Pdx-1 links histone H3-Lys-4 methylation to RNA polymerase II elongation during activation of insulin transcription.
Expression of the insulin gene is nearly exclusive to the beta 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 betaTC3 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.
Francis
Joshua
J
Department of Pharmacology, University of Virginia, Charlottesville 22908, USA.
Chakrabarti
Swarup K
SK
Garmey
James C
JC
Mirmira
Raghavendra G
RG
D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D002460:Cell Line; D002843:Chromatin / Q000737:chemistry / Q000378:metabolism; D047369:Chromatin Immunoprecipitation; D019175:DNA Methylation; D005822:Genetic Vectors; D005982:Glutathione Transferase / Q000378:metabolism; D006367:HeLa Cells; D006657:Histones / Q000737:chemistry* / Q000378:metabolism; D018398:Homeodomain Proteins / Q000737:chemistry / Q000502:physiology*; D006801:Humans; D015151:Immunoblotting; D007150:Immunohistochemistry; D047468:Immunoprecipitation; D007328:Insulin / Q000378:metabolism*; D050417:Insulin-Secreting Cells; D007515:Islets of Langerhans / Q000378:metabolism; D008239:Lysine / Q000737:chemistry; D008745:Methylation; D051379:Mice; D008957:Models, Genetic; D041681:NIH 3T3 Cells; D010766:Phosphorylation; D011401:Promoter Regions, Genetic; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D012319:RNA Polymerase II / Q000737:chemistry* / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012694:Serine / Q000737:chemistry; D015534:Trans-Activators / Q000737:chemistry / Q000502:physiology*; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D014162:Transfection
D002843:Chromatin; D006657:Histones; D018398:Homeodomain Proteins; D007328:Insulin; D020033:Protein Isoforms; D034741:RNA, Small Interfering; D015534:Trans-Activators; C084010:pancreatic and duodenal homeobox 1 protein; D012694:Serine; D005982:Glutathione Transferase; D012319:RNA Polymerase II; D008239:Lysine