Abstract
Biomechanical cues dynamically control major cellular processes but whether genetic variants actively participate in mechano-sensing mechanisms remains unexplored. Vascular homeostasis is tightly regulated by hemodynamics. Exposure to disturbed blood flow at arterial sites of branching and bifurcation causes constitutive activation of vascular endothelium contributing to atherosclerosis, the major cause of coronary artery disease (CAD) and ischemic stroke (IS). Conversely, unidirectional flow promotes quiescent endothelium. Genome-wide association studies have identified chromosome 1p32.2 as one of the most strongly associated loci with CAD/IS; however, the causal mechanism related to this locus remains unknown. Employing statistical analyses, ATAC-seq, and H3K27ac/H3K4me2 ChIP-Seq in human aortic endothelium (HAEC), our results demonstrate that rs17114036, a common noncoding polymorphism at the 1p32.2, is located in an endothelial enhancer dynamically regulated by hemodynamics. CRISPR/Cas9-based genome editing shows that rs17114036-containing region promotes endothelial quiescence under unidirectional flow by regulating phospholipid phosphatase 3 (PLPP3). Chromatin accessibility quantitative trait locus mapping using HAECs from 56 donors, allelic imbalance assay from 7 donors, and luciferase assays further demonstrate that CAD/IS protective allele at rs17114036 in PLPP3 intron 5 confers an increased endothelial enhancer activity. ChIPPCR and luciferase assays show that CAD/IS protective allele at rs17114036 creates a binding site for transcription factor Krüppel-like factor 2, which increases the enhancer activity under unidirectional flow. These results demonstrate for the first time that a human single-nucleotide polymorphism contributes to critical endothelial mechanotransduction mechanisms and suggest that human haplotypes and related cisregulatory elements provide a previously unappreciated layer of regulatory control in cellular mechano-sensing mechanisms.
Significance Statement Biomechanical stimuli control major cellular functions and play critical roles in the pathogenesis of diverse human diseases. Although recent studies have implicated genetic variation in regulating key biological processes, whether human genetic variants contribute to the cellular mechano-sensing mechanisms remains unclear. This study provides the first line of evidence supporting an underappreciated role of genetic predisposition in cellular mechanotransduction mechanisms. Employing epigenomic profiling, genome-editing, and latest human genetics approaches, our data demonstrate that rs17114036, a common noncoding polymorphism implicated in coronary artery disease and ischemic stroke by genome-wide association studies, dynamically regulates endothelial responses to blood flow (hemodynamics) related to atherosclerosis via regulation of an intronic enhancer. The results provide new molecular insights linking disease-associated genetic variants to cellular mechanobiology.
Introduction
Mechanical stimuli regulate major cellular functions and play critical roles in the pathogenesis of diverse human diseases (1). This is especially important in the vasculature, where endothelial cells are activated by local disturbed flow in arterial regions prone to atherosclerosis (2–6), the major cause of coronary artery disease (CAD) and ischemic stroke (IS). The role of biomechanical forces on the non-coding and regulatory regions of the human genome is unexplored. Recent studies demonstrated that the non-coding, non-transcribed human genome is enriched in cisregulatory elements (7). In particular, enhancers are distinct genomic regions that contain binding sites for sequence-specific transcription factors (8). Enhancers spatially and temporally control gene expression with cell type and cell state specific patterns (9). Notably, top-associated human disease-associated single nucleotide polymorphisms (SNPs) are frequently located within enhancers that explicitly activate genes in disease relevant cell types (10). The nature of mechano-sensitive enhancers and their biological roles in vascular functions have not been identified.
Atherosclerotic disease is the leading cause of morbidity and mortality worldwide. Genome-wide association studies (GWAS) identified chromosome 1p32.2 as one of the loci most strongly associated with susceptibility to CAD and IS (11–13). One candidate gene in this locus is PhosphoLipid PhosPhatase 3 (PLPP3, also known as PhosPhatidic-Acid-Phosphatase-type-2B/PPAP2B) which inhibits endothelial inflammation and promotes monolayer integrity by hydrolyzing lysophosphatidic acid (LPA) that activates endothelium (14, 15). Our recent study demonstrated that PLPP3 expression is significantly increased in vascular endothelium by unidirectional flow in vitro and in vivo (14). Moreover, expression quantitative trait locus (eQTL) mapping showed that CAD protective allele at 1p32.2 is associated with increased PLPP3 expression in an endothelium-specific manner (14). However, whether genetic variants and mechano-sensing mechanisms converge on PLPP3 expression is unclear. In addition, causal SNP(s) at locus 1p32.2 remain unknown.
