Date of Award

January 2020

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biomedical Sciences

First Advisor

Sergei Nechaev

Second Advisor

Min Wu

Abstract

Precise regulation of gene expression is essential for maintaining cell homeostasis and survival. Rapid responses to intracellular or extracellular stimuli involve highly regulated genome-wide changes in the transcriptional landscape. However, works on transcriptional regulation thus far has been primarily focused on genes and gene promoters. Consequently, ubiquitous transcription outside of genes has been greatly neglected. Therefore, in order to understand genome-wide transcriptional changes with stimuli, we looked at transcription factor distribution, RNA Polymerase II (Pol II) dynamics, histone changes, chromatin accessibility at both promoters and promoter-distal regions along with changes in gene transcription. With this approach, we were able to create a more comprehensive picture of how cells respond when exposed to stimuli.

Previous studies in mouse and human cells have shown with heat stress at elevated temperature, there is massive genome-wide binding of transcription factor HSF1 (Heat-Shock Factor 1) (Mahat et al., 2016; Vihervaara et al., 2017). Genome-wide analyses revealed there are approximately 280,000 HSF1 binding elements in the human genome. However, with heat-shock, HSF1 binds to less than 4% of these sites indicating HSF1 binding might be context-dependent. To address whether HSF1 binding depends upon stimulus and/or cell-type, we exposed two different cell types, MCF7 breast cancer and K562 chronic myelogenous leukemia cells to a heat-dependent (heat stress at 43°C) and a heat-independent stress (Arsenic) and compared genome-wide changes in HSF1 distribution, histone marks, RNA Pol II and nascent transcription. We found HSF1 binds across the entire human genome during HSR and is a highly sensitive indicator of global heat-shock response (HSR) among the marks we tested. This genome-wide HSF1 binding is independent of stress type but is dependent on cell type. This cell type specific difference in HSF1 binding is more prominent at distal intergenic regions rather than at promoters. The mechanism of this difference in HSF1 binding in different cell types might not be dependent on chromatin accessibility as detected by ATAC-sequencing but might be potentially pre-determined at ground state of cells maintaining cells in a poised state for rapid orchestration of HSR.

We next studied the dynamics of RNA Polymerase (Pol II) regulation and gene expression under different stresses and different human cell types. HSR has been associated with massive repression of gene expression due to elevated temperature (Mahat et al., 2016; Vihervaara et al., 2017). We found this repression is cell type dependent and takes place by distinct cell-specific mechanisms. Regulation of gene expression at the level of Pol II can occur at two steps: (i) during recruitment of Pol II at promoter and (ii) during release of Pol II from promoter-paused site to gene body. We found this mechanism of recruitment versus release at promoter differs between cell types and can, in turn, explain the cell-type specific extent of repression.

These studies indicated that distal intergenic regions or potential enhancer regions might be important regulatory elements showing significant transcriptional changes with stress. Therefore, we next utilized another biological model of IL-1β -induced inflammation in lung adenocarcinoma cells (A549) to investigate the role of enhancers in gene regulation. We found that rapid genome-wide transcriptional changes take place in A549 cells induced with IL-1β. Transcription at genes and their nearest predicted enhancers display similar patterns of expression changes with inflammation. We further found that RNA production at genes and enhancers are not separated by time but are co-transcriptional events.

In conclusion, this dissertation demonstrates that cells undergo rapid and robust genome-wide transcriptional changes with inflammatory or environmental stresses affecting gene-promoters, gene-bodies as well as extragenic regulatory elements such as enhancers. The mechanisms of these transcriptional changes to stress responses in different cell types under different conditions might be determined during cell lineage specification and is probably dependent on the availability of cell-specific transcriptional regulators involved in the recruitment and release of Pol II at gene regulatory elements.

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