region of the DNA in the nucleosome (Cato et al., 2008; Varga-Weisz et al., 1994). This 194

interaction of HMGB1 with the linker region of DNA may require a longer piece of DNA than the 161bp used in our experiment. The 161bp DNA used in our study may explain in part why HMGB1 does not associate as a stable component of the HMGB1 treated nucleosomes.

Together with these observations, it clearly suggests that HMGB1 transiently interacts with the nucleosome by altering the structure of nucleosome to reduce its mobility in an EMSA.

2.2. Influence of HMGB1 on DNase I 10bp Pattern of Canonical Nucleosomes

To determine if the nucleosomes in the presence of 1600 nM HMGB1 differ from the

canonical nucleosomes, DNase I digestion was done. This enzymatic digestion with DNase I

provide information to analyze if the rotational phasing of the DNA has been altered. DNase

I recognizes the minor groove of the DNA. Interestingly, the minor grooves of the DNA in

the canonical nucleosomes are alternatively phased outward and inward about every 5bp (Fig

4). Therefore, DNase I can readily gain access to minor grooves facing outward only, and thus gives a 10bp pattern. If there were changes in the DNA-histone interaction in the canonical nucleosome by the interaction of HMGB1, we might expect some DNA-histone interactions to be weakened in the nucleosome. This would make additional sites more accessible and produce extra cuts in addition to the 10bp pattern.

The canonical nucleosomes shows a characteristic 10bp pattern (Fig 33, Lane 4 and

5) which results from the DNase I cutting every 10bp at which the minor groove is facing outward. The DNase I digestion of nucleosomes in the presence of 400 nM HMGB1 performed (in our lab) by Yaw Sarpong showed no changes to 10bp pattern (data not shown).

However, in the presence of 1600 nM HMGB1 (Fig. 33, Lane 6 and 7), we observed at least

17 additional bands centered over the dyad axis. This altered pattern was further comparable to the DNase I digestion profile of free DNA (Fig. 33, Lane 2 and 3), which suggests that the 195

character of nucleosomal DNA has been altered to permit greater DNase I access to the DNA

backbone and a clear indication of altered structure of remodeled nucleosome or dynamic

nature of the DNA-histone complex in the presence of 1600 nM HMGB1.

An ATP-dependent chromatin remodeling complex SWI/SNF has been reported in a number of studies to remodel the nucleosome structure to facilitate transcription factor binding (Cote et al., 1998; Cote et al., 1994; Imbalzano et al., 1994; Kwon et al., 1994; Utley et al., 1997). Imbalzano reported that hSWI/SNF complex alters the rotational phasing of

DNA in a nucleosome to facilitate the binding of TBP to the TATA box in the nucleosomal

DNA. The ATP-dependent SWI/SNF interacts in the nucleosome and allowed DNase I to produce additional 15bp cuts to the 10bp DNase I pattern (Imbalzano et al., 1994).

Interestingly, HMGB1 at the higher levels (1600 nM) produced a similar pattern of DNase I

profile (Fig 33, Lane 7) as reported for hSWI/SNF (Imbalzano et al., 1994), but without ATP

hydrolysis.

Furthermore, another SWI/SNF related chromatin remodeling complex RSC has also

been shown to alter the nucleosome structure in an ATP-dependent manner (Lorch et al.,

1998; Saha et al., 2002). Interestingly, these ATP-dependent chromatin remodeling

complexes are associated with an HMG box containing proteins, such as BAF57 in SWI/SNF

complex, BAP111 in Drosophila Brahma. BAF57 subunit in the SWI/SNF complex has been

reported to be a critical regulator of ER function in breast cancer cells (Garcia-Pedrero et al.,

2006). Likewise, the HMG-domain protein BAP111 was reported to be important for the

function of the BRM-complex in Drosophila (Papoulas et al., 2001). Therefore, whether

these HMG box proteins can be competed with HMGB1 is an area that should be explored. 196

However, to our interest, a family of factors that facilitate chromatin transcription

(FACT complex) in yeast was reported to reorganize nucleosome structure in an ATP-

independent manner (Rhoades et al., 2004; Ruone et al., 2003). Intriguingly, Nhp6 protein,

which is a subunit in yFACT, also contains a single ~70-residue high mobility group (HMG)

box motif of the type found in HMGB family (Bustin, 2001). Multiple Nhp6 molecules were

reported to facilitate the recruitment of Spt16-Pob3 to form the yFACT complexes. The

yFACT complex was then shown to alter the structure of nucleosome in an ATP-independent

manner (Ruone et al., 2003).

Additionally, HMGB1 has been reported to facilitate the remodeling of the

nucleosome by nucleosome remodeling complexes CHARC and ACF by transiently

interacting at the linker region of the DNA in the nucleosomes and increasing the rate of

activity (Bonaldi et al., 2002). Recently, HMGB1 was reported to enhance the remodeling

activity of SWI/SNF by~ 2-fold, with the acetylated HMGB1 enhancing activity by ~4-fold,

along with facilitating the binding of SWI/SNF to the nucleosomes (Ugrinova et al., 2009).

Altogether, these findings suggest that cells may have evolved early with the ubiquitously

found DNA-architectural protein, HMGB1, to remodel the nucleosome in an ATP-

independent manner and later cells may have evolved to facilitate higher order remodeling by

ATP-dependent remodeling complexes.

3. HMGB1-remodeled Nucleosomes

It is obvious from the DNase I experiment that DNA-histone interactions were weakened.

This suggests that the weakened interaction may facilitate nucleosomal DNA to be more easily

dissociated from nucleosome and replaced by “cold” DNA when competed with excess “cold”

DNA. However, in this competition experiment (Fig. 34), we observed that the altered 197

nucleosome with reduced EMSA mobility reverted back to the position of the canonical

nucleosomes. We reasoned that this could be the result of HMGB1 molecules being sequestered

away from the nucleosomes with the excess “cold” competitor DNA, reducing the effective

concentration of HMGB1 available to remodel the nucleosomes. This ability of the altered

nucleosome state to revert back to the canonical state argues against any DNA loss. Interestingly,

nucleosome remodeled by SWI/SNF were reported to revert back to the canonical state when

competed with excess “cold” oligonucleosomes (Cote et al., 1998), and when competed with

excess “cold” competitor DNA (Schnitzler et al., 1998). On the other hand, the action of

SWI/SNF on nucleosome was suggested to provoke the loss of one or two H2A/H2B dimers from the core particle (Cote et al., 1994; Peterson and Tamkun, 1995).

Together, all these studies suggest that nucleosome is not a static structure, the histone-

histone and DNA-histone interactions constantly breathe and maintain a dynamic equilibrium.

In a given microenvironment, one or more of the energetically most stable forms of

nucleosomes may persist. This dynamic behavior of nucleosomes associated with the

microenvironment possibly make the various important cellular processes like DNA replication,

DNA repair, transcription, gene silencing as well as differential expression of genes in different

tissues and cell types feasible.

In an attempt to isolate the HMGB1-remodeled nucleosomes, 1600 nM HMGB1-treated

nucleosomes were sedimented in 5-30% sucrose linear gradient (Fig. 35 A). Fractions collected

for nucleosomes treated with 1600 nM HMGB1 showed that the sedimentation rate for these

HMGB1-remodeled nucleosomes were greater than that of the canonical nucleosomes (Fig. 29

A). And interestingly, there were two different forms of the remodeled nucleosome (N’/N’’) in

this fraction, as evident two bands of reduced mobility in EMSA (Fig. 35 B). Importantly, these 198

two distinctly different populations of altered nucleosomes, factions in 18% sucrose/TE buffer, are in the presence of little or no HMGB1 because HMGB1 (~25kDa) would not sediment along with nucleosomes (~200kDa) due to the difference in molecular weight. In addition, the antibody supershift assay with anti-HMGB1 to these HMGB1-remodeled nucleosomes (Fig. 38) showed no supershift of the complex.

Intriguingly, although HMGB1 was removed from persistently interacting with the nucleosome during sedimentation, these two (N’/N’’) altered forms of nucleosomes with reduced mobility in EMSA was observed. This contrasts to the experiment (Fig. 34), in which the altered forms of nucleosome reverted back when possibly most of the HMGB1 molecules targeted excess “cold” competitor DNA rather than canonical nucleosomes. The difference in outcome of these two different systems may be attributed to the solution conditions. In the competition experiment (Fig 34), addition of excess “cold” DNA in the reaction system of 1600 nM HMGB1 and nucleosomes changes the solution conditions increasing the ionic strength of the reaction system, due to negative charges of added DNA, in addition to acting as HMGB1

“sink”. However, in the sedimentation experiment (Fig. 35), the ionic strength of the environment is very low because of the low salt buffer gradient used (5-30% sucrose/TE) and furthermore, HMGB1 was slowly taken away from the nucleosomes during the 16 hour sedimentation time in ultracentrifuge. Together it suggests that, 1600 nM HMGB1 persistently interacting with nucleosomes alters the structure of the nucleosomes which are thermodynamically stable in the continuous presence of HMGB1. However, when HMGB1 is removed from interacting with nucleosomes in low ionic strength environment, the nucleosomes are altered to form N’ and N’’, which are thermodynamically stable in low salt buffer in the absence of HMGB1. This suggests that HMGB1 at 1600 nM can remodel the nucleosomes to 199

alter the histone-DNA interactions, and two thermodynamically stable (in 18% sucrose/TE buffer at 4oC) forms of HMGB1-free nucleosomes (N’/N’’) distinctly different from canonical nucleosome can be isolated from 5-30% sucrose linear gradient.

The position of the remodeled nucleosomes in the 5-30% sucrose linear gradient fractions appeared similar to the position for a “nucleosome dimer”, which had been reported as a result of SWI/SNF action on nucleosome (Schnitzler et al., 1998). In this case, it was suggested that the very large SWI/SNF complex had the ability to bind two mononucleosomes, disrupt the histone-DNA interaction and non-covalently link the two mononucleosomes by using the energy from ATP-hydrolysis. Unlike SWI/SNF complex, HMGB1 is small and has no evidence for ATP-hydrolysis activity. Therefore, the possibility of “nucleosome dimers” formed was looked at in a different perspective, in which we explored the formation of nucleosome dimers by histone-histone interaction formed by possible covalent interaction between histones in one nucleosome with histones in the other nucleosome. Cysteine residue at 110th position on

H3 was reported to form a disulfide linkage with another H3 molecule at 110th cysteine residue

(Camerini-Otero and Felsenfeld, 1977). This suggests the possibility of having a covalently linked “nucleosome dimers”, formed by disulfide link between an H3 in two different nucleosomes. To investigate this possibility, we treated the HMGB1-remodeled nucleosomes with increasing concentration of DTT in an effort to reduce any disulfide crosslinks. However, the EMSA profile of HMGB1-remodeled nucleosome treated with increasing concentration of

DTT showed no change (Fig. 47), indicating that these remodeled nucleosomes did not contain disulfide linkages.

In another experiment, we used atomic force microscope (AFM) to try to detect dinucleosomes. However, nucleosomes structure resembling nucleosome dimers was not 200

observed (Fig. 48). In contrast to our study, a nucleosome population remodeled by SWI/SNF appeared as dimers in AFM (Schnitzler et al., 2001). On the reflection of these observations, it was apparent to revisit our procedure to isolate HMGB1-remodeled nucleosomes. The initial isolation of nucleosomes from the 5-30% sucrose linear gradient results in the nucleosomes being in18% sucrose/TE buffer, and in the subsequent preparation of the new forms of nucleosomes (N’/N’’), the canonical nucleosomes were reacted with 1600 nM HMGB1 in this

18% sucrose/TE buffer. This was then mistakenly loaded on top of 5-30% sucrose linear gradient. Loading the sample, which is in 18% sucrose, on top of the 5-30% sucrose linear gradient does not permit it to layer as a sharp distinct band as opposed to the canonical nucleosomes in TE buffer. Therefore, during sedimentation, canonical nucleosomes and 1600 nM HMGB1-treated nucleosomes in two individual tubes in the 5-30% sucrose linear gradients, the canonical nucleosomes and HMGB1-treated nucleosome may not start to sediment from the same point. This could have resulted in a different sedimentation value for canonical nucleosomes and altered nucleosomes, fallaciously indicating the formation of nucleosome dimers. Therefore, in a subsequent experiment to compare the sedimentation velocity of canonical nucleosomes and HMGB1-remodeled nucleosomes, the sucrose concentration was diluted to 0.006% in both solutions of the canonical nucleosomes and the HMGB1-remodeled nucleosomes by dialysis (against TE buffer). A distinct sharp layer was observed while loading these dialyzed samples on top of the 5-30% sucrose linear gradient. This ensured that when these samples were run individually in separate tubes, they sedimented from the same starting point. To our expectation, we observed the similar sedimentation velocity for both the canonical nucleosomes and HMGB1-remodeled nucleosomes (Fig 36 A), with the remodeled nucleosomes detectibly greater. This slight variation in sedimentation value may be, in part, due 201

to the handling technique and preparation of the sucrose gradient. This indicated that the two populations of HMGB1-remodeled nucleosomes (N’/N’’) are altered mononucleosomes, but with two forms, distinctly different from canonical nucleosomes.

3.1. Influence of HMGB1 on EMSA Mobility of Remodeled Nucleosomes

We also explored if these stable forms of HMGB1-free remodeled nucleosomes

(N’/N’’) could be further altered by adding a higher concentrations of HMGB1 to the N’/N’’.

