Molecular characterization of the bHLH protein Mist1

Thai H Tran, Purdue University

Abstract

The development of the pancreas requires both external and internal cues that orchestrate precise transcriptional regulation to ensure that the mature pancreas is capable of carrying out its endocrine and exocrine functions. Failure of the pancreas to develop properly results in diseases that are associated with either the exocrine or endocrine components. To understand the development of the exocrine tissue, we have examined the function and involvement of a basic Helix-Loop-Helix (bHLH) transcription factor known as Mist1, which has been shown to play a role in maintaining the mature acinar cell phenotype. Mice lacking Mist1 exhibit disrupted acinar cell organization and defective zymogen granule trafficking. Experiments designed to elucidate the molecular mechanism(s) of Mist1's function suggests that Mist1 forms both heterodimer as well as homodimer complexes in vitro and in vivo , although the homodimer complex appears to be the preferred DNA binding complex. CASTing experiments have revealed that Mist1 homodimers bind preferentially to a specific DNA E-box consensus site known as the TA-E-box. Expression assays using artificial promoter gene constructs containing the TA-E-box suggest that Mist1 is capable of functioning as a transcriptional activator, and we have mapped the region that is responsible for this activity to be within the bHLH domain. In addition, Mist1 transcriptional activity may be mediated by a specific class of co-activators belonging to the p300/CBP family. These results suggest that Mist1 activates exocrine-specific gene transcription through an acetylation mechanism. However, we also discovered that Mist1 can potentially function as a transcriptional repressor. In an attempt to further investigate Mist1's role in exocrine pancreas development, we employed ChIP-on-Chip assays to identify Mistl target genes. This resulted in 3 potential Mist1 target genes: Chymotrypsin-like factor (CTRL), Neuregulin4 (Nrg4), and Reg3δ. Our analysis of Nrg4 using RT-PCR, immunoblot, and immunohistochemistry assays, coupled with reporter gene analyses, suggested that Nrg4 is, indeed, repressed by Mist1. Future studies will focus on confirming the in vivo interactions between Mist1 and its various protein partners and determining the effects that these complexes have on the overall transcriptional regulation of the Nrg4 gene.

Degree

Ph.D.

Advisors

Konieczny, Purdue University.

Subject Area

Molecular biology

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