INFIMA leverages multi-omics model organism data to identify effector genes of human GWAS variants.

Genome-wide association studies reveal many non-coding variants associated with complex traits. However, model organism studies largely remain as an untapped resource for unveiling the effector genes of non-coding variants. We develop INFIMA, Integrative Fine-Mapping, to pinpoint causal SNPs for diversity outbred (DO) mice eQTL by integrating founder mice multi-omics data including ATAC-seq, RNA-seq, footprinting, and in silico mutation analysis. We demonstrate INFIMA\u27s superior performance compared to alternatives with human and mouse chromatin conformation capture datasets. We apply INFIMA to identify novel effector genes for GWAS variants associated with diabetes. The results of the application are available at http://www.statlab.wisc.edu/shiny/INFIMA/

INFIMA leverages multi-omics model organism data to identify effector genes of human GWAS variants

AbstractGenome-wide association studies reveal many non-coding variants associated with complex traits. However, model organism studies largely remain as an untapped resource for unveiling the effector genes of non-coding variants. We develop INFIMA, Integrative Fine-Mapping, to pinpoint causal SNPs for diversity outbred (DO) mice eQTL by integrating founder mice multi-omics data including ATAC-seq, RNA-seq, footprinting, and in silico mutation analysis. We demonstrate INFIMA’s superior performance compared to alternatives with human and mouse chromatin conformation capture datasets. We apply INFIMA to identify novel effector genes for GWAS variants associated with diabetes. The results of the application are available at http://www.statlab.wisc.edu/shiny/INFIMA/.11Ysciescopu

Gene loci associated with insulin secretion in islets from non-diabetic mice.

Genetic susceptibility to type 2 diabetes is primarily due to β-cell dysfunction. However, a genetic study to directly interrogate β-cell function ex vivo has never been previously performed. We isolated 233,447 islets from 483 Diversity Outbred (DO) mice maintained on a Western-style diet, and measured insulin secretion in response to a variety of secretagogues. Insulin secretion from DO islets ranged \u3e1,000-fold even though none of the mice were diabetic. The insulin secretory response to each secretagogue had a unique genetic architecture; some of the loci were specific for one condition, whereas others overlapped. Human loci that are syntenic to many of the insulin secretion QTL from mouse are associated with diabetes-related SNPs in human genome-wide association studies. We report on three genes, Ptpn18, Hunk and Zfp148, where the phenotype predictions from the genetic screen were fulfilled in our studies of transgenic mouse models. These three genes encode a non-receptor type protein tyrosine phosphatase, a serine/threonine protein kinase, and a Krϋppel-type zinc-finger transcription factor, respectively. Our results demonstrate that genetic variation in insulin secretion that can lead to type 2 diabetes is discoverable in non-diabetic individuals

Identification of direct transcriptional targets of NFATC2 that promote β cell proliferation

The transcription factor NFATC2 induces β cell proliferation in mouse and human islets. However, the genomic targets that mediate these effects have not been identified. We expressed active forms of Nfatc2 and Nfatc1 in human islets. By integrating changes in gene expression with genomic binding sites for NFATC2, we identified approximately 2200 transcriptional targets of NFATC2. Genes induced by NFATC2 were enriched for transcripts that regulate the cell cycle and for DNA motifs associated with the transcription factor FOXP. Islets from an endocrine-specific Foxp1, Foxp2, and Foxp4 triple-knockout mouse were less responsive to NFATC2-induced β cell proliferation, suggesting the FOXP family works to regulate β cell proliferation in concert with NFATC2. NFATC2 induced β cell proliferation in both mouse and human islets, whereas NFATC1 did so only in human islets. Exploiting this species difference, we identified approximately 250 direct transcriptional targets of NFAT in human islets. This gene set enriches for cell cycle–associated transcripts and includes Nr4a1. Deletion of Nr4a1 reduced the capacity of NFATC2 to induce β cell proliferation, suggesting that much of the effect of NFATC2 occurs through its induction of Nr4a1. Integration of noncoding RNA expression, chromatin accessibility, and NFATC2 binding sites enabled us to identify NFATC2-dependent enhancer loci that mediate β cell proliferation.11Nsciescopu

The Transcription Factor Nfatc2 Regulates β-Cell Proliferation and Genes Associated with Type 2 Diabetes in Mouse and Human Islets

