Endothelial cells (ECs) are critical determinants of vascular homeostasis and inflammation, but transcriptional mechanisms specifying their identities and functional states remain poorly understood. ...Here, we report a genome-wide assessment of regulatory landscapes of primary human aortic endothelial cells (HAECs) under basal and activated conditions, enabling inference of transcription factor networks that direct homeostatic and pro-inflammatory programs. We demonstrate that 43% of detected enhancers are EC-specific and contain SNPs associated to cardiovascular disease and hypertension. We provide evidence that AP1, ETS, and GATA transcription factors play key roles in HAEC transcription by co-binding enhancers associated with EC-specific genes. We further demonstrate that exposure of HAECs to oxidized phospholipids or pro-inflammatory cytokines results in signal-specific alterations in enhancer landscapes and associate with coordinated binding of CEBPD, IRF1, and NFκB. Collectively, these findings identify cis-regulatory elements and corresponding trans-acting factors that contribute to EC identity and their specific responses to pro-inflammatory stimuli.
While the immediate and transitory response of breast cancer cells to pathological stiffness in their native microenvironment has been well explored, it remains unclear how stiffness-induced ...phenotypes are maintained over time after cancer cell dissemination in vivo. Here, we show that fibrotic-like matrix stiffness promotes distinct metastatic phenotypes in cancer cells, which are preserved after transition to softer microenvironments, such as bone marrow. Using differential gene expression analysis of stiffness-responsive breast cancer cells, we establish a multigenic score of mechanical conditioning (MeCo) and find that it is associated with bone metastasis in patients with breast cancer. The maintenance of mechanical conditioning is regulated by RUNX2, an osteogenic transcription factor, established driver of bone metastasis, and mitotic bookmarker that preserves chromatin accessibility at target gene loci. Using genetic and functional approaches, we demonstrate that mechanical conditioning maintenance can be simulated, repressed, or extended, with corresponding changes in bone metastatic potential.
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•Mechanical conditioning is associated with distinct cytoskeletal features of cells•Mechanical conditioning is reflected in a temporal profile of chromatin accessibility•RUNX2 promotes bone metastasis via maintenance of mechanical conditioning•Breast tumor mechanical conditioning is associated with bone metastasis in patients
Watson et al. demonstrate that mechanical conditioning by stiff microenvironments in breast tumors is maintained in cancer cells after dissemination to softer microenvironments, including bone marrow. They show that mechanical conditioning promotes invasion and osteolysis and establish a mechanical conditioning (MeCo) score, associated with bone metastasis in patients.
Heterogeneity in endothelial cell (EC) sub-phenotypes is becoming increasingly appreciated in atherosclerosis progression. Still, studies quantifying EC heterogeneity across whole transcriptomes and ...epigenomes in both in vitro and in vivo models are lacking. Multiomic profiling concurrently measuring transcriptomes and accessible chromatin in the same single cells was performed on six distinct primary cultures of human aortic ECs (HAECs) exposed to activating environments characteristic of the atherosclerotic microenvironment in vitro. Meta-analysis of single-cell transcriptomes across 17 human ex vivo arterial specimens was performed and two computational approaches quantitatively evaluated the similarity in molecular profiles between heterogeneous in vitro and ex vivo cell profiles. HAEC cultures were reproducibly populated by four major clusters with distinct pathway enrichment profiles and modest heterogeneous responses: EC1-angiogenic, EC2-proliferative, EC3-activated/mesenchymal-like, and EC4-mesenchymal. Quantitative comparisons between in vitro and ex vivo transcriptomes confirmed EC1 and EC2 as most canonically EC-like, and EC4 as most mesenchymal with minimal effects elicited by siERG and IL1B. Lastly, accessible chromatin regions unique to EC2 and EC4 were most enriched for coronary artery disease (CAD)-associated single-nucleotide polymorphisms from Genome Wide Association Studies (GWAS), suggesting that these cell phenotypes harbor CAD-modulating mechanisms. Primary EC cultures contain markedly heterogeneous cell subtypes defined by their molecular profiles. Surprisingly, the perturbations used here only modestly shifted cells between subpopulations, suggesting relatively stable molecular phenotypes in culture. Identifying consistently heterogeneous EC subpopulations between in vitro and ex vivo models should pave the way for improving in vitro systems while enabling the mechanisms governing heterogeneous cell state decisions.
