INSIGHTS INTO NUCLEAR ARCHITECTURE AND STOCHASTIC GENE EXPRESSION THROUGH THE STUDY OF HOMOLOGOUS CHROMOSOME PAIRING AND TRANSVECTION by Kayla Viets A dissertation submitted to Johns Hopkins University in conformity with the requirements for the degree of Doctor of Philosophy Baltimore, Maryland March 2019 © Kayla Viets 2019 All Rights Reserved Abstract Chromosomes are organized in a complex manner within the nucleus. Their localization relative to activating and repressing nuclear compartments and to other chromosomes can have major effects on gene expression. One important aspect of nuclear architecture involves the physical colocalization, or “pairing” of different alleles of the same gene. Pairing is involved in mammalian processes including genomic imprinting and X-inactivation, but the most well- studied example occurs in Drosophila melanogaster, where homologous chromosomes are paired along their entire lengths throughout interphase. Homologous chromosome pairing in fruit flies facilitates a gene-regulatory phenomenon known as transvection, in which DNA elements on one mutant allele of a gene act between chromosomes on another mutant allele to rescue expression. While homologous chromosome pairing and transvection were first described over 60 years ago, the mechanisms that drive homologous chromosome pairing and transvection across the genome are still unclear. Here, we develop a DNA FISH-based approach to identify button regions across the fly genome that drive pairing. These buttons allow “reconstitution” of genes that are split apart by chromosome rearrangements, suggesting that buttons play a role in maintaining the structural integrity of the genome. Buttons are enriched for topologically associated domains (TADs), indicating that TADs are responsible for homologous chromosome interactions. Using the stochastically expressed spineless (ss) locus as a paradigm, we gain deeper insight into the mechanisms that control transvection. We find that pairing is necessary but not sufficient for ii transvection, and that pairing and transvection are cell-type specific. We also identify the DNA elements required for the separable mechanisms of activation and repression between chromosomes. Furthermore, we find a biological role for transvection in regulating the expression of naturally occurring ss alleles to control photoreceptor patterning. Our work suggests a model in which specialized TADs drive homologous chromosome pairing to facilitate cell-type- specific interchromosomal gene regulation. This work has important implications for our understanding of nuclear architecture-linked diseases including Prader- Willi and Angelman syndromes, breast, renal, and pancreatic cancers, and limb malformations. Primary readers: Xin Chen and Robert J. Johnston, Jr. Secondary readers: Joseph Gall, Karen Reddy, and Yixian Zheng iii Acknowledgements My heartfelt thanks go to all of the members of the Johnston lab who have provided invaluable scientific ideas, experimental advice, and friendship throughout my PhD. I would especially like to thank my PI, Bob Johnston, for his supportive and enthusiastic mentorship, championing of his students, and copious free chocolate. I would also like to thank the many colleagues and collaborators who have contributed to the success of this work by generously providing their feedback, reagents, and time, including Eric Joyce, Ting Wu, Jelena Erceg, Jumana AlHaj Abed, Marco Gallio, James Taylor, Mike Sauria, Max Echterling, Peter DeFord, Andrew Gordus, Claude Desplan, Geraldine Seydoux, Jeff Corden, Judy Kassis, and the Johns Hopkins Tri-Fly labs. Additionally, I would like to thank my thesis committee members, Joe Gall, Karen Reddy, and Yixian Zheng. Their constructive feedback, excellent scientific guidance, and support of my personal and career goals have been essential to my success in graduate school. Finally, I would like to thank my amazing friends and family. My friends have made the last five and a half years in Baltimore some of the best of my life. I am so grateful to have gone through graduate school with such an amazing group of people who support each other through tough scientific times and celebrate each other’s successes. I am also certain that I have the best family around. Without their love, their cheerleading, and their wholehearted support of my career goals throughout my life, I would never have made it as a scientist. I iv am also extremely lucky to have found a wonderful second family in my soon-to- be in-laws, and want to thank them for all their support over the past few years, especially these past few months as I’ve been writing my thesis. Most importantly of all, I want to thank my wonderful fiancé, Alex, for always being there for me through five and a half years of grad school plus five years of long distance. I can’t imagine a better, sweeter, more understanding person to have had by my side for the past five and a half years, and I am so excited to finally start a new adventure together as the Master and the Doctor. v Publications 1) “Probing chromosome pairing and transvection to understand nuclear organization” (Chapter 1) is a review article currently in preparation. 