MULTIPLE APPROACES TO THE STUDY OF STEROIDOGENIC FACTOR 1: IDENTIFICATION OF A NOVEL REGULATORY ELEMENT AND IDENTIFICATION OF NOVEL TARGET GENES APPROVED BY SUPERVISORY COMMITTEE Keith L. Parker, M.D., Ph.D. Robert Hammer, Ph.D. Carole Mendelson, Ph.D. Andrew Zinn, M.D., Ph.D. To Joe “To love and be loved is to feel the sun from both sides” David Viscott MULTIPLE APPROACHES TO THE STUDY OF STEROIDOGENIC FACTOR 1: IDENTIFICATION OF A NOVEL REGULATORY ELEMENT AND IDENTIFICATION OF NOVEL TARGET GENES by NANCY RUTH STALLINGS DISSERTATION Presented to the Faculty of the Graduate School of Biomedical Sciences The University of Texas Southwestern Medical Center at Dallas In Partial Fulfillment of the Requirements For the Degree of DOCTOR OF PHILOSOPHY The University of Texas Southwestern Medical Center at Dallas Dallas, Texas April, 2005 Acknowledgments I would like to thank the many people that have been supportive of me. I have been very fortunate in meeting many great people throughout the years. My list of thanks starts even before enrolling in graduate school-it begins with the wonderful people that helped me adjust to my first “real job” after college. Judy, Diana, Diane, Karen, Carole and Mike---Thank you so much for all you were able to teach me. I was lucky to find a laboratory to do my dissertation research that also had many talented individuals and fantastic co-workers. Through the years I was able to learn a lot from them. Suria, Neil, Gregor, Liping, Marit, Nathan, Kimmie, Anne, Tomo, Tom, Gareth, and Yelena were great co-workers. It was a pleasure working with all of you. And finally my mentor-Keith Parker-who allowed me the freedom to explore my own ideas while giving me the guidance necessary to do the research. On a more personal level, I would also like to thank those outside the lab for their support and encouragement. Tom and Tara for their friendship throughout graduate school and members of the “Trivia Mofia” for endless hours of entertainment outside the lab, and my Mom and Dad for their encouragement—and unwavering belief that I can be successful in anything I try. Most of all, I would like to thank Joe, my husband of ten years. Day in and day out, Joe has been there for me and I am very lucky to have his support and encouragement. Copyright by Nancy Ruth Stallings, 2005 All Rights Reserved MULTIPLE APPROACHES TO THE STUDY OF STEROIDOGENIC FACTOR 1: IDENTIFICATION OF A NOVEL REGULATORY ELEMENT AND IDENTIFICATION OF NOVEL TARGET GENES Publication No. Nancy Ruth Stallings, Ph.D. The University of Texas Southwestern Medical Center at Dallas, 2005 Supervising Professor: Keith L. Parker, M.D., Ph.D. Steroidogenic Factor 1 (SF-1) is an essential component of the hypothalamic- pituitary-adrenal-gonadal axis. SF-1 knockout (KO) mice lack adrenals, gonads, the ventromedial hypothalamic nucleus (VMH), and pituitary gonadotropes. SF-1 is a transcription factor implicated in the regulation of many genes important in endocrine function. Research into the regulation of SF-1 expression, mostly focused on the proximal promoter, has been unable to fully explain the expression pattern of SF-1. I used DNase hypersensitivity mapping to search for novel regulatory regions of the SF-1 genomic region. One region between the 6th and 7th exons of SF-1 had tissue specific DNase I hypersensitivity. Analysis of this region revealed high conservation vi with the human genomic sequence and a smaller region that was also highly conserved in the chicken genomic sequence. Transient transfection assays and electrophoretic mobility shift assays have been employed to investigate this conserved element for enhancer activity. Numerous genes are target genes of SF-1, yet loss of known target genes fail to explain why the adrenals, gonads and VMH fail to develop in SF-1 KO mice. I used an SF-1/eGFP transgene as a reporter in both WT and KO E16.5 embryos. eGFP+ cells from the developing VMH of these mice were collected through the use of FACS. Several potential target genes of SF-1 have been identified and analysis of these genes is an ongoing project. vii PUBLICATIONS Zhao, L., Ikeda, I., Stallings, N.R., Tobet, S.A., Reuter, A.L., and K. L. Parker. CNS- specific Knockout of Steroidogenic Factor 1 Results in a Metabolic Syndrome and Increased Anxiety. In Preparation. Nef, S., Schaad, O., Cederroth, C., Pitetti, J., Stallings, N.R., Descombes, P. Parker, K.L., and Jean-Dominique Vassalli. Gene expression during sex determination reveals a robust female genetic program at the onset of early ovarian development. In Preparation. Stallings, N.R. Reuter, A.L., and K.L. Parker. Identification of a Conserved Intronic Element that Regulates the Cell-selective Expression of Steroidogenic Factor 1. In