ABSTRACT

SIMMONS, STEVEN O’NEAL. Biochemical and Functional Analysis of Homeoprotein

Nkx3.1. (Under the direction of Jonathan M. Horowitz.)

Nkx3.1 is a homeodomain-containing that plays an important role

in the development and differentiation of prostatic epithelia. Loss of Nkx3.1

expression is often an early event in prostate tumorigenesis, and the abundance of Nkx3.1-

negative epithelial cells increases with disease progression. Herein I report that Nkx3.1

collaborates with Sp-family members in the regulation of prostate specific antigen (PSA) in

prostate-derived cells. Nkx3.1 forms protein complexes with Sp dependent on their

respective DNA-binding domains and an amino-terminal segment of Nkx3.1, and negatively

regulates Sp-mediated transcription via Trichostatin A-sensitive and –insensitive

mechanisms. Nkx3.1 DNA-binding activity is not required for trans-repression of Sp

proteins, suggesting that Nkx3.1 regulates Sp-mediated transcription via direct

protein/protein interactions.

I report that Nkx3.1 homeodomain encodes at least one nuclear localization signal

(NLS) as well as sequences that facilitate the association of Nkx3.1 with the nuclear matrix. I

show further that a functionally intact homeodomain is not required for nuclear localization

but is required for the association of Nkx3.1 with the nuclear matrix. In contrast to many transcription factors, I show that Nkx3.1 is associated with mitotic chromatin throughout most, if not all, of mitosis and that a functionally intact Nkx3.1 homeodomain is sufficient to facilitate inclusion within mitotic .

Finally, I report my efforts to identify Nkx3.1 target using a genome-wide

approach. This genome-wide screen for putative Nkx3.1 target genes yielded 42 clones containing novel, human genomic DNA. Ten of these clones harbored unique genomic fragments, while the remaining 32 clones carried sequences that were isolated repeatedly and could be subdivided into three sequence classes. Many of the recovered sequences mapped to locations that are within or near known genes and most carried one or more consensus

Nkx3.1 DNA-binding sites. Further work must be performed to corroborate that the results from this genome-wide screen represent in vivo Nkx3.1 targets. Biochemical and Functional Analysis of Homeoprotein Nkx3.1

by

Steven O. Simmons

A dissertation submitted to the Graduate Faculty of

North Carolina State University

In partial fulfillment of the

Requirements for the degree of

Doctor of Philosophy

Toxicology

Raleigh, NC

2006

Approved by:

Jonathan M. Horowitz

Robert C. Smart

James Mahaffey

Gregg Dean

BIOGRAPHY

Steven O. Simmons born August 5, 1974 Orange, Texas

Education:

1999-Present North Carolina State University, Raleigh, North Carolina Department of Toxicology Laboratory of Jonathan M. Horowitz, Ph.D. Program in Molecular and Cellular Toxicology

1992-1997 Lamar University, Beaumont, Texas B.S. Biology, 1997

Publications:

Spengler M.L., Kennett S.B., Moorefield K.S., Simmons S.O., Brattain M.G., Horowitz J.M. (2005). Sumoylation of internally initiated Sp3 isoforms regulates transcriptional repression via a Trichostatin A-insensitive mechanism. Cell Signal. 17, 153-166.

Simmons S.O., Horowitz J.M. (2006). Nkx3.1 binds and negatively regulates the transcriptional activity of Sp-family members in prostate-derived cells. Biochem. J. 393, 397-409.

Moorefield K.S., Yin H., Nichols T.D., Cathcart C., Simmons S.O., Horowitz J.M. (2006). Sp2 localizes to subnuclear foci associated with the nuclear matrix. Mol. Biol. Cell. 17, 1711-1722.

ii ACKNOWLEDGMENTS

I wish to acknowledge all of the individuals without whom I could not have completed this entire process. First and foremost I wish to thank my wife, Deidra, for her tireless work to keep the bills paid and food on the table in addition to her support as a loving wife. I would not have survived six years of graduate school without her constant encouragement and unconditional love. She has in every sense earned this honor more than me.

I would like to also acknowledge the support of my son, Tanner, who more than anyone on earth has helped to realize what is truly important in life. My son loves me with that special unconditional love that only a son has for his dad, and that love has further transformed my life. Tanner has taught me more in two short years about the nature of God and of life itself than I will ever be able to teach him in ten lifetimes, and for that I am eternally indebted.

I wish to thank my parents Steven Sr. and Kathyrn for their love, encouragement and support over the past six years. Having the opportunity to make my parents proud has no doubt been an additional motivation for me to finish graduate school. I wish my father, who had invested his whole life into me, was alive today to see that investment come to fruition.

My mother provided me with a personal example of achievement, and taught me that I could be anything in spite of whatever circumstances in which I found myself.

I also need to acknowledge the support of my mother- and father-in-law, Carolyn and

Damon. Carolyn made countless trips to Raleigh during our six plus years here, and that made home seem a lot closer for my family, especially for my son who loves his

iii grandparents so much. My father-in-law was instrumental in leading me to Christ before I

matriculated to graduate school and has served a spiritual anchor in my life ever since.

I wish to extend a special acknowledgement to my advisor, Dr. Jonathan Horowitz.

