Project description:Non-coding regions of the genome contain cis-regulatory elements (CREs) that govern transcriptional activity critical for development and physiologic functions of major metabolic tissues, such as islets, adipose, liver, and skeletal muscle. Moreover, they contain the majority of genetic variants associated with risk of type 2 diabetes and quantitative alterations in related metabolic phenotypes. Here, we defined comprehensive, representative CRE maps of these major metabolic tissues in mice of both sexes using ATAC-seq and incorporating genetic variation of the 8 major Collaborative Cross founders and diet-response. Cross-species comparison of these representative maps with those from corresponding human tissues revealed conservation and functional preservation of ~28% of CREs in one or more major metabolic tissues. Importantly, this included an average of 700 (1.8%) cross-species concordant peaks that harbored genetic variants associated with T2D and related metabolic traits, including variants in the CAMK1D/CDC123, C2CD4A/B, and ADCY5 loci. Germline knock-out of the cross-species concordant CRE in ADCY5 yielded mice with impaired fasting glucose, demonstrating the importance of these functionally preserved CREs to glucose homeostasis and genetic risk of type 2 diabetes and progression. Together, the creation and cross-species comparison of comprehensive CRE maps in major metabolic tissues provides a critical resource for variant-to-function studies of type 2 diabetes and an array of related metabolic and cardiovascular traits and demonstrates the power of these mapping strategies to identify conserved metabolic cis-regulatory networks and enable data-driven creation of new preclinical models of diabetes and related metabolic diseases. Sample preparation: ATAC-seq libraries were prepared from adipose, liver, muscle, and islets as described in [Corces et al., 2016 (Nature Genetics) with slight modifications. Adipose, liver, and muscle samples were dissociated into single cell suspensions using MACS Miltenyi Biotec Adipose Tissue (130-105-808), Liver (130-105-807), and Skeletal Muscle (130-098-305) Dissociation Kits. Dissociated cells were counted and 50,000 cells were resuspended in freezing media (10% DMSO, 50% FBS, 40% RPMI) and frozen at -80°C until cells were thawed and washed in cold PBS, and incubated in ATAC lysis buffer. Islets were isolated from total pancreas as described in [Simonett et al., 2021 (JCI)], and 50 islets were rested overnight in RPMI in 37°C incubator. The following day islets were washed in cold PBS, incubated in ATAC lysis buffer, and triturated with 25-gauge needle and centrifuged to generate a crude nuclei pellet, and washed in ATAC lysis buffer. Prepared nuclei from all tissues were transposed using the Tagment DNA TDE1 Enzyme and Buffer kit (Illumina) with the addition of 0.01% digitonin (1x TDE for liver, muscle; 2x TDE for adipose). Transposed DNA was purified using the ChIP DNA Clean & Concentrator Kit (Zymo Research), amplified with 11 cycles of PCR, and cleaned using KAPA Pure Beads (Roche). All ATAC-seq libraries were sequenced at 2 x 100 bp using Illumina NovaSeq 6000. 1. Corces M.R., Buenrostro J.D., Wu B., Greenside P.G., Chan S.M., Koenig J.L., Snyder M.P., Pritchard J.K., Kundaje A., Greenleaf W.J. Lineage-specific and single-cell chromatin accessibility charts human hematopoiesis and leukemia evolution. Nat. Genet. 2016;48:1193–1203. https://www.nature.com/articles/ng.3646 2. Simonett S.P., Shin S., Herring J.A., Bacher R., Smith L.A., Dong C., Rabaglia M.E., Stapleton D.S., Schueler K.L., Choi J., Bernstein M.N., Turkewitz D.R., Perez-Cervantes C., Spaeth J., Stein R., Tessem J.S., Kendziorski C., Keleş S., Moskowitz I.P., Keller M.P., Attie A.D. Identification of direct transcriptional targets of NFATC2 that promote beta-cell proliferation. J Clin Invest. 2021;131(21):e144833. https://doi.org/10.1172/JCI144833.

