BASIC RESEARCH www.jasn.org Chromatin Conformation Links Distal Target Genes to CKD Loci †‡ Maarten M. Brandt,* Claartje A. Meddens, Laura Louzao-Martinez,§ | †‡ † Noortje A.M. van den Dungen, ¶ Nico R. Lansu, ¶ Edward E.S. Nieuwenhuis, †‡ Dirk J. Duncker,* Marianne C. Verhaar,§ Jaap A. Joles,§ Michal Mokry, ¶ and Caroline Cheng*§ *Experimental Cardiology, Department of Cardiology, Thoraxcenter Erasmus University Medical Center, Rotterdam, The Netherlands; and †Department of Pediatrics, Wilhelmina Children’s Hospital, ‡Regenerative Medicine Center Utrecht, Department of Pediatrics, §Department of Nephrology and Hypertension, Division of Internal Medicine and Dermatology, |Department of Cardiology, Division Heart and Lungs, and ¶Epigenomics Facility, Department of Cardiology, University Medical Center Utrecht, Utrecht, The Netherlands ABSTRACT Genome-wide association studies (GWASs) have identified many genetic risk factors for CKD. However, linking common variants to genes that are causal for CKD etiology remains challenging. By adapting self-transcribing active regulatory region sequencing, we evaluated the effect of genetic variation on DNA regulatory elements (DREs). Variants in linkage with the CKD-associated single-nucleotide polymorphism rs11959928 were shown to affect DRE function, illustrating that genes regulated by DREs colocalizing with CKD-associated variation can be dysregulated and therefore, considered as CKD candidate genes. To identify target genes of these DREs, we used circular chro- mosome conformation capture (4C) sequencing on glomerular endothelial cells and renal tubular epithelial cells. Our 4C analyses revealed interactions of CKD-associated susceptibility regions with the transcriptional start sites of 304 target genes. Overlap with multiple databases confirmed that many of these target genes are involved in kidney homeostasis. Expression quantitative trait loci analysis revealed that mRNA levels of many target genes are genotype dependent. Pathway analyses showed that target genes were enriched in processes crucial for renal function, iden- tifying dysregulated geranylgeranyl diphosphate biosynthesis as a potential disease mechanism. Overall, our data annotated multiple genes to previously reported CKD-associated single-nucleotide polymorphisms and provided evidence for interaction between these loci and target genes. This pipeline provides a novel technique for hypothesis generation and complements classic GWAS interpretation. Future studies are required to specify the implications of our dataset and further reveal the complex roles that common variants have in complex diseases, such as CKD. J Am Soc Nephrol 29: ccc–ccc, 2017. doi: https://doi.org/10.1681/ASN.2016080875 CKD is a condition marked by loss of kidney func- functional annotation and explanation of these loci tion, which can lead to ESRD and is associated with a remain an issue. Currently, the functional annota- dramatic increase in cardiovascular disease–related tion of GWAS data is mainly conducted by linking morbidity and mortality.1 On the basis of the latest report of the Center for Disease Control and Pre- Received August 15, 2016. Accepted September 9, 2017. vention (2007–2014), over 15% of the United States M.M.B. and C.A.M. contributed equally to this work. M.M. and population is affected by CKD, and the numbers are C.C. contributed equally to this work. expected to rise. CKD incurs substantial rising Published online ahead of print. Publication date available at medical costs in the United States, with similar de- www.jasn.org. velopments observed globally. Over the last decade, Correspondence: Dr. Caroline Cheng, University Medical Center the findings of multiple genome-wide association Utrecht, PO Box 85500, 3508 GA Utrecht, The Netherlands. studies (GWASs) have established common DNA Email: [email protected] variants as genetic risk factors for CKD.2,3 However, Copyright © 2017 by the American Society of Nephrology J Am Soc Nephrol 29: ccc–ccc, 2017 ISSN : 1046-6673/2902-ccc 1 BASIC RESEARCH www.jasn.org susceptibility loci by spatial proximity to the nearest gene.4 For Significance Statement example, well known single-nucleotide polymorphisms (SNPs) that are associated with CKD include SNPs annotated Genome-wide association studies (GWASs) have identified a num- with ALMS1 and UMOD. ALMS1 is required for medullar ber of genetic regions correlated with development of CKD, but collecting duct ciliogenesis,5 whereas UMOD is involved in establishing causality remains challenging. This study applies a new fl approach to GWAS interpretation: to complement classic annota- the inhibition of calcium oxalate crystallization in renal u- tion on the basis of linear spatial proximity, the principle of tran- ids6 and has an