Organization and regulation of the human insulin locus

我们正在进行涉及胰岛素基因启动子在人胰岛β细胞核内以及最近在人胰岛β细胞系Endo-betaH1细胞核内的远程相互作用的研究。为了检测这种相互作用，我们在人胰岛进行了4C(扩展染色质构象捕获)实验，以定位核内INS启动子与11号染色体上远程位点之间的联系。我们首先证明了胰岛素启动子与11号染色体上选定的基因，包括突触素8(Syt8)基因进行了物理接触。。接触受到添加葡萄糖的刺激，接触增加与SYT8表达增加相关。我们发现SYT8的表达与胰岛素蛋白的分泌呈正相关。接下来，我们将这一分析扩展到60Mb以外的基因ANO1，大约是11号染色体长度的一半，并表明该基因参与了小鼠的葡萄糖代谢控制。然后，我们将我们的4C方法扩展到人类胰岛β细胞系，精确度大大提高。我们在同一染色体(11)上与胰岛素基因相互作用的结果与在胰岛中的结果相似，但我们现在有足够的分辨率来检测与人类基因组中所有其他染色体的相互作用。我们对这些接触进行了研究，强调了它们对调节胰岛素分泌和代谢的意义。 我们的结果表明，胰岛素基因座与其他染色体上的位点之间存在着许多联系。这些站点中的许多都含有1型或2型糖尿病易感基因。干扰胰岛素基因的表达会导致存在于这些基因座的许多联系基因的下调。这使得我们能够确定一个新的参与调节胰岛素分泌的基因座，并表明基因组的大规模组织在调节胰岛素表达方面是重要的。这可能是许多基因调控的一个重要特征，特别是在专门生产大量S少量蛋白质的细胞中。 我们现在把注意力集中在胰岛素基因启动子区域和大约500kb外p方向上的位置之间的相互作用上。这是由早先的一份报告推动的，该报告称，该远处位置的SNP对人类2型糖尿病的易感性具有起源父母的影响，其中父亲等位基因的遗传使易感性增加，母亲等位基因的遗传使易感性降低。我们利用人类胰腺细胞系EnDoc-H1对INS和这些上游位点之间的多个接触进行了广泛的3C图谱绘制。然后，我们在使我们能够区分这两个等位基因的区域中确定了SNPs，并表明这两个等位基因上的接触模式完全不同。这些结果与该区域广泛的亲本特异性基因组结构一致，对胰岛素基因的表达具有重要意义。这些结果表明，除了胰岛素基因座外，基因组的其他区域可能显示出两个等位基因染色质结构的重大差异，这与印记不一定相关，这可能会导致基因表达的等位基因特异性差异。这在一般情况下可能是真的，但特别是在CTCF结合位点附近。

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 then 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. These results show that aside from the insulin locus, other regions of the genome may show major differences in chromatin structure of two alleles, not necessarily related to imprinting, that can result in allele specific differences in gene expression. This may be true quite generally, but particularly in the neighborhood of CTCF binding sites.