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Gene activity patterns associated with pathological cardiac hypertrophy are mediated by specific chromatin modifying factors
thesisposted on 2017-02-28, 23:41 authored by Chang, Lisa Hsiu Chuan
Pathological cardiac hypertrophy is an initial compensatory response of the heart to a range of intrinsic stimuli including arterial hypertension, myocardial infarction and cardiomyopathy whereby prolonged stress often results in congestive heart failure and sudden death. This condition is commonly associated with the reactivation of the foetal gene program. Recent work has uncovered the importance of chromatin remodeling in the control of gene expression in heart disease. We used a transverse aortic constricted (TAC) mouse model to induce hypertrophy. The increased expression of Nppa, Nppb and embryonic Myh7 were inversely correlated with reduced expression of the adult Myh6 and Atp2a2 genes in TAC animals. We examined the regulatory complexes associated with hypertrophy and changes in the transcriptional response using strategies that allowed us to immunopurify soluble chromatin fractions. Chromatin immunopurifications were performed on the left ventricles of SHAM and TAC cardiac tissues. Quantification of the immunopurified chromatin indicated a unique pattern of binding on the promoters of Nppa, Nppb, Myh6 and Myh7 genes. Our findings suggested that the ATP-dependent chromatin remodeling complex, SWI/SNF, could act in a coordinated fashion with histone acetyltransferase (HAT) or histone deacetylase (HDAC) complexes to regulate the expression of these genes in the hypertrophic heart. SWI/SNF complex serves as a co-regulator in the development of pathological cardiac hypertrophy. We observed enrichment of SWI/SNF subunit BRM and p300 HAT during the reactivation of the foetal gene program on the promoters of upregulated Nppa, Nppb and Myh7 genes. We also observed the recruitment of the SWI/SNF subunit BRG1 and HDAC2 on the Myh6 gene which was consistent with its suppressed gene expression in the hypertrophic heart. The data presented indicate that components of SWI/SNF machinery are associated with diverse regulatory mechanism and the suppression and activation of gene expression. Suppression of HDACs is known to blunt pressure-overload cardiac hypertrophy. However, the molecular mechanism behind this blockade remains unclear. We used a broad-spectrum HDAC inhibitor, Trichostatin A (TSA) to investigate hypertrophy prevention in a mouse model of TAC. TSA treatment resulted in the downregulation of Nppa, Nppb and embryonic Myh7 which was intrinsically highly expressed in the hypertrophic heart. The observed changes in gene expression were found to be associated with concurrent release of BRM, H3K9/14 acetylation and recruitment of BRG1, HDAC2 on suppressed Nppa, Nppb and Myh6 gene promoters in the TSA treated TAC animals. This study described the reciprocoal association of SWI/SNF subunits, BRG1 and BRM, with histone modifications correlated with the regulation of cardiac gene transcription in pathological hypertrophy and their regulatory function in response to TSA exposure. To determine changes in the expression of genes mediated by pathological cardiac hypertrophy, we used a global approach using RNA-Seq. In recent years, high-throughput technologies have been developed and rapidly improved to interrogate several aspects of cellular processes. RNA-Seq was used to map global mRNA expression profile, providing a more sensitive approach than microarrays. It allowed the identification of rare transcripts and gene isoforms which the array approach was unable to detect. This was followed by further investigation using bioinformatics resources such as gene enrichment analysis, pathways, and regulatory network analysis. This enabled us to classify the differentially expressed genes and transcript isoforms into functional categories. A major challenge in deciphering the molecular mechanism associated with the development and prevention of cardiac hypertrophy is the identification of regulatory determinants that are thought to regulate gene expression. We have identified that SWI/SNF chromatin remodeling complex, more specifically, BRM was associated with HAT whereas BRG1 was associated with HDAC2 in correlation with gene expression. Further investigation of regulatory determinants mediated gene expression would need to be carried out to provide a useful framework for understanding and distinguishing the regulatory function of SWI/SNF in pathological cardiac hypertrophy. These studies could lead to more precise understanding of heart disease and potential new strategy to personalised therapies to prevent or reverse cardiac hypertrophy and in turn, cardiac failure.