Chromatin Determinants of the Eukaryotic DNA Replication Program by Matthew L. Eaton Program in Computational Biology and Bioinformatics Duke University Date: Approved: David M. MacAlpine, Supervisor Fred S. Dietrich Terrence S. Furey Alexander J. Hartemink Laura N. Rusche Dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Program in Computational Biology and Bioinformatics in the Graduate School of Duke University 2011 ABSTRACT (0715) Chromatin Determinants of the Eukaryotic DNA Replication Program by Matthew L. Eaton Department of Computational Biology and Bioinformatics Duke University Date: Approved: David M. MacAlpine, Supervisor Fred S. Dietrich Terrence S. Furey Alexander J. Hartemink Laura N. Rusche An abstract of a dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Program in Computational Biology and Bioinformatics in the Graduate School of Duke University 2011 Copyright © 2011 by Matthew L. Eaton All rights reserved Abstract The accurate and timely replication of eukaryotic DNA during S-phase is of critical importance for the cell and for the inheritance of genetic information. Missteps in the replication program can activate cell cycle checkpoints or, worse, trigger the genomic instability and aneuploidy associated with diseases such as cancer. Eu- karyotic DNA replication initiates asynchronously from hundreds to tens of thou- sands of replication origins spread across the genome. The origins are acted upon independently, but patterns emerge in the form of large-scale replication timing domains. Each of these origins must be localized, and the activation time deter- mined by a system of signals that, though they have yet to be fully understood, are not dependent on the primary DNA sequence. This regulation of DNA replica- tion has been shown to be extremely plastic, changing to fit the needs of cells in development or effected by replication stress. We have investigated the role of chromatin in specifying the eukaryotic DNA replication program. Chromatin elements, including histone variants, histone mod- ifications and nucleosome positioning, are an attractive candidate for DNA replica- tion control, as they are not specified fully by sequence, and they can be modified to fit the unique needs of a cell without altering the DNA template. The origin recognition complex (ORC) specifies replication origin location by binding the DNA of origins. The S. cerevisiae ORC recognizes the ARS (autonomously replicating sequence) consensus sequence (ACS), but only a subset of potential iv genomic sites are bound, suggesting other chromosomal features influence ORC binding. Using high-throughput sequencing to map ORC binding and nucleosome positioning, we show that yeast origins are characterized by an asymmetric pattern of positioned nucleosomes flanking the ACS. The origin sequences are sufficient to maintain a nucleosome-free origin; however, ORC is required for the precise positioning of nucleosomes flanking the origin. These findings identify local nu- cleosomes as an important determinant for origin selection and function. Next, we describe the D. melanogaster replication program in the context of the chro- matin and transcription landscape for multiple cell lines using data generated by the modENCODE consortium. We find that while the cell lines exhibit similar repli- cation programs, there are numerous cell line-specific differences that correlate with changes in the chromatin architecture. We identify chromatin features that are associated with replication timing, early origin usage, and ORC binding. Pri- mary sequence, activating chromatin marks, and DNA-binding proteins (including chromatin remodelers) contribute in an additive manner to specify ORC-binding sites. We also generate accurate and predictive models from the chromatin data to describe origin usage and strength between cell lines. Multiple activating chro- matin modifications contribute to the function and relative strength of replication origins, suggesting that the chromatin environment does not regulate origins of replication as a simple binary switch, but rather acts as a tunable rheostat to regu- late replication initiation events. Taken together our data and analyses imply that the chromatin contains suffi- cient information to direct the DNA replication program. v To my parents, Jonathan and Mariellen Eaton, who taught me how to think and in so doing taught me everything I know. To my wife-to-be, Dr. Hilary Wade, my reverse complement. vi Contents Abstract iv List of Tables xi List of Figures xii List of Abbreviations and Symbols xiv Acknowledgments xvi 1 Introduction1 1.1 DNA replication and the eukaryotic DNA replication program....3 1.1.1 Overview of the eukaryotic DNA replication program.....4 1.1.2 Regulation of the eukaryotic DNA replication program.... 