University of Pennsylvania ScholarlyCommons Publicly Accessible Penn Dissertations 2012 Higher-Order Chromatin Organization in Hematopoietic Transcription Wulan Deng University of Pennsylvania, [email protected] Follow this and additional works at: https://repository.upenn.edu/edissertations Part of the Biology Commons, Genetics Commons, and the Molecular Biology Commons Recommended Citation Deng, Wulan, "Higher-Order Chromatin Organization in Hematopoietic Transcription" (2012). Publicly Accessible Penn Dissertations. 627. https://repository.upenn.edu/edissertations/627 This paper is posted at ScholarlyCommons. https://repository.upenn.edu/edissertations/627 For more information, please contact [email protected]. Higher-Order Chromatin Organization in Hematopoietic Transcription Abstract Coordinated transcriptional networks underlie complex developmental processes. Transcription factors play central roles in such networks by binding to core promoters and regulatory elements and thereby controlling transcription activities and chromatin states in the genome. GATA1 is a hematopoietic transcription factor that controls multiple hematopoietic lineages by activating and repressing gene expression, yet the in vivo mechanisms that specify these opposing activities are unknown. By examining the composition of GATA1 associated protein complexes in a genetic complementary erythroid cell system as well as through the use of tiling arrays, we found that a multi-protein complex containing SCL/ TAL1, LMO2, Ldb1, and E2A (the SCL complex thereafter) is present at most sites where GATA1 functions as an activator but depleted at most repressive GATA1 sites. Functional interference of the SCL complex selectively impairs activation but not repression by GATA1. These results identify the SCL complex as a critical and consistent determinant of positive GATA1 activity. The SCL complex and GATA1 co-occupy the active &beta-globin promoter and the distant locus control region (LCR), which are juxtaposed into close proximity by chromatin looping. The physical interaction of genes with their cis-regulatory elements are commonly observed in the metazoan genome, however, the molecular architecture of loop formation and the cause-effect relationship between transcription and looping remain unclear. A few lines of evidence suggest Ldb1, one component of the SCL complex, mediates GATA1 induced chromatin looping. To manipulate chromatin loop formation, we employed artificial zinc fingers (ZF)o t tether Ldb1 to the &beta-globin promoter in GATA1 null erythroblasts in which the &beta-globin locus is relaxed and inactive. Remarkably, targeting Ldb1 or only its self-association domain to the &beta-globin promoter substantially activated &beta-globin transcription in the absence of GATA1. Promoter-tethered Ldb1 interacted with endogenous Ldb1 complexes at the LCR to form a chromatin loop, causing recruitment and phosphorylation of RNA polymerase II. ZF-Ldb1 proteins were inactive in primary erythroblasts with LCR deletion, demonstrating that their activities depend on long-range interactions. Our findings establish Ldb1 as critical effector of GATA1-mediated loop formation and indicate that chromatin looping causally underlies gene regulation. Our studies have for the first time forced long-range chromatin looping at a native locus and provide a novel approach to manipulate gene activity in cells. Degree Type Dissertation Degree Name Doctor of Philosophy (PhD) Graduate Group Biology First Advisor Gerd A. Blobel Keywords Artificial Zinc Finger, Beta-Globin, Chromatin Organization, Hematopoiesis, Transcription Subject Categories Biology | Genetics | Molecular Biology This dissertation is available at ScholarlyCommons: https://repository.upenn.edu/edissertations/627 HIGHER-ORDER CHROMATIN ORGANIZATION IN HEMATOPOIETIC TRANSCRIPTION Wulan Deng A DISSERTATION in Biology Presented to the Faculties of the University of Pennsylvania in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy 2012 Supervisor of Dissertation _____________________ Gerd A. Blobel M.D., Ph.D. Professor of Pediatrics Graduate Group Chairperson _______________________ Doris Wagner, Ph.D., Associate Professor of Biology Dissertation Committee Michael L. Atchison, Ph.D., Professor, Department of Animal Biology Marisa S. Bartolomei, Ph.D., Professor of Cell and Developmental Biology Doris Wagner, Ph.D., Associate Professor of Biology Mitchell J. Weiss, M.D., Ph.D., Professor of Pediatrics Jianxin You, Ph.D., Assistant Professor of Microbiology HIGHER-ORDER CHROMATIN ORGANIZATION IN HEMATOPOIETIC TRANSCRIPTION COPYRIGHT 2012 Wulan Deng This work is licensed under the Creative Commons Attribution- NonCommercial-ShareAlike 3.0 License To view a copy of this license, visit