Insulin Promoter in Human Pancreatic Β Cells Contacts Diabetes
Total Page：16
File Type：pdf, Size：1020Kb
Insulin promoter in human pancreatic β cells contacts PNAS PLUS diabetes susceptibility loci and regulates genes affecting insulin metabolism Xing Jiana and Gary Felsenfelda,1 aLaboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892 Contributed by Gary Felsenfeld, April 6, 2018 (sent for review February 21, 2018; reviewed by Peter Fraser and Susan E. Ozanne) Both type 1 and type 2 diabetes involve a complex interplay mosome conformation capture-based methods could detect between genetic, epigenetic, and environmental factors. Our long-range interactions within the same transcription factory (9). laboratory has been interested in the physical interactions, in Type 1 and type 2 diabetes are two distinct disease entities nuclei of human pancreatic β cells, between the insulin (INS) gene (10). In type 1 diabetes (T1D), the patients’ immune system and other genes that are involved in insulin metabolism. We have attacks and destroys the insulin-producing pancreatic β cell; it identified, using Circularized Chromosome Conformation Capture comprises about 5% of all cases of diabetes. Type 2 (T2D) is the (4C), many physical contacts in a human pancreatic β cell line be- most common form of diabetes. An estimated 30.3 million people tween the INS promoter on chromosome 11 and sites on most in the United States, or 9.4% of the population, have type 2 di- other chromosomes. Many of these contacts are associated with abetes, and it remains the seventh leading cause of death in type 1 or type 2 diabetes susceptibility loci. To determine whether the United States (https://www.niddk.nih.gov/health-information/ physical contact is correlated with an ability of the INS locus to diabetes). It is a long-term metabolic disorder that is characterized affect expression of these genes, we knock down INS expression by high blood sugar, insulin resistance, and relative lack of insulin. by targeting the promoter; 259 genes are either up or down- It has multiple causes, including both lifestyle and genetic elements. regulated. Of these, 46 make physical contact with INS.We Recent advances in genome-wide association studies (GWAS) analyze a subset of the contacted genes and show that all are have made it possible to identify genetic variants that are asso- associated with acetylation of histone H3 lysine 27, a marker of ciated with T1D and T2D, and many of them are important for β actively expressed genes. To demonstrate the usefulness of this cell function (11). Nevertheless, GWAS studies have only iden- approach in revealing regulatory pathways, we identify from tified a small fraction of the risks attributable to genetic factors, among the contacted sites the previously uncharacterized gene so that diabetes is still recognized as “a geneticist’s nightmare” SSTR5-AS1 and show that it plays an important role in controlling (12). Our laboratory has been interested in the physical inter- the effect of somatostatin-28 on insulin secretion. These results actions, in nuclei of human pancreatic β cells, between the in- are consistent with models in which clustering of genes supports sulin (INS) gene and other genes that are involved in insulin transcriptional activity. This may be a particularly important mech- metabolism. In earlier work, we used 4C in human pancreatic anism in pancreatic β cells and in other cells where a small subset islets to find contacts between the INS promoter and other genes of genes is expressed at high levels. on chromosome 11 (13, 14). This led to the identification of two pancreatic beta cells | 4C-seq | insulin | gene regulation | somatostatin Significance t is well established that within the nucleus the genome is or- We show that in a human pancreatic β cell line the human in- Iganized into a series of domains of varying scale that can serve sulin gene promoter on chromosome 11 physically contacts to segregate active from inactive chromatin, block inappropriate sites on other chromosomes. Many of these contacted sites interactions between regulatory sites, or in other cases, bring contain type 1 or type 2 diabetes susceptibility loci. We find together regulatory elements widely separated on the genome, that insulin gene expression can affect expression of contacted such as enhancers and promoters (1). Some of these interactions genes on other chromosomes. Some of these genes, in turn, in vertebrates are mediated by pairs of sites separated by as much regulate insulin secretion. These results reveal physical regu- as a megabase of DNA and occupied by the protein CTCF in latory mechanisms in which the level of insulin expression association with the cohesin complex. Such interactions result in controls expression of genes involved in insulin transport and formation of large loop domains within chromosomes. metabolism. We study the properties of one such gene, so- Other kinds of interactions between