Insights into Mbd2 Function Revealed by a Novel Genetic Tagging Approach

Methylation of cytosine is an epigenetic mark essential for many cellular and developmental processes. How methylation is interpreted into transcriptional regulation is not fully understood, but previous studies have found that this process involves the methyl-CpG binding domain (MBD) family of proteins. Three MBD proteins, MeCP2, MBD1 and MBD2, specifically bind methylated cytosines and recruit different co-repressor complexes to regulate transcription and chromatin states. Genetic studies also linked MeCP2 and MBD1 to neurodevelopmental disorders in humans and mice. However, a role for MBD2 in the brain has not been described. In this work, we characterized the phenotypes of mice lacking MBD2. We found that, unlike MeCP2 and MBD1, Mbd2 null mice behave similarly to wildtype littermates, with the exception of mildly altered nesting and locomotor activity and reduced body weight. To investigate the underlying cause of different functional requirements for the MBDs, we generated knockin mice in which endogenous MBD2 and MBD1 are biotin-tagged. We systematically compared the spatiotemporal expression patterns of the MBDs and found that MeCP2, MBD1 and MBD3 are primarily expressed in the brain. In contrast, MBD2 is widely expressed throughout the body at young and adult ages. In addition, the expression of MBD2 is upregulated in adult spleen and small intestine compared to younger ages, while MBD1 and MBD3 are only enriched at early ages in the brain. We also determined that MBD2 interacts with the NuRD complex ubiquitously across tissues. We conclude that MBD2 is likely dispensable for brain function and instead may mediate NuRDrelated functions primarily in peripheral tissues. Our study provides novel genetic tools and reveals new directions to investigate MBD2 functions in vivo. Degree Type Dissertation Degree Name Doctor of Philosophy (PhD) This dissertation is available at ScholarlyCommons: http://repository.upenn. edu/edissertations/2103 Graduate Group Cell & Molecular Biology First Advisor Zhaolan Zhou Subject Categories Genetics This dissertation is available at ScholarlyCommons: http://repository.upenn. edu/edissertations/2103 INSIGHTS INTO MBD2 FUNCTION REVEALED BY A NOVEL GENETIC TAGGING APPROACH Kathleen H. Wood A DISSERTATION in Cell and Molecular Biology Presented to the Faculties of the University of Pennsylvania in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy 2016 Supervisor of Dissertation ______________________ Zhaolan Zhou, Ph.D. Associate Professor of Genetics Graduate Group Chairperson _______________________ Daniel S. Kessler, Ph.D., Associate Professor of Cell and Developmental Biology Dissertation Committee Thomas A. Jongens, Ph.D., Associate Professor of Genetics Marisa S. Bartolomei, Ph.D., Professor of Cell and Developmental Biology Klaus H. Kaestner, Ph.D., Thomas and Evelyn Suor Butterworth Professor in Genetics Christopher J. Lengner, Ph.D., Assistant Professor of Biomedical Sciences INSIGHTS INTO MBD2 FUNCTION REVEALED BY A NOVEL GENETIC TAGGING APPROACH COPYRIGHT 2016 Kathleen Helen Wood This work is licensed under the Creative Commons AttributionNonCommercial-ShareAlike 3.0 License To view a copy of this license, visit: http://creativecommons.org/licenses/by-ny-sa/2.0/ iii ACKNOWLEDGEMENTS I would like to thank all past and present members of the Zhou lab, and especially my advisor Joe, for their support throughout this project. Joe works very hard to ensure we as the members of his lab perform at our best and are well prepared for the next stages of our careers, and I sincerely appreciate all of his guidance. Everyone in this lab has offered me invaluable help and advice, both academic and personal, which allowed me to complete this project successfully. I would especially like to thank Brian Johnson for initiating this project and for our many helpful and encouraging talks in our bay. I also thank Sarah Welsh for her very significant contribution to the MBD1 project, without which this part of the project would not have been completed. Thank you to Dr. Jerry Zhao for his work to analyze high-throughput sequencing data and for helpful discussions. I would like to thank former lab members Drs. Darren Goffin and Judy Wang for their considerable help when I was new in the lab, and for teaching me how to perform behavioral and statistical analyses. I also thank Jun Lee for his assistance with maintaining my mouse colony, which allowed me to focus on completing my experiments. Finally, I especially thank Yolanda Cui for her very hard work to keep our lab running smoothly and maintain our mouse colonies. I would like to acknowledge the help I received from many people outside the Zhou lab. First, I would like to thank the members of my thesis committee, Drs. Tom Jongens, Marisa Bartolomei, Chris Lengner, and Klaus Kaestner, for their invaluable help with this project. I also thank my earlier mentors Dr. Karen Hales and Dr. Tom Schwarz for their guidance before I started this program. Thank you to Dr. Meera Sundaram for her mentorship through the Genetics T32 training grant. Thank you to Dr. Marisa Bartolomei and Dr. Lara Abramowitz for providing Mbd2 and Mbd1 null mice and helping to initiate this project. The labs of Drs. Edward S. Brodkin, Tracy Bale, and Steve Siegel provided expertise and equipment for the behavioral assays. Elizabeth Krizman in Dr. Michael Robinson’s lab performed HPLC experiments and both her and Dr. Robinson provided helpful discussion. I also thank members of Dr. Brian Gregory’s lab, specifically Dr. Ian Silverman, Nate Berkowitz, and Sager Gosai, for their help with RNA iv extraction, high-throughput sequencing library construction, data analysis, and for being very patient TAs for the Python Bootcamp course. Thank you to members of Dr. Klaus Kaestner’s lab, particularly Dr. Karyn Sheaffer and Rinho Kim, for assistance with experimental techniques using the gastrointestinal tract. Finally, I thank all members of Dr. Doug Epstein’s lab for help with staining and imaging techniques and for being great neighbors. I would also like to acknowledge the Neurobehavior Testing Core and Dr. Tim O’Brien for assistance with behavioral assays, the Next-Generation Sequencing Core, the Pathology Core Laboratory at CHOP, the Cell Center stockroom, the members of the Genetics Department, all staff members of the University Laboratory Animal Resources, and the administrative staff of the CAMB program for making this project possible. I want to thank my friends and family for all of their support over these years. My fellow graduate students have encouraged me so much by reminding me that we all have similar struggles through this process. I also could not have completed this project without the support of my many friends both near and far outside of this program, who give me much-needed breaks from science and also remind me why I want to do it. I give a huge thank you to Ian Silverman, who has supported me in every aspect throughout this journey. I can’t imagine how I would have gotten here without his help literally starting from day one. Finally, I am incredibly grateful to my family for their unwavering confidence in me. Thank you for all of your encouragement and guidance over the years.