Project description:Non-coding regions of the genome contain cis-regulatory elements (CREs) that govern transcriptional activity critical for development and physiologic functions of major metabolic tissues, such as islets, adipose, liver, and skeletal muscle. Moreover, they contain the majority of genetic variants associated with risk of type 2 diabetes and quantitative alterations in related metabolic phenotypes. Here, we defined comprehensive, representative CRE maps of these major metabolic tissues in mice of both sexes using ATAC and pcHiC data and incorporating genetic variation of the 8 major Collaborative Cross founders and diet-response. Cross-species comparison of these representative maps with those from corresponding human tissues revealed conservation and functional preservation of ~28% of CREs in one or more major metabolic tissues. Importantly, this included an average of 700 (1.8%) cross-species concordant peaks that harbored genetic variants associated with T2D and related metabolic traits, including variants in the CAMK1D/CDC123, C2CD4A/B, and ADCY5 loci. Germline knock-out of the cross-species concordant CRE in ADCY5 yielded mice with impaired fasting glucose, demonstrating the importance of these functionally preserved CREs to glucose homeostasis and genetic risk of type 2 diabetes and progression. Together, the creation and cross-species comparison of comprehensive CRE maps in major metabolic tissues provides a critical resource for variant-to-function studies of type 2 diabetes and an array of related metabolic and cardiovascular traits and demonstrates the power of these mapping strategies to identify conserved metabolic cis-regulatory networks and enable data-driven creation of new preclinical models of diabetes and related metabolic diseases. Sample preparation: Promotor capture libraries were constructed via the following method. Crosslinked cells were lysed, digested with DpnII, ends filled in using biotin-14-dATP followed by overnight proximity ligation. Ligated samples underwent crosslink reversal, DNA extraction with proteinase K, and biotin removal from unligated fragments. DNA was fragmented to 500-600 bp, column purified, and biotinylated fragments pulled down. Biotinylated fragments were converted into Illumina compatible libraries using the SureSelect XT HS2 DNA System and a custom SureSelect probe set (design ID S3346176) (Agilent Technologies) according to the manufacture’s protocol with the following exception: pre-capture PCR was performed as four 50 ul reactions that were recombined during clean-up. The quality and concentration of the libraries were assessed using the High Sensitivity D5000 ScreenTape (Agilent Technologies) and Qubit dsDNA HS Assay (ThermoFisher), respectively, according to the manufacturers’ instructions. Libraries were sequenced 150 bp paired end on an Illumina NovaSeq 6000.

Project description:A robust system using disease relevant cells to systematically evaluate the role in diabetes for loci identified through genome wide association studies (GWAS) is urgently needed. Toward this goal, we created isogenic mutant human embryonic stem cell (hESC) lines in GWAS-identified candidate diabetes genes including CDKAL1, KCNQ1 and KCNJ11, and used directed differentiation to evaluate the function of derivative human beta-like cells. The mutations did not affect the generation of insulin+ cells, but impaired insulin secretion both in vitro and in vivo, coinciding with defective glucose homeostasis. CDKAL1-/- insulin+ cells also displayed hypersensitivity to lipotoxicity. A high-content chemical screen identified a candidate drug that rescued CDKAL1-/--specific defects by inhibiting the AP1 (FOS/JUN) pathway. These studies establish a platform using isogenic hESCs to evaluate the function of GWAS-identified loci, and identify a drug candidate that rescues gene-specific defects, paving the way to precision therapy of metabolic diseases.A robust system using disease relevant cells to systematically evaluate the role in diabetes for loci identified through genome wide association studies (GWAS) is urgently needed. Toward this goal, we created isogenic mutant human embryonic stem cell (hESC) lines in GWAS-identified candidate diabetes genes including CDKAL1, KCNQ1 and KCNJ11, and used directed differentiation to evaluate the function of derivative human beta-like cells. The mutations did not affect the generation of insulin+ cells, but impaired insulin secretion both in vitro and in vivo, coinciding with defective glucose homeostasis. CDKAL1-/- insulin+ cells also displayed hypersensitivity to lipotoxicity. A high-content chemical screen identified a candidate drug that rescued CDKAL1-/--specific defects by inhibiting the AP1 (FOS/JUN) pathway. These studies establish a platform using isogenic hESCs to evaluate the function of GWAS-identified loci, and identify a drug candidate that rescues gene-specific defects, paving the way to precision therapy of metabolic diseases.

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Identification of cross-species preserved cis-regulatory elements containing type 2 diabetes GWAS variants

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