Copyright by Akshay Anant Bhinge 2009 The Dissertation Committee for Akshay Anant Bhinge Certifies that this is the approved version of the following dissertation: A Functional Genomics Approach To Map Transcriptional and Post- transcriptional Gene Regulatory Networks Committee: Vishwanath Iyer, Supervisor Makkuni Jayaram Krishnendu Roy Scott Stevens Christopher Sullivan A Functional Genomics Approach To Map Transcriptional and Post- transcriptional Gene Regulatory Networks by Akshay Anant Bhinge, M.B.B.S.; M.Tech. Dissertation Presented to the Faculty of the Graduate School of The University of Texas at Austin in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy The University of Texas at Austin August, 2009 Dedication I dedicate this work to my parents who, against great odds, gave me the opportunity to pursue my goals and to Seema, my best friend and the love of my life. Acknowledgements I would like to thank my advisor Dr. Vishwanath Iyer for his incredible patience as he mentored me throughout graduate school. He always made it a priority to make himself available to his students. Every project idea, however wacky, that I have presented to him was granted serious thought and critical comments. Without his constant encouragement, I would not have made it this far. I would also like to thank my committee members for their insightful and helpful suggestions. To my family, I am very grateful. I would not have been able to pursue graduate studies without my parent’s unwavering support or my brother’s motivation. I want to thank Yogish for sparking my interest in science. He has been more like a mentor than just a good neighbor. I have been friends with Ganapathy as far back as I can remember and have turned to him for advice on numerous occasions. I want to thank all my friends from Grant Medical College, IIT-Bombay and IISc. They have made my otherwise drab academic journey a whole lot interesting. The best thing that happened to me in medical school was meeting my wife, Seema. Her love and care has kept me going through the worst. Its very comforting to know there are some aspects of your life you can always count on. I want to thank the present and past members of the Iyer lab for enriching my graduate life, especially Jonghwan Kim from whom I gained a lot of experimental skills. My thanks to Dr. Chris Sullivan and his lab members for their help and guidance in the microRNA project. I would also like to thank Dr. Edward Marcotte for scientific discussions during our weekly lab meetings. His ability to describe complex ideas in the simplest few words never ceases to amaze me. v A Functional Genomics Approach To Map Transcriptional and Post- transcriptional Gene Regulatory Networks Publication No._____________ Akshay Anant Bhinge, Ph.D. The University of Texas at Austin, 2009 Supervisor: Vishwanath Iyer It has been suggested that organismal complexity correlates with the complexity of gene regulation. Transcriptional control of gene expression is mediated by binding of regulatory proteins to cis-acting sequences on the genome. Hence, it is crucial to identify the chromosomal targets of transcription factors (TFs) to delineate transcriptional regulatory networks underlying gene expression programs. The development of ChIP- chip technology has enabled high throughput mapping of TF binding sites across the genome. However, there are many limitations to the technology including the availability of whole genome arrays for complex organisms such human or mouse. To circumvent these limitations, we developed the Sequence Tag Analysis of Genomic Enrichment (STAGE) methodology that is based on extracting short DNA sequences or “tags” from ChIP-enriched DNA. With improvements in sequencing technologies, we applied the recently developed ChIP-Seq technique i.e. ChIP followed by ultra high throughput sequencing, to identify binding sites for the TF E2F4 across the human genome. We vi identified previously uncharacterized E2F4 binding sites in intergenic regions and found that several microRNAs are potential E2F4 targets. Binding of TFs to their respective chromosomal targets requires access of the TF to its regulatory element, which is strongly influenced by nucleosomal remodeling. In order to understand nucleosome remodeling in response to transcriptional perturbation, we used ultra high throughput sequencing to map nucleosome positions in yeast that were subjected to heat shock or were grown normally. We generated nucleosome remodeling profiles across yeast promoters and found that specific remodeling patterns correlate with specific TFs active during the transcriptional reprogramming. Another important aspect of gene regulation operates at the post-transcriptional level. MicroRNAs (miRNAs) are ~22 nucleotide non-coding RNAs that suppress translation or mark mRNAs for degradation. MiRNAs regulate TFs and in turn can be regulated by TFs. We characterized a TF-miRNA network involving the oncofactor Myc and the miRNA miR-22 that suppresses the interferon pathway as primary fibroblasts enter a stage of rapid proliferation. We found that miR-22 suppresses the interferon pathway by inhibiting nuclear translocation of the TF NF-kappaB. Our results show how the oncogenic TF Myc cross-talks with other TF regulatory pathways via a miRNA intermediary. vii Table of Contents List of Tables ....................................................................................................... xiii List of Figures...................................................................................................... xiv Chapter 1: Introduction...........................................................................................1 Eukaryotic transcriptional regulation..............................................................1 How complex are we?............................................................................1 Eukaryotic transcription factors.............................................................1 Regulatory DNA sequences...................................................................2 Transcriptional regulatory networks......................................................2 Role of chromatin ...........................................................................................3 The nucleosome .....................................................................................4 Histone modifications and variants........................................................4 Chromatin dynamics ..............................................................................5 Sequence determinants of nucleosome positioning ......................5 Chromatin remodelers...................................................................6 Post-transcriptional gene regulation ...............................................................6 Discovery ...............................................................................................6 Biogenesis..............................................................................................7 Mode of action .......................................................................................8 TF-miRNA interactions .........................................................................8 Chapter 2: Mapping Genome-wide Transcription Factor Binding Sites Using STAGE ...............................................................................................11 Introduction...................................................................................................11 E2F4 STAGE................................................................................................13 Results...........................................................................................................13 Identifying significant tags ..................................................................15 Scoring genes as putative targets .........................................................16 Discussion.....................................................................................................17 viii Material and Methods ...................................................................................19 ChIP for E2F4......................................................................................19 STAGE using Sanger sequencing........................................................19 STAT1 STAGE.............................................................................................20 Results...........................................................................................................20 STAGE identified STAT1 targets........................................................21 Verification by ChIP-chip and quantitative PCR.................................24 Motif analysis.......................................................................................25 Genes proximal to STAT1 binding sites..............................................29 Identification of c-Myc targets by STAGE..........................................30 Discussion.....................................................................................................31 Materials and Methods..................................................................................32 STAT1 ChIP ........................................................................................32 STAGE using 454 sequencing.............................................................32 Assigning probabilities for tag enrichment..........................................32 STAGE