On treatment of N’/N’’ with 400-1600 nM HMGB1 (Fig. 39), we observed further reduction

in EMSA mobility, the form we called N’’’. Unlike canonical nucleosomes (Fig. 30), there

was little or no difference on the influence of 400 nM or 1600 nM HMGB1 on the remodeled

nucleosomes (N’/N’’) as evidently the mobility on EMSA was not very different (Fig. 39) for

400 nM or 1600 nM treated N’/N’’. This may be due to the fact that nucleosomes are already

altered and 400 nM of HMGB1 was sufficient to produce an effect comparable to 1600 nM

HMGB1. This further suggests that the N’/N’’ are altered forms of the canonical

nucleosomes, which have dramatically reduced the activation energy for further remodeling.

This indicates that HMGB1 may act as a co-remodeler, in a similar way it acts as a co-

activator protein in facilitating activator proteins in transactivation of a gene.

3.2. Composition of Nucleosomes

The canonical nucleosomes prepared by histone exchange and salt dialysis technique

using the histones from oligonucleosomes obtained from limited micrococcal nuclease

digestion of chicken erythrocytes contained all four core histones (H2A, H2B, H3, and H4)

(Fig 54 in appendix III). Antibody supershift assay with α-H2B, α-H3, and α-H4 showed

supershift for the canonical nucleosomes, 1600 nM HMGB1-treated nucleosomes as well as

HMGB1-remodeled nucleosomes (N’/N’’), whereas, to our surprise antibody supershift 202

assay for the 1600 nM HMGB1-treated nucleosomes and HMGB1-remodeled nucleosomes

(N’/N’’) along with the canonical nucleosomes (N) showed no supershift with α-H2A (Fig.

37). This suggests that the epitope for the α-H2A is not exposed and is not detectable in the

supershift assay or that the level of α-H2A was is too low. Antibodies for supershift assay should be 10X greater in concentration than typically used in western blot. Therefore, we concluded that all the nucleosomes (canonical, 1600 nM HMGB1-treated and HMGB1-

remodeled) contained all least three core-histones although the stoichiometry was uncertain.

However, in combination with the findings from experiment with challenging N’/N’’ population with excess “cold” competitor DNA (Fig. 45), the ability of N’/N’’ to convert back to canonical nucleosome state (N) suggests that these altered forms possibly contain all four core histones.

3.3. Characterization of HMGB1-remodeled Nucleosomes

In an attempt to study the in vitro binding of ER/PR to the HMGB1-remodeled nucleosomes (N’/N’’), when HMGB1-remodeled nucleosomes (N’/N’’) were added to ER dilution buffer (at the final concentration of buffer compositions: 40 mM KCl, 5% glycerol,

7.5 mM Tris-HCl, pH 8.0, 0.1 mM EDTA, 0.2 mM DTT, 50 ng/µL BSA, 1 ng/µL poly (dI- dC)), it was observed that both the N’ and N’’ reverted back to the position of the canonical nucleosomes in EMSA, which in this study was reproduced as shown in figure 40, lane 4 and figure 41, lane 8. To investigate which components of the buffer was responsible for this effect, ER dilution buffer was prepared without one of the components and then incubated with N’/N’’. Intriguingly, the additive effect of 40 mM KCl and 1 ng/µL of poly (dI-dC) present in the buffer (Fig. 42, lanes 4 and 6 compared to lane 2) was found to be necessary for the reversion of the altered nucleosomes (N’/N’’) to the canonical nucleosomes. 203

However, the presence of 40 mM KCl or 1 ng/µL poly (dI-dC) alone in the buffer reverted

N’’ to N whereas N’ was not changed (Fig. 42, lanes 7 and 5, 3 compared to lane 2). This suggests that the altered nucleosome structures are unstable in high salt buffer. Therefore, we decided to further characterize the HMGB1-remodeled nucleosomes by challenging it with increasing levels of salt (NaCl), excess “cold” competitor DNA, and heat.

In the presence of increasing NaCl concentration (Fig. 43), the N’’ was converted into

N, while N’ was stable even at 100 mM NaCl. Likewise, in a study that reported persistently

altered nucleosome by the action of RSC-complex in an ATP-dependent manner, 70% of the

altered nucleosome was completely destabilized to free DNA in contrast to only ~20% for

canonical nucleosomes at 0.5M NaCl (Lorch et al., 1998), suggesting the weak histone-DNA interaction in the altered nucleosome. This indicates that although HMGB1-remodeled nucleosomes are stable at -20oC storage temperature in low salt buffer (TE/sucrose (18%)

buffer) for at least months as indicated by the EMSA bands (Figs. 40-44, untreated N’/N’’),

the N’’ population is very sensitive to increasing salt concentration and much more unstable

than the N’ population.

Furthermore, in the presence of increasing amounts of “cold” competitor DNA (Fig.

45), N’’ is completely converted back to N at 100 ng of DNA, where as N’ was converted

back to N only at very high level of “cold” competitor DNA (1000 ng). Additionally, when

N’/N’’ in 18% sucrose/TE buffer was heated to 37oC for increasing times (Fig. 44) N’’ was

converted to N’, while N’ was found stable and unchanged at 37oC for at least 24 hours.

However, in an attempt to examine if any hydrogen bonds were contributing to the stability of N’/N’’ in low salt buffer we treated it with increasing level of urea (up to 1 M) and found to have no effect (Fig. 46). These findings suggest that the HMGB1-remodeled nucleosomes 204

(N’/N’’) are thermodynamically stable in low ionic strength buffer and at 4oC, but N’/N’’

appears to be stabilized by electrostatic interactions, which can be disrupted by higher ionic

strength, excess DNA while heat simply converts N’’ to N’.

We find that the two different forms of altered nucleosome (N’ and N’’) produced by the action of 1600 nM HMGB1 on canonical nucleosomes are stable at 4oC in low salt buffer

and can revert back to the canonical form by high salt or excess competitor DNA in a step

wise manner is a reminiscent of the energy landscape for protein folding (Brooks et al., 1998;

Onuchic et al., 1995). As evident from various experiments to assess the stability of altered

nucleosome (N’/N’’) such as action of heat, higher salt concentration and excess competitor

DNA, N’’ seems to be much more unstable than N’ and resembles a higher energy

nucleosome state in the energy landscape model (fig. 53). Depending on the energy source

and level, N’ is destabilized and it converts either to N’ or all the way back to the

thermodynamically most stable canonical form (N). Heating the altered form N’/N’’ to 37oC

converts N’’ to N’, and N’ is stable at 37oC for at least 24 hours (Fig. 44). This suggests that the minimal input of energy to N’’ is sufficient to reorganize the nucleosome structure to N’.

However, on addition of increasing salt (up to 100 mM NaCl) (Fig. 43), N’’ converted back to N in 15 minutes incubation at 4oC, whereas N’ remained stable for at least 15mins. This further suggested that N’’ is less stable and has a higher energy state in the energy landscape,

and N’ attains the intermediate state between the N’’ and the thermodynamically most stable

state of canonical nucleosome (N). In the presence of excess competitor DNA (Fig. 45), N’’

appears to be progressively converted to N as DNA levels increases to 100 ng while in the

presence of 1000 ng DNA, N’ is finally unstable and is converted to N. Furthermore, due to

additive effect of 40 mM KCl and 1 ng/µL poly (dI-dC) present in final concentration of the 205

ER dilution buffer, both N’’ and N’ was converted to N whereas 40 mM KCl or 1 ng/µL poly

(dI-dC) alone could only revert N’’ to N (Fig. 42). This suggests that the HMGB1 alters the

electrostatic interactions within the canonical nucleosomes to produce N’ and N’’ and that

when placed in low salt buffer, these remodeled states persist.

Overall, our observation shows for the first time that chromosomal protein, HMGB1

remodels the nucleosome in an ATP-independent manner and the remodeled structure can be

maintained in the absence of HMGB1 with the reversion of the altered nucleosome structure

by the action of high salt buffers, excess competitor DNA and heat. Although the altered

nucleosomes are stable in low salt buffer in the absence of HMGB1, HMGB1 is required in

high salt buffer to produce and sustain the remodeled state. The finding that the altered

nucleosomes revert back to the state of canonical nucleosomes on increasing the salt

concentration further supports the notion that the canonical nucleosome form is the

thermodynamically most stable form at physiological salt conditions and remodeled states

are higher energy forms as illustrated in the energy landscape model for the HMGB1-

remodeled nucleosomes (fig. 53).

4. Role of Histone Tails on Nucleosome

The tail domains of the histones which comprises of about 25% of the mass of the core histones (Van Holde, 1989) are largely structurally undefined and contains high proportions of positively charged arginine and lysine residues (Fig. 5). This suggests that a primary binding target of the tail domains is the DNA within the nucleosome. In isolated nucleosomes, the ends of the N-terminal tail domain of H2A were found to contact two sites, at about 5bp

206

Fig 53. Energy Landscape for HMGB1-remodeled Nucleosomes: A model for energy landscape for canonical nucleosomes and the two altered nucleosomes structures, which are

HMGB1 free, and produced by the action of 1600 nM HMGB1 on canonical nucleosomes. The canonical nucleosomes (N, Red circle) are the thermodynamically most stable structure with lowest energy state. The HMGB1-remodeled nucleosomes with altered structure (N’’, Green octagon) are the thermodynamically most unstable structures in high ionic strength buffer with the highest energy state. The HMGB1-remodeled nucleosomes with altered structure (N’,

Orange square) are moderately stable in high salt buffer with intermediate state between the canonical nucleosomes (N) and altered nucleosome structure (N’’) with respect to energy landscape.

207

on each side of the DNA from where the tail emerges from the core, approximately 40bp from

the nucleosomal dyad (Lee and Hayes, 1997). The C-terminal of H2A was found to contact DNA near the dyad axis in the nucleosomal core (Davies and Lindsey, 1991; Lee and Hayes, 1998;

Usachenko et al., 1994) and near the edge of the core when linker DNA is present (Guschin et al., 1998; Lee and Hayes, 1998). In a study that demonstrated UV-laser induced histone-DNA

cross linking of all tail domains to DNA, a significant increase in cross-linking (about 5-fold)

was reported as the length of DNA is extended from 145 to 165bp (Angelov et al., 2001). This

suggests that the native sites or binding modes for most of the tails involve the ends of the DNA

or the “linker” DNA.

Furthermore, removal of N-terminal tail domains of H3/H4 was reported to enhance the

binding of transcription factor TFIIIA to nucleosomal DNA, and similarly allowed binding of

sequence specific small molecules to DNA sites oriented towards the histone surface (Gottesfeld

et al., 2001; Vitolo et al., 2000). Additionally, acetylation of H4 tails seems to be critical for

GAL4 binding to a nucleosome (Vettese-Dadey et al., 1996). These data suggest that H3/H4 tails play a dominant role in regulating DNA accessibility at the nucleosome level. Altogether, it indicates that the DNA within the nucleosomes is at least partially insulated by histone tails and not favorably accessible to the transcription factors or other remodeling complexes.

In a given microenvironment, these positively charged unstructured tail domains possibly interact with the negatively charged DNA to arrive at the thermodynamically most stable structure. NMR experiments have shown that tail domains are bound within isolated nucleosome core particles under low ionic strength conditions (~0.14 M), which are released and mobile in solutions containing moderated concentrations of salt (0.3-0.4 M) (Cary et al., 1978; Hilliard et 208

al., 1986; Smith and Rill, 1989; Walker, 1984). This suggests that the tail binding interactions are

largely electrostatic. In addition, the tail domains contribute marginally to the thermal stability of

the nucleosome core by providing the countercharge to the polyanionic backbone of the core

DNA (Ausio et al., 1989; Gottesfeld and Luger, 2001; Widlund et al., 2000).

Interestingly, the HMGB1, an architectural DNA bending protein has a highly negatively

charged C-terminal acidic tail domain (Fig. 1), which may possibly interact with the positively

charged histone tails in the nucleosome and may dissociate them from DNA and make ERE

more accessible for ER to bind. Recently, C-terminal acidic tail domain (residues 210-214) of

HMGB1 was reported to interact with lysine 36/37 of histone H3, while in native conformation of the HMGB1 molecules, the C-terminal acidic tail domains (residues 195-199) interact with its positively charged HMG-box A (at Lys 2 and residues 70-72) (Kawase et al., 2008). This suggests that at 1600 nM HMGB1, some of the HMGB1 molecules were able to interact with histone tails bound to the DNA within the nucleosomes and unfold the histone tails exposing the

DNA within the nucleosomes, while some of the HMGB1 molecules persistently interacted with the DNA-histone interface at the exposed region and weaken the interactions, which was detectable in DNase I digestion assay (Fig. 33). This weak DNA-histone interaction was not observed when 400 nM HMGB1 was used (Sarpong, 2006), possibly because the level of

HMGB1 was just enough to interact with histone tails and expose the DNA within the nucleosomes.

When the 1600 nM HMGB1-treated nucleosomes were then sedimented in a 5-30% sucrose linear gradient sedimentation, the HMGB1 molecules were separated from the nucleosomes and the unfolded histone tails may have started folding randomly back on the DNA such that the two thermodynamically stable forms of nucleosomes structures (N’/N’’) at low 209

ionic strength buffer (TE/sucrose), distinctly different from canonical nucleosomes were detected. Therefore, to explore this possibility nucleosomes were reconstituted from histones in which the N-terminal tails have been cleaved off by trypsin digestion and the influence of

HMGB1 protein was investigated.

