Author ORCID Identifier

0000-0003-1250-7864

Date of Award

2024

Document Type

Thesis (Ph.D.)

Department or Program

Cancer Biology

First Advisor

Xiaofeng Wang

Second Advisor

Steve Leach

Third Advisor

Brock Christensen

Abstract

Cell identity is defined by the epigenome, whereby chromatin regulators work in concert to promote gene expression programs that serve a cell’s specialized purpose. Mutations in chromatin regulators are amongst the most frequent drivers of human disease, underscoring the importance of understanding their activities in maintaining cell identity and tissue function. In particular, mutations in subunits of the evolutionarily conserved SWI/SNF chromatin remodeling complexes drive diseases across human tissues in both development and adult tissue maintenance5. Three major SWI/SNF complexes exist: BAF, PBAF, and GBAF, which differ in their composition and genomic targeting but share an ATP-dependent catalytic activity to bind and mobilize nucleosomes. SWI/SNF complexes control chromatin access at cis-regulatory elements known as enhancers, promoters, and insulators which are dense in transcription factor binding sites and undergo dynamic regulation by lineage-specific chromatin regulators. Thus, the sheer biochemical complexity and genome wide functions have made it difficult to grasp the rules that govern SWI/SNF functions in vivo. Despite their enrichment at lineage-specific regulatory elements, SWI/SNF chromatin functions have been largely investigated in cancer cell lines that suffer from already compromised genomes and epigenomes. No deep investigation has been made into their homeostatic contributions to somatic stem cells, which underpin disease origins. We sought to address these gaps in knowledge using CRISPR-Cas9 gene editing and state-of-the-art chromatin assays in human colon organoids. Colorectal cancers and inflammatory bowel diseases frequently incur ARID1A mutations in the colon epithelium. This presented a relevant paradigm to model BAF complex function in lineage competent adult stem cells. Using organoid models, we identified robust cellular and molecular evidence for the pathogenic relevance of ARID1A loss-of-function in inflammatory bowel diseases and colorectal cancers. We also revealed temporal dynamics of BAF complex cooperation with lineage-specific transcription factors NFIX, ELF3, HNF4A, CDX2, and AP-1 during colonic stem cell renewal and differentiation. Loss of ARID1A impaired stem cell differentiation to both absorptive and secretory lineages. These phenotypes are further inspected by single cell RNA and chromatin accessibility profiles showing loss of open chromatin at cell-type-specific enhancers. Taken together, these studies underscore ARID1A as a crucial regulator of the colonic epithelium and support a pleiotropic disease driving mechanism for ARID1A loss of function in human disease.

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