Characterization of Boundary Element-Associated Factors BEAF-32A and BEAF-32B and Identification of Novel Interaction Partners in Drosophila Melanogaster S

Louisiana State University LSU Digital Commons LSU Doctoral Dissertations Graduate School 2016 Characterization of Boundary Element-Associated Factors BEAF-32A and BEAF-32B and Identification of Novel Interaction Partners in Drosophila Melanogaster S. V. Satya Prakash Avva Louisiana State University and Agricultural and Mechanical College Follow this and additional works at: https://digitalcommons.lsu.edu/gradschool_dissertations Recommended Citation Avva, S. V. Satya Prakash, "Characterization of Boundary Element-Associated Factors BEAF-32A and BEAF-32B and Identification of Novel Interaction Partners in Drosophila Melanogaster" (2016). LSU Doctoral Dissertations. 617. https://digitalcommons.lsu.edu/gradschool_dissertations/617 This Dissertation is brought to you for free and open access by the Graduate School at LSU Digital Commons. It has been accepted for inclusion in LSU Doctoral Dissertations by an authorized graduate school editor of LSU Digital Commons. For more information, please [email protected]. CHARACTERIZATION OF BOUNDARY ELEMENT-ASSOCIATED FACTORS BEAF-32A AND BEAF-32B AND IDENTIFICATION OF NOVEL INTERACTION PARTNERS IN DROSOPHILA MELANOGASTER A Dissertation Submitted to the Graduate Faculty of the Louisiana State University and Agricultural and Mechanical College in partial fulfillment of the requirements for the degree of Doctor of Philosophy in The Department of Biological Sciences by S. V. Satya Prakash Avva B. Tech., ICFAI University, 2007 August 2016 I dedicate this to my father Late. Dr. Siva Rama Krishna Avva. You will always be in my thoughts. I remember the days when you came home from practice late in the night and still found the time to pick up a book. This vision of you, keeps me motivated, more than you could have ever imagined. ii ACKNOWLEDGEMENTS I take this opportunity to express my profound sense of gratitude to my advisor, Dr. Craig M. Hart, for his invaluable guidance, and for allowing me to carry out this project in his lab. He has generously taken out his valuable time on numerous occasions to discuss the projects and also to give my ideas and thoughts a proper direction. I would like to thank my committee members, Dr. David Donze, Dr. Patrick DiMario, Dr. Joomyeong Kim and Dr. Rendy Kartika for providing their thoughtful suggestions on my project, during our meetings. Particularly, I am very grateful for Dr. Donze’s help in yeast-two-hybrid assays and also for allowing me to use his lab facilities and reagents. I would also like to thank Dr. DiMario for lending valuable reagents. I extend my gratitude to Dr. William E. Wischusen, for enhancing my experience of the graduate program through various leadership and teaching duties. My sincere thanks to Dr. David Burk and Dr. Hollie Hale-Donze, for their timely help in fluorescent microscopy experiments, and Ms. Ying Xiao for her guidance in SEM. My lab mates have been a huge part of my experience in the graduate program. I have shared many fond memories with them. Shraddha Shrestha, Yu (Alex) Ge, Keller McKowen, Mukesh Maharjan, Dr. Peter Klein and Yuankai Dong have all been very supportive in my research endeavours and other wise. I like to thank them all for inculcating a sense of comradery among the lab members and also for their involvement in creating a conducive environment for learning and doing thoughtful research. LBRN summer undergrad, Janice Jacobi, has put in a lot of effort in making clones for yeast two-hybrid assays, in chapter three. I am very thankful for her assistance. Many other undergrads at LSU have worked in this lab with me who have been very helpful. iii Trace Favre, Luis Guerrero, Charles Lewis, and Armand Jacques to just name a few among many. I hope I made their time in the lab worthwhile. I thank all the scholars and staff of LSU, who have talked and discussed about their research work, and providing a stimulating and intellectual environment. Finally, it’s hard to ink all the heart felt feelings for my parents. I am always indebted to them, for being a source of inspiration to pursue studies in the field of biology, and for extending their moral support in all forms, throughout my professional and personal life. iv TABLE OF CONTENTS ACKNOWLEDGEMENTS……………………………………………………………...iii ABSTRACT…....………………………………………………………………………..vii CHAPTER ONE: GENOME ORGANIZATION, CHROMATIN DOMAINS AND INSULATOR ELEMENTS: RELATIONSHIP BETWEEN STRUCTURE AND FUNCTION……………………………………………………………………………….1 Introduction…………………………………………………….……..…….………….….1 Histone Modifications in Gene Regulation and Chromatin Structure.…………….……...3 Chromatin Loop Domain Model………………………………………………….……….7 Regulatory Elements…………………………………………………………….………...8 Insulator (Boundary) Elements……………………………………………………………9 Insulator (Architectural) Proteins….