Employing statistical analyses, ATAC-seq, H3K27ac ChIP-seq, H3K4me2 ChIP-seq, luciferase assays, and CRISPR/Cas9-based genome editing, we report that rs17114036-containing genomic region at 1p32.2 causatively promotes endothelial expression of phospholipid phosphatase 3 (PLPP3) and governs the athero-resistant endothelial phenotype under unidirectional flow by functioning as a mechano-sensitive endothelial enhancer. Using HAECs isolated from a cohort of human subjects, we performed transcriptome analyses and chromatin accessibility quantitative trait locus (caQTL) mapping showing nucleotide-specific epigenetic and transcriptomic effects of rs17114036 in humans. Allelic imbalance assays, ChIP-PCR, and luciferase assays collectively demonstrate that due to a single base pair change, the CAD/IS protective allele at rs17114036 confers increased activity of an endothelial intronic enhancer that is dynamically activated by unidirectional blood flow and transcription factor Krüppel-like factor 2 (KLF2). This is the first report elucidating underlying molecular mechanisms related to CAD/IS locus 1p32.2 and linking human disease-associated genetic variants to critical mechano-transduction mechanisms. The new molecular insights suggest that human genetic variants provide a novel layer of molecular control by which cells convert physical stimuli into biological signaling via tissue-specific enhancers.
Results
Bayesin Refinement and Conditional and Joint multiple-SNP analyses predict rs17114036 and rs2184104 are putative causal SNPs located in CAD/IS locus 1p32.2
Rs17114036 is the tag SNP used in most CAD/IS GWAS (11–13) and in our eQTL mapping (14); however, there are forty-four common SNPs in high linkage disequilibrium (LD) (r2>0.8) with rs17114036, and any of these SNPs could conceivably be a causal variant. To predict possible causal SNPs at the 1p32.2 locus, we conducted two statistical analyses. First, we employed a Bayesian statistical approach to assign posterior probabilities and credible sets of SNPs that refine the association signals of GWAS-detected loci (16). Second, we applied conditional and joint association analysis using summary-level statistics of GWAS data to predict causal variants (17). Using Bayes’ theorem in the cohort of forty-five SNPs at 1p32.2, we identified fifteen SNPs with >95% posterior probability to be causal (Fig. 1A and Supplementary Table 1). Using the approximate conditional and joint association analysis, we identified seven 1p32.2-associated SNPs to be possible causal (Fig. 1A and Supplementary Table 1). Only two SNPs, rs17114036 and rs2184104, were predicted to be causal by both methods.