<div><p>Human genome-wide association studies (GWAS) have shown that genetic variation at >130 gene loci is associated with type 2 diabetes (T2D). We asked if the expression of the candidate T2D-associated genes within these loci is regulated by a common locus in pancreatic islets. Using an obese F2 mouse intercross segregating for T2D, we show that the expression of ~40% of the T2D-associated genes is linked to a broad region on mouse chromosome (Chr) 2. As all but 9 of these genes are not physically located on Chr 2, linkage to Chr 2 suggests a genomic factor(s) located on Chr 2 regulates their expression in <i>trans</i>. The transcription factor <i>Nfatc2</i> is physically located on Chr 2 and its expression demonstrates <i>cis</i> linkage; <i>i</i>.<i>e</i>., its expression maps to itself. When conditioned on the expression of <i>Nfatc2</i>, linkage for the T2D-associated genes was greatly diminished, supporting <i>Nfatc2</i> as a driver of their expression. Plasma insulin also showed linkage to the same broad region on Chr 2. Overexpression of a constitutively active (ca) form of <i>Nfatc2</i> induced β-cell proliferation in mouse and human islets, and transcriptionally regulated more than half of the T2D-associated genes. Overexpression of either ca-Nfatc2 or ca-Nfatc1 in mouse islets enhanced insulin secretion, whereas only ca-Nfatc2 was able to promote β-cell proliferation, suggesting distinct molecular pathways mediating insulin secretion <i>vs</i>. β-cell proliferation are regulated by NFAT. Our results suggest that many of the T2D-associated genes are downstream transcriptional targets of NFAT, and may act coordinately in a pathway through which NFAT regulates β-cell proliferation in both mouse and human islets.</p></div

NFAT transcriptionally regulates a T2D GWAS genes in mouse and human islets.

<p>Heat maps illustrate the change in the expression of T2D-associated GWAS candidate genes in mouse (<b>A</b>, <b>B</b> and <b>C</b>) and human (<b>D</b>) islets in response to the overexpression of ca-Nfatc1, ca-Nfatc2 or GFP. For mouse islets, only those GWAS genes with a posterior probability (<i>PP</i>) > 0.99 of being differentially regulated by one or both of ca-NFATs are shown. For human islets, GWAS genes were selected from those showing robust regulation in mouse islets. Gene expression was determined by RNA-sequencing or qPCR in mouse and human islets respectively. Z-scores were computed from expression values for each gene across all samples (15 for mouse and 9 for human), and are shown relative to GFP (average Z-score for GFP = 0), which ranged from -3 to +3. Blue indicates reduced expression; red, increased expression; white, no change. Mouse genes are grouped according to their differential regulation (<b>A</b>), versus those that showed roughly equivalent suppression (<b>B</b>), or induction (<b>C</b>) in response to the two ca-NFATs. In <b>D</b>, * indicates <i>P</i> < 0.05 for human genes showing differential regulation by ca-Nfatc1 or ca-Nfatc2, relative to GFP.</p

NFAT triggers β-cell proliferation in mouse and human islets.

<p>Adenoviruses (Ad) were used to overexpress constitutively active (ca) <i>Nfatc1</i> or <i>Nfatc2</i> in human and mouse islets; Ad-LacZ was used as the negative control. Cellular proliferation was monitored by incorporation of [³H]-thymidine into islet DNA (<b>A</b>), FACS-based analysis of cell cycle phases (<b>B</b>), and incorporation of BrdU (white arrow heads) into islet cells that were co-stained for insulin or glucagon to identify β-cells and α-cells respectively (<b>C</b>). Thymidine incorporation measurements were conducted on 5 and 3 separate mouse and human islet preparations, respectively. Immunofluorescent images are representative of >30 islets (BrdU) per adenoviral treatment collected from 5 mice, or 3 human islet preparations. FACS analysis of mouse islets was performed on three separate occasions, each using a pool of ~300 islets per mouse (B6) collected from 5 or more mice per adenoviral treatment; analysis of human islets was performed on 4 separate human donor preparations, each with >6000 islets per adenoviral treatment. *, <i>P</i> < 0.05 relative to LacZ for N ≥ 3. Scale bars in C, 25 μm.</p

Nfatc1 <i>vs</i>. Nfatc2-mediated gene regulation in mouse islets.

<p>Whole-islet RNA-sequencing was used to profile gene expression 48 hr after overexpression of ca-Nfatc1, ca-Nfatc2 or GFP (negative control). Genes that were differentially expressed (DE) were classified into one of 4 distinct patterns (relative to GFP): <b>A</b>, DE in response to ca-Nfatc1 only (518 genes); <b>B</b>, DE in response to ca-Nfatc2 only (1580 genes); <b>C</b>, equally DE for ca-Nfatc1 and ca-Nfatc2 (2293 genes); and <b>D</b>, unequally DE for ca-Nfatc1 and ca-Nfatc2 (2621 genes). Gene sets enriched with cell cycle regulatory transcripts are highlighted in red. Expression values for all genes and isoforms are contained within <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006466#pgen.1006466.s016" target="_blank">S7 Table</a>.</p