Organization and regulation of the human insulin locus

National Institute Of Diabetes And Digestive And Kidney Diseases

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Abstract

We are carrying out studies of long range interactions involving the insulin gene promoter within the nucleus of human pancreatic islet beta cells and recently within the nucleus of the human pancreatic beta cell line, EndoC-betaH1 To detect such interactions, we performed 4C (extended chromatin conformation capture) experiments in human islets, to map contacts within the nucleus between the Ins promoter and distant sites on chromosome 11. We showed first that the insulin promoter makes physical contact with selected genes on chromosome 11, including the synaptotagmin 8 (Syt8) gene. . Contact was stimulated by addition of glucose, and increased contact was associated with increased SYT8 expression. We showed that SYT8 expression is positively coupled to insulin protein secretion. We next extended this analysis to the gene ANO1, 60 Mb away, about half the length of chromosome 11, and showed that this gene is involved in control of glucose metabolism in mice. We have now extended our 4C methods, with greatly increased precision, to a human islet beta cell line. Our results for interactions on the same chromosome (11) with the insulin gene are similar to those in islets, but we now have sufficient resolution to detect interactions with all other chromosomes in the human genome. We have investigated these contacts with emphasis on significance for regulation of insulin secretion and metabolism. Our results show that there are many contacts between the insulin gene locus and sites on other chromosomes. Many of these sites contain Type 1 or Type 2 diabetes susceptibility loci. Interfering with insulin gene expression results in down regulation of many of the contacted genes present at these loci. This has allowed us to identify a novel locus that is involved in regulation of insulin secretion, and demonstrates that large scale organization of the genome is important in regulation of insulin expression. This is likely to be an important feature in regulation of many genes, particularly in cells specialized to produce large amounts of s small number of proteins. We have now focused our attention on the interactions between the insulin gene promoter region and sites about 500 Kb away in the p direction. This was prompted by an earlier report that a snp in that distant site had a parent of origin effect on Type2 diabetes susceptibility in humans, in which inheritance on the paternal allele confers increased susceptibility and inheritance on the maternal allele confers decreased susceptibility. We made use of the human pancreatic cell line, EndoC-H1 to carry out extensive 3C mapping of multiple contacts between INS and these upstream sites. We then identified snps in the region that allowed us to distinguish between the two alleles, and showed that the pattern of contacts on the two alleles was entirely different. These results are consistent with extensive parent of origin specific genome organization in this region with important implications for insulin gene expression. There are also important implications for regulation of expression genome wide, because our results show that even in regions of the genome not considered to be imprinted, two alleles may be imbedded in quite different chromatin structures and may have different patterns of expression.