19 1.1.3 Genomic tools for analysis of replication............ 28 1.2 Chromatin................................. 37 1.2.1 Overview of chromatin...................... 38 1.2.2 Genomic tools for analysis of chromatin............ 43 1.3 Regulation of the replication program by chromatin.......... 45 1.3.1 Nucleosome positioning..................... 46 1.3.2 Chromatin features and origin localization........... 48 1.3.3 Chromatin features and origin initiation............ 50 1.4 Thesis roadmap.............................. 53 vii 2 Conserved Nucleosome Positioning Defines Replication Origins in S. cerevisiae 57 2.1 Introduction................................ 57 2.2 Results................................... 59 2.2.1 Identification of functional ACSs................ 59 2.2.2 Nucleosome positioning at origins of replication........ 61 2.2.3 Nucleosome depletion at origins is encoded by sequence... 65 2.2.4 ORC is necessary and sufficient for precise nucleosome posi- tioning............................... 70 2.3 Discussion................................. 74 2.3.1 Model for ORC specificity.................... 76 2.3.2 NFR architecture......................... 77 2.3.3 Connection to origin activity and extension to metazoans... 78 2.4 Methods.................................. 79 2.4.1 Data deposition.......................... 79 2.4.2 Yeast strains and growth conditions............... 79 2.4.3 Chromatin immunoprecipitation and mononucleosome prepa- ration............................... 80 2.4.4 Reconstituted chromatin assembly............... 80 2.4.5 High-throughput sequencing.................. 80 2.4.6 Mapping ORC binding sites by ChIP-seq............ 81 2.4.7 Comparison of ORC ChIP-seq peaks with prior ChIP-chip peaks 81 2.4.8 Identification of functional ACS matches............ 82 2.4.9 Mapping nucleosome positions................. 83 2.4.10 Visualization of nucleosomes.................. 84 2.4.11 Nucleosome positioning at genomic features.......... 85 2.4.12 Construction of the nr-ACS set................. 86 viii 2.4.13 Sequence analysis around genomic features.......... 87 2.4.14 Purification of ORC and ABF1.................. 88 3 Chromatin signatures of the Drosophila replication program 89 3.1 Introduction................................ 89 3.2 Results................................... 92 3.2.1 Characterization of the Drosophila replication program in three cell lines.......................... 92 3.2.2 ORC enrichment at genic elements............... 96 3.2.3 Diverse chromatin marks define the replication program... 97 3.2.4 Potential cis and trans-acting elements directing ORC associ- ation................................ 103 3.2.5 Predicting early origin usage from the chromatin landscape. 110 3.3 AWG analysis............................... 118 3.3.1 Chromatin state 3........................ 118 3.3.2 HOT spots............................. 121 3.4 Discussion................................. 123 3.5 Methods.................................. 127 3.5.1 Cell growth............................ 127 3.5.2 ChIP-seq.............................. 127 3.5.3 Read mapping and peak calling................. 127 3.5.4 Replication timing........................ 128 3.5.5 Early origin mapping...................... 128 3.5.6 DNA extraction.......................... 129 3.5.7 BrdU immunoprecipitation................... 129 3.5.8 Array hybridization and analysis................ 130 3.5.9 Meta-peaks............................ 130 ix 3.5.10 ORC at genic loci......................... 131 3.5.11 SVM analysis........................... 131 3.5.12 Motif analysis........................... 132 3.5.13 Chromatin enrichment heatmaps................ 132 3.5.14 Regression Analysis........................ 133 3.5.15 Data accession.......................... 135 4 Conclusion 136 4.1 Summary and Discussion......................... 136 4.2 Future Directions............................. 142 5 Appendix A – Aneuploidy analysis 146 5.1 Results................................... 148 5.1.1 Model............................... 148 5.1.2 Interpreting calls......................... 149 5.1.3 Initial results........................... 150 5.1.4 Future directions......................... 151 5.2 Conclusion................................ 154 Bibliography 156 Biography 185 x List of Tables 3.1 Pearson correlation of whole-genome replication timing between cell lines.................................. 93 3.2 Top ORC binding SVM features by F-score................ 109 xi List of Figures 1.1 ARS1....................................7 1.2 The chorion locus............................. 11 1.3 Origin licensing and activation.....................