http://creativecommons.org/licenses/by-ny-sa/2.0/ DEDICATION I would like to dedicate this work to my family. Thank Mom and Dad for having worked so hard to support my education throughout the years, and for having made the decision of supporting me to go abroad for this adventure. My Ph.D. journey would not have started at the first place without their openness and great love. Thank them for the discipline and determination they have taught me. Thank my sisters and brother for their love and support. iii ACKNOWLEDGMENTS I am grateful to my thesis adviser, Dr. Gerd Blobel, for his continued guidance with countless stimulating discussions, enthusiastic encouragement to overcome difficulties, tremendous support with ample opportunities, and kindness and patience of assuring me my success when I have doubts. He has seen and helped me to grow as a scientist. I would like to thank my dissertation committee members, Drs. Doris Wagner, Michael Atchison, Marisa Bartolomei, Mitchell Weiss, and Jianxin You for their support and constructive insights. I would like to thank all the members of the Blobel lab for providing a lovely, sincere, and collaborative environment. I am grateful to all members past and present for their inspiration and support, especially Chris Vakoc, Janine Lamonica, Stephan Kadauke, and Amy Campbell. I would also like to thank the neighboring Weiss lab and Tong lab. I would like to thank my collaborators, Ann Dean and Jongjoo Lee from the National Institutes of Health, and Philip Gregory and Andreas Reik from Sangamo Biosiences, and Ross Hardison, Yong Cheng and Ying Zhang from Penn State University. Last but not least, I would like to thank my dear friends. Pursuing a Ph.D. is a challenging path, especially in a foreign country. It would not be possible for me to finish this journey without them. I am grateful to many of them, including Yuting Zhao, Yanshan Fang, Yao Zhang, Yao Yao, Li Yang, Zhongqiang Chen, Shu Lin, Bing He, and Xuan Geng, for their company and support throughout the years. iv ABSTRACT HIGHER-ORDER CHROMATIN ORGANIZATION IN HEMATOPOIETIC TRANSCRIPTION Wulan Deng Gerd A. Blobel Coordinated transcriptional networks underlie complex developmental processes. Transcription factors play central roles in such networks by binding to core promoters and regulatory elements and thereby controlling transcription activities and chromatin states in the genome. GATA1 is a hematopoietic transcription factor that controls multiple hematopoietic lineages by activating and repressing gene expression, yet the in vivo mechanisms that specify these opposing activities are unknown. By examining the composition of GATA1 associated protein complexes in a genetic complementary erythroid cell system as well as through the use of tiling arrays, we found that a multi- protein complex containing SCL/TAL1, LMO2, Ldb1, and E2A (the SCL complex thereafter) is present at most sites where GATA1 functions as an activator but depleted at most repressive GATA1 sites. Functional interference of the SCL complex selectively impairs activation but not repression by GATA1. These results identify the SCL complex as a critical and consistent determinant of positive GATA1 activity. The SCL complex and GATA1 co-occupy the active β-globin promoter and the distant locus control region (LCR), which are juxtaposed into close proximity by chromatin looping. The physical interaction of genes with their cis-regulatory elements are commonly observed in the v metazoan genome, however, the molecular architecture of loop formation and the cause- effect relationship between transcription and looping remain unclear. A few lines of evidence suggest Ldb1, one component of the SCL complex, mediates GATA1 induced chromatin looping. To manipulate chromatin loop formation, we employed artificial zinc fingers (ZF) to tether Ldb1 to the β-globin promoter in GATA1 null erythroblasts in which the β-globin locus is relaxed and inactive. Remarkably, targeting Ldb1 or only its self-association domain to the β-globin promoter substantially activated β-globin transcription in the absence of GATA1. Promoter-tethered Ldb1 interacted with endogenous Ldb1 complexes at the LCR to form a chromatin loop, causing recruitment and phosphorylation of RNA polymerase II. ZF-Ldb1 proteins were inactive in primary erythroblasts with LCR deletion, demonstrating that their activities depend on long-range interactions. Our findings establish Ldb1 as critical effector of GATA1-mediated loop formation and indicate that chromatin looping causally underlies gene regulation. Our studies have for the first time forced long-range chromatin looping at a native locus and provide a novel approach to manipulate gene activity in cells. vi TABLE OF CONTENTS DEDICATION