4.1. Influence of HMGB1 on EMSA Mobility of Tailless Nucleosomes (Nucleosomes in

which the N-terminal histone tails have been cleaved off by trypsin digestion)

To explore the influence of HMGB1 on the structure of tailless nucleosomes,

homogenous population of tailless nucleosomes were treated with increasing amounts of

HMGB1 (400, 800, and 1600 nM), in which we observed a progressive reduction in EMSA

mobility of tailless nucleosomes (Fig. 49), suggesting the altered sate of nucleosomes. In

comparison to the influence of increasing concentration of HMGB1 on tailed or tailless

nucleosomes (Fig. 30 lane 3), a distinct reduction in mobility was observed for tailless

nucleosomes even at 400 nM HMGB1 (Fig. 49, lane 2). However, at higher HMGB1

concentrations (800 nM and 1600 nM), both tailed and tailless nucleosomes exhibited

significant reduction in mobility on EMSA, therefore, HMGB1 had similar effect (Fig. 30,

lanes 4 and 5, Fig. 49, lanes 3 and 4). This was interesting because, when tails were removed,

HMGB1did not have to perform an additional work to unravel the histone tails bound to the

DNA within the nucleosome and expose the DNA for further interaction. Therefore, HMGB1

may have gained an easy access to the exposed DNA within the nucleosome to interact with

and weaken the DNA-histone interaction to alter the nucleosome structure. Further

supporting this idea, my colleague (Yaw Sarpong) found that (data not shown), when tails are

removed the binding affinity of ER was dramatically reduced (KD=50 nM) which was about

the same for ER binding to canonical nucleosomes in the presence of 400 nM HMGB1. In 210

addition, the binding affinity of ER to the tailless nucleosome was further decreased in the

presence of 400 nM HMGB1 (KD~20 nM). Taken together, it indicates that HMGB1 alters

the DNA-histone interaction in the nucleosomes by first exposing the DNA within the

nucleosome by interacting with histone tails through its C-terminal acidic tail domain, and

altering the structure of nucleosome to facilitate the binding of transcription factors.

Unexpectedly, also when 1600 nM HMGB1-treated tailless nucleosome were sedimented in 5-30% linear sucrose gradient, two different forms of altered nucleosomes, which we referred as (∆T) N’ and (∆T) N’’, of reduced EMSA mobility were detected (Fig.

50 B). Interestingly, in comparison to the EMSA mobility of these altered tailless nucleosomes with the altered canonical nucleosomes, it was observed that the EMSA mobility of the tailless nucleosomes both altered and not treated with HMGB1 exhibited a more reduced mobility than the tailed nucleosome (Fig. 50 B). This may be attributed to the change in mass, charge and structure of the tailless nucleosomes compared to canonical nucleosomes. Although, we observed the difference in EMSA mobility for the complete set of tailless nucleosomes, the two altered forms of nucleosome, (∆T) N’ and (∆T) N’’, and the

two altered forms of nucleosomes with the tails intact, N’ and N’’, have a similar

sedimentation rate (Fig. 50 A), with the tailless slightly greater. This small observed

difference in sedimentation rate may be real, but may be also attributed to the possible

variations while making 5-30% sucrose linear gradient in two individual tubes for

sedimentation of 1600 nM HMGB1-treated tailless nucleosomes and nucleosomes with tails

intact. Overall, our observations suggests that, the two altered forms of nucleosomes, N’ and

N’’, produced by the action of 1600 nM HMGB1 are due to the global change in DNA- histone interactions within the nucleosome and possibly not due to the random folding of 211

histone tails back to the nucleosome core when persistently interacting HMGB1 is taken

away.

5. Binding Affinity of PR to PRE at Nucleosomal Level

Previously, in our lab we have studied the binding of PR to PRE both at DNA level and nucleosomal level, and it was found that PR had a strong binding affinity to free DNA (KD=2.5 nM) while it did not show any binding (PR as high as 140 nM) at the nucleosomal level, even in the presence of 400 nM HMGB1 (Sarpong, 2006). Therefore, we tried to determine if PR binds to the PRE in a nucleosome in the presence of higher concentration of HMGB1 (1600 nM). Also, since we were able to isolate the altered form of nucleosomes (N’ and N”), we thought PR could possibly bind to this population of nucleosomes as the DNA-histone interactions were already weakened. We find that (Fig. 52), PR did not bind to the nucleosome as well as to the altered population of nucleosome (N’/N’’), even at a high concentration of PR (200 nM) and in the presence of 1600 nM HMGB1. However, HMGB1 at 400 nM showed an obvious influence on

PR binding to free DNA even at very low concentration of PR, as low as 0.02 nM (Fig. 51). The possibility of PR not binding to PRE in the altered form of nucleosome could be due to the reversion of N’/N’’ to N in the ER dilution buffer (Fig. 40, lane 4 and Fig. 41, lane 8).

In apparent contrast to our observations, O’Malley’s lab showed that PR can bind to the nucleosomal DNA with a definite preference for sites near the nucleosome boundary and DNA regions least constrained by histones (Pham et al., 1992). However, close analysis of their data shows a dissociation of free DNA from the nucleosome in the EMSA profile, which could probably have participated in the binding of PR rather than the PRE at the nucleosome level. In our case, we virtually did not have any dissociation of free DNA from the nucleosome preparations. In addition, calculation of the KD for his work suggests that his KD is ~250 nM. 212

Largely, from our observations (Sarpong, 2006), the binding of PR, GR and ER to their hormone response elements is found very different although they share the same family of the steroid hormone receptor. It seems that PR requires more than the DBD for stable interaction and may need to include other interactions with DNA that might require wrapping partially around the DNA. However, this seems more likely the case with GR as it shows a strong affinity to GRE at the nucleosome level as well (Li and Wrange, 1995; Sarpong, 2006). This suggests that PR would probably have to wrap the DNA, which is not feasible with our nucleosome model, which is prepared from 161bp 2G2 containing four very strong nucleosome positioning sequences

(NPS) with PRE in between the two adjacent NPS. Nucleosomes made with only two NPS, one on each side of the PRE but further away from the PRE site may help answer this question by

HMGB1 proteins weakening the DNA-histone interactions in the vicinity of PRE at the nucleosome, which is probably already weaker than that for nucleosome positioning sequences, and allowing more room for PR to interact with DNA by wrapping around it to from the PR/PRE complex. 213

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APPENDIX I

Table 29: Levels of HMGB1 Expression on Tissues/Tumor/Cell lines

Tissue/Tumor/Cell line Source Method HMGB1 level Subcellular Refs location Tissue Spleen P Cell fractionation High N,C (Mosevitsky et al., 1989) Liver P Cell fractionation Low C Adult Brain P Cell fractionation Low C Thymus P Perchloric acid, CM-Sephadex High in young rats Low in old rats nd (Prasad and Thakur, fractionation, acetic acid-urea 1990) polyacrylamice gel electrophoresis Mammary gland R Northern High in pregnant and nonpregnant nd (Brezniceanu et al., mice, low in lactating mice and during 2003) gland involution Testis P Immunohistochemistry Highh in spermatogonia, low in N (Muller et al., 2004) spermatocytes, absent in spermatids Brain P Cell fractionation High in perinatal brain, low in adult C,M (Merenmies et al., brain 1991) Brain cortex R mRNA profiling High after damage nd (Kobori et al., 2002) Peripheral nervous P Immunohistochemistry, High in , Schwann cells N,C,M (Daston and Ratner, system western blotting 1991) Endothelial cells P Immunohistochemistry High N (Degryse et al., 2001) Smooth muscle cells P Immunohistochemistry Low N,C (Degryse et al., 2001) Monocytes, P Immunofluorescense High N in resting (Bonaldi et al., 2003; macrophages cells, C in Gardella et al., 2002) activated cells P, protein; R, mRNA; N, nuclear localization; C, cytoplasmic localization; M, plasma membrane localization; nd, not determined 234

Table 29: Levels of HMGB1 Expression on Tissues/Tumor/Cell lines (Contd…)

Tissue/Tumor/Cell line Source Method HMGB1 level Subcellular Refs location Tumors Human R Northern Higher in cancer tissue than in normal nd (Kawahara hepatocarcinoma tissue et al., 1996) Gastric mucosa and R Northern Higher in cancer tissue than in normal nd (Xiang et al., colon tissue 1997) Breat carcinoma P Western Higher in human breast carcinomas than nd (Brezniceanu in normal tissue et al., 2003) Smooth muscle P 2D gel analysis and mass Higher in tumours with KIT mutation nd (Choi et al., tissue of gastro- spectrometry 2003) intestinal tract Mammary and R Substractive cDNA cloning, Higher in metastatic carcinomas nd (Nestl et al., pancreatic Norther 2001) adenocarcinoma Cell lines Neuroblastoma P Acetic acid/urea/PAGE Low in differentiated, high in nd (Seyedin et undifferentiated al., 1981) N18 neuroblastoma P Immunofluorescence C, M, (Kobori et filopodia al., 2002) Muscle cell line R Northern Low in myotubes, high in myoblasts nd (Begum et al., 1990) Leukamiea cell lines P Perchloric acid extraction and Higher in myeloid than in lymphoid cells, nd (Cabart et SDS-PAGE Lower in irreversibly differentiated cells al., 1995) Melanocytic cells P Western, immunochemistry Hiher in melanoma than in melanocytes nd (Poser et al., 2003) U937 monocytic P Immunofluorescence High N in resting (Bonaldi et cells cells, C in al., 2003) activated cells P, protein; R, mRNA; N, nuclear localization; C, cytoplasmic localization; M, plasma membrane localization; nd, not determined

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APPENDIX II EREs Found in Estrogen Responsive Genes (Adapted from Bourdeau et al., 2004, O'Lone et al., 2004)

Table 30: Consensus ERE (cERE): 15bp Inverted Palindrome 5’-AGGTCAnnnTGACCT-3’ Estrogen ERE Sequences Genes Organism UniGene # References Responsive Region 5’ to 3’ Vitellogenin A2 Frog NA* -334 tcAGGTCAcagTGACCTga (Klein-Hitpass et al., 1988) GREB1 Human Hs.438037 -9517 AGGTCAtcaTGACCT (Ghosh et al., 2000) SUOX Human Hs.16340 -7499 AGGTCAcagTGACCT (Charpentier et al., 2000) GAPD Human Hs. 169476 -21207 AGGTCAaaaTGACCT (Revillion et al., 2000) * NA, Not available

Table 31: Minimal Consensus ERE (cERE): 13bp Inverted Palindrome 5’-GGTCAnnnTGACC-3’ Estrogen ERE Sequences Genes Organism UniGene # References Responsive Region 5’ to 3’ GREB1 Human Hs.438037 -1583 gGGTCAttcTGACCt (Ghosh et al., 2000) NRIP1 Human Hs. 155017 -706 gGGTCActtTGACCc (Soulez and Parker, 2001) Greb1 Mouse Mm. 218957 -3289 aGGTCAggaTGACCc (Bourdeau et al., 2004) Nrip1 Mouse Mm. 20895 -636 aGGTCAtttTGACCc (Bourdeau et al., 2004) EFP Human Hs. 1579 +23088 gGGTCAtggTGACCc (Inoue et al., 1993) EBAG9 Human Hs. 9222 +6(-64) gGGTCAgggTGACCt (Watanabe et al., 1998) COX7A2L Human Hs. 423404 +383(+443) gGGTCAaggTGACCc (Watanabe et al., 1998) Ebag9 Mouse Mm. 287896 -4886 gGGTCAgggTGACCt (Bourdeau et al., 2004) Cox7A2l Mouse Mm. 30072 +336 gGGTCAcctTGACCc (Bourdeau et al., 2004) Agt Mouse Mm.285467 -2379(-2742) aGGTCAcctTGACCc (Congiu et al., 1992) OXT Human Hs. 113216 -2465 aGGTCAgccTGACCg (Bourdeau et al., 2004) ADFP Human Hs. 3416 +147 aGGTCAggtTGACCa (Monroe et al., 2003) GAL Human Hs. 278959 -8992 tGGTCAggcTGACCt (Ormandy et al., 1998) COX7A2L Human Hs. 423404 +381 gGGTCAcctTGACCc (Watanabe et al., 1998) CPM Human Hs. 334873 -2066 aGGTCActcTGACCc (Monroe et al., 2003) KIAA0153 Human Hs. 82563 +4055 tGGTCAcgaTGACCt (Soulez and Parker, 2001) GNRHR Human Hs. 73064 +3727 gGGTCAtcaTGACCa (Kang et al., 2001) ADFP Human Hs. 3416 147 AGGTCAggtTGACCA (Monroe et al., 2003)

