Thesis

Investigation of promoter histone deacetylation as a mechanism for cyclic adenosine monophospate-stimulated repression of microRNA-375

Type 2 Diabetes (T2D) is a disease which affects a large percentage of the United States and the risk of developing T2D is increasing according to recent CDC estimates. T2D can be fatal, and although patients are living longer with it, the costs of caring for this disease are burdensome on both the patients and the health care system. Better understanding of the aberrations in cell signaling during T2D could lead to insights that may restore proper signaling. Investigations of genetic dysregulation in these patients has implicated the overexpression of the small regulatory RNA microRNA(miR)-375. These miRNA are post-transcriptional regulators that function through translational silencing, and two of the genes which miR-375 silences are involved in the pancreatic β-cell viability and insulin exocytosis (phosphoinositide-dependent kinase-1 and myotrophin respectively). The factors which govern the activation and repression of miR-375 are not well known, but it is downregulated by the cyclic adenosine monophosphate (cAMP)/ protein kinase A (PKA) pathway through an unknown mechanism. Since cAMP activates PKA, which then activates histone deacetylase (HDAC) 1 and 2 through phosphorylation, deacetylation is a possible mechanism for repression during this pathway. This study has identified two regions of the miR-375 upstream promoter which are deacetylated at histone H3 lysine 9/14 corresponding with decreased transcriptional initiation by cAMP induction. Observed cAMP-induced deacetylation does not cause overall changes in the chromatin structure of the miR-375 upstream promoter as analyzed by a micrococcal nuclease protection assay. The necessity of HDAC in this process is still unclear due to the broad effects of HDAC inhibition by HDAC inhibitor trichostatin A. While some regions of the promoter are deacetylated during cAMP signaling, the moderate changes it confers suggest that it may only be partially responsible for the repression of miR-375.

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