Pdx-1 regulation of the INGAP promoter involves sequestration of NeuroD into a non-DNA-binding complex

Pancreas. 2010 Jan;39(1):64-70. doi: 10.1097/MPA.0b013e3181baa0cc.

Abstract

Objective: Islet neogenesis-associated protein (INGAP) can enhance beta-cell mass to offset progression of diabetes. Identifying how transcription factors regulate INGAP gene expression could reveal key checkpoints governing islet neogenesis.

Methods: Protein complex interactions at the INGAP promoter were detected using a beta-galactosidase reporter, these protein-DNA complexes being validated in competitive electrophoresis mobility shift assays. The relevance of the revealed promoter interactions was confirmed in small interfering RNA (siRNA) gene knockdown studies.

Results: Pdx-1 negatively regulates stimulation of the INGAP promoter by Pan-1/NeuroD. Independently, Pdx-1, Pan-1, and NeuroD bind to the INGAP promoter as revealed by electrophoresis mobility shift assay studies. In combination, Pdx-1 selectively displaces NeuroD from a DNA-binding complex with Pan-1 to form a non-DNA-binding unit. The importance of this interaction is shown in HIT cells that have a forced reduction of Pdx-1 expression. In siRNA/Pdx-1-depleted HIT cells, the interaction of Pan-1/NeuroD with the INGAP promoter is increased 6-fold. Furthermore, endogenous INGAP expression is detected in Pdx-1-depleted cells.

Conclusions: These data reveal a dynamic interaction between Pdx-1, NeuroD, and Pan-1 for the regulation of INGAP promoter activity. Modulating molecular regulators of DNA expression may be a consideration in diabetic therapies that translate exogenous stimuli into new endogenous beta-cell mass.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Antigens, Neoplasm / genetics
  • Basic Helix-Loop-Helix Transcription Factors / genetics
  • Basic Helix-Loop-Helix Transcription Factors / metabolism*
  • Biomarkers, Tumor / genetics
  • Blotting, Western
  • Cell Line
  • Cell Line, Tumor
  • Cricetinae
  • DNA / genetics
  • DNA / metabolism
  • Electrophoretic Mobility Shift Assay
  • Homeodomain Proteins / genetics
  • Homeodomain Proteins / metabolism*
  • Humans
  • Insulinoma / genetics
  • Insulinoma / metabolism
  • Insulinoma / pathology
  • Lectins, C-Type / genetics
  • Nerve Tissue Proteins / genetics
  • Nerve Tissue Proteins / metabolism
  • Pancreatic Neoplasms / genetics
  • Pancreatic Neoplasms / metabolism
  • Pancreatic Neoplasms / pathology
  • Pancreatitis-Associated Proteins
  • Promoter Regions, Genetic / genetics*
  • Protein Binding
  • RNA Interference
  • Reverse Transcriptase Polymerase Chain Reaction
  • TCF Transcription Factors / genetics
  • TCF Transcription Factors / metabolism
  • Trans-Activators / genetics
  • Trans-Activators / metabolism*
  • Transcription Factor 7-Like 1 Protein
  • Transfection

Substances

  • Antigens, Neoplasm
  • Basic Helix-Loop-Helix Transcription Factors
  • Biomarkers, Tumor
  • Homeodomain Proteins
  • Lectins, C-Type
  • NEUROD1 protein, human
  • Nerve Tissue Proteins
  • Pancreatitis-Associated Proteins
  • REG3A protein, human
  • TCF Transcription Factors
  • TCF7L1 protein, human
  • Trans-Activators
  • Transcription Factor 7-Like 1 Protein
  • pancreatic and duodenal homeobox 1 protein
  • NeuroD protein
  • DNA