Integrated Functional Genomic Analysis Enables Annotation of Kidney Genome-Wide Association Study Loci
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BASIC RESEARCH www.jasn.org Integrated Functional Genomic Analysis Enables Annotation of Kidney Genome-Wide Association Study Loci Karsten B. Sieber,1 Anna Batorsky,2 Kyle Siebenthall,2 Kelly L. Hudkins,3 Jeff D. Vierstra,2 Shawn Sullivan,4 Aakash Sur,4,5 Michelle McNulty,6 Richard Sandstrom,2 Alex Reynolds,2 Daniel Bates,2 Morgan Diegel,2 Douglass Dunn,2 Jemma Nelson,2 Michael Buckley,2 Rajinder Kaul,2 Matthew G. Sampson,6 Jonathan Himmelfarb,7,8 Charles E. Alpers,3,8 Dawn Waterworth,1 and Shreeram Akilesh3,8 Due to the number of contributing authors, the affiliations are listed at the end of this article. ABSTRACT Background Linking genetic risk loci identified by genome-wide association studies (GWAS) to their causal genes remains a major challenge. Disease-associated genetic variants are concentrated in regions con- taining regulatory DNA elements, such as promoters and enhancers. Although researchers have previ- ously published DNA maps of these regulatory regions for kidney tubule cells and glomerular endothelial cells, maps for podocytes and mesangial cells have not been available. Methods We generated regulatory DNA maps (DNase-seq) and paired gene expression profiles (RNA-seq) from primary outgrowth cultures of human glomeruli that were composed mainly of podo- cytes and mesangial cells. We generated similar datasets from renal cortex cultures, to compare with those of the glomerular cultures. Because regulatory DNA elements can act on target genes across large genomic distances, we also generated a chromatin conformation map from freshly isolated human glomeruli. Results We identified thousands of unique regulatory DNA elements, many located close to transcription factor genes, which the glomerular and cortex samples expressed at different levels. We found that ge- netic variants associated with kidney diseases (GWAS) and kidney expression quantitative trait loci were enriched in regulatory DNA regions. By combining GWAS, epigenomic, and chromatin conformation data, we functionally annotated 46 kidney disease genes. Conclusions We demonstrate a powerful approach to functionally connect kidney disease-/trait–associated loci to their target genes by leveraging unique regulatory DNA maps and integrated epigenomic and ge- netic analysis. This process can be applied to other kidney cell types and will enhance our understanding of genome regulation and its effects on gene expression in kidney disease. J Am Soc Nephrol 30: ccc–ccc, 2019. doi: https://doi.org/10.1681/ASN.2018030309 In the past 15 years, large-scale genome-wide asso- ciation studies (GWAS) have successfully identified genetic variants associated with a wide variety of Received March 23, 2018. Accepted December 26, 2018. measurable traits and human diseases, including Published online ahead of print. Publication date available at those related to kidney function.1–3 The minority www.jasn.org. of GWAS variants localize to protein-coding Correspondence: Dr. Shreeram Akilesh, Department of Ana- sequences (exemplified by APOL14); most lie in tomic Pathology, University of Washington, Box 356100, 1959 nonprotein-coding genomic sequences, which NE Pacific Street, Seattle, WA 98195. Email: [email protected] compose .98% of the human genome.5 Regulatory Copyright © 2019 by the American Society of Nephrology J Am Soc Nephrol 30: ccc–ccc, 2019 ISSN : 1046-6673/3003-ccc 1 BASIC RESEARCH www.jasn.org DNA elements, which encompass promoters, enhancers, and Significance Statement insulators, are small segments of the genome where DNA bind- ing proteins recognize specific DNA sequence features leading to The absence of high-resolution epigenomic maps of key kidney cell the recruitment of histone modifying complexes, displacement types has hampered understanding of kidney-specific genome of nucleosomes, and opening of nuclear chromatin.6 Active reg- regulation in health anddisease. Kidney-associatedgenetic variants, identified in genome-wide association studies, are concentrated in ulatory DNA elements are often located in nonprotein-coding accessible chromatin regions containing regulatory DNA elements. sequences7,8 and appear to functionally and physically associate The authors describe the generation and initial characterization of with their target gene promoters over large genomic distances, paired DNA maps of these regulatory regions and gene expression often skipping intervening genes.8 Furthermore, GWASvariants profiles of cells from primary human glomerular and cortex cultures. frequently localize to regulatory DNA elements.5,9–14 Because By integrating analyses of genetic and epigenomic data with ge- nome-wide chromatin conformation data generated from freshly the chromatin accessibility