期刊
MOLECULAR ENDOCRINOLOGY
卷 20, 期 12, 页码 3133-3145出版社
ENDOCRINE SOC
DOI: 10.1210/me.2006-0126
关键词
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资金
- NIDDK NIH HHS [R01 DK060581, R01 DK060581-05, T32 DK0732025, R01 DK60581] Funding Source: Medline
- NIGMS NIH HHS [T32 GM007055] Funding Source: Medline
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 (beta TC3) vs. pancreatic ductal cells (mPAC). We demonstrate that Pdx-1 occupies the endogenous insulin promoter in beta TC3 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, nucleosomerich 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.
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