evolutionary role in protection from urinary scriptionaldysregulationisusedtoidentifysiteswhereCKD-associated tract infections.7 Because these SNPs are located in coding variation colocalizes with DNA regulatory elements. The study de- regions of genes with important renal protective functions, scribestheidentificationof304candidategenesthatphysicallyinteract it is conceivable that the genetic variation marked by these with regulatory elements that colocalize with 39 common variants associated with CKD. Future studies will be required to verify the SNPs affects both genes, contributing to CKD pathogenesis. findings of this screening pipeline, but the method could help to de- For many of the CKD-associated susceptibility loci that are not termine the causal roles that common variants play in complex dis- directly located in or near protein coding regions, the causal eases, such as CKD. contribution to disease etiology is far less straightforward. New insights brought by epigenetic research have revealed the prevalence of DNA regulatory elements (DREs), such as target gene expression, leading to disease or other phenotypes enhancers and repressors, located in both coding- and non- (Figure 1, C and D). This was shown previously for the SNP protein-coding DNA regions (Figure 1A).8 These DREs play a rs12913832, which was shown to modulate human pigmentation crucial role in regulating gene expression in a cell-specific by affecting the enhancer regulation of the OCA2 promoter.10 manner. Enhancer elements regulate transcription of their Systematic mapping of the target genes of DREs that overlap target genes through three-dimensional (3D) chromatin in- with known CKD-associated SNPs could greatly improve our un- teractions with transcriptional start sites (TSSs) (Figure 1B). derstanding of the complex genetics of CKD. Importantly, DREs can regulate expression levels of gene targets Here, we used self-transcribing active regulatory region over a distance up to thousands of kilobase pairs,9 far exceeding the sequencing (STARR-seq) to evaluate the potential effect of current standard distance for GWAS annotation. Common genet- CKD-associated genetic variation on transcriptional regula- ic variation in DREs could be a causative factor in dysregulation of tion.Inaproofofprincipleapproach,weclonedputativeDREs located on the same linkage disequilibrium (LD) block as the CKD-associated SNP rs11959928 from 20 donors in STARR-seq reporter plasmids. This approach enabled us to study the effect of all variants found on this susceptibility region in the donor pool on enhancer activity in primary human renal proximal tubular epithelial cells (HRPTECs), human renal glomerular endo- thelial cells (HRGECs), and the human embryonic kidney cell line HEK293a. The findings of this experiment illustrated how regulatoryfunctioncouldbeaffectedbycom- mon small variants, thereby highlighting the relevance of studying downstream target genes of DREs overlapping with disease- associated susceptibility regions to add an additional layer to post-GWAS analysis. Subsequently, we used circular chromo- some conformation capture sequencing (4C-seq) to identify putative candidate genes for CKD by examining 3D interac- Figure 1. Genetic variation in DREs could be a causative factor in dysregulation of tions between DREs that colocalize with distal target gene expression. (A) Many of the susceptibility loci that are not located in CKD susceptibility loci and their target protein coding regions overlap with DREs, such as enhancers and repressors. (B) DREs play a crucial role in regulating gene expression in a cell-specific manner by modulating genes. This allowed us to study long-range 3D chromatin interactions and increasing spatial proximity of DREs with TSSs, thereby regulation of target gene promoters by regulating transcription of genes on a nonlinear DNA scale. (C) Distal transcriptional crosslinking the folded and interacting activity of DREs could be compromised by (D) genetic variation (represented by co- DRE segments followed by two restriction- localization with disease-associated SNP). ligation steps of the DNA strands and 2 Journal of the American Society of Nephrology J Am Soc Nephrol 29: ccc–ccc,2017 www.jasn.org BASIC RESEARCH Figure 2. STARR-seq analysis illustrates the effect of CKD-associated genetic variation on transcriptional regulation. (A) The STARR-seq reporter principle is on the basis of a reporter plasmid containing a minimal promotor followed by a cloned candidate enhancer se- quences. The activity of each enhancer is reflected by its ability to transcribe itself. ORF, Open Reading Frame. (B) Putative DREs, identified by H3K4Me1, H3K27Ac, and DNAse clusters (human umbilical vein endothelial cells are in blue and human epidermal keratinocytes