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Mechanism of insulin gene regulation by the pancreatic transcription factor Pdx-1: application of pre-mRNA analysis and chromatin immunoprecipitation to assess formation of functional transcriptional complexes.

Publication ,  Journal Article
Iype, T; Francis, J; Garmey, JC; Schisler, JC; Nesher, R; Weir, GC; Becker, TC; Newgard, CB; Griffen, SC; Mirmira, RG
Published in: J Biol Chem
April 29, 2005

The homeodomain factor Pdx-1 regulates an array of genes in the developing and mature pancreas, but whether regulation of each specific gene occurs by a direct mechanism (binding to promoter elements and activating basal transcriptional machinery) or an indirect mechanism (via regulation of other genes) is unknown. To determine the mechanism underlying regulation of the insulin gene by Pdx-1, we performed a kinetic analysis of insulin transcription following adenovirus-mediated delivery of a small interfering RNA specific for pdx-1 into insulinoma cells and pancreatic islets to diminish endogenous Pdx-1 protein. insulin transcription was assessed by measuring both a long half-life insulin mRNA (mature mRNA) and a short half-life insulin pre-mRNA species by real-time reverse transcriptase-PCR. Following progressive knock-down of Pdx-1 levels, we observed coordinate decreases in pre-mRNA levels (to about 40% of normal levels at 72 h). In contrast, mature mRNA levels showed strikingly smaller and delayed declines, suggesting that the longer half-life of this species underestimates the contribution of Pdx-1 to insulin transcription. Chromatin immunoprecipitation assays revealed that the decrease in insulin transcription was associated with decreases in the occupancies of Pdx-1 and p300 at the proximal insulin promoter. Although there was no corresponding change in the recruitment of RNA polymerase II to the proximal promoter, its recruitment to the insulin coding region was significantly reduced. Our results suggest that Pdx-1 directly regulates insulin transcription through formation of a complex with transcriptional coactivators on the proximal insulin promoter. This complex leads to enhancement of elongation by the basal transcriptional machinery.

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Published In

J Biol Chem

DOI

ISSN

0021-9258

Publication Date

April 29, 2005

Volume

280

Issue

17

Start / End Page

16798 / 16807

Location

United States

Related Subject Headings

  • Transcription, Genetic
  • Trans-Activators
  • Time Factors
  • Temperature
  • Reverse Transcriptase Polymerase Chain Reaction
  • RNA, Small Interfering
  • RNA, Messenger
  • RNA
  • Protein Structure, Tertiary
  • Promoter Regions, Genetic
 

Citation

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Iype, T., Francis, J., Garmey, J. C., Schisler, J. C., Nesher, R., Weir, G. C., … Mirmira, R. G. (2005). Mechanism of insulin gene regulation by the pancreatic transcription factor Pdx-1: application of pre-mRNA analysis and chromatin immunoprecipitation to assess formation of functional transcriptional complexes. J Biol Chem, 280(17), 16798–16807. https://doi.org/10.1074/jbc.M414381200
Iype, Tessy, Joshua Francis, James C. Garmey, Jonathan C. Schisler, Rafael Nesher, Gordon C. Weir, Thomas C. Becker, Christopher B. Newgard, Steven C. Griffen, and Raghavendra G. Mirmira. “Mechanism of insulin gene regulation by the pancreatic transcription factor Pdx-1: application of pre-mRNA analysis and chromatin immunoprecipitation to assess formation of functional transcriptional complexes.J Biol Chem 280, no. 17 (April 29, 2005): 16798–807. https://doi.org/10.1074/jbc.M414381200.
Iype T, Francis J, Garmey JC, Schisler JC, Nesher R, Weir GC, Becker TC, Newgard CB, Griffen SC, Mirmira RG. Mechanism of insulin gene regulation by the pancreatic transcription factor Pdx-1: application of pre-mRNA analysis and chromatin immunoprecipitation to assess formation of functional transcriptional complexes. J Biol Chem. 2005 Apr 29;280(17):16798–16807.

Published In

J Biol Chem

DOI

ISSN

0021-9258

Publication Date

April 29, 2005

Volume

280

Issue

17

Start / End Page

16798 / 16807

Location

United States

Related Subject Headings

  • Transcription, Genetic
  • Trans-Activators
  • Time Factors
  • Temperature
  • Reverse Transcriptase Polymerase Chain Reaction
  • RNA, Small Interfering
  • RNA, Messenger
  • RNA
  • Protein Structure, Tertiary
  • Promoter Regions, Genetic