Expression and function of corticotropin-releasing hormone in anthropoid primate placenta A dissertation submitted to the Graduate School of the University of Cincinnati in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Graduate Program of Molecular and Developmental Biology of the College of Medicine by Caitlin E. Dunn-Fletcher B.S. Indiana University, Bloomington, Indiana, 2011 Committee Chair: Louis Muglia, M.D., Ph.D. Helen Jones, Ph.D. Raphael Kopan, Ph.D. Rolf Stottmann, Ph.D. Sing Sing Way, M.D., Ph.D. Matthew Weirauch, Ph.D. Abstract Pregnancy and parturition are intricately regulated to ensure successful reproductive outcomes. However, the factors that control gestational length in humans and other anthropoid primates remain poorly defined. Variations in maternal plasma corticotropin-releasing hormone (CRH) concentrations, arising from placental synthesis, have been associated with pathological alterations of gestation length in humans. Although hypothalamic CRH expression is conserved in all vertebrates, placental CRH expression is specific to anthropoid primates. Using comparative genomic analysis, we discovered a correlation between genomic integration of retroviral long terminal repeat transposon-like human element 1B (THE1B) and placental expression of CRH. Here, we show the endogenous retroviral element THE1B selectively controls placental expression of CRH that, in turn, influences gestational length and birth timing. Placental expression of CRH and subsequently prolonged gestational length were found in two independent strains of transgenic mice carrying a 180-kb human bacterial artificial chromosome (BAC) DNA that contained the full length of CRH and extended flanking regions, including THE1B. Restricted deletion of THE1B silenced placental CRH expression and normalized birth timing in these transgenic lines. Furthermore, we revealed an interaction at the 5′ insertion site of THE1B with distal-less homeobox 3 (DLX3), a transcription factor required for placental development. Together, these findings suggest that retroviral insertion of THE1B into the anthropoid primate genome may have initiated expression of CRH in placental syncytiotrophoblasts via DLX3 and that this placental CRH is sufficient to alter the timing of birth. ii iii Acknowledgements The work presented here has been strengthened and refined by the guidance of my thesis committee. I would like to thank the members of my committee for their time and expertise, and for the many lively discussions that helped shape this work. Special thanks to my mentor, Louis Muglia, for providing the right balance of leadership and freedom for me to explore my own ideas and develop as a scientist. I especially appreciated the opportunities he provided to share our work with others and truly partake in collaborative team science. I would also like to thank the members of the Muglia Lab and our collaborators and core facilities at Cincinnati Children’s Hospital Medical Center for stepping in with technical knowledge, advice, and instructions that helped me overcome many roadblocks throughout this project. I have greatly appreciated the privilege of training at an institution that actively encourages the sharing of skills and knowledge. Much of the work presented here would not have been possible without the willingness of others to demonstrate techniques, share protocols, provide resources, and generously give of their own time. I would especially like to recognize the undergraduate students who went above and beyond what was expected of them and were instrumental to the success of this work. Thank you to Katri, Lauren, Libbey, Sammy, and Shivani for sticking with me while I developed as a mentor. Special thanks to Katie Bezold Lamm, Kayleigh Swaggart, and the other past members of the Bae for all the little things (and some big things) that made this work possible and enjoyable. Thanks for all the feedback and the Starbucks runs. We did it! Finally, I would like to thank my family for their help and support. To my parents and siblings, thank you for encouraging me to pursue my interests and providing a sense of normalcy when iv the process seemed unpredictable. To Jon, my teammate, thank you for all of your scientific and non-scientific contributions. The best collaboration is ours (including Harper, Dora, and Abe). v Table of Contents Abstract ii Acknowledgements iv List of Figures and Tables xiii List of Abbreviations xv Chapter I: Introduction 1 The onset of normal parturition 2 Structure and function of the uterus 2 Structure and function of the placenta 5 Hormonal transition to parturition: role of progesterone 6 Hormonal transition to parturition: role of prostaglandins 8 Hormonal transition to parturition: role of oxytocin 