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Table 32: Imperfect EREs Estrogen ERE Sequences Genes Organism UniGene # References Responsive Region 5’ to 3’ AGT Human Hs. 19383 -10(-25) AGGGCAtcgTGACCC (Zhao et al., 1999) Bcl2 Mouse Mm. 233518 279 TGGTCCatcTGACCC (Bourdeau et al., 2004) C3 Human Hs. 284394 -226(-237) AGGTGGcccTGACCC (Fan et al., 1996) C3 Human Hs. 284394 -9210 GGGTCTttgTGACCG (Bourdeau et al., 2004) C3 Mouse Mm. 19131 -208 ATCTGGcctTGACCC (Bourdeau et al., 2004) Casp7 Mouse Mm. 298737 1458 GTGTCAactTGACCA (Bourdeau et al., 2004) COX7A2L Human Hs. 423404 -5246 AGGTCAggaTGTCCA (Bourdeau et al., 2004) Cstd Mouse Mm. 231395 -8157 AGGCCAatcTGACCT (Bourdeau et al., 2004) CTSD Human Hs. 343475 -125(-270) GGGCCGggcTGACCC (Augereau et al., 1994) CTSD Human Hs. 343475 -8763 GGGCCAccaTGACCC (Bourdeau et al., 2004) EFP Human Hs. 1579 22583 AGGGCAgggTGACCT (Bourdeau et al., 2004) F12 Human Hs. 1321 -30(-47) AGGGCAgctTGATCT (Citarella et al., 1993) F12 Mouse Mm. 42224 -75 GAGCAAgctTGACCA (Bourdeau et al., 2004) GAD2 Human Hs. 231829 -564 AGGTCGcagTGACCT (Bourdeau et al., 2004) Gad2 Mouse Mm. 4784 -349 AGGTCAcagCGACCT (Bourdeau et al., 2004) Gapd Mouse Mm. 288146 -745 AGGTCAggaTGCCCT (Bourdeau et al., 2004) Igfbg4 Mouse Mm. 233799 -6697 AGATCAcgcTGACCT (Bourdeau et al., 2004) IGFBP4 Human Hs. 1516 -4124 AGGTCAttgTGACCC (Soulez and Parker, 2001) LTF Human Hs. 437457 -81(-358) AGGTCAaggCGATCT (Teng et al., 1992) LTF Human Hs. 437457 -1166 AGGTGAgtcTGACCA (Bourdeau et al., 2004) Ltf Mouse Mm. 282359 -340(-342) AGGTCAaggTAACCC (Lee et al., 1995) LY6E Human Hs. 77667 -499 GGGACAagaTGACCT (Soulez and Parker, 2001) OVGP1 Human Hs. 1154 -156(-170) GGGTCActgTGACTC (Agarwal et al., 2002) OVGP1 Human Hs. 1151 200 GGGTCCctcTGACCT (Bourdeau et al., 2004) Ovgp1 Mouse Mm. 5110 866 AGGTCAggaTGTCTG (Bourdeau et al., 2004) Ovpg1 Mouse Mm. 5110 -110(-110) CGGTCAttgTGACTC (Takahashi et al., 2000) OXT Human Hs. 1132216 -166(-168) CGGTGAcctTGACCC (Richard and Zingg, 1990) Oxt Mouse Mm. 16745 -210 CGATGAcctTGACCC (Bourdeau et al., 2004) Oxt Mouse Mm. 16745 -5424 TGGTCAccgTGATCC (Bourdeau et al., 2004) pS2/TFF1 Human Hs. 350470 -392(-406) AGGTCAcggTGGCCA (Berry et al., 1989)

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Table 32: Imperfect EREs (Contd…) Estrogen ERE Sequences Genes Organism UniGene # References Responsive Region 5’ to 3’ SCNNIA Human Hs. 446415 -559 AGGTCAgccTCACCC (Soulez and Parker, 2001) TERT Human Hs. 439911 -2687(-2677) TGGTCAggcTGATCT (Kyo et al., 1999) TERT Human Hs. 439911 -5663 GGGACAgagTGACCC (Bourdeau et al., 2004) Tff1 Mouse Mm. 2854 -462 CTGTCAtctTGTCCA (Bourdeau et al., 2004) Trim25 Mouse Mm. 248445 17499 AGGGCAgggTGACCT (Bourdeau et al., 2004) TSHB Human Hs. 406687 -4099 AGGTCAgctTGACAT (Gurr et al., 1986) Tshb Mouse Mm. 110730 -535 ATGTCAaacTGACCT (Bourdeau et al., 2004) AHSG Human Hs. 324746 -3185 GGATCAaacTGACCC (Hashimoto et al., 1991) AIM1 Human Hs. 422550 363 GGGTCAcgcCCACCC (Inoue et al., 2002) AKR1C4 Human Hs. 177687 1657 GGGTCAattTAACCA (Inoue et al., 2002) ALPP Human Hs. 284255 -6033 GGATCAcagCGACCC (Bhat and Pezzuto, 2001) AR Human Hs. 99915 -599 GGGTAGaaaTGACCT (Apparao et al., 2002) ART3 Human Hs. 24976 -9809 GGGTCAcagCAACCT (Pedram et al., 2002) ASS Human Hs. 160786 585 GGGTGActcTGACCT (Charpentier et al., 2000) BDKRB2 Human Hs. 250882 -3298 TGGTCTgtcTGACCT (Monroe et al., 2003) BCL2L11 Human Hs. 84063 -6090 GGGTGAtctCGACCT (Pedram et al., 2002) B1CD1 Human Hs. 412020 929 AGGTCAcccTGACTA (Pedram et al., 2002) BIRC3 Human Hs. 127799 -6658 AGGGCAtatTGACCT (Monroe et al., 2003) BTEB1 Human Hs. 150557 -9843 AGGTCActgCAACCT (Monroe et al., 2003) C11off8 Human Hs. 432000 4209 AGGTGAtatTGACCA (Monroe et al., 2003) CALCR Human Hs. 640 927 GGGTCAgtaTGACAG (Frasor et al., 2003) CASP8 Human Hs. 243491 1374 AGGCCAtctTGACCT (Monroe et al., 2003) CBFA2T3 Human Hs. 110099 -2378 GGGTCAgccTGACGC (Frasor et al., 2003) CCBP2 Human Hs. 24286 -1509 AGGTCTttaTGACCT (Frasor et al., 2003) CD33 Human Hs. 83731 82 GGGTCAagcTGACCC (Monroe et al., 2003) CD34 Human Hs. 374990 -8306 AGGTCAaatTCACCC (Soulez and Parker, 2001) CD69 Human Hs. 82401 -9834 GGGTCAacaTGACTG (Pedram et al., 2002) CD86 Human Hs. 27954 -7718 GGGTGGgagTGACCC (Pedram et al., 2002) CDC27 Human Hs. 406631 -6134 TGGTCAggcTGGCCT (Monroe et al., 2003)

238

Table 32: Imperfect EREs (Contd…) Estrogen ERE Sequences Genes Organism UniGene # References Responsive Region 5’ to 3’ CDC6 Human Hs. 405958 2208 AGGTCActcTGAACT (Inoue et al., 2002; Lobenhofer et al., 2002) CDR1 Human Hs. 446675 -9550 TGGTCAccaTCACCT (Pedram et al., 2002) CEBPB Human Hs. 99029 -3454 GGGTCAaatTGAACA (Inoue et al., 2002) CITED2 Human Hs. 82071 2468 AGCTCAtcaTGACCT (Pedram et al., 2002) COL18A1 Human Hs. 413175 -7884 TTGTCAaagTGACCC (Monroe et al., 2003) COMPT Human Hs. 240013 -8302 AGGTCAtccTGCCCT (Xie et al., 1999) CP Human Hs. 282557 -3500 AGGTCActcTGACTA (Pedram et al., 2002) CPM Human Hs. 334873 -2066 AGGTCActcTGACCC (Monroe et al., 2003) CRHR1 Human Hs. 417628 -7603 AGGTCAgggTGGCCA (Pedram et al., 2002) CSPG2 Human Hs. 434488 -8225 GGGTGGgagTGACCC (Pedram et al., 2002) CST4 Human Hs. 56319 -7706 AGGTCAaatTGTCCC (Monroe et al., 2003) CTBP1 Human Hs. 196083 2874 GGGTTAccaCGACCC (Soulez and Parker, 2001) CXCL2 Human Hs. 75765 2628 AGGTCTttaTGACCT (Pedram et al., 2002) DC6 Human Hs. 443163 -6888 AGGCCAgagTGACCA (Seth et al., 2002a) DCC Human Hs. 172562 -9826 GGGGCAgccTGACCA (Monroe et al., 2003) DD1T3 Human Hs. 355867 -751 AGATCAgccTGACCA (Pedram et al., 2002) DHCR24 Human Hs. 75616 2615 TTGTCAtttTGACCT (Inoue et al., 2002) DIM1 Human Hs. 433683 -6657 GGGTCAcgaAGACCA (Seth et al., 2002a) DNAH8 Human Hs. 172101 -1457 AGGTCAgttTGTCCT (Monroe et al., 2003) DSCAM Human Hs. 49002 -9891 AGGCCAtttTGACCT (Pedram et al., 2002) DTNA Human Hs. 255526 -2020 AGATCAaggTGACCT (Monroe et al., 2003) DTR Human Hs. 799 -1080 GGGTCAgtcTGGCCC (Pedram et al., 2002) E2IG2 Human Hs. 31500 -711 AGATCAgccTGACCA (Charpentier et al., 2000) E2GI5 Human Hs. 5243 -8234 AGATCAgccTGACCA (Charpentier et al., 2000) EDEM Human Hs. 154332 -7338 TGGTCAgtgTGAACC (Seo et al., 1998) EGFR Human Hs. 77432 3406 AAGTCActtTGACCC (Shimomura et al., 1998) EGLN2 Human Hs. 324277 -8489 GGCTCActgCGACCT (Seth et al., 2002a) (Apparao et al., 2002; Pedram EGR3 Human Hs. 74088 2335 GGGGCGggtTGACCC et al., 2002)

239

Table 32: Imperfect EREs (Contd…) Estrogen ERE Sequences Genes Organism UniGene # References Responsive Region 5’ to 3’ ELA2A Human Hs. 21 -7130 AGGGCAgagTGACCA (Monroe et al., 2003) F12 Human Hs. 1321 -30 TGGTCAagcTGCCCT (Citarella et al., 1993) FBP1 Human Hs. 360509 4639 AGGCCAgttCGACCC (Inoue et al., 2002) FBP2 Human Hs. 61255 112 GGGTCAgggTGAGCA (Kuang et al., 1998) FGF9 Human Hs. 111 -204 TGGTCAttcTGATCT (Tsai et al., 2002) FHR-4 Human Hs. 194776 85 ATGTCAttcTGATCT (Pedram et al., 2002) FKBP4 Human Hs. 848 1065 AGGGCAtctTGACCT (Charpentier et al., 2000; Kumar et al., 2001) FOS Human Hs. 25647 -3411 AAGTCAcccTGACCT (Apparao et al., 2002; Duan et al., 1998) FOSL2 Human Hs. 301612 -7343 TGGGCAagtTGACCT (Inoue et al., 2002) FOXO1A Human Hs. 170133 391 CGGCCAtggTGACCC (Monroe et al., 2003) GAB2 Human Hs. 30687 -3399 AGGTCAgtgTGACCA (Daly et al., 2002) GAS6 Human Hs. 437710 1780 GGGTCAcccCCACCC (Monroe et al., 2003) GNRHR Human Hs. 73064 3727 GGGTCAtcaTGACCA (Kang et al., 2001) H11 Human Hs. 111676 -7364 AGGTCTttgTGACCT (Lobenhofer et al., 2002) H2AFZ Human Hs. 119192 2777 CTGTCAaaaTGACCT (Charpentier et al., 2000) H3F3A Human Hs. 181307 -4907 AGGCCAgccTGACCA (Charpentier et al., 2000) HM13 Human Hs. 444601 2754 GGGTCAaggTGTCCA (Pedram et al., 2002) HMGB1 Human Hs. 434102 -5191 AGCTCActgCGACCT (Inadera et al., 2000) HPS1 Human Hs. 404568 2429 GGGGCAgccTGACCA (Monroe et al., 2003) HSPCA Human Hs. 446579 -3795 AGGGCAatcTGACCA (Charpentier et al., 2000) HSPD1 Human Hs. 79037 -4771 TAGTCAgtgTGACCT (Kuang et al., 1998) IDH2 Human Hs. 5337 -4929 AGCTCActaTGACCT (Inoue et al., 2002) IFRD2 Human Hs. 315177 1836 GGCTCActgCGACCT (Pedram et al., 2002) IL1R2 Human Hs. 25333 -6432 GGGACAaggTGACCC (Pedram et al., 2002) INPP5A Human Hs. 408063 1410 AGGCCGgtcTGACCT (Monroe et al., 2003) ISG20 Human Hs. 105434 -7373 AGGTCAcacAGACCT (Pentecost, 1998) JUNB Human Hs. 400124 -2609 GGGTCAcagGGACCC (Pedram et al., 2002) KCNIP2 Human Hs. 97044 3108 TGGTGAgaaTGACCT (Inoue et al., 2002)

240

Table 32: Imperfect EREs (Contd…) Estrogen ERE Sequences Genes Organism UniGene # References Responsive Region 5’ to 3’ KIAA0062 Human Hs. 301743 -4025 AGGTCGcagTGAGCC (Inoue et al., 2002) KIAA0073 Human Hs. 1191 -764 AGGTCAtttTGACAA (Monroe et al., 2003) KIAA0864 Human Hs. 430725 -4360 AGGTTGcagTGACCT (Oesterreich et al., 2001) KRT6B Human Hs. 432677 -2049 TGGCCAattTGACCT (Inoue et al., 2002) KRTHB3 Human Hs. 482506 -8336 GGGTCAcagAGCCA (Seth et al., 2002b) L3MBTL Human Hs. 300863 3633 GGGTCCtgtTGACCA (Pedram et al., 2002) MAP1B Human Hs. 103042 -2800 AGGTGAaatTGACCA (Inoue et al., 2002) MATN1 Human Hs. 150366 -8045 GGGTCAggaCCACCC (Fawell et al., 1990) MGC8407 Human Hs. 145156 -7041 AGGTCAgggTGACAT (Soulez and Parker, 2001) MJD Human Hs. 419756 -518 AGATCAgccTGACCA (Monroe et al., 2003) MSX2 Human Hs. 89404 -9630 AGGTCAgaaTGATCA (Soulez and Parker, 2001) MTHFD2 Human Hs. 154672 -4241 GCCTCActgTGACCT (Inoue et al., 2002) MYBL2 Human Hs. 179718 3441 AGGTGGagcTGACCT (Frasor et al., 2003) NAFATC3 Human Hs. 172674 -2740 GGGTCCagaTGACCT (Pedram et al., 2002) NME1 Human Hs. 118638 -5636 TGGTCAgacTGATCT (Charpentier et al., 2000) NPM1 Human Hs. 411098 -1876 AGGTCAcagTGAGCC (Skaar et al., 1998) NR4A1 Human Hs. 78518 3478 TGTTCActcTGACCT (Pedram et al., 2002) NUCB2 Human Hs. 423095 -8257 GGGTCActtTGACCC (Inoue et al., 2002) P2RY6 Human Hs. 16362 4581 AGGTCAgccTGGCCA (Pedram et al., 2002) PA2G4 Human Hs. 374491 -5274 GGCTCAccgTGACCT (Charpentier et al., 2000) PDE4B Human Hs. 188 4409 GGGTCAttcTTACCC (Pedram et al., 2002) PDK4 Human Hs. 8364 557 AGGTGGcctTGACCT (Monroe et al., 2003) POLA2 Human Hs. 201897 -2268 AGGTCGaggAGACCT (Frasor et al., 2003) PPP2R4 Human Hs. 400740 -267 GGGTCAgtaCCACCC (Prange-Kiel et al., 2001) PRC1 Human Hs. 344037 4225 AGGTTGcagTGACCT (Monroe et al., 2003) PSMB4 Human Hs. 89545 -1786 GGGTCGcagTGAGCC (Charpentier et al., 2000) PTGR3 Human Hs. 527970 -5403 GGGACAgagTGACCT (Frasor et al., 2003) PTMA Human Hs. 459927 4361 AGGCCGccgTGACCT (Martini and Katzenellenbogen, 2001) PTPN2 Human Hs. 446126 -9369 AGGTCGcagTGAGCC (Pedram et al., 2002) PTPRO Human Hs. 160871 -6656 AGGTCGctgTGACCT (Pedram et al., 2002)

