Date of Award

Spring 1-1-2016

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Chemistry & Biochemistry

First Advisor

Xuedong Liu

Second Advisor

Robin D. Dowell

Third Advisor

Thomas Cech

Fourth Advisor

James Goodrich

Fifth Advisor

Dylan Taatjes

Abstract

Histone deacetylases (HDACs) regulate gene expression through deacetylation of histones and non-histone proteins. Histone deacetylase inhibitors (HDACIs) are known to alter gene expression by both up- and down-regulation of protein coding genes in normal and cancer cells. However, the mechanisms that are responsible for activation and repression of gene expression remain obscure. To understand the underlying mode of action of HDACIs, I used genome wide chromatin immunoprecipitation sequencing (CHIP-seq) to determine dose-dependent changes in histone acetylation and methylation marks in HCT116 cells treated with increasing concentrations of the natural product largazole, a class I and class IIb selective HDAC inhibitor. Changes in mRNA expression were also measured by RNA-seq under similar conditions. I found that cells exposed to low nanomolar concentrations (GI50) triggered a decrease in mRNA accumulation.

Largazole’s effect on transcription is likely associated with its activity on enhancer elements rather than on gene bodies. Low dose largazole exposure elevates the activity of ~1600 poised enhancers characterized by increased H3K9/27ac and H3K4me2 marks in addition to RNA pol II recruitment, suggesting class I HDACs are involved in maintaining the repressive state of poised enhancers. Mid to high dose largazole appears to directly repress transcription of many genes, an effect correlated with increased RNA Pol II pausing at the promoters of most actively transcribed genes. Under such conditions, I found ~800 putative enhancers become decommissioned with the features of loss of H3K9/27ac, reduction of H3K4me2 and the decrease of RNA Pol II occupancy. A significant number of primed (~47%) and decommissioned enhancers (~22%) display the recognition motif for the AP-1 transcription factor. Therefore, HDAC inhibitors sculpt the enhancer landscapes in a dose-dependent manner to impact gene expression and cytostatic responses.

Collectively, the data presented here challenge traditional assumptions that HDACI-driven chromatin hyperacetylation of promoters and gene regions results in positive stimulation of transcription. This study represents the most comprehensive analysis to date showing largazole dose dependent genome-wide acetylation changes and has helped identify the activation of a large cis-regulatory network as a novel output from HDAC inhibition.

Share

COinS