CAD/IS-associated SNP rs17114036 is located in an enhancer element (chr1:56497123-56497188) in human aortic endothelial cells
Both rs17114036 and rs2184104 are located in non-coding regions. To probe the regulatory functions of these two SNPs in vascular endothelium, we performed Assay of Transposase Accessible Chromatin with whole genome sequencing (ATAC-seq) as well as H3K27ac and H3K4me2 chromatin immuno-precipitation with whole genome sequencing (ChIP-seq) in human aortic endothelial cells (HAECs). ATAC-seq is a high-throughput, genome-wide method to define chromatin accessibility that correlates with precise measures of transcription factor binding (18). The combination of H3K27ac and H3K4me2 ChIP-seq marks were used to identify active enhancers. Since the human PLPP3 gene is expressed from the minus strand in the annotated human genome, we use alleles in the minus strand at rs17114036 and rs2184104 in this manuscript. It is important to note that since CAD/IS risk alleles at rs17114036 (T) and rs2184104 (A) are major alleles in all ethnic groups (70-99% frequency) (19), our experiments, unless specified otherwise, were conducted in HAEC lines from donors who carry major alleles at rs17114036 and rs2184104. As demonstrated in Fig. 1B, rs17114036 in the intron 5 of the PLPP3 resides in an enhancer-like element (chr1:56962213-56963412, UCSC VERSION hg19) identified by ATAC-seq and H3K27ac/H3K4me2 ChIP-seq in HAECs. Encyclopedia of DNA Elements (ENCODE) also reported a DNase hypersensitive site and a H3K27ac peak in a ~1kb region enclosing rs17114036 in human umbilical vein endothelial cells (HUVEC) (7) (Supplemental Fig. 1). Notably, this region does not exhibit enhancer-like marks in other ENCODE cell types, such as K562, GM12878, and NHEK cells (Supplemental Fig. 1). In contrast, the other putative causal SNP, rs2184104, is located ~120kb downstream of the PLPP3 transcription start site at a location that lacks enhancer-like features (Fig. 1B). ENCODE data are consistent with our findings, and also signify an inactive chromatin domain surrounding rs2184104 (Supplemental Fig 1). Supplemental Fig. 2 shows the ATAC-seq and H3K27ac/H3K4me2 tracks in HAECs at 1p32.2 locus. The enhancer activity of chr1:56496541-56497740 was experimentally demonstrated by a luciferase reporter assay (Fig. 1C). Plasmid transfection was first validated in HAECs using electroporation of pmaxGFP-expressing constructs (Supplemental Fig. 3). A 1200 bp DNA sequence corresponding to human chr1:56962213-56963412 was cloned upstream of firefly luciferase that was driven by a minimal promoter. Reporter assays demonstrated that insertion of this putative enhancer region with major allele T at rs17114036 significantly increased the luciferase activity in HAECs (Fig. 1C). We further cloned these putative enhancer elements in a luciferase vector that contains the human PLPP3 promoter. Endogenous human PLPP3 promoter led to a 7.9 fold higher luciferase activity in HAECs when compared with the vector with minimal promoter (Fig. 1C). Moreover, insertion of the putative enhancer elements resulted in a 2.14-fold increase in luciferase activity when compared with the vector with only PLPP3 promoter (Fig. 1C). Minimal enhancer activities were detected when the constructs were expressed in the nonendothelium cell line HEK 293 (Supplemental Fig. 4). ATAC-seq, H3K27ac/H3K4me2 ChIP-seq, and luciferase assays altogether demonstrate that chr1:56962213-56963412 functions as an enhancer in HAECs.
CRISPR/Cas9-mediated deletion of rs17114036-containing genetic locus reduces PLPP3 expression in human aortic endothelium
To determine the causal role of rs17114036-containing genomic locus in regulating endothelial PLPP3 expression, the bacterial CRISPR-associated protein-9 nuclease (CRISPR/Cas9) system was used to selectively delete a ~66 bp genomic region (chr1:56962783-56962849) enclosing rs17114036 in HAECs. A pair of guide RNAs (Fig. 2A) was designed according to the method described by Ran et al (20). We pre-assembled Cas9-guide RNA ribonucleoprotein (RNP) complex by incubating guide RNAs with recombinant S. pyogenes Cas9 (21), followed by the delivery to cells using cationic liposome transfection reagents. To improve the efficiency of the CRISPR/Cas9-mediated deletion, cells were reverse-transfected by the RNP complex four times before flow cytometry assays to sort single cells (Fig. 2B). Immortalized HAECs (carrying major alleles at rs17114036) with high proliferating capacity were used for single cell clonal isolation that selects a genetically-identical cell line. Among 459 HAEC colonies we grew to confluency, PCR assays detected 17 lines with ~66 bp genetic deletion in PLPP3 intron 5 enclosing rs17114036 (Supplemental Fig. 5A). DNA deletion was further confirmed by TA-cloning and Sanger sequencing (Supplemental Fig 5B). Endothelial PLPP3 expression is significantly reduced in the genome-edited cells when compared to teloHAECs that underwent CRISPR/Cas9 treatment and single cell clonal isolation but showed no sign of deletion at chr1:56962783-56962849 (Fig 2C). In addition, deletion of this putative enhancer in PLPP3 intron 5 resulted in an increase of LPA-induced E-selectin expression (Fig 2D) and leukocyte adhesion (Fig 2E), in agreement with the anti-inflammatory/adhesive role of endothelial PLPP3 (14, 15). Moreover, trans-endothelial electrical resistance (TER) detected increased monolayer permeability in rs17114036-deleted HAECs (Fig 2F), consistent with PLPP3’s role in maintaining endothelial monolayer integrity (14, 15). These results demonstrate that deletion of the rs17114036-containing region in HAECs causatively reduces PLPP3 expression and promotes endothelial activation.