241

Table 32: Imperfect EREs (Contd…) Estrogen ERE Sequences Genes Organism UniGene # References Responsive Region 5’ to 3’ PTTG2 Human Hs. 511755 -3351 GGATCAagcTGACCA (Pedram et al., 2002) PTX3 Human Hs. 2050 844 AGGCCAggcTGACCA (Monroe et al., 2003) QPCT Human Hs. 79033 190 GGGTCAgccCTACCT (Monroe et al., 2003) RARA Human Hs. 361071 -8627 GTGTCAccgTGACCC (Apparao et al., 2002; Soulez and Parker, 2001) RBBP8 Human Hs. 437224 -9688 AGGTCActgCAACCT (Soulez and Parker, 2001) RFC4 Human Hs. 35120 -3314 AGGTCActgCAACCT (Frasor et al., 2003) RPS6KA1 Human Hs. 149957 -5213 TGGGCAagcTGACCC (Inoue et al., 2002) SCD Human Hs. 119597 -7982 GGGGCAgtaTGACCC (Inoue et al., 2002) SERPINA5 Human Hs. 76353 3152 AGCTCAgtgTGACCC (Soulez and Parker, 2001) SERPINA6 Human Hs. 1305 1967 GGGTCTttgTGACCC (Soulez and Parker, 2001) SF3A3 Human Hs. 77897 -8338 CGGACAtgaTGACCC (Charpentier et al., 2000) SLC29A1 Human Hs. 25450 4242 CGGTCAcgtTGACCT (Soulez and Parker, 2001) SLC7A5 Human Hs. 184601 4963 AAGTCAgaaTGACCT (Inoue et al., 2002; Soulez and Parker, 2001) SLK Human Hs. 105751 -3049 AGGTCActgCAACT (Frasor et al., 2003) SNRK Human Hs. 79025 -1342 AGGTCAgagTGATCC (Pedram et al., 2002) SNRPA Human Hs. 173255 200 AGGTCGcagTGACCT (Soulez and Parker, 2001) SOS1 Human Hs. 326392 -3457 TGGGCAtgtTGACCT (Pedram et al., 2002) SP4 Human Hs. 2982 -1480 GGGTCActtCCACCT (Pedram et al., 2002) SPRY1 Human Hs. 436944 138 GGGTCAgccAGACCG (Pedram et al., 2002) SPRY2 Human Hs. 18676 1041 GGGTCAgccCGAGCT (Pedram et al., 2002) STAG1 Human Hs. 138263 -4456 CAGTCActaTGACCT (Monroe et al., 2003) STK6 Human Hs. 250822 3170 GGGTCAattCCACCT (Frasor et al., 2003)

242

Table 32: Imperfect EREs (Contd…) Estrogen ERE Sequences Genes Organism UniGene # References Responsive Region 5’ to 3’ TAC1 Human Hs. 2563 167 GGGTCAcccCGCCCC (Pedram et al., 2002) TCF2 Human Hs. 408093 996 AGGTCAatgTGTCCG (Pedram et al., 2002) TFF1 Human Hs. 350470 -392 TGGCCAccgTGACCT (Inoue et al., 2002; Monroe et al., 2003) TFP12 Human Hs. 438231 -4126 TGGTCAtgtTTACCT (Soulez and Parker, 2001) THBD Human Hs. 2030 -2152 GGGTGGacaTGACCC (Pedram et al., 2002) TOB1 Human Hs. 178137 -3159 AGATCAgctTGACCA (Soulez and Parker, 2001) TOPBP1 Human Hs. 91417 -1262 TGATCAcctTGACCT (Monroe et al., 2003) TP53 Human Hs. 408312 -1529 AGGTCGatcTGTCCT (Qin et al., 2002) TPO Human Hs. 71304 -609 CAGTCAtggTGACCT (Pedram et al., 2002) TRIM31 Human Hs. 493275 -288 AGATCAgaaTGACCG (Monroe et al., 2003) TSHB Human Hs. 406687 -4099 AGGTCAgctTGACAT (Gurr et al., 1986) VDR Human Hs. 2062 -3231 GGGTCActtTGTCCC (Duque et al., 2002) VGEF Human Hs. 73793 -9667 AGGCCActgTGACCC (Frasor et al., 2003) VTN Human Hs. 2257 -4209 AGGTCAcaaTGAGCT (Lobenhofer et al., 2002) VWF Human Hs. 440848 -9547 AGGTCAcagCGAGCC (Monroe et al., 2003) WNT1 Human Hs. 248164 -9908 GGGTCAagaTGATCT (Katoh, 2003) WNT10B Human Hs. 91985 3215 GGGTCAagaTGATCT (Kirikoshi and Katoh, 2002) XPNPEP1 Human Hs. 3900623 -799 AGGTTGcagTGACCT (Pedram et al., 2002) XRCC4 Human Hs. 150930 -7225 AGGTTAtctTGACCA (Monroe et al., 2003) ZDHHC4 Human Hs. 5268 -9951 GGGTCAcctTGCCCG (Seth et al., 2002a) ZNF17 Human Hs. 185796 3231 GGATCAttgCGACCT (Pedram et al., 2002) ZNF9 Human Hs. 2110 2272 AGGTCGcagTGAGCC (Charpentier et al., 2000) ZNF91 Human Hs. 8597 -5023 ATGTCAcagTGACCC (Levenson et al., 2002)

243

Table 33: Multiple EREs Estrogen ERE Sequences Genes Organism UniGene # References Responsive Region 5’ to 3’ -3928 TGGTCAtacTGACCT ADCY9 Human Hs. 20196 (Frasor et al., 2003) 1856 AAGTCAcacTGACCC -3824 AGGTCAagaTGTCCT ADORA1 Human Hs. 77867 (Soulez and Parker, 2001) -1171 AGGTTAgggTGACCT -9620 TGGTCActcTGACCT ALP1 Human Hs. 37009 -419 AGGTCTggcTGACCC (Monroe et al., 2003) 2170 AGGACAcgcTGACCC -9916 AGGTCAcagCCACCT ARHC Human Hs. 179735 -9815 AGGCCAgaaTGACCA (Inoue et al., 2002) -3315 GGGTCAgggTGCCCC -2600 GGGTCAtgtCCACCT ATP2B2 Human Hs. 89512 (Pedram et al., 2002) -358 GGGTCAgggTGCCCC -6019 TGGTCAggaGGACCC AX1N1 Human Hs. 512765 (Soulez and Parker, 2001) -5940 AGGTCAgtgTGACTG -3158 AGGTGAagcTGACCC B4GALT1 Human Hs. 396798 (Soulez and Parker, 2001) 3242 AGGTCAcacTAACCC 195 TGGTCGccaGGACCC BCL2 Human Hs. 79241 (Perillo et al., 2000) +319(+276) TGGTCCccaTGACCC -6502 TGGTGAcagTGACCT BIRC5 Human Hs. 1578 (Frasor et al., 2003) 4986 GGCTCActgCGACCT -4500 AGGTCAtgcGGACCT BUB1 Human Hs. 287472 (Frasor et al., 2003) -3360 AGGCCAgccTGACCA -9210 GGGTCTttgTGACCG C3 Human Hs. 284394 (Monroe et al., 2003) -226 GGGTCAgggCCACCT -8603 CGGTCActgTAACCT CALM2 Human Hs. 425808 (Charpentier et al., 2000) 1287 ACGTCAagaCGACCT

244

Table 33: Multiple EREs (Contd…) Estrogen ERE Sequences Genes Organism UniGene # References Responsive Region 5’ to 3’ -486 GGGTCAgggTGAACT -392 GGGTCGgggTGAACT -312 GGGTCAgggTGAACT CASP7 Human Hs. 9216 -272 GGGTCAgggTGAACT (Lobenhofer et al., 2002) -232 GGGTCAgggTGAACG -195 TGGTCAggTGAACT -141 GGGTCAgggTGAACT -6690 TGGCCAggcTGACCT CAV1 Human Hs. 74034 (Charpentier et al., 2000) 2616 GGGTCAgccTGCCCT -3061 GGGCCAgtgTGACCT CNR1 Human Hs. 75110 (Pedram et al., 2002) 2628 AGGTCAggaTGAGCA -8302 AGGTCAtccTGCCCT COMT Human Hs. 240013 (Xie et al., 1999) -1798 TGGCCActgTGACCT -5246 AGGTCAggaTGTCCA COX7A2L Human Hs. 423404 (Watanabe et al., 1998) 381 GGGTCAcctTGACCC -8763 GGGCCAccaTGACCC (Lobenhofer et al., 2002; CTSD Human Hs. 343475 -125 GGGTCAgccCGGCCC Soulez and Parker, 2001) -4324 GGGTCAcgaAGACCA DLG5 Human Hs. 500245 (Lobenhofer et al., 2002) 1303 AGGCCAatcTGACCA -9101 TGGTCAggcTGATCT Human Hs. 96055 (Zhang et al., 2000) -3250 TGGTCAacaTGACCT -7843 AGTTCAgcaTGACCA E2IG4 Human Hs. 8361 (Charpentier et al., 2000) 1377 GGGCCGgccTGACCC -7418 CTGTCAcccTGACCC EDG2 Human Hs. 75794 257 GGGGCGcacTGACCT (Monroe et al., 2003) 1826 GGATCAcctTGACCT

245

Table 33: Multiple EREs (Contd…) Estrogen ERE Sequences Genes Organism UniGene # References Responsive Region 5’ to 3’ 1913 AGGTCCtgtTGACCT EIF5A Human Hs. 310621 (Charpentier et al., 2000) 3296 AGGTCAattTGAGCC -1664 AGGTCAttgTCACCT EPO Human Hs. 2303 (Pedram et al., 2002) 2697 GGGACAggaTGACCT -3472 AGGTTAcagTGACCC FGF2 Human Hs. 284244 (Piotrowicz et al., 2001) 4284 AGGTGGgccTGACCC -59 GGGTCAagcCGGCCC G6PD Human Hs. 80206 (Soulez and Parker, 2001) 4556 AGGTCAtgcTGAGCT -8992 TGGTCAggcTGACCT GAL Human Hs. 278959 (Ormandy et al., 1998) -4905 GGGTCActgGGACCC -8031 AGGTGGcagTGACCC GAPD Human Hs. 169476 -5870 CGGTCCccaTGACCC (Revillion et al., 2000) -738 AGGACAtcgTGACCT -3702 CGGGCAtggTGACCC GCH1 Human Hs. 86724 (Monroe et al., 2003) 3788 AGGTCAggaTCACCA -3049 TGGCCAggcTGACCT GHRHR Human Hs. 767 4741 AGGTCAtgtGGACCA (Monroe et al., 2003) -1619 TGGTCAggcTGGCCT -9517 AGGTCAtcaTGACCT GREB1 Human Hs. 438037 (Ghosh et al., 2000) -1583 GGGTCATttcTGACCT -8553 GGGTCAgtcTGAGCT GTF21 Human Hs. 408507 -6471 AGATCAgccTGACCA (Inoue et al., 2002) -2171 AGCTCAtgtTGACCT -6954 AGGTCAgacCTACCT HMOX2 Human Hs. 284279 (Tschugguel et al., 2001) -2454 GGGTCCcagTGACCT -6710 AGGTCTttgTGACCT HPCAL1 Human Hs. 3618 (Monroe et al., 2003) 4293 AGGTTGctgTGACCC

