Chromatin interaction maps of human arterioles reveal new mechanisms for the genetic regulation of blood pressure

Abstract

Arterioles are small blood vessels located just upstream of capillaries in nearly all tissues. The constriction and dilation of arterioles regulate tissue perfusion and are primary determinants of systemic blood pressure (BP). Abnormalities in arterioles are central to the development of major diseases such as hypertension, stroke, and microvascular complications of diabetes. Despite the broad and essential role of arterioles in physiology and disease, current knowledge of the functional genomics of arterioles is largely absent, partly because it is challenging to obtain and analyze human arteriole samples. Here, we report extensive maps of chromatin interactions, single-cell expression, and other molecular features in human arterioles and uncover new mechanisms linking human genetic variants to gene expression in vascular cells and the development of hypertension. Compared to large arteries, arterioles exhibited a higher proportion of pericytes which were strongly associated with BP traits. BP-associated single nucleotide polymorphisms (SNPs) were enriched in chromatin interaction regions in arterioles, particularly through enhancer SNP-promoter interactions, which were further linked to gene expression specificity across tissue components and cell types. Using genomic editing in animal models and human induced pluripotent stem cells, we discovered novel mechanisms linking BP-associated noncoding SNP rs1882961 to gene expression through long-range chromatin contacts and revealed remarkable effects of a 4-bp noncoding genomic segment on hypertension in vivo. We anticipate that our rich data and findings will advance the study of the numerous diseases involving arterioles. Moreover, our approach of integrating chromatin interaction mapping in trait-relevant tissues with SNP analysis and in vivo and in vitro genome editing can be applied broadly to bridge the critical gap between genetic discoveries and physiological understanding.

Figure 1.. The cellular composition and properties of human arterioles exhibit distinct features compared with the aorta.

A. UMAP plot showing major cell types identified in arteriole and thoracic aorta. B. Dot plot displaying marker gene expression across different cell types. C. Relative proportions of pericytes to ECs and VSMCs to ECs in arteriole and thoracic aorta, shown separately. D. Heatmap showing pairwise gene expression correlations across cell types in arteriole and thoracic aorta. E. Dot plot showing significant associations between cell types and blood pressure-related traits from MAGMA cell typing analysis. Cell type abbreviations: EC (endothelial cell), LEC (lymphatic endothelial cell), VSMC (vascular smooth muscle cell).

Figure 2.. Chromatin contact regions in human arterioles contain DNA regulatory elements and correlate with greater gene expression.

A. Venn diagrams showing the overlap between loops identified in arterioles and EC-denuded arterioles (EDA) by Micro-C at 4 kb, 8 kb, and 16kb resolutions. B. Venn diagrams showing the overlap between interactions identified in arterioles and EDA by pan-promoter Capture Micro-C at 10 kb and 20 kb resolutions. C. Bar plot showing the number of loops with regulatory elements present in one or both chromatin contact regions in arterioles and EDA across resolutions. “None,” “one,” and “both” indicate the presence of regulatory elements in neither, one, or both interacting regions, respectively. D. Number of chromatin interactions classified by interaction type: promoter-promoter (PP), enhancer-promoter (EP), enhancer-enhancer (EE), enhancer-transcription factor binding site (ET), promoter-transcription factor binding site (PT), and transcription factor binding site-transcription factor binding site (TT). Categories are not mutually exclusive. E. Expression levels of genes proximal to chromatin interactions (within 5 kb upstream or downstream of contact regions), grouped by interaction type (PP, EP, EE, ET, PT, TT). Controls include genes proximal to regulatory regions, including promoters, enhancers, or transcription factor binding sites, that were not in contact regions based on our data. F. Boxplot showing gene expression levels grouped by the number of chromatin interactions involving gene promoter regions. Spearman correlation was applied.

Figure 3.. Chromatin interactions with gene promoters, DNA methylation, and mRNA abundance of representative genes essential to arteriolar function.

A. AGTR1 (Angiotensin II Receptor Type 1). B. EDN1 (Endothelin 1). C. NOS3 (Nitric Oxide Synthase 3). Linux-based IGV was used to plot chromatin interactions based on pan-promoter capture Micro-C, RNA abundance based on poly(A)-dependent RNA-seq, and methylation levels based on RRBS. Only chromatin interactions with the promoter of the gene of interest are shown. The chromatin contact regions were compared with the regulatory elements as defined by Ensembl for overlaps. E, enhancer; T, transcriptional factor binding site; C, CCCTC-binding factor (CTCF) binding site; O, open chromatin region.

Figure 4.. Chromatin contact regions in human arterioles are enriched for SNPs associated with blood pressure and stroke.

