ABSTRACT FLORES VERGARA, MIGUEL ANGEL. Diversity of Scaffold/Matrix Attachment Regions (S/MARs) in Arabidopsis is Revealed by Analysis of Sequence Characteristics, Nucleosome Occupancy, Epigenetic Marks, and Gene Expression. (Under the direction of Dr. George C. Allen and Dr. William F. Thompson.) Eukaryotic chromatin is organized as independent loops of varying sizes. Following histone extraction with lithium diiodosalicylate (LIS), these loops can be visualized as a DNA halo anchored to the nuclear matrix structure. As a basic unit, the loop is thought to be essential for DNA replication, transcription and chromosomal packaging. The formation of each loop is dependent on a specific chromatin segment that must function as an anchor to the nuclear matrix. Sequences that attach specifically to the nuclear matrix have been termed scaffold/matrix attachment regions (S/MARs). Since only a limited number of putative S/MARs have been characterized so far, their role in genomic structure and function is not well understood. Thus, a more global analysis is necessary to answer a variety of questions such as: How are S/MARs distributed across the genome? Are S/MARs associated with different genomic features and are S/MARs typically AT-rich, as previously suggested? What is the nucleosomal organization at S/MAR sequences and do they define regions of accessible chromatin? Are S/MARs associated with specific epigenetic features such as certain histone modifications or DNA methylation? What role do S/MARs play in transcriptional regulation? I have approached these questions by mapping the S/MARs on Arabidopsis chromosome 4 (chr4) using a high-resolution tiling array. We find that Arabidopsis S/MARs can be divided into five clusters based on their location in relation to other previously identified genomic features and show that the S/MARs are a diverse group of elements with multiple functions within the genome. While many of the Arabidopsis chr4 S/MARs appear to define structural domains, others are significantly enriched at transcription start sites (TSS) of genes and have a highly significant association with genes that encode transcription factors (TFs). Our data, along with additional genomic information for S/MARs from other organisms, will help our understanding of the roles that different types of S/MARs play within the nucleus. © Copyright 2011 by Miguel Angel Flores Vergara All Rights Reserved Diversity of Scaffold/Matrix Attachment Regions (S/MARs) in Arabidopsis is Revealed by Analysis of Sequence Characteristics, Nucleosome Occupancy, Epigenetic Marks, and Gene Expression. by Miguel Angel Flores Vergara A dissertation submitted to the Graduate Faculty of North Carolina State University in partial fulfillment of the requirements for the Degree of Doctor of Philosophy Plant Biology Raleigh, North Carolina 2011 APPROVED BY: _______________________________ ______________________________ William F. Thompson George C. Allen Co-chair of Advisory Committee Co-chair of Advisory Committee ________________________________ ________________________________ Linda Hanley-Bowdoin Dominique Robertson DEDICATION This thesis is dedicated to the memory of my Grandfather Angel Flores Patiño and Grandmother Carolina Vergara Sosa for the profound effect they had on my life. For of him, through him, and to him, are all things: to whom be glory for ever. Pro Rege. ii BIOGRAPHY Miguel Angel Flores Vergara was born February 26, 1975 in Chalcatzingo, Morelos, Mexico. He was raised by his grandparents and lived on a farm for the first eighteen years of his life. He received his elementary school education at General Mariano Matamoros school in Chalcatzingo, followed by high school education at Professor Manuel Noceda Barrios High School at Jonacatepec Morelos. He received his Bachelor of Science in Agronomy from Metropolitan Autonomous University in Mexico City, Mexico in 1998. In June, 2002, he received a Master of Science degree in Plant Biotechnology from Center for Research and Advanced Studies of the National Polytechnic Institute (CINVESTAV-IPN) at Irapuato, Mexico, for studies on plant- geminivirus interactions. In September, 2002, he joined the laboratory of Dr. Dominique Robertson as technician in the Department of Plant Biology, North Carolina State University (NCSU). Miguel was admitted to the Department of Plant Biology at NCSU in 2005, as a candidate for the degree of Doctor of Philosophy. He has undertaken his research under the guidance of Dr. William F. Thompson and Dr. George C. Allen. Miguel married Yokiko Hiromoto on April 12, 2008. They plan to have children. iii ACKNOWLEDGMENTS Although it would be impossible to list all of the people who have influenced the work described here, I would like to acknowledge those who have had the greatest impact. I am especially grateful to my Ph.D. advisors Dr. George C. Allen and Dr. William F. Thompson for their support and untiring efforts as true teachers. I want to thank the members of my committee, Drs. Linda Hanley-Bowdoin and Dominique Robertson for their guidance and support. I would also like to acknowledge Dr. Steve Spiker for thoughtful discussions that were truly the motivation for much of this work. I would also like to acknowledge Dr. Pete Pascuzzi for his contribution in the analysis of the microarray data, processing of the figures and his comments and guidance on this work. I want to thank my friends and co- workers Yokiko Hiromoto, Mariana Franco, Trino Ascencio, Tae-Jin Lee, Sandeep Kumar, Randy Shultz, Emily Markham, Sergei Krasynanski, Roselyn Hatch, Donna Evans, Dolores Sowinski, Gerson Dominguez, and Shingo Nagaya for their encouragement. Special thanks go out to my Mom Edith and Dad Alberto, and siblings Willy, Ale, and Beto for all their love, support and encouragement. I want to thank my uncles Migue, Milo, Rosalio, and Javier as well as my aunts Roge, Gloria, Chela, Duli, and Rocio for all their love. Finally I want to thank that one person who, more than anyone else, is responsible for my completing this degree. Yokiko, you have stood by me through everything with your love. Thank you. This work was supported by a grant from the National Science Foundation. iv TABLE OF CONTENTS LIST OF TABLES....................................................................................................................................... vii LIST OF FIGURES....................................................................................................................................viii LIST OF ABBREVIATIONS......................................................................................................................ix LIST OF ABBREVIATIONS (CONTINUED) ........................................................................................x CHAPTER 1.....................................................................................................................................................1 High-Order Chromatin Organization .........................................................................................................................1 The Nuclear Matrix.............................................................................................................................................................3 S/MAR Sequence Characteristics..................................................................................................................................6 The S/MAR Binding Assay ............................................................................................................................................11 S/MARs and Gene Transcription................................................................................................................................14 S/MARs and DNA Replication.....................................................................................................................................17 Mapped S/MARs in Metazoans (Large Scale).......................................................................................................19 Mapped S/MARs in Plants ............................................................................................................................................22 Goals of this Thesis.....................................................................................................................................................25 REFERENCES.............................................................................................................................................................29 CHAPTER 2.................................................................................................................................................. 49 INTRODUCTION .............................................................................................................................................................49 MATERIALS AND METHODS .....................................................................................................................................53 Plant Materials and Growth Conditions............................................................................................................53 Isolation of Nuclei.......................................................................................................................................................53 Preparation of Nuclear Halos and Characterization of Proteins............................................................54 Analysis of nuclear matrix proteins by gel