Role of chromatin accessibility in the occupancy and transcription of the insulin gene by the pancreatic and duodenal homeobox factor 1.
The pancreatic and duodenal homeobox factor 1 (Pdx-1) is a Hox-like transcription factor that is responsible for the activation of the insulin gene. Previous studies have demonstrated the interaction in vitro of Pdx-1 with short (20-40 nucleotide) DNA fragments corresponding to A boxes of the insulin promoter. Precisely how Pdx-1 binds to DNA in the complex milieu of chromatin, however, has never been studied. In this study, we explored how Pdx-1-DNA interactions might be influenced by chromatin accessibility at the insulin gene in beta-cells (betaTC3) vs. pancreatic ductal cells (mPAC). We demonstrate that Pdx-1 occupies the endogenous insulin promoter in betaTC3 cells but not in mPAC cells, a finding that is independent of the intracellular Pdx-1 protein concentration. Based on micrococcal nuclease protection assays, the difference in promoter binding between the two cell types appears to be secondary to chromatin accessibility at predicted Pdx-1 binding sites between bp -126 to -296 (relative to the transcriptional start site) of the insulin promoter. Binding studies using purified Pdx-1 and reconstituted chromatin in vitro suggest that the positioning of a nucleosome(s) within this crucial region of the promoter might account for differences in chromatin accessibility. Consistent with these observations, fluorescence colocalization studies show that Pdx-1 does not occupy regions of compacted, nucleosome-rich chromatin within the nucleus. Our findings suggest a model whereby insulin transcription in the beta-cell is at least partially facilitated by enhanced chromatin accessibility within a crucial regulatory region between bp -126 to -296, thereby permitting occupancy by transactivators such as Pdx-1.
Francis
Joshua
J
Department of Pharmacology, University of Virginia, Charlottesville, Virginia 22908, USA.
Babu
Daniella A
DA
Deering
Tye G
TG
Chakrabarti
Swarup K
SK
Garmey
James C
JC
Evans-Molina
Carmella
C
Taylor
David G
DG
Mirmira
Raghavendra G
RG
D000107:Acetylation; D000818:Animals; D045744:Cell Line, Tumor; D002843:Chromatin / Q000737:chemistry / Q000378:metabolism*; D004247:DNA / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D022041:Euchromatin / Q000737:chemistry / Q000378:metabolism; D005786:Gene Expression Regulation; D006570:Heterochromatin / Q000737:chemistry / Q000378:metabolism; D006657:Histones / Q000378:metabolism; D018398:Homeodomain Proteins / Q000032:analysis / Q000378:metabolism*; D007328:Insulin / Q000235:genetics*; D050417:Insulin-Secreting Cells / Q000378:metabolism*; D051379:Mice; D009707:Nucleosomes / Q000737:chemistry / Q000378:metabolism; D011401:Promoter Regions, Genetic; D015534:Trans-Activators / Q000032:analysis / Q000378:metabolism*; D014158:Transcription, Genetic; D015533:Transcriptional Activation
D002843:Chromatin; D022041:Euchromatin; D006570:Heterochromatin; D006657:Histones; D018398:Homeodomain Proteins; D007328:Insulin; D009707:Nucleosomes; D015534:Trans-Activators; C084010:pancreatic and duodenal homeobox 1 protein; D004247:DNA