Covalent Histone Modifications Underlie the Developmental Regulation of Insulin Gene Transcription in Pancreatic β Cells

Abstract

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...

Pdx-1 Links Histone H3-Lys-4 Methylation to RNA Polymerase II Elongation during Activation of Insulin Transcription

“…However, the recent discovery of LSD1, an H3-K4 demethylase (31), and the recognition that differentially modified histone variant proteins can be introduced during nonreplicative phases of the cell cycle (32) suggest the possibility that histone methylation may be more dynamic than previously thought. Methylation of H3-K4 has been associated with active genes in euchromatic loci, including insulin (15). In this regard, out data show that siRNA-mediated knockdown of Pdx-1 results in both the attenuation of insulin transcription and the partial depletion of dimethylated H3-K4 at the insulin promoter and coding regions.…”

“…As shown in Fig. 6A, when a cDNA encoding Pdx-1 is cotransfected into NIH3T3 cells with a reporter plasmid containing tandem copies of the E2/A3/A4 insulin mini-enhancer driving luciferase (20), 15-fold activation of luciferase activity is observed. No activation is observed, however, upon cotransfection of the reporter with either a cDNA encoding wild-type Set9 or a catalytically inactivate mutant of Set9 (H297A and Set9m, see Ref.…”

“…In earlier studies, we demonstrated that this enzyme occupied the endogenous proximal insulin promoter in ␤TC3 cells in a pattern consistent with the dimethylation of H3-K4 (15). To determine whether Set9 is also expressed in normal pancreatic tissue, we performed immunohistochemical localization of Set9 in fixed, serial sections of mouse pancreas.…”

“…For example, Set1 (a histone H3-Lys-4 (H3-K4) methyltransferase) and Set2 (an H3-Lys-36 methyltransferase) are found in association with initiation-phosphorylated (Ser(P)-5) and elongation-phosphorylated (Ser(P)-2) isoforms of pol II, respectively (12,13). Studies in higher eukaryotic systems are also beginning to suggest that histone methylation patterns may indeed correlate with the activity of pol II (14,15). These findings also emphasize that histone Lys methylation is not necessarily a stable epigenetic marker of active genes but may play a more dynamic role in the acute regulation of gene transcription.…”

Egr-1 binds the GnRH promoter to mediate the increase in gene expression by insulin

“…Quantitative PCR was performed as detailed above using the following primers to amplify a 200 bp region in the proximal GnRH promoter, sense 5'-GACCAGCAGGTGTTGCAAT-3', Antisense, 5'-TCGAGCTTCCGTTGGTAGGT-3' or a 150 bp region of in the distal GnRH promoter, sense5'-CAACCATCTCTAGTGGGATC-3', antisense 5'-GCCTCGAACTCAGAAATCTG-3'. Relative DNA levels were calculated using the ΔΔCt method with input DNA as internal control (Chakrabarti et al, 2003).…”