An Investigation of Regulatory and Chromatin Drivers of Cancer

Abstract

Chromatin and its regulatory elements are crucial for controlling gene expression and maintaining cellular identity. Chromatin, a dynamic structure made of DNA and histone proteins, is organized into nucleosomes. Modifications to histones and chromatin architecture regulate DNA accessibility for transcriptional machinery, thus influencing gene activity. Besides core histone modifications, various regulatory elements like promoters, enhancers, and silencers coordinate gene expression patterns essential for development and cellular function. Disruption of these processes, whether through mutations in non-coding regulatory regions or changes in chromatin-modifying proteins, can lead to cancer progression. This thesis investigates the impact of mutations in regulatory elements and chromatin regulators in cancer, with a focus on breast cancer and Germinal Center B-cell-like Diffuse Large B-cell Lymphoma (GCB-DLBCL). The first part of the thesis explores non-coding regulatory elements in breast cancer. Traditionally, cancer research concentrated on mutations in protein-coding genes, but there is increasing awareness of the significant role non-coding mutations play in tumorigenesis by influencing gene expression. Integrating multi-omic techniques such as ATAC-seq, ChIP-seq, RNA-seq, and promoter-capture HiC (PcHiC) across breast cancer cell lines, we characterized the landscape of active and repressive transcriptional regulatory elements. Our study revealed, cell-type specificity, subtype clustering, and bi-functionality between active and repressive distal elements. Notably, we found an enrichment of non-coding mutations at an active enhancer looping to ADGRG6, a gene previously found to be a driver of bladder cancer. The second part of this thesis focuses on GCB-DLBCL, and specifically the EZH2-mutant subtype. EZH2, the catalytic subunit of PRC2, is often mutated in GCB-DLBCL, causing aberrant tri-methylation of histone H3K27 and excessive gene repression. Our research identified AEBP2, an accessory protein of PRC2, as a novel genetic dependency in EZH2-mutant GCB-DLBCL via genome-wide CRISPR-Cas9 screens. Functional studies showed that AEBP2 knockdown led to increased global levels of H3K27me3 and decreased H3K27me2, highlighting AEBP2's complex role in regulating PRC2 activity. These findings suggest that targeting AEBP2 alongside EZH2 inhibitors may offer a new therapeutic strategy for overcoming resistance in lymphoma treatment. In summary, I believe that these results advance our understanding of how chromatin modifications and regulatory elements drive cancer progression. By elucidating the roles of non-coding regulatory mutations in breast cancer and a novel dependency of GCB-DLBCL, this research provides new insights into the molecular mechanisms underlying these diseases.