246

Table 33: Multiple EREs (Contd…) Estrogen ERE Sequences Genes Organism UniGene # References Responsive Region 5’ to 3’ -9233 AGGTCGcagTGAGCC HSPA8 Human Hs. 180414 (Charpentier et al., 2000) -2702 GGCTCActgCGACCT -7227 TGGTCAcgcTGGCCT -6805 GGGCCAttcTGACCC HSPB1 Human Hs. 76067 (Charpentier et al., 2000) 1428 AGGGCAcacTGACCG 4951 GGGTCAggcTGGCCC -7195 AGCTCAgccTGACCC IER3 Human Hs. 76095 (Soulez and Parker, 2001) -5668 TGGGCAaatTGACCT -4124 AGGTCAttgTGACAC (Inoue et al., 2002; Soulez and IGFBP4 Human Hs. 1516 273 CGGTCAtgcTGCCCC Parker, 2001) -2298 GGCTCActgTGACCT IL8RB Human Hs. 846 (Monroe et al., 2003) 3946 TGGTCAgccGGACCC -8211 AAGTCAccaCGACCC INHBB Human Hs. 1735 (Pedram et al., 2002) -3738 AGGCCAgctTGACCC -6253 GGGTCAccaTGGCCC (Lee et al., 1999) -6096 AGGTCAccaTGTCCC (Sanchez et al., 2002) IRS1 Human Hs. 390242 -462 AGGTAAgacTGACCC (Mauro et al., 2001) 1921 GGGTCAgccCCACCT (Oesterreich et al., 2001) -6389 GGCTCActgCGACCT ITPK1 Human Hs. 408429 (Soulez and Parker, 2001) -2279 AGGCCAgccTGACCA -9574 GGCTCActgTGACCA KIAA0153 Human Hs. 82563 2042 AGATCAgccTGACCA (Soulez and Parker, 2001) 4055 TGGTCAcgaTGACCT -9590 AGGTCAggcTGACGA KRT19 Human Hs. 309517 -7850 AGGTCAggaTGGCCC (Choi et al., 2000) 2484 AGGTCTtacTGACCT 2161 GGGCCAatgTGACCC KRT7 Human Hs. 23881 (Seth et al., 2002b) 2659 TGGTCAttgTGAGCC

247

Table 33: Multiple EREs (Contd…) Estrogen ERE Sequences Genes Organism UniGene # References Responsive Region 5’ to 3’ -1074 AGATCAgccTGACCA -265 AGGTCGtccTGACTC LDHA Human Hs. 2795 (Charpentier et al., 2000) 3181 AGGTGGcagTGACCC 4478 TGGTCAataTCACCT -5009 AGGTTGcagTGACCC LOC91012 Human Hs. 458450 (Soulez and Parker, 2001) -882 AGGTCGcagTGAGCC -1166 AGGTGAgtcTGACCA LTF Human Hs. 437457 (Teng et al., 1992) -81 AGATCGcctTGACCT -9749 GGGTCAgacTGGCCC -499 GGGACAagaTGACCT LY6E Human Hs. 77667 (Soulez and Parker, 2001) -298 CGGTCAcccCGACCT 3400 AGGTCAggcCCACCC -5633 AGCTCAaagTGACCC MMP12 Human Hs. 1695 (Monroe et al., 2003) -3556 TGGTCAtctTGACTC -2578 AGGTCTttaTGACCT -801 GGGTCTttgTGACCT MMP2 Human Hs. 367877 (Moalli et al., 2002) 2295 GGGTCTttgTGACCC 3822 AGGTGAtctTGACCA -4618 AGGTCAaggCGTCCC NOS3 Human Hs. 446303 (Kleinert et al., 1998) -1593 CTGTCAcctTGACCC -6833 AGGTCGcttTGACAC NPR2 Human Hs. 78518 -2519 AGGACActgCGACCT (Monroe et al., 2003) 705 GGGTCGctgTGTCCC -1929 GGGTCAgggCGCCCC NR4A3 Human Hs. 279522 (Pedram et al., 2002) 3743 GGGCCGtagTGACCC -156 GAGTCAcagTGACCC (Agarwal et al., 2002) OVGP1 Human Hs. 1154 200 GGGTCCctcTGACCT (Monroe et al., 2003)

248

Table 33: Multiple EREs (Contd…) Estrogen ERE Sequences Genes Organism UniGene # References Responsive Region 5’ to 3’ -2596 AGGTCAtcgTGCCCA OXT Human Hs. 113216 -2465 AGGTCAgccTGACCG (Richard and Zingg, 1990) -166 CGGTGAcctTGACCC -9730 TGGTCAggcTGGCCT (Shimomura et al., 1998) PCNA Human Hs. 78996 -5397 AGGTCAaaaTTACCA (Frasor et al., 2003) -2035 GGCTCActgCGACCT PDE4C Human Hs. 4372111 (Monroe et al., 2003) 460 CGGTCAcacAGACCC -3298 AGGTCAcaaAGACCT PKMYT1 Human Hs. 77783 (Frasor et al., 2003) -722 TGGTCAcctAGACCC -8067 AGGTTGcagTGACCT PRKAR1A Human Hs. 280342 (Lobenhofer et al., 2002) 3744 ATGTCAttgTGACCT 1396 TGGTCGgagTGACCT PRKCB1 Human Hs. 349845 (Monroe et al., 2003) 2434 GGGGCAaagTGACCA -5945 CGGTCTttcTGACCT PTPRU Human Hs. 19718 (Pedram et al., 2002) 3505 TGGCCAgccTGACCT 2869 GGCTCActgCGACCT RAN Human Hs. 10842 (Charpentier et al., 2000) 3007 TGGTCAggcTGATCT -7698 TGGTCAggcTGATCT RFP Human Hs. 440382 -5598 AGGCCAgccTGACCA (Charpentier et al., 2000) -522 AGGTTGcagTGACCC 581 GGGTCAcgtTGGCCA -4605 GAGTCAttgTGACCA SCNN1A Human Hs. 446415 (Soulez and Parker, 2001) -559 AGGTCAgccTCACCC -8602 GGGGCAttcTGACCA SDS Human Hs. 511764 (Monroe et al., 2003) -1020 AGGTGGcccTGACCT -2501 GGGTCAgggTGCCCA SHMT2 Human Hs. 75069 (Inoue et al., 2002) -1575 AGATCAgccTGACCA

249

Table 33: Multiple EREs (Contd…) Estrogen ERE Sequences Genes Organism UniGene # References Responsive Region 5’ to 3’ -9528 AGGTCTttgTGACCT SMC2L1 Human Hs. 119023 (Pedram et al., 2002) -8678 TGCTCAgctTGACCT -8128 GGGTCGtgaTGAGCC SSNA1 Human Hs. 18528 -1829 CGGTCTcccTGACCC (Kang et al., 2001) 3468 AGGTCAttcTGACTG -7499 AGGTCAcagTGACCT SUOX Human Hs. 16340 306 AAGTCAgtcTGACCC (Charpentier et al., 2000) 1718 GGGTCAgggTGAGCT -5663 GGGTCActcTGACCC TERT Human Hs.439911 (Wang et al., 2002) -2687 AGATCAgccTGACCA (Soulez and Parker, 2001; -8655 AGGTCTttaTGACCT TFF3 Human Hs. 82961 Wang et al., 2002) -4315 GAGTCAtcaCGACCT (Gruvberger et al., 2001) -6502 AGGTCAaaaGGACCT TNFRSF8 Human Hs. 1314 (Pedram et al., 2002) -2999 GGGTGActcTGACCC -4705 TTGTCAaatTGACCC TPBG Human Hs. 82128 (Soulez and Parker, 2001) 820 TGGTGAgccTGACCT -7640 GGGTCAtctTGAACA TPX1 Human Hs. 2042 (Monroe et al., 2003) -3900 AGGTCTttaTGACCT 305 GGGCCGcgcTGACCC USF2 Human Hs. 454534 (Seth et al., 2002a) 2061 GGGTCCcccTGACCA -2526 AAGTCGgagTGACCT WISP2 Human Hs. 194679 (Seth et al., 2002a) -452 GGGTCAcacCCACCT 1839 GGGTCActgCAACCT WNT9B Human Hs. 326420 (Kirikoshi et al., 2001) 3736 GGGTTGcacTGACCC -9478 GGCTCAcagCGACCT ZNF230 Human Hs. 193583 (Pedram et al., 2002) 82 GGGTCGcaaCGACCC -6059 AGGTCAtgaTGAGCA ZNF75 Human Hs. 355015 (Pedram et al., 2002) -62 GGGTCAcatTGACAA 250

Table 34: cHEREs Estrogen Responsive ERE Sequences Genes Organism References Region 5’ to 3’ Ha-ras Human Exon 1,-1709 TGACC (Pethe and Shekhar, 1999) (Van Dijck and Verhoeven, Hepatic α2u Protein Human -594 GGTCA 1992) Osteopontin gene Mouse NA* TCAAGGTCA (Vanacker et al., 1999) Progesterone receptor Human 571 TGACC (Petz and Nardulli, 2000) Retinoic acid receptorα-1 (hRARα-1) Human NA GGTCA (Rishi et al., 1995) Transforming growth factorα (hTGFα) Human -216 GGTCA (El-Ashry et al., 1996) Prolactin Rat -1573 GGTCA (Anderson and Gorski, 2000)

Table 35: Multiple HEREs Estrogen Responsive ERE Sequences Genes Organism References Region 5’ to 3’ (Hyder et al., c-fos Mouse NA Cluster of seven GGTCA 1992) (Burbach et Oxytocin Rat NA CCAGGcggTGACCtTGACC al., 1994) Two GGTCA (Kraus et al., Progesterone receptor Rat NA Two TGACC 1994) (Pethe and c-H-ras1 Human 49 to 78 ggttctgGATCAgctggatGGTCAgcgcactctt Shekhar, 1999) tggtcgTGACCAtgaggttatgtttggtatgaaaaGGTCAcattta (Treilleux et ERα (P1 Promoter) Human -892 (N422)cccagGGTCAtcctatg al., 1997) gGGTCA(N )aGGTCA(N )gGGTCA(N )GGTC 159 59 29 (Watanabe et NMDA, 2D subunit Human 3' UTR A(N )gGGTCAggGGTCA(N )TGACCa(N )gGG 7 15 25 al., 1998) TCA(N30)GGTCA gGGTCA(N )aGGTCA(N )gGGTCAtcgaggtct(N (Watanabe et Rat NMDA, 2D subunit Rat NA 99 47 2 3)gGGTCa(N23)gGGTCA(N34)gGGTCA al., 1999) (Zhang and Lactoferrin Human -441 tcaaGGTCAtc Teng, 2000) -736 taaaGGTCA (Vanacker et Osteopontin Mouse -708 tcagGGTCA al., 1999) -571 tccaGGTCA 251

Table 35: Multiple HEREs (Contd…) Estrogen Responsive ERE Sequences Genes Organism References Region 5’ to 3’ Four AGGTCA motifs separated Ovalbumin gene promoter Chicken NA* from each other by more than 100 bps (Kato et al., 1992)

-267 AGGTCA (van de Stolpe et al., Corticotropin releasing hormone Human -682 AGGTCA 2004) -735 AGGTCA -1162 GGTCA -361 GGTCA Galanin gene Human (Kofler et al., 1995) -122 GGTCA

Heat shock protein (HSP27) Human -104 GGGCGG(n)9GGGTCA (Porter et al., 1997) Intron,1516, 1571 TGACC HMG1 Human Intron 168 GGTCA (Borrmann et al., 2001) -149 GGTCA -103 TGACC (Richard and Zingg, Oxytocin Human TGACC 1990) Platelet activating factor receptor AGGTCA (Martini and Human NA (PARF) gene AGGTCA Katzenellenbogen, 2001) Proteinase inhibitor-9 (hPI-9) Human TGACC(n)13TGACC (Krieg et al., 2001) -750 TGACC (Martini and Prothymosin α (hPTα) Human -1051 TGACC Katzenellenbogen, 2001) Na+/H+ exchange regulatory factor Human From -2845 to -19 13 GGTCA sites (Ediger et al., 2002) (hNHE-RF) Mouse Mammary tumor virus RGGTCA (Aumais et al., 1996) Mouse NA (MMTV) RGGTCA *NA, Not available

252

Table 36: Variant Spacer EREn Estrogen Responsive ERE Sequences Genes Organism References Region 5’ to 3’ Na+/H+ exchange regulatory Human -1631 aGGTCATGACCt (Ediger et al., 2002) NMDA, 2D subunit Human 3' UTR gGGTCAggGGTCA (Watanabe et al., 1998) -252 (El-Ashry et al., 1996; Vyhlidal TGFα Human ggGGTCAgctgTGCCCcg et al., 2000) LH B Rat -1173 atGGACAgatggTGTCCcg (Shupnik and Rosenzweig, 1991)

253

254

APPENDIX III

Preparation of Oligonucleosomes from Chicken Erythrocytes

1. Isolation of Erythrocytes from Chicken Blood

About 500 mL of chicken blood was centrifuged at 2000 rpm for 5 minutes (GSA rotor)

to separate the plasma for the cells. The cells were then washed five times with isolation buffer A

(0.85% NaCl, 0.01% methiolate, 6 mM Na2HPO4 pH 7.0) and pelleted at 2000 rpm for 5

minutes. The supernatant was then discarded. About 250 mL of suspended cells were recovered

at the end of the centrifugation. Aliquots of 10 mL were suspended with 10% glycerol and stored

at -80oC. All work with the isolations was performed at 4oC.

2. Isolation of Nuclei from Chicken Erythrocytes

About 15 mL of cell suspension was thawed to 4oC and homogenized in a glass

homogenizer with Teflon pestle for 30 seconds at 4oC in 25 mL of RSB buffer (reticulocyte

standard buffer) 10 mM Tris-HCl pH 7.2, 10 mM NaCl, 3 mM MgCl2 containing 0.5% IGEPAL,

nonionic detergent to lyse the cell membrane. The solution was made 0.2 mM PMSF (protease

inhibitor) from 0.2 M stock in isopropanol immediately before use. The resulting suspension was

centrifuged at 5000 rpm SS34 rotor for 5 minutes at 4oC to pellet the nuclei. The nuclei were washed with RSB buffer containing PMSF and IGEPAL by suspending in a total volume of 25 mL. The subsequent washes were without the detergent and contained only buffer/PMSF. The nuclei were washed twice with 20 mL of RSB buffer and centrifuged at 5000 rpm and the supernatant discarded. The cells were finally washed with RSB alone and the final nuclei pellet was resuspended in a total of 5 mL with RSB buffer. The final concentration in terms of DNA was determined by measuring the A260 of nuclei dissolved in 2 M NaCl / 5 M urea, with the same solution as the blank. Double stranded DNA concentration was calculated by assuming 1 O.D. at 255

260 nm was equivalent to 50 µg DNA per mL. The preparation yields 5 mL of nuclei suspension

and a DNA concentration of 15,000-16500 µg DNA per mL (300-330 O.D. units).

