Date of Award

2022

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Graduate Group

Cell & Molecular Biology

First Advisor

Kenneth S. Zaret

Abstract

The hundreds of cell types that compose human body tissues contain identical genomes yet vary considerably in their gene activity and protein content. While the underlying blueprints that define these diverse cell types exist in all cells, a given cell’s specific transcriptomic and proteomic landscape is controlled by an incredibly complex system of gene regulatory complexes and epigenetic marks which promote activation of cell-type specific gene expression profiles and silence alternative cell fate genes. However, the mechanism by which specific genomic loci are silenced in different cell types is still not well understood. Here I strove to understand the vital contribution of tightly compacted heterochromatin on cell identity at specific loci. I studied how 97 unique heterochromatin-associated human proteins play a role in heterochromatin-based gene and repetitive element silencing. As H3K9me3 can block cellular reprogramming, we used a human-induced fibroblast to hepatocyte (hiHep) reprogramming model to uncover diverse genes and repetitive elements silenced by each protein. We conducted RNA-seq experiments both with and without the addition of hiHep reprogramming factors. We identified four unique clusters of these proteins based on their common targets of repression, including differences in H3K9me3 and H3K27me3 marks across genes derepressed by each cluster. In an additional study, I investigated how KRAB domain-containing zinc finger proteins (KRAB-ZFPs) might silence hepatic genes in alternate lineages. I identified six putative liver lineage antagonistic KRAB-ZFPs. A mostly unstudied primate-specific KRAB-ZFP known as ZNF695 became the top candidate for allowing hundreds of genes, including hepatic genes, to be derepressed following knockdown in the hiHep reprogramming model and facilitated the widespread loss of H3K9me3. I mapped ZNF695 localization throughout the genome and tied these patterns to the observed gene expression changes. Additionally, we were able to see that depletion of ZNF695 in hiHeps transplanted in a diseased liver mouse model were able to produce higher levels of human albumin that hiHeps without the knockdown. Together, these findings greatly expanded our knowledge of H3K9me3-based repression in humans and provided a novel picture of ZNF695 regulatory capabilities.

Files over 3MB may be slow to open. For best results, right-click and select "save as..."

Share

COinS