review. Segal, J.P., Stallings, N.R., Lee, C.E., Zhao, L., Socci, N.D., Viale, A., Harris, T.M., Soares, M., Childs, G., Elmquist, J., Parker, K.L., and J.M. Friedman. (2005). Use of Laser-Capture Microdissection for the Identification of Marker Genes for the Ventromedial Hypothalamic Nucleus. Journal of Neuroscience 25: 4181-4188. Davis, A.M., Seney, M.L., Stallings, N.R., Zhao, Z., Parker, K.L., and S.A. Tobet. (2004). Loss of steroidogenic factor 1 alters cellular topography in the mouse ventromedial nucleus of the hypothalamus. Journal of Neurobiology 60: 424-436. Cui, S., Ross, A., Stallings, N., Parker, K.L., Capel, B., and S E. Quaggin. (2004). Disrupted Gonadogenesis in Pod1 Mutant Mice. Development 131: 4095-105. Stallings, N.R., Hanley, N.A., Zhao, L., Majdic, G., Bakke, M., and K.L. Parker. (2002) Development of a Transgenic Green Fluorescent Protein Lineage Marker for Steroidogenic Factor 1. Molecular Endocrinology 16: 2360-2370. Shet, M.S., McPhaul, M., Fisher, C.W., Stallings, N.R., and R.W. Estabrook. (1997). Metabolism of the antiandrogenic drug (Flutamide) by human CYP1A2. Drug Metabolism & Disposition 25: 1298-303. viii TABLE OF CONTENTS PUBLICATIONS............................................................................................................................................ VIII TABLE OF CONTENTS.................................................................................................................................. IX FIGURE AND TABLE LIST ...........................................................................................................................XII ABBREVIATIONS......................................................................................................................................... XIII CHAPTER 1 INTRODUCTION TO STEROIDOGENIC FACTOR 1 .....................................................1 1.1 IDENTIFICATION OF STEROIDOGENIC FACTOR 1.........................................................................1 A Common Element in the Promoter of Several Cytochrome P450 Genes...................................................1 Genomic organization of the Nr5a1 locus ....................................................................................................1 1.2 EXPRESSION OF SF-1..............................................................................................................4 SF-1 is expressed in a cell-and tissue-selective manner ...............................................................................4 1.3 CHARACTERIZATION OF SF-1 KO MICE ....................................................................................5 Global SF-1 knockout mice fail to develop gonads, adrenals, and the ventromedial hypothalamic nucleus5 Tissue specific KO of SF-1............................................................................................................................8 1.4 TRANSCRIPTIONAL REGULATION OF SF-1 ...............................................................................10 Global Regulation of SF-1 Expression .......................................................................................................10 Tissue and Cell Specific Regulation of SF-1 Expression ............................................................................12 1.5 SF-1 TARGET GENES.............................................................................................................13 SF-1 binding sites .......................................................................................................................................13 Target Genes of SF-1 can be in steroidogenic and non-steroidogenic cells...............................................13 1.6 SF-1 PROTEIN ......................................................................................................................15 SF-1 has the protein structure of a typical nuclear hormone receptor.......................................................15 Phosphorylation and acetylation of SF-1 regulate transcriptional activity................................................16 Crystal Structures of SF-1 reveal a potential ligand for SF-1 ....................................................................17 Activation of transcription by SF-1 is modified by co-activators and co-repressors..................................17 Co-Activators..............................................................................................................................................19 Co-repressors..............................................................................................................................................23 1.7 LIVER RECEPTOR HOMOLOG 1 ................................................................................................26 1.8 HUMAN MUTATIONS OF SF-1