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. This approach has now been successful in identifying a set of genes distributed throughout the genome and detected as in contact with the insulin promoter, that are down regulated by inhibition of insulin expression. One of these has not previously been implicated in the regulation of insulin secretion.
|Jian, Xing; Felsenfeld, Gary (2018) Insulin promoter in human pancreatic ? cells contacts diabetes susceptibility loci and regulates genes affecting insulin metabolism. Proc Natl Acad Sci U S A 115:E4633-E4641|
|Xu, Zhixiong; Lefevre, Gaelle M; Gavrilova, Oksana et al. (2014) Mapping of long-range INS promoter interactions reveals a role for calcium-activated chloride channel ANO1 in insulin secretion. Proc Natl Acad Sci U S A 111:16760-5|
|Xu, Zhixiong; Felsenfeld, Gary (2012) Order from chaos in the nucleus. Mol Cell 48:327-8|
|Xu, Z; Lefevre, G M; Felsenfeld, G (2012) Chromatin structure, epigenetic mechanisms and long-range interactions in the human insulin locus. Diabetes Obes Metab 14 Suppl 3:1-11|
|Xu, Zhixiong; Wei, Gang; Chepelev, Iouri et al. (2011) Mapping of INS promoter interactions reveals its role in long-range regulation of SYT8 transcription. Nat Struct Mol Biol 18:372-8|
|Mutskov, Vesco; Felsenfeld, Gary (2009) The human insulin gene is part of a large open chromatin domain specific for human islets. Proc Natl Acad Sci U S A 106:17419-24|