Unidirectional flow increases the enhancer activity at chr1:56497123-56497188 in vascular endothelium
Given the critical role of hemodynamics in controlling endothelial PLPP3 transcription (14), we tested whether blood flow regulates the enhancer activity of chr1:56962213-56963412. ATAC-seq and H3K27ac ChIP-seq were conducted in HAECs subjected to “athero-protective” unidirectional flow representing wall shear stress in human distal internal carotid artery or “athero-susceptible” flow mimicking hemodynamics in human carotid sinus (22). ATAC-seq captured an increased open chromatin region at chr1:56962213-56963412 in HAECs under unidirectional flow compared with cells under disturbed flow (Fig 3A). H3K27ac ChIP-seq indicated an increased enhancer activity of chr1:56962213-56963412 in HAECs under unidirectional flow (Fig 3A). In addition, genetic deletion of the rs17114036-containing region by CRISPR/Cas9 significantly impaired unidirectional flow-induced PLPP3 expression in HAECs (Fig 3B). These results collectively demonstrate that enhancer activity of chr1:56962213-56963412 is dynamically activated by the athero-protective unidirectional flow to regulate endothelial PLPP3.
CAD/IS protective allele C at rs1711403 confers a higher enhancer activity of chr1:56497123-56497188
GWAS have linked the minor allele C at rs17114036 at 1p32.2 to reduced CAD/IS susceptibility (11–13) and our eQTL mapping described increased PLPP3 expression in HAECs with minor allele C (14). We investigated the genotype-dependent effect of rs17114036 on the enhancer activity of chr1:56962213-56963412 by ATAC-seq and luciferase assays. In addition to HAEC lines carrying major (risk) allele T at rs17114036, we conducted ATAC-seq in HAECs isolated from donors who are heterozygous (T/C) (~20% of Europeans) at rs17114036, allowing us to perform chromatin accessibility quantitative trait locus (caQTL) mapping. caQTL was recently developed to detect between-individual signaling in cis-regulatory element as a function of genetic variants (23). ATAC-seq detected significantly increased numbers of reads corresponding to rs17114036-containing region in HAEC lines that contain one CAD protective allele (T/C) when compared with HAECs from donors homozygous of CAD risk allele (T/T) (Fig 4A), supporting increased chromatin accessibility associated with C allele at rs17114036. In addition, we conducted RNA-seq analysis in these cells demonstrating that there is a strong correlation between enhanced chromatin accessibility in rs17114036-containing region and increased mRNA levels of PLPP3 in HAECs (Fig 4B), further suggesting that chr1:56962213-56963412 functions as an enhancer in promoting endothelial PLPP3 transcription. Moreover, ATAC-seq experiments in HAEC lines heterozygous at rs17114036 further allow us to determine whether the chromosome with C at rs17114036 exhibits higher chromatin accessibility at chr1:56962213-56963412 when compared to the chromosome with T allele. This is achieved by the allelic imbalance (AI) analysis which assigns next generation sequencing reads overlapping heterozygous sites to one chromosome or the other for allele-specific signals (24). ATAC-seq detected reads enriched from the C-containing chromosome compared to that with T allele in HAECs heterozygous at rs1711403 (Fig. 4C), further supporting the increased chromosome accessibility associated with C allele at rs17114036. Lastly, luciferase assays were conducted to support the genotype-dependent enhancer activity of chr1:56962213-56963412. Replacement of T allele with C allele led to a much higher luciferase activity (~5.2 fold, C vs T) in endothelium (Fig. 4D). Taken together, these results demonstrate that CAD protective allele C at rs17114036 confers a higher enhancer activity of chr1:56962213-56963412 to promote PLPP3 expression in vascular endothelium.
