Organization and regulation of the human insulin locus

我们正在对人类胰岛细胞中胰岛素基因位点及其邻域的组蛋白修饰进行研究，试图确定可能影响胰岛素基因表达的长期调节影响。我们利用胰岛素启动子和其他基因组位点之间的长距离物理接触在细胞核内的测量。为了检测这种相互作用，我们在人类胰岛上进行了4C（扩展染色质构象捕获）实验，绘制了细胞核内Ins启动子与11号染色体上远端位点之间的接触图谱。我们选择了Syt8 （synaptotagmin 8）基因作为进一步研究的候选基因，该基因与胰岛素位点一样，在附近有一个CTCF结合位点。我们发现Ins- syt8接触是存在的，它们被添加葡萄糖刺激，当Ins启动子被阻断以沉默Ins基因活性时被抑制。接触增加与Syt8表达增加相关。CTCF的缺失导致接触缺失和Syt8（不包括Ins）表达缺失。此外，我们发现Syt8的消耗导致胰岛胰岛素分泌减少。这些结果揭示了导致耦合调节控制的远程相互作用的物理网络。这类调控网络似乎在整个基因组中都存在。我们现在发现了一个具有类似行为的不同网站。Syt8位点距离Ins只有300kb，而新位点距离Ins大约60mb，大约是11号染色体长度的一半。这个新位点位于基因ANO1附近，该基因编码依赖钙的氯离子通道。对于Syt8，胰岛素基因与ANO1的物理接触增加，并且ANO1的表达也随着葡萄糖的加入而增加。此外，干扰ANO1表达会减少胰岛素分泌。我们还发现，ANO1拷贝被删除的小鼠表现出葡萄糖代谢缺陷。结果表明，涉及胰岛素基因的长期相互作用对于胰岛素分泌的调节和其他涉及胰岛素功能的调节过程是重要的。

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.