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Under the Direction of Jonathan M 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 transcription factor that plays an important role in the development and differentiation of prostatic epithelia. Loss of Nkx3.1 protein 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 proteins 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 chromosomes. Finally, I report my efforts to identify Nkx3.1 target genes 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
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