distant sites, often on matostatin receptor 5 antisense (SSTR5-AS1), and show that it different chromosomes, arise from the clustering of transcribed regulates SSTR5 expression, which affects insulin secretion. genes. In an early example (2), it was shown that the Hbb-b1 β Analysis of insulin contacts thus may reveal new insulin hemoglobin gene in mouse erythroid cells colocalizes at tran- metabolic pathways. scription factories (for a recent review on transcription factories, Author contributions: G.F. designed research; X.J. performed the research; X.J. and G.F. see ref. 3) with other expressed genes. These include the analyzed data; and X.J. and G.F. wrote the paper. erythroid-specific gene Eraf; the Uroporphyrinogen III synthe- Reviewers: P.F., The Babraham Institute; and S.E.O., University of Cambridge. tase gene Uros, essential for heme biosynthesis (both on the same The authors declare no conflict of interest. chromosome as Hbb-b1); and Hba, the gene for hemoglobin α, Published under the PNAS license. on a different chromosome. In this and other cases, the clusters – Data deposition: The data reported in this paper have been deposited in the Gene Ex- included coregulated genes (4 7). Other transcription factory- pression Omnibus (GEO) database, https://www.ncbi.nlm.nih.gov/geo (accession no. mediated interchromosomal interactions have been reported in GSE112346). mouse B lymphocytes between the Myc gene on chromosome 1To whom correspondence should be addressed. Email: [email protected]. 15 and the Igh gene on chromosome 12 (4), and in human en- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. dothelial cells, which in response to TNF-α organize target genes 1073/pnas.1803146115/-/DCSupplemental. CELL BIOLOGY to form “NFκB factories” (8). It has been suggested that chro- Published online April 30, 2018. www.pnas.org/cgi/doi/10.1073/pnas.1803146115 PNAS | vol. 115 | no. 20 | E4633–E4641 Downloaded by guest on September 29, 2021 genes, synaptotagmin 8 (SYT8) and anoctamin 1 (ANO1), such contacts often mark sites near T1D or T2D susceptibility ∼300 kb and 68 Mb away, respectively, from the INS locus, that loci (17–22). we showed contacted the INS promoter. These physical contacts A large number of these sites include genes whose expression were correlated with expression of INS: Blocking INS promoter is sensitive to the level of INS transcriptional activity. Consistent activity resulted in decreased contact between the promoter and with our earlier observations in human islets, many of these both SYT8 and ANO1, as well as their deceased expression. In genes are part of regulatory mechanisms for insulin secretion. addition, we showed that both SYT8 and ANO1 proteins pro- Among these we identified SSTR5-AS1, (somatostatin receptor mote secretion of insulin. Thus, these interactions constitute a 5 antisense) located on chromosome 16. Down-regulation of this physical feedback transvection-like mechanism in which contacts previously uncharacterized gene results in down-regulation of with the highly active INS promoter stimulate expression of the nonoverlapping gene SSTR5 (somatostatin receptor 5). genes associated with insulin metabolism. It appeared that these Through this mechanism, SSTR5-AS1 indirectly controls the extent of SSTR5 signaling with SST28 (somatostatin-28), and regulatory pathways might be relevant to normal and abnormal β this, in turn, affects the known inhibitory effect of SST on cell function. At least 40% of the genes associated with T1D insulin secretion. susceptibility loci are expressed in human islets and β cells (15). Similarly, many loci known to harbor common SNPs contributing Results β to T2D contain genes associated with cell or islet function (11). Local Interactions on Chromosome 11. The EndoC-βH1 cell line is Here, we take advantage of the availability of a human pan- an insulin-secreting cell line derived from human pancreatic β β β creatic cell line, EndoC- H1 (16), to collect much higher cells (16). To explore the interactions between the INS locus and density 4C data without the complicating presence of signals other genomic sites, we used these cells to carry out 4C-seq ex- from the other kinds of cells present in islets. We show that in periments (23), with a site near the INS promoter as anchor (Fig. EndoC-βH1 cells, the INS promoter makes many strong cell 1A). The bait sequence contains the PDX1-binding elements type-specific contacts over large distances. The increased se- A3 and A5, the enhancer core element (binding region for his- quencing depth allows us not only to detect interactions within tone H3 lysine 27 acetyltransferase p300), the T1D susceptibility- chromosome 11, on which INS resides, but also to identify many linked microsatellite (VNTR), along with some other elements contacts of INS with sites on other chromosomes. We find that (24). We focused initially on contacts between INS and sites on A B C rs7111341 rs2334499 2Kb 50Kb 4 DE rs2334499 Fig. 1.