3. Micrococcal Nuclease

Micrococcal nuclease was purchased from Worthington. The protein was 74% purified and contained 25,986 U/mg protein (15000 units total).

4. Proteinase K

Proteinase K was from Tritirachium album. A unit will hydrolyze urea denatured hemoglobin to produce color equivalent to 1.0 micromole (181 µg) of tyrosine per minute at pH

7.5 at 37oC.

5. Preparation of Samples for Micrococcal Nuclease Digestion.

The total volume of 3 mL of nuclei were used in the digestion in which the concentration of DNA was adjusted to 5000 µg/mL (100 O.D) and digested with 75 units/mL micrococcal nuclease at 37oC.

Table 37. Table for volumes and concentrations for MNase digestion

Initial Volume measured Final concentration (µL) concentration DNA 14,000 µg/mL 1072 µL 5000 µg/mL CaCl2 100 mM 30 µL 1 mM RSB 1893 µL Micrococcal 15 U/ µL 5 µL 75 units nuclease The reaction was stopped by the addition of cold 1/10 volume of 100 mM EDTA pH 8.0.

6. Monitoring Micrococcal Nuclease Digestion by % Acid Solubility Produced

The percent acid soluble product was used to measure the extent of DNA digestion in

chromatin. There is a direct correlation between the percent soluble product and the size of DNA

cut by MNase. When the percent acid solubility reaches above 30%, the predominant size of 256

DNA (mononucleosomes) is about 200bp (Jeff Hayes, MS thesis). The concentration of nuclei

was adjusted to 5000 µg/mL (100 O.D) in a total volume of 3 mL. This was then treated with 75

units/mL micrococcal nuclease. About 200 µL was sampled at various times (10-60 minutes) and added to 4.8 mL of ice cold 1M NaCl/1M HClO4 while vortexing. The mixture was centrifuged

0 at 27,000 x g for 10 min at 4 C and the A260 of the supernatant checked for acid soluble DNA.

The % acid solubility was calculated as:

%AS = A260 supernatant x dilution factor x100

A260 original digest x 1.4 hyperchromicity factor

7. DNA Extraction Following Micrococcal Nuclease Digestion.

DNA was phenol extracted from the nuclease digestion by to determine the average DNA

lengths that were cut by the MNase. During the above digestion, at various times (10-60 minutes), 200 µL samples were taken and 20 µL of 10 mM EDTA was added to stop the reaction. The sample was then treated with 20 µL of 10X Protease K digestion buffer (10X 10 mM Tris-HCl pH 7.5, 0.5 % SDS, 1 mM EDTA pH 8.0) and 10 µL Proteinase K (stock concentration of 23 mg/mL) at a final concentration of 1 mg/mL. The solution was incubated for

3 hours at 37oC.

The samples were extracted with an equivalent volume of phenol:chloroform:isoamyl

alcohol (50:24:1), using phenol that was equilibrated with 0.1 M Tris-HCl pH 7.5. The tube

contents were vortexed to an emulsion, and the phases separated in the microfuge. The upper,

aqueous phase was removed while the protein interface was left intact and discarded with the

lower organic phase on the second extraction. This extraction was repeated and the aqueous

phase was then extracted 2 times in a similar manner with an equal volume of

chloroform:isoamyl alcohol (24:1). The sample was made 0.25 M sodium acetate, 0.01 M MgCl2 257

by addition of 1/10 volume of a 10X stock solution (Jeff Hayes, MS thesis). Exactly 2 volumes

of 95% (-20oC) ethanol was added, the samples vortexed and the DNA precipitated overnight at -

20oC. The DNA was centrifuged (12,000 x g, 30min) and the supernatant carefully removed.

The pellets were then washed twice by vortexing with 70% ethanol at 4 0C and then air dried.

The pellet was then dissolved in TE buffer. The A260 was measured and the DNA concentration

was adjusted to 0.5-1.0 µg/mL. The DNA fragments were then separated by electrophoresis on native 4% polyacrylamide gels at 170V for 1.45 hours and the gel stained with ethidium bromide

(1 µg/mL) in TBE. The stained gels were viewed under UV irradiation and photographed using the Foto/Convertible Dual Transilluminator setup in the Department of Biological Science, 5th

floor.

8. Sucrose Gradient Centrifugation

Another strategy to determine the extent of digestion by micrococcal nuclease was to fractionate

the digestion products by sucrose gradient centrifugation. Five to thirty per cent sucrose gradient

(5 mL) was prepared in gradient buffer (10 mM Tris-HCl pH 7.5, 1 mM EDTA, 0.2 mM PMSF)

with a gradient maker. A total of 5mL were poured into centrifuge tubes for SWTi55. These

were then left in the refrigerator at 4 0C to diffuse to linearity for about 8-10 hours (Jeff Hayes,

MS thesis). About 250 µg of the micrococcal nuclease digestion (50 µL from 5000 µg DNA per

mL) was loaded carefully on to the top of the gradient. The samples were centrifuged at 38,000

rpm for 11 hours at 4oC in a SW55Ti rotor. Fractions of about 250 µL were collected (with the

ISCO Density Gradient Fractionator) by poking the bottom of the tube and a 50% sucrose

solution in gradient buffer (10 mM Tris-HCl pH 7.5, 1 mM EDTA, 0.2 mM PMSF) to push up

the gradient fractions. The flow rate was 0.75 mL/min. The samples were then extracted with

phenol:chloroform and precipitated in 95% ethanol. The precipitate was washed twice with 75% 258

ethanol and then air dried. The DNA was dissolved in TE buffer and run on 4% polyacrylamide gel.

9. Preparation of Oligonucleosomes as a Source of Histone Proteins.

9.1. Isolation of H1-free oligonucleosomes

Nuclei were isolated as above except that additional washes were made with a buffer

B (15 mM Tris-HCl, pH 7.5; 15 mM NaCl; 15 mM 2-mercaptoethanol; 60 mM KCl; 0.5 mM

spermidine; 0.15 mM spermine) containing 0.34 M sucrose.The concentration of nuclei (5

mL total), was adjusted to 5000 µg DNA / mL and 50 µL of 100 mM CaCl2 was added to a

final concentration of 1 mM. The solution was incubated at 37oC and then 75 U/mL

micrococcal nuclease was added, followed by 4 min incubation at 37oC. The 4 minutes was

used because, preliminary runs indicated that four minutes was sufficient to produce

oligonuleosome. The reaction is stopped by adding 100 mM EDTA pH 7.5, to a final

concentration of 2.2 mM. The nuclear suspension is then centrifuged at 4000 x g for 5 min at

4oC. The supernatant is discarded and the nuclear pellet is suspended in 5 mL of 0.2 mM

EDTA. Then 810 µL of 4 M NaCl was added slowly while vortexing to make a final

concentration of 0.65 M NaCl. The suspension was homogenized, and re-centrifuged for 5

min at 4000 x g at 4oC. At this step, the nuclei lyses. The pellet formed after centrifugation is

viscous and is discarded. The supernatant (5 mL) that contains soluble donor chromatin was

transferred into a fresh tube. It is important to keep the NaCl concentration at 650 mM in

order to dissociate histone H1. The core histones remain bound to the DNA at this NaCl

concentration.

259

9.2. Sepharose CL-4B Column preparation

Sepharose CL-4B (Sigma cat # 035K1722) was washed three times with a buffer

containing 650 mM NaCl, 5 mM Tris-HCl pH 7.5 to get rid of the fines. The slurry was then

degassed for 30 minutes. The column was then poured (2.5 cm in diameter and 30 cm in

length) making sure that there are no breaks or bubbles in the tube. This was then washed

with equilibrating buffer (650 mM NaCl, 5 mM Tris-HCl pH 7.5, in addition to 0.2 mM 2- mercaptoethanol added just before use). The clear supernatant (4 mL of about 50 OD) was applied onto a 200 mL Sepharose CL-4B column. The column was eluted with a flow rate of about ten drops per minute, for 90 drops per fraction. About 2.5 mL fractions were collected per tube. The DNA concentration was determined to be 115 µg/mL. The fractions that

contained oligonucleosomes were pulled together and 200 µL aliquots stored at -80oC.

9.3. Sepharose Column Chromatography

Sepharose column (gel permeation) chromatography separates molecules according to

size. This method is used to separate H1 and non histone protein and oligonucleosomes which are used as a source of chromatin donor (Wrange, 1998). At high salt concentration of

650 mM, histone H1 dissociates from the rest of the core histones. Long oligonucleosomes, because of the large size, are eluded first at that high salt concentration of 0.65 M with the core histone proteins intact. The presence of histone proteins in the fractions was checked by

18% SDS-PAGE.

260

Figure 54. Core histones in nucleosomes: An 18% SDS-PAGE, stained with Coomassie blue, showing core-histones from nucleosome fraction collected from the sepharose CL-4B column which were prepared by micrococcal nuclease digestion of chicken erythrocytes: Lane 1: Core histones, Lane 2: Protein molecular weight marker.

261

APPENDIX IV

Preparation of Tailless Oligonucleosomes

Nuclei were isolated from chicken erythrocytes. About 500 mL of chicken blood was centrifuged at 2000 rpm for 5 minutes (GSA rotor) to separate the plasma for the cells. The cells were then washed five times with isolation buffer A (0.85% NaCl, 0.01% methiolate, 6 mM

Na2HPO4 pH 7.0) and pelleted at 2000 rpm for 5 minutes. Aliquots of 10 mL were suspended with 10% glycerol and stored at -80oC. About 15 mL of cell suspension was thawed to 4oC and homogenized in a glass homogenizer with Teflon pestle for 30 seconds in 25 mL of RSB buffer

(reticulocyte standard buffer) 10 mM Tris-HCl pH 7.2, 10 mM NaCl, 3 mM MgCl2 containing

0.5% IGEPAL, nonionic detergent to lyse the cell membrane. The solution was made 0.2 mM

PMSF from 0.2 M stock in isopropanol immediately before use. The resulting suspension was centrifuged at 5000 rpm SS34 rotor for 5 minutes at 4oC to pellet the nuclei. The nuclei were washed with RSB buffer containing PMSF and IGEPAL by suspending in a total volume of 25 mL. The subsequent washes were without the detergent and contained only buffer/PMSF. The nuclei were washed twice with 20 mL of RSB buffer and centrifuged at 5000 rpm and the supernatant discarded. The cells were finally washed with buffer B (15 mM Tris-HCl, pH 7.5; 15 mM NaCl; 15 mM 2-mercaptoethanol; 60 mM KCl; 0.5 mM spermidine; 0.15 mM spermine) containing 0.34 M sucrose. The concentration of nuclei (5 mL total), was adjusted to 5000 µg

DNA/mL and 50 µL of 100 mM CaCl2 was added to a final concentration of 1 mM. The solution was incubated at 37oC and then 75 U/mL micrococcal nuclease was added, followed by 4 min incubation at 37oC. The reaction is stopped by adding 100 mM EDTA pH 7.5, to a final concentration of 2.2 mM. The nuclear suspension is then centrifuged at 4000 x g for 5 min at

4oC. The supernatant is discarded and the nuclear pellet is suspended in 5 mL of 0.2 mM EDTA. 262

Then 810 µL of 4 M NaCl was added slowly while vortexing to make a final concentration of

650 mM NaCl. The suspension was homogenized, and re-centrifuged for 5 min at 4000 x g at

4oC. At this step, the nuclei lyses. The pellet formed after centrifugation is viscous and is discarded and the supernatant containing the chromatin is saved. It is important to keep the NaCl concentration at 650 mM in order to dissociate histone H1. The core histones remain bound to the DNA at this NaCl concentration.

The supernatant was transferred onto a Sepharose CL-4B column equilibrated with a buffer containing 650 mM NaCl, 5 mM Tris-HCl pH 7.5, with 0.2 mM 2-mercaptoethanol added just before use. The column was eluted with a flow rate of about ten drops per minute, for 90 drops per fraction. About 2.5 mL fractions were collected per tube. An SDS gel was run to determine the purity of the histones.

The chromatin depleted of H1(1 mg/mL) was treated with 0.5 µg/mL trypsin, for 30 min at 37oC and the reaction stopped by adding a trypsin inhibitor (Sigma, T9003) to a final

concentration of 10 µg/mL. The cleaved tails and the inhibitor were separated from the rest of

the chromatin with Sepharose CL-4B. A gel of the tailless histones was run with core histones to

test the purity of the histone proteins, and stained with Coomassie blue.

