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 are carrying out 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 find 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 are now extending our 4C methods, with greatly increased precision, in a human islet beta cell line. 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 50 Mb away, about half the length of chromosome 11. This new site is in a locus associated with Type I diabetes susceptibility. Our analysis strongly suggests that defects in the expression or function of the nearby gene may affect insulin secretion, and we are exploring this possibility in detail.

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