STRUCTURE-FUNCTION ANALYSIS OF CXXC FINGER PROTEIN 1 Courtney Marie Tate Submitted to the faculty of the University Graduate School in partial fulfillment of the requirements for the degree Doctor of Philosophy in the Department of Biochemistry and Molecular Biology, Indiana University April 2009 Accepted by the Faculty of Indiana University, in partial fulfillment of the requirements for the degree of Doctor of Philosophy. __________________________ David G. Skanik, Ph.D.-Chair __________________________ Robert M. Bigsby, Ph.D. Doctoral Committee __________________________ Joseph R. Dynlacht, Ph.D. February 19, 2009 __________________________ Ronald C. Wek, Ph.D. ii Acknowledgements First, I would like to thank my advisor, Dr. David Skalnik, for his mentorship throughout my graduate career. He has been an outstanding advisor, and I appreciate his time, patience, and the guidance he has given me for my thesis project. I also appreciate his advice and suggestions he has given for my future career. I would like to express my gratitude to the members of my committee: Dr. Joseph Dynlacht, Dr. Ronald Wek, and Dr. Robert Bigsby. I am grateful to all for their time, guidance, and suggestions concerning my research project. I would also like to thank Dr. Melissa L. Fishel for her help and collaboration with the DNA damage aspect of my project. I am grateful to the NIH for three years of fellowship support for an Infectious Disease Training Grant through Dr. Janice Blum. I would like to thank Dr. Janice Blum for her interest in my project and for alerting me to conferences and workshops to enhance my graduate studies. I would also like to thank Dr. Kristin Chun for her advice with my projects and with improving my presentations. I am grateful to the Deparment of Education for support my first year of graduate studies through a GAANN (Graduate Assistance in Areas of National Need) fellowship. I also need to acknowledge the past and present members of the Skalnik Lab, the environment was an enjoyable place to carry out my research, and the interactions, both scientific and personal, were crucial for my success at Indiana University. For this, I am grateful to Dr. Jeong-Heon Lee, Dr. Suzanne Young, Dr. Jill Butler, Erika Dobrota, Dr. Raji Muthukrishnan, and Patricia Pick-Franke. I would like to thank the lab members for their friendship, support, advice, and help. iii Finally, I need to sincerely thank my family for their love, support, and encouragement. I appreciate my parents, Jerry and Sherree, for ingraining a solid foundation of hard work and dedication in me. My parents have provided me with everything I have ever needed to be where I am today and have always been there for me. Also, I want to thank my parents for believing in me and encouraging me to pursue my dreams. I am grateful to know that I can always count on my family for help, and comforted to know that I will always have their love and support. I would particularly like to thank my grandparents (Mary and Ralph), my brothers (Ryan and Dustin), and my aunts (Teta Jeannie and Teta Karen) for their love, support, and interest in my project. I am grateful to Giancarlo for his love, support, colorful suggestions to explain the unexpected results of some of my experiments, and patience with me while carrying out my thesis research and writing. I would also like to thank the rest of my family, Giancarlo’s family, and my friends for their support and encouraging me to relax, have fun, and enjoy life. I am indebted to my family for their support and indispensable role in my achievements, for this, I dedicate this work to them. iv Abstract Courtney Marie Tate STRUCTURE-FUNCTION ANALYSIS OF CXXC FINGER PROTEIN 1 This dissertation describes structure-function studies of CXXC finger protein 1 (Cfp1), encoded by the CXXC1 gene, in order to determine the functional significance of Cfp1 protein domains and properties. Cfp1 is an important regulator of chromatin structure and is essential for mammalian development. Murine embryonic stem (ES) cells lacking Cfp1 ( CXXC1 -/-) are viable but demonstrate a variety of defects, including hypersensitivity to DNA damaging agents, reduced plating efficiency and growth, decreased global and gene-specific cytosine methylation, failure to achieve in vitro differentiation, aberrant histone methylation, and subnuclear mis-localization of Setd1A, the catalytic component of a histone H3K4 methyltransferase complex, and tri- methylated histone H3K4 (H3K4me3) with regions of heterochromatin. Expression of wild-type Cfp1 in CXXC1 -/- ES cells rescues the observed defects, thereby providing a convenient method to assess structure-function relationships of Cfp1. Cfp1 cDNA expression constructs were stably transfected into CXXC1 -/- ES cells to evaluate the ability of various Cfp1 fragments and mutations to rescue the CXXC1 -/- ES cell phenotype. These experiments revealed that expression of either the amino half of Cfp1 (amino acids 1-367) or the carboxyl half of Cfp1 (amino acids 361-656) is sufficient to rescue the hypersensitivity to DNA damaging agents, plating efficiency, cytosine and histone methylation, and differentiation defects. These results reveal that Cfp1 contains redundant functional domains for appropriate regulation of cytosine methylation, v histone methylation, and in vitro differentiation. Additional studies revealed that a point mutation (C169A) that abolishes DNA-binding activity of Cfp1 ablates the rescue activity of the 1-367 fragment, and a point mutation (C375A) that abolishes the interaction of Cfp1 with the Setd1A and Setd1B histone H3K4 methyltransferase complexes ablates the rescue activity of the 361-656 Cfp1 fragment. In addition, introduction of both point mutations (C169A and C375A) ablates the rescue activity of the full-length Cfp1 protein. These results indicate that retention of either DNA- binding or Setd1 association of Cfp1 is required to rescue hypersensitivity to DNA damaging agents, plating efficiency, cytosine and histone methylation, and in vitro differentiation. In contrast, confocal immunofluorescence analysis revealed that full- length Cfp1 is required to restrict Setd1A and histone H3K4me3 to euchromatic regions. David G. Skalnik, Ph.D. - Chair vi Table of Contents LIST OF TABLES ................................................................................................. xiv LIST OF FIGURES................................................................................................. xv ABBREVIATIONS................................................................................................. xx FOCUS OF DISSERTATION ........................................................................... xxiii INTRODUCTION ................................................................................................... 1 I. Chromatin Structure and Epigenetics........................................................ 1 II. Cytosine Methylation............................................................................... 5 II. DNA Methyltransferase Enzymes ............................................................ 8 III. Methyl CpG Binding Proteins ................................................................ 14 V. Heterochromatin .................................................................................... 16 VI. Histone Modifications............................................................................ 17 VII. Histone Methylation............................................................................... 20 VIII. Histone Methylation and RNA Polymerase II......................................... 24 IX. ATP-dependent Chromatin Remodeling................................................. 26 X. Epigenetic Cross-talk............................................................................. 27 XI. Epigenetics and Disease......................................................................... 29 XII. Chromatin Structure and DNA Repair.................................................... 33 XIII. DNA Base Excision Repair.................................................................... 38 XIV. Apurinic/Apyrimidinic Endonuclease (Ape1/Ref-1)............................... 41 XV. CXXC Finger Protein 1 (Cfp1)............................................................... 42 METHODS ............................................................................................................ 53 I. Cell Culture............................................................................................ 53 vii II. Transient Transfection............................................................................ 53 III. Stable Transfection ................................................................................ 54 IV. Construction of Plasmids........................................................................ 55 1. Construction of hCfp1 pcDNA3.1/Hygro constructs........................... 55 2. Construction of hCfp1/pcDNA3-Myc and hDNMT1/ pcDNA3-FLAG constructs................................................................. 58 V. Plasmid Purification and Transformation................................................ 58 1. Plasmid Transformation.................................................................... 58 2. Minipreps.......................................................................................... 59 3. Maxipreps......................................................................................... 59 VI. Site-directed Mutagenesis .....................................................................