? Insulin production in the ? cell is controlled primarily at the level of transcription, a process which is regulated through the action of transcription factors specifically expressed in the ? cell and via histone modification of insulin gene chromatin structure. The overall goal of this project will be to further investigate the process whereby insulin gene activation occurs and to define acute and chronic changes that occur in chromatin structure in response to nutrients such as glucose. I hypothesize that transcription of the insulin gene is regulated, in part, through changes in chromatin, which are mediated by Pdx-1, a member of the homeobox class of transcription factors. I further hypothesize that in states of disease, like diabetes mellitus, there are defects at the chromatin level that alter gene expression patterns.
In Aim 1 of this proposal, I will characterize the role of Pdx-1 in glucose-stimulated insulin gene transactivation, using Pdx-1 deletion studies in whole animals and isolated human islets. In this aim, I will establish how Pdx-1 functionally links extracellular glucose levels to insulin transcription, histone modifications, and metabolic control.
Aim 2 will characterize the interactions of Pdx-1 with novel components of the basal transcriptional machinery in the ? cell, concentrating initially on the interaction of Pdx-1 with Paf1, a protein that associates with RNA polymerase II. Other novel interacting proteins will be identified using MALDI-TOF mass spectrometry, and interactions will be verified by mammalian two-hybrid assay. The biological relevance of these interactions in the ? cell will be tested using siRNA and chromatin immunoprecipitation assays.
In Aim 3, I will characterize defects in histone modification and turnover in hyperinsulinemic and hypoinsulinemic rodent models of Type 2 diabetes and human islets chronically exposed to high glucose, using chromatin analysis techniques, such as the micrococcal digest assay and chromatin immunoprecipitation. Relevance: Diabetes mellitus (DM) is a metabolic disease that results from either a complete or relative deficiency of the hormone insulin. DM currently affects 20.8 million people in the U.S. and is increasing in incidence. The cost of caring for this disease is enormous and exceeds over 100 billion dollars annually. In this proposal, the precise mechanisms by which the pancreas produces the hormone insulin will be explored at the level of the gene encoding insulin. Research of this type has the potential to guide the development of new therapies for DM. ? ? ?
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