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

Tessy Iype, Joshua Francis, James C. Garmey, Jonathan C. Schisler, Rafael Nesher, Gordon C. Weir, Thomas C. Becker, Christopher B. Newgard, Steven C. Griffen, Raghavendra G. Mirmira

Research output: Contribution to journalArticle

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Abstract

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.

Original languageEnglish (US)
Pages (from-to)16798-16807
Number of pages10
JournalJournal of Biological Chemistry
Volume280
Issue number17
DOIs
StatePublished - Apr 29 2005

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Chromatin Immunoprecipitation
RNA Precursors
Gene expression
Chromatin
Transcription Factors
Insulin
Transcription
Genes
Half-Life
Messenger RNA
Machinery
Insulinoma
RNA Polymerase II
RNA-Directed DNA Polymerase
Reverse Transcriptase Polymerase Chain Reaction
Islets of Langerhans
Adenoviridae
Small Interfering RNA
Real-Time Polymerase Chain Reaction
Pancreas

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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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. / Iype, Tessy; Francis, Joshua; Garmey, James C.; Schisler, Jonathan C.; Nesher, Rafael; Weir, Gordon C.; Becker, Thomas C.; Newgard, Christopher B.; Griffen, Steven C.; Mirmira, Raghavendra G.

In: Journal of Biological Chemistry, Vol. 280, No. 17, 29.04.2005, p. 16798-16807.

Research output: Contribution to journalArticle

Iype, Tessy ; Francis, Joshua ; Garmey, James C. ; Schisler, Jonathan C. ; Nesher, Rafael ; Weir, Gordon C. ; Becker, Thomas C. ; Newgard, Christopher B. ; Griffen, Steven C. ; Mirmira, Raghavendra G. / 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. In: Journal of Biological Chemistry. 2005 ; Vol. 280, No. 17. pp. 16798-16807.
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abstract = "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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AU - Schisler, Jonathan C.

AU - Nesher, Rafael

AU - Weir, Gordon C.

AU - Becker, Thomas C.

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AU - Griffen, Steven C.

AU - Mirmira, Raghavendra G.

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