263

APPENDIX V

MEDIA AND BUFFERS

LB (Luria-Bertani) Broth (Per Liter) Tryptone……………………….. 10g Yeast Extract…………………... 5g NaCl…………………………… 5g Adjust pH to 7.5 with NaOH

LB (Luria-Bertani) Agar (Per Liter) Tryptone……………………….. 10g Yeast Extract…………………... 5g NaCl…………………………… 5g Agar……………………………. 15g Adjust pH to 7.5 with NaOH

Antibiotics were added after cooling the media to ~ 55oC

(Typically antibiotic was added to 50-100μg/mL final concentration in the media for plasmid selection)

SOC Media (Per Liter) Tryptone……………………….. 20g Yeast Extract…………………... 5g 1M NaCl……………………….. 10mL 1M KCl………………………... 2.5mL

1M MgCl2……………………… 10mL

1M MgSO4…………………….. 10mL 1M Glucose……………………. 20mL (The final concentration of the components was: 2% Trptone, 0.5% yeast extract, 10mM

NaCl, 2.5mM KCl, 10mM MgCl2, 10mM MgSO4 and 10mM Glucose)

264

Buffers for Western Blot 10X Running Buffer Tris Base 30.3 g. f.c* [250 mM, pH 8.3] Glycine 144.2 g. f.c [1.92 M] dH2O 1.0 L Store at 4°C

1X Running Buffer 10X Running Buffer 100 mL dH2O 890 mL 10% SDS 10 mL f.c[0.1%]

1X Transfer Buffer 10X Running Buffer 100mL [Tris Base 25 mM, pH 8.3, Glycine, 192mM] Methanol 200mL [20% Methanol] dH2O 700mL

10X TBS 1M Tris (pH 7.5) 500 mL f.c. [0.5 M] NaCl 87.66 g f.c. [1.5 M] Bring volume to 1L with sterile distilled water Store at 4°C

1X TBST 10X TBS 100 mL Tween 20 1mL f.c [0.1%] dH2O 898 mL Store at 4°C

* f.c., final concentration.

265

Buffer used in cell culture

10 X PBS (Phosphate Buffered Saline) NaCl……………….. 80 g KCl……………….. 2 g

Na2HPO4………… 14.4 g

KH2PO4………….. 2.4 g Adjust pH of PBS Buffer Solution to 7.4 with HCl. Bring volume to 1 liter, autoclave or sterilize by filtration.

266

APPENDIX VI Sequences for pGL2-3cERE-TATA-Inr-Luc

The green highlight shows the three tandem cEREs, the yellow highlight shows the TATA rich sequence in the TATA box, and the purple highlight shows the Inr sequence. The orange highlight is Xho I restriction site and the blue highlight is Hind II restriction site. The underlined sequence shows the sites for forward and reveres primer to amplify the sequences within Xho I and Hind III sites.

CCCGGGAGGTACCGAGCTCTTACGCGTGCTAGCTCGAGATCTAGGTCACAGTGACCTGCGGATC CGCAGGTCACTGTGACCTAGATCCGCAGGTCACTGTGACCTAGATCTGATATCATCGATGAATT CGGGCTATAAAAGGGGGTGGGGGGAGCTCGGCCCTCATTCTGGAGACGGATCCTCTAGAGTCG ACCTGCAGGCATGCAAGCTTGGCATTCCGGTACTGTTGGTAAAATGGAAGACGCCAAAAACATA AAGAAAGGCCCGGCGCCATTCTATCCTCTAGAGGATGGAACCGCTGGAGAGCAACTGCATAAGG CTATGAAGAGATACGCCCTGGTTCCTGGAACAATTGCTTTTACAGATGCACATATCGAGGTGAA CATCACGTACGCGGAATACTTCGAAATGTCCGTTCGGTTGGCAGAAGCTATGAAACGATATGGG CTGAATACAAATCACAGAATCGTCGTATGCAGTGAAAACTCTCTTCAATTCTTTATGCCGGTGTT GGGCGCGTTATTTATCGGAGTTGCAGTTGCGCCCGCGAACGACATTTATAATGAACGTGAATTG CTCAACAGTATGAACATTTCGCAGCCTACCGTAGTGTTTGTTTCCAAAAAGGGGTTGCAAAAAAT TTTGAACGTGCAAAAAAAATTACCAATAATCCAGAAAATTATTATCATGGATTCTAAAACGGATT ACCAGGGATTTCAGTCGATGTACACGTTCGTCACATCTCATCTACCTCCCGGTTTTAATGAATAC GATTTTGTACCAGAGTCCTTTGATCGTGACAAAACAATTGCACTGATAATGAATTCCTCTGGATC TACTGGGTTACCTAAGGGTGTGGCCCTTCCGCATAGAACTGCCTGCGTCAGATTCTCGCATGCC AGAGATCCTATTTTTGGCAATCAAATCATTCCGGATACTGCGATTTTAAGTGTTGTTCCATTCCA TCACGGTTTTGGAATGTTTACTACACTCGGATATTTGATATGTGGATTTCGAGTCGTCTTAATGT ATAGATTTGAAGAAGAGCTGTTTTTACGATCCCTTCAGGATTACAAAATTCAAAGTGCGTTGCTA GTACCAACCCTATTTTCATTCTTCGCCAAAAGCACTCTGATTGACAAATACGATTTATCTAATTTA CACGAAATTGCTTCTGGGGGCGCACCTCTTTCGAAAGAAGTCGGGGAAGCGGTTGCAAAACGCT TCCATCTTCCAGGGATACGACAAGGATATGGGCTCACTGAGACTACATCAGCTATTCTGATTACA CCCGAGGGGGATGATAAACCGGGCGCGGTCGGTAAAGTTGTTCCATTTTTTGAAGCGAAGGTTG TGGATCTGGATACCGGGAAAACGCTGGGCGTTAATCAGAGAGGCGAATTATGTGTCAGAGGACC TATGATTATGTCCGGTTATGTAAACAATCCGGAAGCGACCAACGCCTTGATTGACAAGGATGGA TGGCTACATTCTGGAGACATAGCTTACTGGGACGAAGACGAACACTTCTTCATAGTTGACCGCTT GAAGTCTTTAATTAAATACAAAGGATATCAGGTGGCCCCCGCTGAATTGGAATCGATATTGTTAC AACACCCCAACATCTTCGACGCGGGCGTGGCAGGTCTTCCCGACGATGACGCCGGTGAACTTCC CGCCGCCGTTGTTGTTTTGGAGCACGGAAAGACGATGACGGAAAAAGAGATCGTGGATTACGTG GCCAGTCAAGTAACAACCGCGAAAAAGTTGCGCGGAGGAGTTGTGTTTGTGGACGAAGTACCGA AAGGTCTTACCGGAAAACTCGACGCAAGAAAAATCAGAGAGATCCTCATAAAGGCCAAGAAGGG CGGAAAGTCCAAATTGTAAAATGTAACTGTATTCAGCGATGACGAAATTCTTAGCTATTGTAATA CTGCGATGAGTGGCAGGGCGGGGCGTAATTTTTTTAAGGCAGTTATTGGTGCCCTTAAACGCCT GGTGCTACGCCTGAATAAGTGATAATAAGCGGATGAATGGCAGAAATTCGCCGGATCTTTGTGA AGGAACCTTACTTCTGTGGTGTGACATAATTGGACAAACTACCTACAGAGATTTAAAGCTCTAAG GTAAATATAAAATTTTTAAGTGTATAATGTGTTAAACTACTGATTCTAATTGTTTGTGTATTTTAG 267

ATTCCAACCTATGGAACTGATGAATGGGAGCAGTGGTGGAATGCCTTTAATGAGGAAAACCTGT TTTGCTCAGAAGAAATGCCATCTAGTGATGATGAGGCTACTGCTGACTCTCAACATTCTACTCCT CCAAAAAAGAAGAGAAAGGTAGAAGACCCCAAGGACTTTCCTTCAGAATTGCTAAGTTTTTTGA GTCATGCTGTGTTTAGTAATAGAACTCTTGCTTGCTTTGCTATTTACACCACAAAGGAAAAAGCT GCACTGCTATACAAGAAAATTATGGAAAAATATTCTGTAACCTTTATAAGTAGGCATAACAGTTA TAATCATAACATACTGTTTTTTCTTACTCCACACAGGCATAGAGTGTCTGCTATTAATAACTATGC TCAAAAATTGTGTACCTTTAGCTTTTTAATTTGTAAAGGGGTTAATAAGGAATATTTGATGTATA GTGCCTTGACTAGAGATCATAATCAGCCATACCACATTTGTAGAGGTTTTACTTGCTTTAAAAAA CCTCCCACACCTCCCCCTGAACCTGAAACATAAAATGAATGCAATTGTTGTTGTTAACTTGTTTA TTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTT TCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGGATCCGTCG ACCGATGCCCTTGAGAGCCTTCAACCCAGTCAGCTCCTTCCGGTGGGCGCGGGGCATGACTATC GTCGCCGCACTTATGACTGTCTTCTTTATCATGCAACTCGTAGGACAGGTGCCGGCAGCGCTCTT CCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCA CTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCA AAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCC GCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACT ATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCG CTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCAATGCTCACGCTG TAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTT CAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACT TATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTAC AGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCT CTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCG CTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGA AGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTT TGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAA TCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACC TATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTA CGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACC GGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCA ACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGT TAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTA TGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAA AAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCAC TCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTG ACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCC CGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAA ACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCC ACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAAC AGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTC TTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAA TGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACG CGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACAC TTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGC TTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCT CGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTT TTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAAC ACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTT 268

AAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGTTTACAATTT CCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATT ACGCCAGCCCAAGCTACCATGATAAGTAAGTAATATTAAGGTACGTGGAGGTTTTACTTGCTTTA AAAAACCTCCCACACCTCCCCCTGAACCTGAAACATAAAATGAATGCAATTGTTGTTGTTAACTT GTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCAT TTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATGGTACTGTAACTG AGCTAACATAA

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APPENDIX VII Table 38: Calculations of Nucleosome Reconstitution Buffer Compositions

No. of moles = concentration x volume

No. of moles [moles] Solutions Volume Tris- NaCl EDTA PMSF* HCl TE buffer (10 mM Tris-HCl, pH -7 -8 10 µL - 1x10 1x10 - 8.0, 1 mM EDTA) 5 M NaCl 8 µL 4 x 10-5 - - - 10X RC buffer (150 mM Tris-HCl pH 7.5, 2 mM 4 µL - 6x10-7 8x10-9 8x10-9 EDTA, 2 mM PMSF) Sepharose column buffer 650 mM NaCl, 5 mM Tris-HCl pH 28 µL 2x10-5 1x10-7 - - 7.5 Total 40 µL* 6x10-5 8x10-7 1.8x10-8 8x10-9 * DNA in TE buffer was lyophilized; therefore, the volume is dried while the ingredients still remain.

Therefore, the final concentrations are:

Volume Concentrations Solutions NaCl Tris- EDTA PMSF* HCl Final buffer compositions 40 µL 2M 20 mM 0.5 mM 0.2 mM

A total of 270 µL of 1X RC buffer was added during a step wise dilution.

No. of moles [moles] Solutions Volume Tris- NaCl EDTA PMSF* HCl Total 40 µL 6x10-5 8x10-7 1.8x10-8 8x10-9 1X RC buffer (15 mM Tris-HCl pH 7.5, 0.2 mM EDTA, 0.2 mM PMSF) 270 µL - 4x10-6 5x10-8 5x10-8

Final Total 310 µL 6x10-5 5x10-6 6.8x10-8 5.8x10-8

The final concentration of buffer in the reconstituted nucleosomes is:

Volume Concentrations Solutions NaCl Tris- EDTA PMSF* HCl Final buffer compositions 310 µL 200 mM 20 mM 0.2 mM 0.2 mM *Original concentration. 270

Table 39: Final concentration of each component of the reaction buffer when remodeled nucleosomes were treated with different buffers

Buffer Components Final Concen. Effect TE/sucrose (18%) buffer Tris-HCl 10 mM EDTA 1 mM N’/N’ (10 mM Tris-HCl pH 8, 1 mM EDTA, No Change 18% Sucrose) Sucrose 18% 1X ER dilution buffer Tris-HCl 10 mM KCL 40 mM (80 mM KCl, 10% glycerol, 15 mM Tris- EDTA 0.6 mM HCl pH 8.0, 0.2 mM EDTA, 0.4 mM DTT 0.2 mM N/N’’N DTT, 100 ng/µL BSA, 2 ng/µL poly (dI- poly (dI-dC) 1 ng/µL dC)) Sucrose 9% Glycerol 5% 10X DNase I buffer Tris-HCl 20 mM MgCl2 2.5 mM N’/N’ CaCl 0.5 mM (100 mM Tris-HCl, pH 7.5, 25 mM 2 No Change MgCl2, 5 mM CaCl2) EDTA 0.9 mM Sucrose 16.2% 10X Exo III buffer Tris-HCl 9 mM Tris-Ac 33 mM KAc 66 mM (330 mM Tris-acetate, pH 7.8, 660 mM MgAc 10 mM N’’N KAc, 100 mM MgAc, 5 mM DTT) EDTA 0.9 mM DTT 0.5 mM Sucrose 16.2% 10X Exo III buffer (Fisher Scientific) Tris-HCl 80 mM MgCl 0.6 mM 2 N’’N (660 mM Tris-HCl, pH 8, 6 mM MgCl2) EDTA 0.9 mM Sucrose 16.2% Note: The reaction was performed at 1X final concentration of all the buffers except for ER dilution buffer, which was at 0.5X. The final reaction volume was 10 µL.

APPENDIX VIII

Detail map of DNase I 10bp pattern on nucleosome (N) + 1600 nM HMGB1

Figure 55. Detail map of DNase I 10bp pattern on nucleosome (N) + 1600 nM HMGB1. A schematic 161bp 2G2 DNA fragment

showing 32P labeling at EcoR I end and central 71bp DNA expanded with individual nucleotides with 10bp pattern ( ), 17 additional

DNase I sensitive sites ( ) when nucleosomes (n2G2) is treated with 1600 nM HMGB1. ( • ) represent the dyad axis, and the bar

( ) represents the GRE site. The pink shaded nucleotides represent AT-rich region with the minor groove toward the histones and the major groove directed the outside. Green shaded nucleotides are GC-rich region with the major groove toward the histone core.

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