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

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.