Development and application of chemical genomic approaches to study the factors mediating heterochromatin maintenance and organization

Abstract

A single genome can give rise to a diversity of phenotypes, as evidenced by the existence of over 200 distinct cell types containing the same genetic material within the human body. Many factors, ranging from DNA methylation and histone modifications to the overall three-dimensional organization of DNA in the nucleus, have been shown to influence the differential gene expression patterns that allow cells to perform their specialized functions. It has become increasingly appreciated that these factors do not act in isolation but instead exhibit complex interdependent relationships that are tightly controlled to ensure proper cellular function. To fully uncover the complexity of this regulation, new integrative approaches paired with precise perturbations are needed to probe the relationship between structure and function not only at a biochemical level, such as how small molecules impact chromatin regulator binding and catalysis, but also at a genome-wide level, such as how the epigenomic landscape and nuclear organization influence gene expression.

To address these interdependencies, in this thesis, I describe the development and application of two approaches. This first can be leveraged to provide an integrative view of three features of epigenome structure in a single workflow. The second enables the systematic discovery of drug resistance mutations to disentangle the diverse functions of epigenomic regulators. Specifically, in Chapter 2, I discuss our efforts towards the development of an approach that can jointly measure DNA-protein interactions and DNA conformation by imprinting information about protein occupancy as a mutational signature that is detectable through sequencing. In Chapter 3, I describe Lamina-Inducible Methylation and Hi-C (LIMe-Hi-C) that leverages this strategy to simultaneously measure DNA conformation, nuclear lamina association, and endogenous DNA methylation. We subsequently apply this approach with chemical inhibition of DNA methyltransferase 1 (DNMT1) and Polycomb repressive complex 2 (PRC2), the complex responsible for installing the facultative heterochromatic histone modification, H3K27me3. Our studies reveal that chromatin compartmentalization and lamina association are distinctly regulated. In addition, through genetic and chemical perturbations, we discover an unexpected structural antagonism between lamina association and facultative heterochromatin that has implications on gene expression.

In Chapter 4, I describe our efforts applying CRISPR-suppressor scanning to systematically identify drug resistance mutations in the histone demethylase, Lysine-Specific histone Demethylase 1 (LSD1). This work led to the identification of a mutation that abolishes LSD1 catalytic activity but still confers a robust survival advantage. By using this mutation to study the catalytic versus non-catalytic functions of LSD1, we demonstrate how drug resistance mutations more generally can serve as powerful tools to disentangle the multifaceted roles of chromatin regulators.

In Chapter 5, I suggest future applications of the LIMe-Hi-C methodology as well as experiments that could enhance our understanding of the antagonism between facultative heterochromatin and lamina association. Finally, I discuss the potential of integrating our knowledge of mutations, such as those discovered through CRISPR-suppressor scanning, with genome structure mapping studies to elucidate the influence of epigenomic regulators, including PRC2, on the many layers of chromatin organization.