Organization and regulation of the human insulin locus

我们正在进行的研究组蛋白修饰的胰岛素基因位点及其附近的人胰岛细胞，在试图确定可能影响胰岛素基因表达的长期调控的影响。 我们利用测量胰岛素启动子和其他基因组位点之间的细胞核内的长距离物理接触。 为了检测这种相互作用，我们在人类胰岛中进行了4C（一种扩展的染色质构象捕获）实验，以绘制Ins启动子和11号染色体上远端位点之间的细胞核内接触。 我们选择了一个单一的候选人，synaptotagmin 8（Syt 8）基因，它像胰岛素基因座，有一个附近的CTCF结合位点进行进一步的研究。 我们发现，Ins-Syt 8接触存在，它们被葡萄糖刺激，当Ins启动子被阻断以沉默Ins基因活性时被抑制。 增加的接触与增加的Syt 8表达相关。 CTCF的消耗导致接触的丧失和Syt 8（但不是Ins）表达的丧失。 此外，我们发现Syt 8的缺失导致胰岛分泌胰岛素减少。 这些结果揭示了一个物理网络的远程相互作用，导致耦合的监管控制。 这类调控网络似乎遍布整个基因组。 我们现在发现了一个具有类似行为的不同网站。 Syt 8基因座距离Ins仅约300 kb，而新位点距离约60 Mb，约为11号染色体长度的一半。 这个新的位点位于基因ANO 1附近，该基因编码钙依赖性氯离子通道。 对于Syt 8，胰岛素基因和ANO 1之间的物理接触增加，并且ANO 1表达也在添加葡萄糖时增加。 此外，干扰ANO 1表达会降低胰岛素分泌。 我们还发现，其中一个拷贝的ANO 1已被删除的小鼠显示出葡萄糖代谢的缺陷。 这些结果表明，涉及胰岛素基因的长程相互作用对于调节胰岛素分泌以及可能对于涉及胰岛素功能的其他调节过程是重要的。 我们现在已经将我们的4C方法扩展到人类胰岛β细胞系，精度大大提高。我们在同一条染色体（11）上与胰岛素基因相互作用的结果与胰岛中的结果相似，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。 我们正在研究这些接触，重点是调节胰岛素分泌和代谢的意义。我们最近发现了受胰岛素基因表达敲低影响的基因，这些基因存在于我们的接触图上。

We are carrying out studies of the histone modifications over the insulin gene locus and its neighborhood in human islet cells, in an attempt to identify long range regulatory influences that may affect insulin gene expression. We made use of measurements of long range physical contacts within the nucleus between the insulin promoter and other genomic sites. To detect such interactions we performed 4C (an 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 selected for further study a single candidate, the synaptotagmin 8 (Syt8) gene, which like the insulin locus, has a nearby CTCF binding site. We found that Ins-Syt8 contacts are present, that they are stimulated by addition of glucose and inhibited when the Ins promoter is blocked to silence Ins gene activity. Increased contact is correlated with increased Syt8 expression. Depletion of CTCF results in loss of contact and loss of Syt8 (but not Ins) expression. Furthermore, we show that depletion of Syt8 results in decreased secretion of insulin from the islets. These results reveal a physical network of long range interactions that leads to coupled regulatory control. It seemed likely that these kinds of regulatory networks occur throughout the genome. We have now found a different site with similar behavior. Whereas the Syt8 locus is only about 300 kb away from Ins, the new site is about 60 Mb away, about half the length of chromosome 11. This new site is near the gene ANO1, which codes for a calcium-dependent chloride channel. As for Syt8, physical contact between the insulin gene and ANO1 increases and ANO1 expression also increases on addition of glucose. Furthermore, interfering with ANO1 expression decreases insulin secretion. We have also found that mice in which one copy of ANO1 has been deleted show defects in glucose metabolism. The results suggest that long range interactions involving the insulin gene are important for regulation of insulin secretion and perhaps for other regulatory processes involving insulin function. 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 are investigating these contacts with emphasis on significance for regulation of insulin secretion and metabolism. We have recently identified genes that are affected by knockdown of insulin gene expression, and that are present on our contact map.