Gene Regulation and Chromatin Structure of Mammalian Olfactory Receptors The Harvard community has made this article openly available. Please share how this access benefits you. Your story matters Citation Tan, Longzhi. 2018. Gene Regulation and Chromatin Structure of Mammalian Olfactory Receptors. Doctoral dissertation, Harvard University, Graduate School of Arts & Sciences. Citable link http://nrs.harvard.edu/urn-3:HUL.InstRepos:41129184 Terms of Use This article was downloaded from Harvard University’s DASH repository, and is made available under the terms and conditions applicable to Other Posted Material, as set forth at http:// nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of- use#LAA Gene regulation and chromatin structure of mammalian olfactory receptors A dissertation presented by Longzhi Tan to The Committee on Higher Degrees in Systems Biology in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the subject of Systems Biology Harvard University Cambridge, Massachusetts April 2018 © 2018 Longzhi Tan All rights reserved. Dissertation Advisor: Professor Xiaoliang Sunney Xie Longzhi Tan Gene regulation and chromatin structure of mammalian olfactory receptors Abstract Mammals sense odors by expressing the gene family of olfactory receptors (ORs). Despite the massive family size — around 1,000 OR genes in the mouse genome and 400 in human, each sensory neuron randomly expresses one, and only one, OR. This phenomenon, termed the “one- neuron-one-receptor” rule, underlies both odor sensing in the nose and the formation of an odor map in the brain. However, it remains a mystery how this rule is established. Combining theoretical modeling, single-cell transcriptomics, spatial transcriptomics, and single-cell 3D genome structures, we investigated the regulation of OR genes during neuronal development. We identified a fundamental kinetic constraint in a recent model of epigenetic OR regulation, uncovered a surprising phenomenon of transient multi-OR expression in immature neurons, created by far the most comprehensive spatial map of mouse ORs in the nose, and revealed for the first time 3D genome structures of single diploid human and mouse cells including olfactory sensory neurons. Our interdisciplinary approach provided valuable insights into the molecular mechanism behind the “one-neuron-one-receptor” rule of OR expression; and our methods could be widely applicable to other systems where gene regulation and chromatin structure underlie important physiological functions. iii Table of Contents !! Abstract (iii) !! Table of Contents (iv) !! Acknowledgements (v) !! Chapter 1: Introduction (1) !! Chapter 2: Modeling the kinetics of gene regulation of olfactory receptors (10) !! Chapter 3: Single-cell transcriptomic sequencing of olfactory sensory neurons (34) !! Chapter 4: Mapping the expression zones of nearly all mouse olfactory receptors (55) !! Chapter 5: Reconstructing the 3D genomes of single diploid human and mouse cells (71) iv Acknowledgements I would like to thank my thesis advisor Xiaoliang Sunney Xie for being a fantastic mentor. He is absolutely awesome in every way. He is perhaps best known for his incredible technologies; but at heart he cares the most about scientific discoveries, about which I share the same passion. He gave me tremendous freedom in research subjects, experimental approaches, and work hours; but whenever I need his help, he gives me the best advice and points me to the best resources. I feel extremely fortunate to work with Sunney — a visionary scientist and a wonderful human being. I would also like to thank !! current and past members of the Xie lab including Chenghang Chuck Zong, Jun Yong, Alec Chapman, Chongyi Chen, Dong Xing, Patricia Purcell, Lin Song, Chi-Han Chang, Xu Zhang, Zi Hertz He, Dan Fu, Fa-Ke Frank Lu, Yuntao Steve Mao, Wenlong Yang, Haisong Liu, Shufang Wang, David Feng Lee, Zheng Yan, Huiyi Chen, Ziqing Winston Zhao, Sabin Mulepati, Asaf Tal, Jenny Lu, Ang Li, Shasha Chong, Minbiao Ji, Lei Huang, Luoxing Xiong, Yaqiong Tang, Yi Yin, Guangyu Gavin Zhou, Bo Zhao, Yuanzhen Suo, Wenting Cai, Liyun Jessica Sang, Yunlong Richard Cao, Yan Gu, David Suter, Rahul Roy, Sijia Lu, Larry Valles, Sarah Quilty, Tracey Schaal, and Tony Jia, !! my collaborators Steven Liberles, Sabina Berretta, Stavros Lomvardas, Heng Li, and Fred Alt, and members of their labs including Qian Li, Anne Boyer-Boiteau, Kevin Monahan, Jerome Kahiapo, and Pei-Chi Peggy Wei, !! my dissertation examining committee Xiaowei Zhuang, Adam Cohen, and Tim Mitchison, !! my dissertation advisory committee (DAC) Catherine Dulac, Yang Shi, and Steven Liberles, !! my preliminary qualifying exam 2 (PQE2) committee Sean Megason, Yang Shi, Catherine v Dulac, and Tim Mitchison, !! my preliminary