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Computational methods for studying gene regulation and genome organization using high-throughput DNA sequencing

  • Author(s): Bonora, Giancarlo A
  • Advisor(s): Plath, Kathrin
  • Pellegrini, Matteo
  • et al.
Abstract

The full sequencing of the human genome ushered in the genomics era and laid the foundation for a more comprehensive understanding of gene regulation and development. But, since the DNA sequence represents only one aspect of the genomic information housed within the nucleus, the question of exactly how it is utilized to direct developmental programs and tissue-specific gene expression is still an open one. However, rapid advances in high-throughput DNA sequencing (HTS) technologies over the past decade have allowed biologists to begin to tackle the question on a genomic scale. HTS has been coupled to bisulfite conversion of DNA for assessing cytosine methylation (bisulfite sequencing), to chromatin immunoprecipitation for ascertaining genomic locations bound by specific factors or found in a particular chromatin state (ChIP-seq), to the isolation of transcripts for the measurement of gene expression (RNA-seq), and to methods of chromosome conformation capture for the identification of genome-wide DNA-DNA interactions (4C-seq and Hi-C). The focus of my doctoral research has been the development of novel bioinformatics approaches to analyze the data produced by these technologies in order to shed light on how distinct cell identities are established and maintained. Here, I present highlights of this work in six chapters. Chapter 1 presents a study investigating DNA methylation changes going from the differentiated to pluripotent state, which shows that changes predominantly occur late in the process and are strongly associated with changes to chromatin state. Chapter 2 introduces methylation-sensitive restriction enzyme bisulfite sequencing (MREBS) as a method for assessing precise differential DNA methylation at cost comparable to RRBS, while providing additional information over a coverage area more comparable to WGBS. Chapter 3 presents a study showing that inhibition of ribonucleotide reductase decreased DNA methylation genome-wide by enhancing the incorporation of a cytidine analog into DNA. Chapter 4 describes a study showing that, for genes important to leaf senescence, temporal changes in expression closely matched changes to two histone modifications. Chapter 5 reviews cutting-edge research exploring the link between regulatory networks and genome organization. Chapter 6 describes a study showing that regulators responsible for cell identity contribute to cell type-specific genome organization.

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