A. Bar plot showing the percentage of SNPs covered by Micro-C or pan-promoter Capture Micro-C in arterioles and endothelium-denuded arteriole (EDA), grouped by different arteriole-related traits. Red dashed lines indicate the average SNP coverage rate from non-arteriole-related traits (Methods). A one-sided binomial test was used to assess the significance of the higher SNP coverage rates in arteriole-related traits. B. Bar plot showing the density of BP-associated SNPs within 1 Mb loop contact regions across our Micro-C data in arterioles and EDA, compared to Hi-C data from the tibial artery and aorta (ENCODE). Red dashed lines represent the average SNP density from the Hi-C data of the tibial artery and aorta. C. Bar plot showing the distance distribution of SNP-gene pairs capture by Micro-C or pan-promoter Capture Micro-C in arteriole and EDA, grouped by distance categories. SNPs associated with arteriole-related traits (as listed in Fig. 4A) were included. D. Pathway enrichment results of genes associated with arteriole-related SNPs through chromatin interactions captured by pan-promoter Capture Micro-C, shown separately for arteriole and EDA. E. Schematic of SNP-gene interaction patterns grouped by SNP bin location in pan-promoter Capture Micro-C, along with the corresponding interaction numbers in arterioles and EDA. F. Percentage of covered SNPs based on the regulatory elements in which the SNPs are located, calculated separately for promoter bins and distal bins. Red dashed lines represent the overall coverage rate of all SNPs. A Fisher’s exact test was used to assess the significance of SNP coverage in specific regulatory groups, followed by Benjamini-Hochberg (BH) correction for p-values. Only significant results with an odds ratio > 1 are highlighted with asterisks. G. Dot plot showing the relationship between enhancer SNP-gene promoter interactions and corresponding gene expression. Only interactions specifically identified in arterioles or EDA, and genes with significant expression differences between arterioles and EDA (BH-corrected p-value < 0.05, absolute log2 fold change > 0.5) were included. Consistent results are highlighted with a red background. Notable SNP-gene pairs are labeled, and the numbers for SNP-gene pairs in each condition are shown in the corners of each quadrant. H. Dot plot showing the enrichment of arteriole-related SNP-interacting genes in cell type-specific genes using a Fisher test. SNP-interacting genes were grouped by interaction type, including promoter-enhancer SNP-interacting genes, promoter-interacting genes, and all-interacting genes (faceted by columns), and by the occurrence of interactions in arterioles only, EDA only, or both (faceted by rows). I. Rank plot of genes significantly higher expressed in fibroblast compared to all other cells (Bonferroni-corrected p-value < 0.05 by Wilcoxon test, ordered by log2 fold change). Genes having chromatin interactions involving arteriole-related SNPs, identified via pan-promoter Capture Micro-C, were highlighted and colored based on the occurrence in samples. Top 10 genes were label. J. UMAP plots showing the expression pattern of MEG3 and SEMA4A across cell types in arterioles, based on snRNA-seq data. Statistical significance is indicated as follows: *p < 0.05, **p < 0.01, and **p < 0.001.

Figure 5.. BP-associated noncoding SNP rs1882961 interacts with NRIP1 promoter 119 kbp away and influences NRIP1 expression.

A. Prioritization of SNP-gene pairs for experimental analysis. B. The genomic region containing rs1882961 interacted with NRIP1 promoter 119 kbp away from it in human endothelium-denuded arterioles, based on pan-promoter capture Micro-C analysis. C. Local gene organization around rs1882961 in the human genome. D. Deletion of DNA segment containing rs1882961. 39b, original iPSC cell line; X33 and X85, rs1882961-deleted cell lines. E. Reconstitution of the rs1882961 locus containing either homozygous low BP allele or high BP allele. 39b, original iPSC cell line; X85, rs1882961-deleted cell line; Y series, cell lines with reconstituted low-BP rs1882961 allele; Z series, cell lines with reconstituted high-BP rs1882961 allele. See Supplementary Fig. S8 for sequence confirmation. F. Homozygous high-BP allele of rs1882961, reconstituted in hiPSCs, increased NRIP1 and SAMSN1 expression in isogenic hiPSC-derived vascular smooth muscle cells (iVSMCs). N = 15; *, p < 0.05, unpaired t-test. G. Region-capture Micro-C analysis showed greater chromatin interactions between the rs1882961 region and the promoters of SAMSN1 and NRIP1 in iVSMCs with the high-BP allele of rs1882961, compared to iVSMCs with the low-BP allele. Each square color box in the zoom-in images is 5 kbp x 5 kbp.

Figure 6.. Deletion of a 4-bp noncoding genomic segment containing the rs1882961 orthologous site attenuates salt-induced hypertension in the Dahl salt-sensitive (SS) rat.

A. Comparative mapping and deletion of a 4-bp rat genomic segment containing the rs1882961 orthologous site. See Supplementary Fig. S10 for additional detail and confirmation of deletion. The deletion of the 4-bp noncoding genomic segment in the SS rat attenuated salt-induced hypertension. B. Mean arterial pressure (MAP). C. Systolic blood pressure (SBP). D. Diastolic blood pressure (DBP). E. Pulse pressure. F. Heart rate. G. Hourly averages of MAP on days 12 to 14 on a 4% NaCl high salt diet. WT, wild-type littermate SS rats; Δrs961, SS-Δrs1882961−/− rats. N = 7 for WT and 8 for Δrs961. #, p < 0.05, ##, p < 0.01 for WT vs. Δrs961 by two-way RM ANOVA; *, p < 0.05 vs. WT by Holm-Sidak test. H. Nrip1 expression in the mesentery was lower in SS-Δrs1882961−/− rats compared to WT. N = 4–9. Data shown as Mean ± SEM. *, p<0.05, Two-way ANOVA followed by Holm-Sidak test.