CAD/IS protective C allele at rs1711403 promotes flow-induced, KLF2-mediated enhancer activity of chr1:56497123-56497188
We further examined whether the genetic variants at rs17114036 modulate the flow-induced enhancer activity of chr1:56962213-56963412. Luciferase assays detected an increase of the enhancer activity of chr1:56962213-56963412 (with protective C allele at rs17114036) in cells under 18-hr unidirectional flow when compared to disturbed flow (Fig 5A). In addition to the ATAC-seq experiments in HAEC homozygous at rs17114036 under flow (Fig. 3), we performed ATAC-seq analysis in four HAEC lines heterozygous at rs17114036 under 24-hr unidirectional flow, in order to perform open chromatin allelic imbalance (AI) analysis. Supplemental Fig. 6 demonstrates that in all four selected HAEC lines heterozygous at rs17114036, unidirectional flow increases ATAC-seq peaks in the proposed enhancer region in PLPP3 intron 5, in agreement with increased ATAC-seq reads in rs17114036-containging region (Fig. 5B). Moreover, allelic imbalance analysis showed an enrichment of ATAC-reads from the chromosome harboring the protective C allele (Fig. 5B). In contrast, ATAC-seq detected no allelic imbalance at rs6421497, a common SNP in high LD with rs17114036 (Supplemental Fig. 7). Indeed, the protective allele C at rs17114036 creates a CACC box that is a binding site for KLF2 which mediates the flow sensitivity of a cohort of endothelial genes including PLPP3 (14, 25-27). We then tested whether KLF2 dynamically activates this rs17114036-containing enhancer and if rs17114036 alleles impact the KLF2-mediated enhancer activity. First, the affinity of KLF2 to the rs17114036-containing locus was determined by KLF2 chromatin immunoprecipitation polymerase chain reaction (ChIPPCR) assays in HAECs carrying a protective allele at rs17114036, showing a physical binding of KLF2 to the CACC sites in the PLPP3 promoter and at rs17114036 (Fig. 5C). Enhancer activities of chr1:56962213-56963412 was further determined in HAECs as a function of KLF2 expression. Constructs of enhancer (chr1:56962213-56963412) and PLPP3 promoter were co-transfected with KLF2-overexpressing plasmids. Luciferase assays detected a 2.9 fold increase of luciferase activity in the T allele-containing construct as the result of KLF2 overexpression (Fig. 5D). Moreover, KLF2 overexpression led to a 4.7 fold increase of luciferase activity when T allele was substituted by the protective allele C at rs17114036 (Fig. 5D). Collectively, KLF2 ChIPPCR and luciferase assays demonstrate that CAD/IS protective allele C at rs17114036 confers a higher KLF2-dependent enhancer activity of chr1:56962213-56963412 in vascular endothelium.
Discussion
Although it is proposed that genetic and environmental factors jointly influence the risk of most common human diseases, the interplay between genetic predisposition and biomechanical cues at the molecular level is poorly understood. The biology underlying the majority of CAD and IS GWAS loci remains to be elucidated (28). Most of the CAD and IS SNPs reside in the noncoding genome. Gupta et al. recently reported that the non-coding common variant at rs9349379, implicated in CAD by GWAS, regulates endothelin 1 (EDN1) expression in endothelium (29). Atherosclerotic lesions preferentially develop at elastic arteries where vascular endothelial cells are activated by local disturbed flow (2–6). As of now, it remains unknown whether disease-associated genetic variants contribute to mechano-sensing mechanisms by which cells sense and convert biomechanical stimuli to biological signaling. Our results here elucidate the convergence of CAD/IS genetic predisposition and mechano-transduction mechanisms in endothelial PLPP3 expression. Statistical analyses, whole-genome chromatin accessibility/enhancer marks, genome editing, enhancer assays, chromatin accessibility QTL (caQTL) mapping, and allelic imbalance (AI) assay collectively demonstrate that CAD/IS locus 1p32.2 harbors a mechano-sensitive endothelial enhancer that regulates PLPP3 expression. Moreover, CAD/IS protective allele at rs17114036 confers an increased enhancer activity that is dynamically regulated by unidirectional flow and transcription factor KLF2 (Fig. 5E).