2) “Mechanisms of Photoreceptor Patterning in Vertebrates and Invertebrates” (Chapter 2) is a review article that was published in Trends in Genetics in 2016. 3) “TADs pair homologous chromosomes to promote interchromosomal gene regulation” (Chapter 3) is a research article that is in revision at Developmental Cell. It was also posted to bioRxiv in 2018. 4) “Activating and repressing stochastic gene expression between chromosomes” (Chapter 4) is a research article that is in preparation for submission to Developmental Cell. 5) “The mir-279/996 cluster represses receptor tyrosine kinase signaling to determine cell fates in the Drosophila eye” (Appendix 1) is a research article that was published in Development in 2018. 6) “Regulatory logic driving stable levels of defective proventriculus expression during terminal photoreceptor specification in flies” (Appendix 2) is a research article that was published in Development in 2017. 7) “The insulator protein BEAF-32 is required for Hippo pathway activity in the terminal differentiation of neuronal subtypes” (Appendix 3) is a research article that was published in Development in 2016. vi Table of Contents Abstract……………………………………………………………………………..ii Acknowledgements……………………………………………………………….iv Publications………………………………………………………………………...vi Chapter 1: Introduction: Probing chromosome pairing and transvection to understand nuclear organization……………………………………………….1 1.1: Abstract………………………………………………………………….1 1.2: Introduction……………………………………………………………...2 1.3: Interchromosomal interactions and their role in gene expression...4 1.4: Homologous chromosome pairing in Drosophila provides insight into nuclear organization………………………………………………7 1.5: Homologous chromosome interactions facilitate interchromosomal gene regulation…………………………………..13 1.6: Conclusions……………………………………………………………..22 Chapter 2: Mechanisms of photoreceptor patterning in vertebrates and invertebrates………………………………………………………………………...42 2.1: Abstract…………………………………………………………………..43 2.2: Retinas are patterned in stochastic/regionalized, regionalized, and ordered mosaics…………………………………………………..43 2.3: The unique retinal patterning of different organisms has evolved to suit their environments and behaviors…………………………….48 2.4: The gene regulatory networks controlling PR specification share functional and sequence-level homologs…………………………….48 vii 2.5: Gradients of signaling molecules determine regionalized retinal development…………………………………………………………….54 2.6: Retinal development proceeds through waves of differentiation…..55 2.7: Looping of DNA elements regulates cone subtypes………………...57 2.8: Concluding Remarks……………………………………………………58 2.9: Author Contributions…………………………………………………….65 Chapter 3: TADS pair homologous chromosomes to promote interchromosomal gene regulation……………………………………………...68 3.1: Abstract…………………………………………………………………..68 3.2: Introduction………………………………………………………………69 3.3: Results……………………………………………………………………72 3.4: Discussion………………………………………………………………..86 3.5: Materials and Methods………………………………………………….88 3.6: Author Contributions……………………………………………………110 Chapter 4: Activating and repressing stochastic gene expression between chromosomes……………………………………………………………………….170 4.1: Abstract…………………………………………………………………..170 4.2: Introduction………………………………………………………………171 4.3: Results……………………………………………………………………174 4.4: Discussion……………………………………………………………….184 4.5: Materials and Methods…………………………………………………188 viii 4.6: Author Contributions……………………………………………………201 Chapter 5: Unpublished data……………………………………………………..224 5.1: Button pairing is position-dependent………………………………….224 5.2: Identification of additional buttons across the Drosophila genome…………………………………………………………………..225 5.3: Polycomb mutations affect pairing between copies of ss…………..226 5.4: Investigating the effects of condensin mutations on pairing between ss copies……………………………………………………..227 5.5: Individual DNA element deletions do not affect ss pairing…………228 5.6: Deletion of the interior of the ss TAD reduces pairing between copies of ss……………………………………………………………..228 5.7: ss nuclear localization changes with ss expression state………….229 5.8: Materials and Methods…………………………………………………231 Chapter 6: Conclusions and Future Directions………………………………247 6.1: The study of pairing and transvection provides insights into nuclear organization…………………………………………………..247 6.2: Perspectives and future work…………………………………………249