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Induction of Protein Deletion Through In Utero Electroporation to Define Deficits in Neuronal Migration in Transgenic Models
Published on: January 12, 2015
Pdx-1 regulation of the INGAP promoter involves sequestration of NeuroD into a non-DNA-binding complex
David A Taylor-Fishwick1, Wenjing Shi, Laura Hughes
1Department of Medicine, Eastern Virginia Medical School, Norfolk, VA, USA. taylord@evms.edu
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.
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