Dysregulation of mechano-sensing mechanisms contributes to the etiology of a wide range of human diseases in cardiovascular, pulmonary, orthopedic, muscular, and reproductive systems (1). The genetic basis of these complex human diseases has been strongly suggested by GWAS but the interplay between genetic variants and mechano-sensing mechanisms has not been investigated. Our data provide the first line of evidence supporting the genetic regulation of mechano-transduction mechanisms in complex human diseases and suggest an underappreciated role of genetic predisposition in cellular mechano-sensing processes.
Transcriptional enhancers orchestrate the majority of cell-type-specific patterns of gene expression (8) and play key roles in development, evolution, and disease (30) which are tightly regulated by mechanical cues (1). Our data provide new molecular evidence that the non-coding genome actively participates in cellular mechanotransduction mechanisms that are influenced by human genetic variances. In addition to the flow-regulation of the specific locus 1p32.2, our results provide one of the first datasets to systematically determine the mechanosensitive chromatin accessibility and putative enhancer regions at the whole-genome scale in vascular endothelium. It is important to note that most of the epigenome studies including ENCODE were conducted in cells without physiological or pathophysiological mechanical stimuli, such as HUVEC under static (no flow) conditions (7). Since major endothelial functions are tightly and dynamically regulated by hemodynamics (2–4), our whole-genome epigenome profiling in HAECs under athero-relevant flows may benefit future studies to investigate mechanical regulation of the non-coding genome in vascular cells. Indeed, we have applied Model-based Analysis of ChIP-Seq (MACS2) (31) and HOMER differential analysis (32) which unbiasedly identified rs17114036-containing locus as one of the 36,965 open chromatin sites that are activated by unidirectional flow (Supplementary Fig. 8).
Mechano-sensitive transcription factors have been proposed as major regulators to determine endothelial functions relevant to atherogenesis. For instance, nuclear factor-κB and HIF-1α mediate gene sets associated with pro-inflammatory, pro-coagulant, and glycolytic endothelial phenotypes under disturbed flow while Krüppellike factors and nuclear factor erythroid 2–like 2 regulate gene networks promoting the quiescent endothelial phenotype under unidirectional flow (26, 27, 33-36). However, the interaction between flow-sensitive transcription factors and disease-associated genetic predisposition in vascular functions has not been suggested. Our results here demonstrate that a genetic variant can influence important endothelial functions via a non-coding enhancer region recognized by the mechano-sensitive transcription factor KLF2. These results are consistent with emerging evidence showing top-scoring disease-associated SNPs are frequently located within enhancers explicitly active in disease-relevant cell types (10). Moreover, the data suggest that disease-associated genetic variants, via modulation of transcription factor binding, may regulate the enhancer activities dynamically responding to biomechanical cues that are instrumental to key cellular processes.
GWAS related to atherosclerotic diseases have suggested previously unsuspected loci, genes, and biology involved in lipoprotein metabolism (28), resulting in the development of new cholesterol-lowering therapies (37). Despite that dyslipidemia is a major systemic risk factor of CAD and ischemic stroke, atherosclerotic lesions largely initiate and develop at arterial regions of atypical vascular geometry associated with disturbed flow. Previous studies demonstrated that cellular mechano-transduction mechanisms, particularly endothelial responses to hemodynamics, causatively contribute to the focal nature of atherosclerotic lesions (2–6). Our studies here demonstrate that genetic variants not only contribute to inter-individual variation in plasma lipid concentrations (38) but also endothelial responses to blood flow. Indeed, genetic variants at rs17114036 predict CAD susceptibility independent of traditional systemic risk factors such as cholesterol and diabetes mellitus (11, 12). Recent GWAS identified 15 new CAD risk loci near genes of key functions in endothelial, smooth muscle, and white blood cells (39), further highlighting the potential importance of genetic contribution to the arterial-wall-specific mechanisms in atherogenesis. Our results indicate that CAD genetic predisposition and disturbed flow converge to inhibit endothelial PLPP3 expression and restoration of endothelial PLPP3 in atherosusceptible regions may provide an attractive approach for future arterial wall-based atherosclerosis therapy complementary to current pharmacological treatments targeting systemic risk factors.