of some regulatory DNA elements isolated human glomeruli, they physically and functionally con- – can be very cell type–specific,7,9,15 17 one mechanism by which nected 42 kidney genetic loci to 46 potential target genes. Apply- genetic variants can contribute to disease risk is by altering the ing this approach to other kidney cell types is expected to regulation of gene expression in a cell type–specific manner enhanceunderstanding of genome regulationand its effects ongene (Supplemental Figure 1).18 Delineating the cell type–specific expression in kidney disease. gene regulatory networks for multiple important kidney cell types will therefore be paramount to dissecting kidney disease mechanisms. (protocol #1297). Approximately 1 cm3 portions of unin- Enzymatic reporters, such as deoxyribonuclease I (DNase I)19,20 volved kidney cortex (from the pole furthest from the tumor and the Tn5 transposase,21 when combined with next-generation mass) were harvested and transported in RPMI medium on sequencing methods, efficiently identify regions of open chroma- ice. These tissues were then minced with a sterilized razor tin that are associated with regulatory DNA elements. However, blade and the fragments were placed in 20 ml of prewarmed these methodologies have only been applied to the study of a few RPMI medium (without serum) supplemented with Accutase kidney cell types and chromatin accessibility maps are lacking for (diluted 1:10; Sigma), collagenase P (100 mg/ml; Roche), and many important kidney cell types such as podocytes, mesangial trypsin/EDTA (0.25% solution diluted 1:10; Gibco). The tis- cells, distal tubule cells, peritubular microvascular endothelial sue fragments were digested at 37°C for 20 minutes with vig- cells, pericytes, and resident immune cells (e.g.,macrophages orous agitation. For glomerular core isolation, the softened and dendritic cells). To begin to approach this problem, we re- tissue fragments were mashed through a #60-gauge sterilized port the generation of high-resolution chromatin accessibility steel mesh using the bottom of sterilized glass beaker. This maps (DNase-sequencing [DNase-seq]) and paired gene expres- treatment stripped the glomerular cores of their Bowman’s sion profiles (RNA-sequencing [RNA-seq]) of primary cultures capsules. The isolated glomerular cores passed through the of human glomerular outgrowth cells (mixed cultures of po- mesh and were collected on a #140-gauge steel mesh placed docytes and mesangial cells). To enable comparative analysis of below. Tubules were disrupted sufficiently that they did not cell type–specific features, we also generated datasets from pri- collect on the #140-gauge steel mesh. The isolated glomerular mary human renal cortical cultures treated in similar fashion. cores were washed extensively with sterile room temperature To understand the basis of long-range interactions between PBS and were then transferred into a tissue culture flask with regulatory DNA elements and their target genes, we prewarmed culture medium (RPMI supplemented with 10% generated a chromatin conformation (Hi-C) map from freshly FBS and insulin-transferrin-selenite+ [ITS+] supplement; isolated human glomeruli. We then integrated these diverse Corning). For the primary culture of cortical cells, after digestion measures of genome function to connect kidney GWAS loci of a separate cortical tissue fragment (as described above), the to their potential target genes, thereby gaining new insights pieces were spun down and macerated in a petri dish using a into the genome regulatory mechanisms that underlie kidney sterile plunger from a 5 ml syringe. These softened tissue frag- phenotypes and disease. ments were then transferred into tissue culture flasks with pre- warmed medium containing 10% FBS and ITS+ as described above. After 2–3 days (for cortex cultures) and 10–14 days METHODS (for glomerular outgrowth cultures), the tissue fragments were decanted and the adherent cells were fed with fresh medium. Generation and Characterization of Primary At this stage, primary cortex cells grew rapidly and had an epi- Glomerular and Cortex Cultures thelioid morphology, whereas primary glomerular outgrowth Kidney tissues were obtained from patients undergoing radi- colonies grew more variably with a mixture of epithelioid and cal nephrectomy for renal tumors with informed consent for spindled cells. Glomerular outgrowth cells were used at first pas- DNA sequencing obtained before surgery. Patient character- sage for all experiments (typically approximately 2–3 weeks istics and resulting dataset features are listed in Supplemental after initial isolation). Cortex outgrowth cells were subcultured Table 1. The study and consent forms were approved by the at 1:4 when they reached 80% confluence,andusedwithintwo University of Washington’s Institutional Review Board passages