The University of San Francisco USF Scholarship: a digital repository @ Gleeson Library | Geschke Center Master's Theses Theses, Dissertations, Capstones and Projects Winter 12-22-2014 Epigenetic Regulation of Nuclear Hormone Receptor DAX-1 Michael B. Heskett University of San Francisco, [email protected] Follow this and additional works at: https://repository.usfca.edu/thes Part of the Biochemistry, Biophysics, and Structural Biology Commons, Biology Commons, Cell and Developmental Biology Commons, and the Genetics and Genomics Commons Recommended Citation Heskett, Michael B., "Epigenetic Regulation of Nuclear Hormone Receptor DAX-1" (2014). Master's Theses. 116. https://repository.usfca.edu/thes/116 This Thesis is brought to you for free and open access by the Theses, Dissertations, Capstones and Projects at USF Scholarship: a digital repository @ Gleeson Library | Geschke Center. It has been accepted for inclusion in Master's Theses by an authorized administrator of USF Scholarship: a digital repository @ Gleeson Library | Geschke Center. For more information, please contact [email protected]. TABLE OF CONTENTS LIST OF ABBREVIATIONS 3 LIST OF FIGURES 6 ABSTRACT 8 CHAPTER 1: INTRODUCTION 9 CHAPTER 2: DETERMINATION OF DAX EXPRESSION 22 CHAPTER 3: DETERMINATION OF DAX-1 METHYLATION STATUS 35 CHAPTER 4: DAX-1 INTERACTION WITH EPIGENETIC FACTORS 60 CHAPTER 5: SUMMARY 77 REFERENCES 79 2 LIST OF ABBREVIATIONS 5-mC - 5-methylCytosine AHC - Adrenal Hypoplasia Congenita AR - Androgen Receptor ATCC - American Type Culture Collection bp - base pairs cDNA - complimentary Deoxyribonucleic Acid CG - Cytosine and Guanine ChIP - Chromatin Immunoprecipitation CpG - Cytosine phosphate Guanine CXXC - Cysteine - any amino acid - any amino acid - Cysteine DAX-1/NR0B1 - Dosage sensative sex reversal, Adrenal hypoplasia congentia, on chromosome X, gene 1/ Nuclear Receptor subfamily 0 group B member 1 DNA - Deoxyribonucleic Acid DNMT - DNA Methyltransferase DSS - Dosage Sensitive Sex Reversal DBD - DNA Binding Domain ER(α/β) - Estrogen Receptor (alpha/beta) ER+ - Estrogen Receptor Positive ES - Embryonic Stem EWAS - Epigenome Wide Association Studies 3 FAS - Fatty Acid Synthase GAPDH - Glyceraldehyde 3-Phosphate Dehydrogenase H3 - Histone H3 HMT - Histone Methyl Transferase HPA - Hypothalamic/Pituitary/Adrenal HRE - Hormone Response Element IgG - Immunoglobulin G LBD - Ligand Binding Domain LRH-1 - Liver Receptor Homolog -1 LXR - Liver X Receptor LxxLL - Leucine - any amino acid - any amino acid - Leucine - Leucine MBD1 - Methyl CpG binding Domain protein 1 MeCP2 - Methyl CpG binding Protein 2 N-CoR - Nuclear Receptor Co-Repressor 1 NCBI - National Center for Biotechnology Information NR - Nuclear Receptor or Nuclear Hormone Receptor PCR - Polymerase Chain Reaction qPCR - quantitative Polymerase Chain Reaction RNA - Ribonucleic Acid RNAPII - RNA Polymerase II RXR - Retinoid X Receptor 4 SF-1 - Steroidogenic Factor - 1 SHP - Small Heterodimeric Partner SMRT - Silencing Mediator for Retinoid or Thyroid hormone receptors / Nuclear Receptor Co- Repressor 2. SRC - Steroid Receptor Coregulators TDF- Testis Determining Factor TET - Ten-Eleven Translocation 5 LIST OF FIGURES Figure 1-1. The accepted model of nuclear receptor action. 10 Figure 1-2. The structure of a typical nuclear receptor. 12 Figure 1-3. The retioid X receptor complex. 14 Figure 1-4. The structure of the unusual nuclear hormone receptor DAX-1. 16 Figure 1-5. Alingment of human and mouse DAX-1 amino acid sequences. 17 Figure 1-6. A simplified model of epigenetic regulation by DNA methylation. 20 Figure 2-1. Relative DAX-1 expression in many tissue types. 23 Figure 2-2. DAX-1 PCR expression analysis in MCF7, MCF10F, and A549. 28 Figure 2-3. DAX-1 quantitative PCR expression analysis: MCF7 and MCF10F. 29 Figure 2-4. DAX-1 quantitative PCR expression analysis: MCF7 and A549. 30 Figure 2-5. DAX-1 quantitative PCR expression analysis: MCF10F and A549. 31 Figure 2-6. DAX-1 expression data from the Cancer Cell Line Encyclopedia. 33 Figure 3-1. Methylation of Cytosine by DNA Methyltransferase. 36 Figure 3-2. Bisulfite Sequencing scheme. 38 Figure 3-3. CpG site analysis of the entire DAX-1 gene region. 46 Figure 3-4. Alignment of human and mouse DAX-1 coding sequences. 47 6 Figure 3-5. Comparison of mouse and human DAX-1 CpG islands. 48 Figure 3-6. Methylation sensitive restriction digest of DAX-1 genomic DNA. 49 Figure 3-7. Locations of three bisulfite sequencing fragments. 51 Figure 3-8. Bisulfite sequencing fragment 1. 52 Figure 3-9. Bisulfite sequencing fragment 2. 53 Figure 3-10. Bisulfite sequencing fragment 3. 54 Figure 3-11. DAX-1 expression in mouse stem and somatic cell lines. 57 Figure 3-12. Methylation analysis of DAX-1 in mouse stem and somatic cell lines. 58 Figure 4-1. Epigenetic modifications and the organization of the eukaryotic chromosome. 61 Figure 4-2. Three proposed mechanisms for modulation of gene expression by DNA methylation. 63 Figure 4-3. Cytosine Methylation Cycle. 66 Figure 4-4. Chromatin immunoprecipitation targeting MeCP2 on DAX-1. 71 Figure 4-5. Proposed model of DAX-1 epigenetic regulation mechanism by MeCP2. 