Our studies demonstrate that the latest human genetics approaches such as caQTL mapping (23), allelic imbalance assay (24), and CRISPR-based assays (20, 21) are powerful tools to investigate possible genetic contributions to cellular mechanotransduction. Miao et al. recently applied CRISPR-Cas9 to achieve high efficiency of a 10 kb deletion of an enhancer region in bulk HUVEC (40). In this study we expanded the applications of CRISPR-based techniques to investigate key vascular functions. Isogenic adult aortic endothelial lines subjected to CRISPR-based deletion were successfully selected to determine the causal role of ~66bp genomic region in regulating endothelial PLPP3 expression. Nevertheless, one limitation here is that we are still unable to replace this human SNP at rs17114036 in adult aortic endothelium even though we have tried various methods to promote homology directed repair during CRISPR-Cas9 gene editing (21). This will be the subject of a future study. Nevertheless, caQTL mapping (23) and allelic imbalance assay (24) provide complementary approaches detecting at the single nucleotide resolution that CAD/IS protective allele at rs17114036 confers a higher enhancer activity at the PLPP3 intron 5. This study demonstrated that human haplotypes and related cis-regulatory elements provide an important layer of molecular controls by which cells convert physical stimuli into biological signaling.
Methods
Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq)
ATAC-seq was performed as previously described (18) using Tn5 transposase (Illumina, San Diego CA). Libraries were sequenced on an Illumina HiSeq 4000 according to manufacturer’s specifications by the Genomics Core Facility at the University of Chicago. The reads were aligned to the UCSC hg19 genome using Bowtie2 (41). ATAC-seq were conducted in HAECs under static conditions or subjected to 24-hr unidirectional flow or disturbed flow.
Chromatin accessibility quantitative trait locus (caQTL) mapping and Allelic Imbalance
caQTL mapping was performed to test for association between genotype at rs17114036 and chromatin accessibility measured by ATAC-seq. We pulled genotypes for HAEC donors from our previous study (42) and imputed linked alleles using IMPUTE2 and SHAPEIT as we published previously (43). Association testing between ATAC-seq tags at the rs17114036 enhancer and genotype were performed using the Combined Haplotype Test in WASP (24).
To perform allelic imbalance (AI) analysis that assigns next generation sequencing reads overlapping heterozygous sites to one chromosome or the other, we quantified ATAC-seq tags at the rs17114036 enhancer using HOMER’s annotatePeaks function to express the log2 normalized tags in this region.
CRISPR Cas9-mediated deletion of enhancer in teloHAECs
The CRISPR reagents were adapted from the Alt-R system from IDT (IDT, Coralville, IA). The guide RNAs were designed using an online tool at http://crispr.mit.edu/ to minimize off targeting effects using two guides to create a ~66 bp deletion. The guide RNAs were made by annealing the tracrRNA to the sgRNA. Cas9-guide RNA ribonucleoprotein (RNP) complex by incubating guide RNAs with recombinant S. pyogenes Cas9, followed by the delivery to cells using Lipofectamine RNAiMAX (Thermo). For each successive treatment the reagent amounts were scaled relative to the size of the destination vessel to compensate for the number of cells in the reaction. The volumes for each part of the reaction was increased 4x when treating cells from the 96-well to a 6-well, and 16x when moving from the 6-well to the T-75 flask.
Detailed methods are available in the supplemental materials.
Funding support
This work was funded by the NIH grants R01 HL136765 (Y.F.), R01 HL138223 (Y.F.), R00 HL123485 (C.R.), R00 HL121172 (M.C.), F32 HL134288 (D.W.), T32 EB009412 (D.H.), and T32 HL007381 (M.K.) as well as American Heart Association BGIA7080012 (Y.F.)