The transcriptional repressor Nkx6.1 also functions as a deoxyribonucleic acid context-dependent transcriptional activator during pancreatic beta-cell differentiation: evidence for feedback activation of the nkx6.1 gene by Nkx6.1.

In the pancreas, the NK homeodomain transcription factor Nkx6.1 is required for the development of beta-cells and is believed to function as a potent repressor of transcription upon binding to A/T-rich sequences within the promoter region of target genes. Because the nkx6.1 promoter itself contains several such sequences, we considered the possibility that the expression level and restricted pattern of the nkx6.1 gene might be precisely regulated by one or more homeodomain transcription factors, including Nkx6.1 itself. In this report, we identify a novel beta-cell-specific enhancer element in the nkx6.1 gene between -157 and -30 bp (relative to the transcriptional start site) that harbors a conserved A/T-containing sequence flanked by G/C-rich stretches. Although the islet homeodomain-containing activator Pdx-1 was unable to stimulate transcription of a reporter gene through this enhancer element in mammalian cell lines, strikingly, Nkx6.1 robustly activated transcription through direct interaction with the A/T-rich sequence in this element. We demonstrate that this activation is indeed transcriptional in nature (and not secondary to translational effects) and is mediated by a modular acidic sequence within the COOH-terminal domain of Nkx6.1. We show by EMSAs that Nkx6.1 binds to the beta-cell-specific enhancer in vitro and by chromatin immunoprecipitation assays that Nkx6.1 natively occupies this region in vivo in betaTC3 cells. We therefore conclude that Nkx6.1 is a bifunctional transcription factor that serves to maintain the specific expression of its own gene during beta-cell differentiation while simultaneously effecting broader gene repression events.

Covalent histone modifications underlie the developmental regulation of insulin gene transcription in pancreatic beta cells.

Histone modifying enzymes contribute to the activation or inactivation of transcription by ultimately catalyzing the unfolding or further compaction, respectively, of chromatin structure. Actively transcribed genes are typically hyperacetylated at Lys residues of histones H3 and H4 and hypermethylated at Lys-4 of histone H3 (H3-K4). To determine whether covalent histone modifications play a role in the beta cell-specific expression of the insulin gene, we performed chromatin immunoprecipitation assays using anti-histone antibodies and extracts from beta cell lines, non-beta cell lines, and ES cells, and quantitated specific histone modifications at the insulin promoter by real-time PCR. Our studies reveal that the proximal insulin promoter is hyperacetylated at histone H3 only in beta cells. This hyperacetylation is highly correlated to recruitment of the histone acetyltransferase p300 to the proximal promoter in beta cells, and is consistent with the role of hyperacetylation in promoting euchromatin formation. We also observed that the proximal insulin promoter of beta cells is hypermethylated at H3-K4, and that this modification is correlated to the recruitment of the histone methyltransferase SET7/9 to the promoter. ES cells demonstrate a histone modification pattern intermediate between that of beta cells and non-beta cells, and is consistent with their potential to express the insulin gene. We therefore propose a model in which insulin transcription in the beta cell is facilitated by a unique combination of transcription factors that acts in the setting of an open, euchromatic structure of the insulin gene.