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  cell-specific expression of the insulin gene, we performed chromatin immunoprecipitation assays using anti-histone antibodies and extracts from  cell lines, non- 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  cells. This hyperacetylation is highly correlated to recruitment of the histone acetyltransferase p300 to the proximal promoter in  cells, and is consistent with the role of hyperacetylation in promoting euchromatin formation. We also observed that the proximal insulin promoter of  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  cells and non- cells, and is consistent with their potential to express the insulin gene. We therefore propose a model in which insulin transcription in the  cell is facilitated by a unique combination of transcription factors that acts in the setting of an open, euchromatic structure of the insulin gene.The pancreatic  cell is exclusively responsible for the synthesis and secretion of insulin. The production of insulin appears to be governed by constraints imposed at the level of transcription of the gene encoding insulin (Ins), 1 and involves an intricate interplay between transcription factors that are known to function as transactivators of the gene. In recent years, specific DNA elements within the proximal ϳ400 base pairs (bp) of the Ins promoter have been mapped precisely and shown to be bound by several major classes of transactivating transcription factors, including homeodomain factors (Pdx1, Lmx1.1), basic helix loop helix factors (NeuroD1, E47), and bZip factors (mMafA) (see Ref. 1 for review). In addition, coactivators such as p300 (by virtue of its interaction with Pdx1, NeuroD1, and E47) have also been suggested to contribute to Ins gene regulation (2, 3). Thus, it is hypothesized that the unique combination of ubiquitous and cell type-specific factors within the  cell results in the transcription of the Ins gene (3-5). However, this hypothesis alone cannot explain why heterologous expression of  cell factors results in activation of the endogenous Ins gene in only limited, "responsive" cell types (e.g. liver, pancreatic ductal cells, and embryonic and intestinal stem cells) (6 -10). Although this finding suggests that "unresponsive" cell types are still missing some critical genetic component (transcription facto...
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.
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