10 Corticotropin-releasing hormone (CRH) in human pregnancy 11 Identification of CRH in placenta of anthropoid primates 11 Clinical utility of CRH as a biomarker of birth timing 12 Interplay of CRH with other pathways in parturition 14 A forward-thinking approach to the study of CRH in parturition 15 Figures 16 Figure 1-1. Progression from Phase 0 through Phase I of labor………………………….16 References 18 Chapter II: Anthropoid primate-specific retroviral element THE1B controls expression of CRH in placenta and alters gestation length 28 vi Introduction 29 Results 30 CRH expression and retroviral element THE1B presence are concordant in anthropoid primate placenta 30 THE1B LTRs may form a coordinated regulatory network in anthropoid primate placenta 31 THE1B-CRH transgenic mice express human CRH in placenta 32 Placental expression of human CRH delays parturition in transgenic mice 33 Deletion of THE1B by CRISPR/Cas9 eliminates placental CRH expression 34 Deletion of THE1B by CRISPR/Cas9 restores wild-type gestation length 35 Transcription factor DLX3 interacts with THE1B 5’ insertion site 35 DLX3 binding to THE1B may drive CRH expression in syncytiotrophoblasts 36 Discussion 36 Materials and Methods 39 Human and rhesus tissue collection, RNA-seq, and transcriptome analysis 39 PCR screening for THE1B-CRH fusion transcript in human and rhesus 40 Relative co-expression analysis of THE1B-associated genes 40 THE1B capture array and analysis 40 Annotation of human placenta-enriched genes 41 Association analysis between human placenta-enriched genes and THE1B subfamily 41 ChIP-seq for histone modifications in human term placenta 42 Ethics statement 42 Establishment of transgenic mouse lines 42 Mouse tissue collection and RNA isolation 43 qPCR for human CRH expression in transgenic mice 44 Localization of human CRH by immunohistochemistry/immunofluorescence 44 Quantification of gestation length in transgenic mice 45 vii Mouse uterus and placenta RNA-seq and analysis 45 Measurement of serum progesterone 45 Measurement of uterine prostaglandin F2α 46 Measurement of serum corticosterone 46 Binding site prediction of placental TFs 46 Electrophoretic mobility shift assay for binding at THE1B 46 ChIP and qPCR 47 Statistical analysis 48 Figures 49 Figure 2-1. THE1B is a candidate enhancer for placenta-specific regulation of CRH and other genes……………………………………………………………………………………...49 Figure 2-2. BAC transgenic mice exhibit placental CRH expression and delayed parturition, which are eliminated by THE1B deletion……………………………………….51 Figure 2-3. THE1B 5’ insertion site creates novel binding site for transcription factor DLX3……………………………………………………………………………………………..53 Supporting Information 56 Figure 2-S1. THE1B near CRH is not associated with classical enhancer chromatin marks…………………………………………………………………………………………….56 Figure 2-S2. Post-term birth phenotype is independent of progesterone and contractile- associated protein changes but may involve prostaglandin F2α………………………….58 Figure 2-S3. Hypothalamic-pituitary-adrenal axis function in transgenic mice is comparable to control………………………………………………………………………….60 Figure 2-S4. TRIM55 expression is altered by deletion of THE1B from the human BAC………………………………………..…………………………………………………….61 Figure 2-S5. Gestation length of litters hemizygous for Tg(CR2) is not significantly different from wild-type control………………………………………………………………..62 Table 2-S1. THE1B-CRH novel splice site is conserved in anthropoid primate species.63 References 64 viii Chapter III: Human CRH induces expression changes in mouse uterus in late gestation 70 Introduction 71 Results 72 Transgenic mice expressing placental CRH demonstrate altered gene expression in uterus 72 Glucocorticoid pathway signaling in uterus is altered by CRH expression 72 CRH production in placenta alters uterine prostaglandin balance 73 CRH-dependent changes to Rho pathway may affect myometrial contractility 73 Genes associated with vesicle-mediated transport are differentially expressed in Tg(BAC1) uterus 74 Selenoproteins are downregulated in Tg(BAC1) uterus 75 Discussion 75 Materials and Methods 77 Mouse uterus tissue collection and RNA isolation 77 Uterus RNA-seq and analysis 77 Hierarchical clustering of uterine samples 78 Measurement of serum corticosterone 78 Measurement of uterine prostaglandin F2α 78 Determination of significantly differentially expressed genes 78 Identification of overrepresented pathways with PANTHER database 79 Figures and Tables 80 Figure 3-1. Tg(BAC1) uterine expression clusters separately from nontransgenic and Tg(CR1)………………………………………………………………………………………….80 Figure 3-2. Tg(BAC1) glucocorticoid receptor and cochaperone expression is elevated in uterus with no changes to serum corticosterone……………………………………………81 ix Figure 3-3. Decreased