72 Figure 4-6. Chromatin immunoprecipitation targeting MeCP2 and MBD1 on DAX-1. 73 Figure 4-7. Proposed model of DAX-1 epigenetic regulation by MBD1. 75 7 ABSTRACT DAX-1 (NR0B1) is an orphan nuclear receptor that plays a key role in the development and maintenance of steroidogenic tissue in mammals. Dax-1 is also expressed in mouse embryonic stem (ES) cells and is required to maintain pluripotency. Duplication of the X-chromosome in the region containing the NR0B1 gene results in sex reversal, and mutations in NR0B1 cause adrenal hypoplasia congenita. DAX-1 has been observed to act as a corepressor of other nuclear receptors including androgen receptor (AR), estrogen receptor (ER), and steroidogenic factor 1 (SF-1). In addition to pluripotent ES cells, DAX-1 is primarily expressed in select tissues of the body such as testes, ovaries, adrenal cortex and breast. In some cases, changes in DAX-1 expression may serve as an indicator of aberrant growth. For example, DAX-1 expression is greatly reduced in ER+ breast cancer patient samples and several ER+ cell lines; however the mechanism leading to this change in DAX-1 expression is unknown. Here, we propose that expression of DAX-1 may be governed by epigenetic mechanisms. We sought to determine whether promoter region CpG island methylation and expression of DAX-1 were inversely related , and if the loss of DAX-1 expression in the breast cancer cell line MCF-7 is due to epigenetic mechanisms. Using gene expression assays and bisulfite sequencing, we have confirmed the relationship between methylation status and DAX-1 expression in MCF7 and A549 cancer cell lines. We have further analyzed the DAX-1 CpG island to observe the presence of epigenetic readers MeCP2 and MBD1 that link methylation to gene repression, suggesting DAX-1 is expressed and regulated differentially by epigenetic mechanisms in the cancer cell lines MCF7 and A549. 8 CHAPTER 1: INTRODUCTION Nuclear Receptors Nuclear Receptors (NRs) are a large group of transcription factors that are characterized by their ability to bind steroid hormones and other lipophilic non-steroid hormones and regulate gene expression by directly binding to the promoter and regulatory regions of specific target genes1 (Fig. 1-1). Nuclear receptors play a major role in differential gene expression and are found in diverse animal species from invertebrates to primates2. Evidence suggests that nuclear receptors may have first appeared around the same time animals appeared in the fossil record, approximately 635 million years ago2. There exists a loose correlation between the number and complexity of tissue types and the number of NR genes an organism has, ranging from only a few NR genes in sponges to 48 distinct NR genes in humans. In humans, these 48 receptors of varied structure and function comprise the nuclear receptor superfamily. Nuclear receptor ligands include steroid hormones such as estrogen and testosterone, Vitamin A compounds 2 known as retinoids, tyrosine-based hormones known as thyroid hormones, and Vitamin D3 . Many nuclear receptors have no known ligand, named orphan receptors, and have a reduced or non-existent ligand binding pocket3. Orphan receptors are found in every metazoan species, and have diverse functions that do not require initiation by ligand binding.3. All vertebrate orphan nuclear hormone receptors contain ligand binding domains (LBDs). However, the LBDs of orphan nuclear receptors mediate multiple other functions besides ligand binding such as dimerization and coactivator interaction, allowing the orphan receptors to regulate gene expression and endocrine responses without direct binding of a ligand. 9 Figure 1-1. A typical nuclear receptor binds a hormone ligand, dissociates heat shock proteins, homodimerizes, then translocates to the nucleus where it directly binds to a hormone response elementon the DNA in association with coactivators and RNA polymerase to affect the levels of gene expression. Created by Boghog2. Nuclear_receptor_action. Accessed 9/10/2014 at http://upload.wikimedia.org/wikipedia/commons/3/3f/Nuclear_receptor_action.png 10 Nuclear receptors were first discovered to have modular substructures by Wrange et al in 19784. Since then, the structure of nuclear hormone receptors has been elucidated. A typical nuclear receptor contains an N-terminal domain of varied function, a zinc finger DNA binding domain (DBD) that is important in mediating direct contact to the DNA, a flexible hinge region, a ligand binding domain (LBD) containing a ligand binding pocket, and a C-terminal domain5 (Fig. 1-2). Nuclear receptors are responsible for a wide variety of physiological functions including cell growth and differentiation, fatty acid synthesis, and cancer initiation and progression6,7,8. During mammalian development, estrogen receptor (ER) is expressed in reproductive tissue and upon binding to estrogens stimulates cell proliferation by activating transcription of cyclins6. In stem cells, orphan nuclear receptors such as LRH-1, SF-1, and DAX-1 play an important role for the maintenance of pluripotency7.