UNIVERSITY of CALIFORNIA, SAN DIEGO Allele-Specific Gene
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UNIVERSITY OF CALIFORNIA, SAN DIEGO Allele-specific Gene Regulation in Humans A dissertation submitted in partial satisfaction of the requirements for the degree Doctor of Philosophy in Chemical Engineering by Nathaniel David Maynard Committee in Charge: Professor Bing Ren, Chair Professor Pao Chau Professor Xiang-Dong Fu Professor Xiaohua Huang Professor Bernhard Palsson Professor Jan Talbot 2008 © Nathaniel David Maynard, 2008 All rights reserved. The dissertation of Nathaniel David Maynard is approved, and it is acceptable in quality and form for publication on microfilm: _______________________________________________ _______________________________________________ _______________________________________________ _______________________________________________ _______________________________________________ _______________________________________________ Chair University of California, San Diego 2008 iii To my girls–Jane, Cate, and Anna In memory of Grampy, Rose, and Gram iv TABLE OF CONTENTS Signature Page………………………………………………………………………...iii Dedication……………………………………………………………………………..iv Table of Contents………………………………………………………………………v List of Figures……………………………………………………………………...…vii List of Tables………………………………………………………………………...viii Acknowledgements……………………………………………………………………ix Vita…………………………………………………………………………………...xii Abstract………………………………………………………………………….......xiii Chapter 1 Introduction ............................................................................................... 1 1.1 The Human Genome......................................................................................... 2 1.2 Gene Transcription........................................................................................... 5 1.3 Chromatin Structure and Gene Regulation........................................................ 7 1.4 CCCTC-binding factor (CTCF) ........................................................................ 8 1.5 Genomic Variation ........................................................................................... 9 1.6 ChIP-chip....................................................................................................... 11 1.7 Allele-specific Gene Regulation ..................................................................... 13 1.7.1 Imprinting................................................................................................ 14 1.7.2 X-Chromosome Inactivation.................................................................... 15 1.7.3 Random Monoallelic Expression (Allelic Exclusion) ............................... 17 1.8 Differential Allelic Expression ....................................................................... 18 1.9 Allele-specific Protein–DNA Interactions....................................................... 21 Chapter 2 Digital Expression Profiling......................................................................25 2.1 Allele-specific Expression Studies.................................................................. 27 2.2 Current Methods for Discovering and Monitoring Allele-Specific Gene Expression............................................................................................................ 29 2.3 Results............................................................................................................ 30 2.3.1 Ratio Mix Test......................................................................................... 32 2.3.2 RNA Digital Expression Profiling (RDEP) .............................................. 32 2.3.3 ACTG1 and EDN1 Transcript Copy Numbers.......................................... 33 2.3.4 RDEP Summary ...................................................................................... 34 2.3.5 Using RDEP for Screening Allele-specific Expression............................. 35 2.4 Discussion...................................................................................................... 36 2.5 Acknowledgments.......................................................................................... 37 2.6 Material and Methods..................................................................................... 38 v Chapter 3 Genome-wide Mapping of Allele-specific Protein–DNA Interactions in Human Cells .............................................................................................................59 3.1 Introduction.................................................................................................... 60 3.2 Results............................................................................................................ 64 3.2.1 Identification of Allele-specific Interactions of RNAP in the Human Genome............................................................................................................ 64 3.2.2 RNAP Interacts with Promoters of Imprinted Genes in an Allele-specific Manner............................................................................................................. 65 3.2.3 Validation of Identified Allele-specific RNAP Binding Sites ................... 68 3.2.4 Correlation of RNAP Binding to Promoters and Allele-specific Gene Expression........................................................................................................ 69 3.3 Discussion...................................................................................................... 70 3.4 Acknowledgements ........................................................................................ 72 3.5 Material and Methods..................................................................................... 74 Chapter 4 Conclusions ............................................................................................103 4.1 RDEP and ADEP...........................................................................................104 4.2 ChIP-SNP Going Forward.............................................................................105 4.3 Future Work..................................................................................................108 4.3.1 CTCF ChIP-SNP in IMR90 ....................................................................108 4.3.2 RNAP ChIP-SNP in Human Embryonic Stem Cells................................109 4.4 Acknowledgements .......................................................................................111 References ..............................................................................................................112 vi LIST OF FIGURES Figure 1-1. Building Blocks of the Human Organism. ............................................. 23 Figure 1-2. Experimental Scheme for ChIP-chip ..................................................... 24 Figure 2-1. Allele-specific Expression..................................................................... 47 Figure 2-2. Padlock Probes and Rolling Circle Amplification.................................. 48 Figure 2-3. Slide Images Before and After Streptavidin Collapse ............................ 49 Figure 2-4. RDEP Experimental Scheme.................................................................. 50 Figure 2-5. ACTG1 and EDN1 3’ Targeting............................................................. 51 Figure 2-6. RDEP Dynamic Range Sample ............................................................. 52 Figure 2-7. ADEP Probe Design.............................................................................. 53 Figure 3-1. Outline of the Procedure for ChIP-SNP................................................. 81 Figure 3-2. ChIP-SNP Analysis of RNAP Binding in Human Fibroblasts................ 82 Figure 3-3. Histogram of Fold Change of Allele-specific Enriched SNPs ................. 83 Figure 3-4. Identification of Allelic-specific Binding of RNAP to the Genomic DNA in Human Fibroblasts ....................................................................................... 84 Figure 3-5. RNAP Allele-specific Binding at Known Imprinted Genes.................... 85 Figure 3-6. Validation of RNAP ChIP-SNP Results by Sequencing......................... 86 Figure 3-7. A Comparison of RNAP ChIP-SNP Ratios with Sequencing Ratios...... 87 vii LIST OF TABLES Table 2-1. Circle Ratio Mix Results ........................................................................ 54 Table 2-2. EDN1 Induction Results ......................................................................... 55 Table 2-3. RDEP Quantification of ACTG1 and EDN1 Transcripts Levels .............. 56 Table 2-4. ACTG1/EDN1 Ratios from RDEP Analysis............................................ 57 Table 2-5. ADEP Dilution Series Results ................................................................ 58 Table 3-1. RNAP Allele-specific Enriched SNPs and the Nearest RefSeq Genes..... 88 Table 3-2. Allele-specific Bound RefSeq Genes and sno-microRNAs within +/- 5 kb from 466 AS-enriched SNPs............................................................................. 98 Table 3-3. GO Results of Human Imprinted Genes and 466 AS-enriched SNPs....... 99 Table 3-4. Predicted Imprinted Mouse Genes Overlapping with RNAP AS-enriched SNPs ...............................................................................................................100 Table 3-5. Allele-specific Transcription Profiling Results.......................................101 Table 3-6. Transcribed Genes with Heterozygous Enriched SNPs within its Promoter ........................................................................................................................102 viii ACKNOWLEDGEMENTS