Role of chromatin accessibility in the occupancy and transcription of the insulin gene by the pancreatic and duodenal homeobox factor 1

Joshua Francis, Daniella A. Babu, Tye G. Deering, Swarup K. Chakrabarti, James C. Garmey, Carmella Evans-Molina, David G. Taylor, Raghu Mirmira

Research output: Contribution to journalArticle

13 Citations (Scopus)

Abstract

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 β-cells (βTC3) vs. pancreatic ductal cells (mPAC). We demonstrate that Pdx-1 occupies the endogenous insulin promoter in βTC3 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 β-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.

Original languageEnglish (US)
Pages (from-to)3133-3145
Number of pages13
JournalMolecular Endocrinology
Volume20
Issue number12
DOIs
StatePublished - Dec 2006
Externally publishedYes

Fingerprint

Chromatin
Insulin
Genes
Nucleosomes
DNA
Nuclease Protection Assays
Micrococcal Nuclease
Trans-Activators
Nucleic Acid Regulatory Sequences
pancreatic and duodenal homeobox 1 protein
Genetic Promoter Regions
Transcriptional Activation
Transcription Factors
Nucleotides
Fluorescence
Binding Sites
Proteins

ASJC Scopus subject areas

  • Molecular Biology
  • Endocrinology, Diabetes and Metabolism

Cite this

Role of chromatin accessibility in the occupancy and transcription of the insulin gene by the pancreatic and duodenal homeobox factor 1. / Francis, Joshua; Babu, Daniella A.; Deering, Tye G.; Chakrabarti, Swarup K.; Garmey, James C.; Evans-Molina, Carmella; Taylor, David G.; Mirmira, Raghu.

In: Molecular Endocrinology, Vol. 20, No. 12, 12.2006, p. 3133-3145.

Research output: Contribution to journalArticle

Francis, Joshua ; Babu, Daniella A. ; Deering, Tye G. ; Chakrabarti, Swarup K. ; Garmey, James C. ; Evans-Molina, Carmella ; Taylor, David G. ; Mirmira, Raghu. / Role of chromatin accessibility in the occupancy and transcription of the insulin gene by the pancreatic and duodenal homeobox factor 1. In: Molecular Endocrinology. 2006 ; Vol. 20, No. 12. pp. 3133-3145.
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