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

Abstract

Expression of the insulin gene is nearly exclusive to the ␤ cells of the pancreatic islets. Although the sequence-specific transcription factors that regulate insulin expression have been well studied, the interrelationship between these factors, chromatin structure, and transcriptional elongation by RNA polymerase II (pol II) has remained undefined. In this regard, recent studies have begun to establish a role for the methylation of histone H3 in the initiation or elongation of transcription by pol II. To determine a role for the transcriptional activator Pdx-1 in the maintenance of chromatin structure and pol II recruitment at the insulin gene, we performed small interfering RNA-mediated knockdown of Pdx-1 in ␤TC3 cells and subsequently studied histone modifications and pol II recruitment by chromatin immunoprecipitation. We demonstrated here that the 50% fall in insulin transcription following knockdown of Pdx-1 is accompanied by a 60% fall in dimethylated histone H3-Lys-4 at the insulin promoter. H3-Lys-4 methylation at the insulin promoter may be mediated, at least partially, by the methyltransferase Set9. Immunohistochemical analysis revealed that Set9 is expressed in an islet-enriched pattern in the pancreas, similar to the pattern of Pdx-1 expression. The recruitment of Set9 to the insulin gene appears to be a consequence of its direct interaction with Pdx-1, and small interfering RNA-mediated knockdown of Set9 attenuates insulin transcription. Pdx-1 knockdown was also associated with an overall shift in the recruitment of pol II isoforms to the insulin gene, from an elongation isoform (Ser(P)-2) to an initiation isoform (Ser(P)-5). Our findings therefore suggest a model whereby Pdx-1 plays a novel role in linking H3-Lys-4 dimethylation and pol II elongation to insulin transcription.During pancreatic development, endocrine and exocrine cell types differentiate from a common endodermal precursor cell via a tightly coordinated sequence of transcriptional events (1, 2). The Hox-like transcription factor Pdx-1 is thought to initiate this cascade by regulating specific genes within the endodermal precursor cell (3, 4). The disruption of pdx-1 expression during development, either by targeted knockout in mice (5, 6) or homozygous mutations in humans (7), can be catastrophic, resulting in the failure of pancreas formation and consequent development of diabetes. Within the mature pancreas, Pdx-1 is necessary for the maintenance and function of endocrine cell types within the islets of Langerhans. In the islet ␤ cell, Pdx-1 is believed to activate a number of critical genes involved in glucose sensing and insulin production, including glucokinase, glut2, and insulin (3,8).Expression of the insulin gene is almost exclusive to the islet ␤ cells. By using small interfering RNA (siRNA) 2 and chromatin immunoprecipitation (ChIP) in isolated mouse islets, we recently demonstrated that Pdx-1 is a direct activator of insulin transcription upon binding to A box DNA elements in the 5Ј-regulatory region of the gene (the ins...

A feat of metabolic proportions: Pdx1 orchestrates islet development and function in the maintenance of glucose homeostasis

“…Interestingly, although Pdx1 itself does not appear to have intrinsic abilility to either modify histones or directly alter chromatin structure, its depletion in β cell lines results in the loss of histone H3-Lys4 dimethylation (a mark of active, open chromatin) at the insulin gene. This result may be secondary to the recruitment of the histone methyltransferase Set7/9 by Pdx1 [147]. Similarly, the glucose-stimulated increase in histone H4 acetylation (another marker of active, open chromatin) at the insulin gene in both cell lines and islets by Pdx1 is consistent with its recruitment of the histone acetyltransferase p300 [145,153,154].…”

“…Our laboratory has demonstrated that in the absence of Pdx1, the RNA polymerase II complex continues to be recruited to the insulin promoter, but transcriptional elongation by the complex is impaired [68]. Consistent with this observation, it appears that Pdx1 is necessary to ensure that the Ser5-phosphorylated isoform (the "initiation isoform") is appropriately converted to the Ser2-phosphorylated isoform (the "elongation isoform") [147]. These findings raise the possibility that the linkage between the Pdx1 transcriptional complex and the basal transcriptional machinery may involve an as yet unidentified kinase.…”

“…Apart from interaction with transcription factors, Pdx1 also appears to recruit transcriptional co-activators (proteins that bridge Pdx1 to the basal transcriptional machinery), including CBP/ p300 [68,113,141,145,146], Set7/9 [147], Bridge-1 [148], PCIF1 [149], and histone deacetylases (HDACs) [150]. Once recruited, cofactors are believed to serve as a physical or functional "bridge" that allows Pdx1 to communicate with components of the basal transcriptional machinery.…”

Methyltransferase Set7/9 regulates p53 activity by interacting with Sirtuin 1 (SIRT1)

“…For example, Set7/9 interacts with Pdx1, an insulin transactivator, and catalyzes Pdx1 methylation. Although the interaction of Pdx1 with Set7/9 is able to recruit Set7/9 to histone H3 and induce H3K4 methylation (28,29), the methylation of Pdx1 is not directly associated with changes in Pdx1 function. In addition, although the methylation of DNMT1 leads to a decrease in the half-life of DNMT1, this modification is also not directly related to changes in DNMT1 methyltransferase activity (21,30).…”

Suppression of the Pancreatic Duodenal Homeodomain Transcription Factor-1 (Pdx-1) Promoter by Sterol Regulatory Element-binding Protein-1c (SREBP-1c)

“…p300 or CBP overexpression did not rescue the inhibition of PDX-1 transactivation by SREBP-1c (data not shown). Frances et al (49) have reported that PDX-1 physically associates with and recruits the H3-K4 methyltransferase SET9 to the insulin gene. Interaction of PDX-1 with SET9 may be required for the transactivation of the Pdx-1 promoter, and SREBP-1c may squelch the recruitment of SET9 to PDX-1.…”