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The role of chromatin modifiers and DNA methylation in transcription regulation during cardiac hypertrophy

thesis
posted on 2017-02-09, 03:11 authored by Ooi, Jenny Ying Ying
At the cellular level, the hypertrophied myocardium is characterized by profound changes in gene transcription. Published literature suggests that histone acetylation/methylation as well as SWI/SNF chromatin remodeling factors may play a role in transcription regulation during cardiac hypertrophy. In addition, histone deacetylase inhibitors (HDACi) are able to attenuate cardiac hypertrophy and the gene program that accompanies the stressed heart. In this thesis, the role of chromatin modification as well as DNA methylation in gene regulation was explored in animal models of cardiac hypertrophy by defining protein-protein and protein-DNA complexes. SWI/SNF protein complexes were examined using coimmunoprecipitation with the SWI/SNF determinant, BRG1, coupled to mass spectrometry (ms). Due to the unexpected limitation of MALDI-ToF-tof ms and the abundance of contractile proteins in the heart, nuclear proteins of interest, including BRG1, could not be detected. An alternative approach was to examine protein-DNA complexes using chromatin immunoprecipitation (ChIP). Histone H3 lysine 9/14 acetylation (H3K9/K14ac) was characterized by massive parallel sequencing (ChIP-Seq), whereas gene expression changes determined by microarray. A mouse model of transverse aortic constriction (TAC) allowed investigation of cardiac hypertrophy, which was attenuated by the HDAC inhibitor, Trichostatin A (TSA). In response to TAC, the majority of genes have reduced H3K9/K14ac content on their promoters. Although TSA induced an increase in global H3K9/K14ac, gene promoters were also hypoacetylated. Differential H3K9/K14ac on a promoter did not necessarily correspond to alterations in gene expression during TAC or TSA prevention. We next focused on gene regulatory epigenetic changes on SERCA2a (a gene target identified from H3K9/K14ac ChIP-Seq). In response to TAC-induced-pathological hypertrophy, reduction in active histone marks as well as enrichment of repressive marks was consistent with suppression of SERCA2a gene expression. In the constitutively active PI3K animals (model of physiological hypertrophy), reduced histone H3 lysine 9 trimethylation (H3K9me3) levels were also associated with SERCA2a gene activation. Taken together, these experimental results suggest that the contribution of H3K9/K14ac to gene expression is not straightforward in the stressed heart. In addition, the data suggests a surprising complexity of gene regulation events that go beyond the traditional view of HDACi mediated histone hyperacetylation in cardiac hypertrophy.

History

Principal supervisor

Assam El-Osta

Year of Award

2013

Department, School or Centre

Medicine

Campus location

Australia

Course

Doctor of Philosophy

Degree Type

DOCTORATE

Faculty

Faculty of Medicine Nursing and Health Sciences

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