Jon took a big risk in bringing me into his laboratory since I did not have the best credentials upon arriving at NCSU. Despite my background, Jon provided me with an intellectually rigorous environment that challenged me daily, even through these final days. Jon’s superior grantsmanship insured that funding interruptions never occurred in my six years in his laboratory, no small feat in today’s academic climate. Jon gave my project much needed direction when all hope seemed lost. He also challenged me to excel beyond graduate school, and I intend to work diligently beyond his tutelage to honor his enormous investment in my life.

I would like to thank Dr. Robert Smart, who provided me with much needed advice

and encouragement along the way. Dr. Smart was extremely helpful in seeking and finding

the right postdoctoral appointment to further my career, and I could not have completed this

passage without his support.

Additionally I would like to thank my committee members Drs. James Mahaffey and

Gregg Dean who have made themselves very available to me. Their participation on my

graduate committee is much appreciated.

I would also like to extend a special thanks to my fellow members of the Horowitz

laboratory throughout these years, especially Dr. Scott Moorefield who took virtually every

step of this journey with me. All of you provided invaluable fellowship and advice throughout my tenure in the Horowitz laboratory and served as a well of encouragement and support. All of you made coming to work everyday fun for me.

iv Most importantly, I wish to acknowledge my personal Lord and Savior, Jesus Christ.

He not only took my place on Calvary’s cross to pay my sin debt and enable me to fellowship

eternally with God, but He also is the central force of my life. Christ renews my mind and replenishes my spirit daily and teaches me to keep a heavenly, not earthly perspective. Lord,

forgive me for not making the absolute most of my opportunity here at NCSU, for not always

serving as a lantern reflecting Your love to those whom I could influence, for not living

perfectly as You do, for not being worthy of Your mercy or Your grace. I thank you, Lord,

for these individuals whom you placed in my path to help me through this stage of my life. I

pray that you would richly bless them for having invested so much of their time, their talent

and their treasure into one of your humble servants. In Your holy name, Amen.

v TABLE OF CONTENTS

List of Tables ...... x

List of Figures...... xi

List of Abbreviations ...... xiv

Chapter I: Nkx3.1 and Prostate Cancer ...... 1

1.1 Prostate cancer ...... 2

1.2 Prostate anatomy and tumor classification ...... 3

1.3 Prostate cancer initiation and progression ...... 7

1.4 Genes involved in hereditary prostate cancers...... 9

1.5 Genes involved in sporadic prostate cancers ...... 14

1.6 Cloning and characterization of Nkx3.1 ...... 25

1.7 Nkx3.1: Roles in development...... 29

1.8 Functional analysis of Nkx3.1 ...... 36

1.9 Nkx3.1 and prostate cancer...... 44

1.10 Functional interactions between homeodomain proteins and zinc-“finger” proteins...... 58

1.11 Conclusions and perspectives ...... 62

Chapter II: Materials and Methods ...... 63

2.1 Cell culture...... 64

2.2 Plasmids ...... 64

2.3 Transient transfections...... 74

2.4 Luciferase assays ...... 75

2.5 Chloramphenicol acetyltransferase (CAT) assays...... 75

vi 2.6 Expression and purification of GST-fusion proteins ...... 76

2.7 Cell extracts ...... 78

2.8 Column filtration chromatography ...... 79

2.9 Protein/DNA-binding assays ...... 80

2.10 In vitro transcription/translation and protein/protein-binding assays ...... 82

2.11 Antibodies...... 83

2.12 Western blotting...... 84

2.13 DNA sequencing...... 85

2.14 Indirect immunofluorescence...... 86

2.15 Chromatin immunoprecipitation...... 88

Chapter III: Nkx3.1 Antagonizes Sp-mediated Transcriptional Activity in

Prostate-Derived Cells...... 91

3.1 Nkx3.1 antagonizes Sp-mediated transcriptional activity ...... 97

3.2 Nkx3.1 represses Sp-mediated transcription via TSA-sensitive and-insensitive mechanisms...... 102

3.3 Putative alternately-spliced Nkx3.1 variants exhibit diminished capacity antagonize

Sp-mediated transcription ...... 104

3.4 A distal portion of the human PSA promoter is required for Nkx3.1 to suppress

Sp-mediated transcription in prostate-derived cells...... 107

3.5 Nkx3.1 DNA-binding activity is not required to down-regulate Sp-mediated transcription ...... 109

3.6 Two non-contiguous portions of Nkx3.1 are each sufficient to antagonize Sp- mediated transcription...... 115

vii Chapter IV: Nkx3.1 Forms Specific Protein Complexes with Sp-family members...... 121

4.1 Nkx3.1 forms specific protein complexes with Sp-family members in vitro...... 123

4.2 The Nkx3.1 homeodomain physically interacts with the zinc-“fingers” of Sp-family members...... 127

4.3 The DNA-binding domains of Nkx3.1 and Sp-proteins physically interact in vivo...... 128

4.4 Recombinant human Nkx3.1 binds its cognate DNA-binding sequence specifically in vitro...... 133

4.5 Physical interactions between the DNA-binding domains of Nkx3.1 and Sp- proteins do not disrupt their capacity to bind DNA...... 137

4.6 DNA-binding sites for both Nkx3.1 and Sp-proteins are required for complex formation...... 141

Chapter V: Nkx3.1 Binds the Nuclear Matrix and Associates with Condensed

Chromatin throughout Mitosis...... 143

5.1 The Nkx3.1 homeodomain carries at least one nuclear localization signal...... 145

5.2 Nkx3.1 binds to the nuclear matrix of transfected COS cells...... 153

5.3 The Nk