……………………………………………………..11 Topologically Associated Domains (TADs)…………………………….…………….…14 Boundary Element Associated Factor-32 (BEAF-32).....………………………………..19 Research Objectives………………………………………………………………….…..23 References……………………………………………………………………..…………24 CHAPTER TWO: DETERMINATION OF THE ESSENTIAL DOMAINS INSIDE BEAF-32 PROTEINS…………………………………………………………………....37 Introduction………………………………………………………………………………37 Materials and Methods…………………………………………………………………...40 Results…………………………………………………………………………………....44 Discussion………………………………………………………………………………..51 References………………………………………………………………………………..54 CHAPTER THREE: IDENTIFICATION OF NOVEL PHYSICAL INTERACTION PARTNERS OF BEAF-32B PROTEIN AND TESTING THEIR SIGNIFICANCE USING A GENETIC SCREEN….………….…..………………………………………………...57 Introduction………………………………………………………………………...…….57 Materials and Methods…………………………………….……………………………..62 Results……………………………………………………….………………….………..68 Discussion…………………………………………………….………………………….79 References…………………………………………………….………………………….86 CHAPTER FOUR: FLUORESCENT STUDIES ON BEAF-32A AND BEAF-32B PROTEINS.…..……………………………………………………………………….....94 Introduction…..…………………………………………………………………………..94 Fluorescent Labelling of BEAF……………..……………………………………………96 Materials and Methods…………………………………………………………………...98 Results…………………………………………………………………………………..101 Discussion…..…………………………………..………………………………………107 References………………………………………………………………………………109 v CHAPTER FIVE: YEAST TWO-HYBRID TEST FOR PHYSICAL INTERACTIONS BETWEEN INSULATOR PROTEINS………...............................................................115 Introduction……………………………………………………………………………..115 Materials and Methods………………………………………………………………….118 Results…………………………………………………………………………………..119 Discussion…..…………………………..……………………………………………....122 References………………………………………………………………………………126 CHAPTER SIX: CONCLUDING REMARKS………….…………………….……….132 References………………………………………………………………………………139 VITA…………………………………………………..………………………………..142 vi ABSTRACT Regulatory elements are DNA sequences which have specialized activities that coordinate the functions of the genome. Promoters, enhancers, locus control regions, boundary elements (or insulator elements) are examples of DNA sequences that have regulatory properties. In transgenic assays insulator elements have been shown to block communication between regulatory regions, such as enhancers and promoters, when placed between these sequences and also protect genes from position effects when bracketing them, thereby affecting gene expression. Insulator sequences are bound by insulator proteins that direct the function of these sequences. One such insulator protein is the Boundary Element Associated Factor-32 (BEAF-32), a 32 kDa protein which was originally found to bind to the scs’ insulator sequence in the 87A heat shock locus of the Drosophila genome. BEAF-32 has two isoforms: 32A and 32B. BEAF was immunolocalized to numerous binding sites across the Drosophila genome. This was substantiated by various genome-wide mapping experiments, which have identified from 1800 to 6000 BEAF binding sites across the genome. Hence, BEAF-32 likely plays an important role in chromatin organization and gene regulation in combination with other proteins in the nucleus. However, it is not clear how BEAF-32 affects genome organization and gene regulation. We characterized essential domains in the BEAF-32 protein and identified protein partners, some of which include Transcription Factors (TFs). We further mapped the interaction regions inside BEAF and these TFs. We then attempted Fluorescent Recovery After Photobleaching (FRAP) to assess the dynamics of BEAF-32 on polytene chromosomes and also observed banding patterns, with the help of fluorescent protein labels, and evaluated its behavior during mitosis in early embryos. Finally, results obtained vii with BEAF prompted us to test for physical interactions between various insulator proteins and to check contradictory reported results from the literature to document interactions. viii CHAPTER ONE: GENOME ORGANIZATION, CHROMATIN DOMAINS AND INSULATOR ELEMENTS: RELATIONSHIP BETWEEN STRUCTURE AND FUNCTION Introduction Genomes in higher eukaryotes are tightly packed inside the nucleus. The level of compaction of a genome is very dramatic, for example: in humans the length of the entire genome is about 2 meters (m) when stretched from end to end. However the average diameter of a human nucleus is around only 10 m which roughly translates into a compaction rate in the order of 105 (FUENTES-MASCORRO et al. 2000). This could be suggestive of an idea that the DNA is randomly arranged inside the nucleus to optimize the packaging of the genome. But as it turns out processes such as DNA replication, repair, recombination and gene expression are highly regulated mechanisms inside the spatially

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