qualifying exam 1 (PQE1) committee Andrew Murray, Michael Desai, and Johan Paulsson, !! my student symposium coach Ethan Garner, !! my rotation advisors Angela Depace, George Church, and Jack Szostak, and members of their labs including Tara Martin, Je Hyuk Jay Lee, Aaron Engelhart, and Anders Björkbom, !! my PhD program directors Tim Mitchison and Andrew Murray, classmates Daniel Flicker, Abigail Groff, John Ingraham, Yunxin Joy Jiao, Farhan Kamili, Peter Koch, Matthieu Landon, Martin Lukacisin, Eran Mick, Alex Ng, and Elizabeth Van Itallie, and administrators Sam Reed and Liz Pomerantz, !! my Neurobiology summer course directors Graeme Davis and Timothy Ryan, the rest of the faculty and assistants, and classmates Antiño Allen, Katie Ferguson, Huong Ha, Natalie Kaempf, Michael Kienzler, Kyle Lyman, Dimphna Meijer, Anne Olsen, Johnny Saldate, Eric Schreiter, Chung Yiu Jonathan Tang, Prahatha Venkatraman, and Christina Whiteus, and !! my past advisors Wit Busza, Jeff Gore, and Pardis Sabeti, !! my parents Lierong Li and Yilun Tan. This work was supported by an NIH Director's Transformative Research Award (5R01EB010244), an NIH Director’s Pioneer Award (5DP1CA186693), and funding from Beijing Advanced Innovation Center for Genomics at Peking University to Xiaoliang Sunney Xie, a Harvard Brain Initiative Collaborative Seed Grant to Fred Alt and Xiaoliang Sunney Xie, an NIH Research Project Grant (5R01DC013289) to Steven Liberles, and an HHMI International Student Research Fellowship to me. vi Chapter 1 Introduction 1 The mammalian nose detects an enormous number of odors; at the molecular level, these odorants are recognized by a large family of proteins, termed olfactory receptors (ORs) (Buck and Axel 1991). ORs are G-protein-coupled receptors (GPCRs) and constitute the largest gene family in many organisms. For example, the human and mouse genomes encode ~ 400 and ~ 1,000 functional OR genes, respectively (Godfrey, Malnic, and Buck 2004, Malnic, Godfrey, and Buck 2004). The large numbers of ORs enable mammals to sense the diverse chemical world around them. In the mammalian nose, ORs are expressed in a striking pattern, termed the “one-neuron-one- receptor” rule (Figure 1.1) (Mombaerts 2004). In the main olfactory epithelium (MOE) of rodents, each olfactory sensory neuron (OSN) randomly chooses one, and only one, OR gene for expression (Ressler, Sullivan, and Buck 1993, Vassar, Ngai, and Axel 1993), and for each OR gene only one of the maternal and paternal alleles (Chess et al. 1994). This phenomenon is also observed in insects (Vosshall et al. 1999) and in fish (Barth, Dugas, and Ngai 1997). OSNs that express the same OR then converge their axons to a few stereotypical spots, termed glomeruli, in the olfactory bulb (OB) of the brain (Ressler, Sullivan, and Buck 1994, Vassar et al. 1994). The “one-neuron-one-receptor” rule therefore ensures the proper sense of smell in the nose and its transduction to the brain. 2 Figure 1.1. Schematic of the “one-neuron-one-receptor” rule of OR expression. Each OSN randomly expresses one, and only one, OR gene. It remains a longstanding mystery how the “one-neuron-one-receptor” rule can be achieved despite the intrinsic stochasticity of gene expression. In particular, each neuron has to continuously transcribe a single OR gene while specifically and strictly silencing all ~ 1,000 others, any leakage of which would add up to a considerable number of undesired OR transcripts. Transgenic experiments revealed that once an OR gene is chosen for expression, its protein product will elicit a feedback that silences all other OR genes (Serizawa et al. 2003, Lewcock and Reed 2004). However, it is unclear how this feedback is achieved and how it avoids silencing the chosen OR. Recent studies suggested an epigenetic mechanism, termed “silencing the de-silencer”, that might explain how a chosen OR avoids silencing itself. In this proposed mechanism, all OR genes are initially silenced by a repressive histone methylation, H3K9me3, according to chromatin immunoprecipitation (ChIP) experiments (Magklara et al. 2011). A transcriptional activator protein KDM1A (also known as LSD1), which could demethylate H3K9 (Shi et al. 2004), is transiently expressed in neuronal progenitors and de-silences a single OR gene (Lyons 3 et al. 2013). Expression of this OR then triggers the unfolded protein response (UPR), which in turn silences the Kdm1a gene and thus prevents de-silencing of any additional OR genes (Dalton, Lyons, and Lomvardas 2013). However, it remains unclear whether this mechanism is sufficient to explain the “one-neuron-one-receptor” rule. In Chapter 2, we theoretically modeled the kinetics of “silencing the de-silencer” during the OR choice. Under minimal assumptions, we