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.
Taylor-Fishwick
David A
DA
Department of Medicine, Eastern Virginia Medical School, Norfolk, VA, USA. taylord@evms.edu
Shi
Wenjing
W
Hughes
Laura
L
Vinik
Aaron
A
D000818:Animals; D000951:Antigens, Neoplasm / Q000235:genetics; D051792:Basic Helix-Loop-Helix Proteins / Q000235:genetics / Q000378:metabolism*; D014408:Biomarkers, Tumor / Q000235:genetics; D015153:Blotting, Western; D002460:Cell Line; D045744:Cell Line, Tumor; D006224:Cricetinae; D004247:DNA / Q000235:genetics / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007340:Insulinoma / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D037181:Lectins, C-Type / Q000235:genetics; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism; D010190:Pancreatic Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D000073718:Pancreatitis-Associated Proteins; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011485:Protein Binding; D034622:RNA Interference; D020133:Reverse Transcriptase Polymerase Chain Reaction; D051820:TCF Transcription Factors / Q000235:genetics / Q000378:metabolism; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*; D058125:Transcription Factor 7-Like 1 Protein; D014162:Transfection
D000951:Antigens, Neoplasm; D051792:Basic Helix-Loop-Helix Proteins; D014408:Biomarkers, Tumor; D004247:DNA; D018398:Homeodomain Proteins; D037181:Lectins, C-Type; D009419:Nerve Tissue Proteins; D000073718:Pancreatitis-Associated Proteins; D051820:TCF Transcription Factors; D015534:Trans-Activators; D058125:Transcription Factor 7-Like 1 Protein; C093970:NEUROD1 protein, human; C000612141:REG3A protein, human; C544912:TCF7L1 protein, human; C084010:pancreatic and duodenal homeobox 1 protein; C093564:Neurogenic differentiation factor 1