Single-cell sequencing have been widely used to characterize cellular heterogeneity. Sample multiplexing where multiple samples are pooled together for single-cell experiments, attracts wide ...attention due to its benefits of increasing capacity, reducing costs, and minimizing batch effects. To analyze multiplexed data, the first crucial step is to demultiplex, the process of assigning cells to individual samples. Inaccurate demultiplexing will create false cell types and result in misleading characterization. We propose scDemultiplex, which models hashtag oligo (HTO) counts with beta-binomial distribution and uses an iterative strategy for further refinement. Compared with seven existing demultiplexing approaches, scDemultiplex achieved great performance in both high-quality and low-quality data. Additionally, scDemultiplex can be combined with other approaches to improve their performance.
Combinatorial signaling by proinflammatory cytokines synergizes to exacerbate toxicity to cells and tissue injury during acute infections. To explore synergism at the gene-regulatory level, we ...investigated the dynamics of transcription and chromatin signaling in response to dual cytokines by integrating nascent RNA imaging mass spectrometry, RNA sequencing, amplification-independent mRNA quantification, assay for transposase-accessible chromatin using sequencing (ATAC-seq), and transcription factor profiling. Costimulation with interferon-gamma (IFNγ) and tumor necrosis factor alpha (TNFα) synergistically induced a small subset of genes, including the chemokines CXCL9, -10, and -11. Gene induction coincided with increased chromatin accessibility at non-coding regions enriched for p65 and STAT1 binding sites. To discover coactivator dependencies, we conducted a targeted chemogenomic screen of transcriptional inhibitors followed by modeling of inhibitor dose-response curves. These results identified high efficacy of either p300/CREB-binding protein (CBP) or bromodomain and extra-terminal (BET) bromodomain inhibitors to disrupt induction of synergy genes. Combination p300/CBP and BET bromodomain inhibition at half-maximal inhibitory concentrations (subIC50) synergistically abrogated IFNγ/TNFα-induced chemokine gene and protein levels.
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•IFNγ/TNFα synergistically induce a subset of proinflammatory transcripts•STAT1/p65 co-bind at enhanced accessible elements in response to IFNγ/TNFα•Synergistic transcription is vulnerable to combined CBP/p300 and BET inhibition•Open chromatin and STAT1/p65 binding are unaffected by CBP/p300 and BET inhibition
Components of the immune system; Cell biology; Systems biology; Genomics; Transcriptomics; Chemogenomics
The identification of causal variants and mechanisms underlying complex disease traits in humans is important for the progress of human disease genetics; this requires finding strategies to detect ...functional regulatory variants in disease-relevant cell types. To achieve this, we collected genetic and transcriptomic data from the aortic endothelial cells of up to 157 donors and four epigenomic phenotypes in up to 44 human donors representing individuals of both sexes and three major ancestries. We found thousands of expression quantitative trait loci (eQTLs) at all ranges of effect sizes not detected by the Gene-Tissue Expression Project (GTEx) in human tissues, showing that novel biological relationships unique to endothelial cells (ECs) are enriched in this dataset. Epigenetic profiling enabled discovery of over 3,000 regulatory elements whose activity is modulated by genetic variants that most frequently mutated ETS, AP-1, and NF-kB binding motifs, implicating these motifs as governors of EC regulation. Using CRISPR interference (CRISPRi), allele-specific reporter assays, and chromatin conformation capture, we validated candidate enhancer variants located up to 750 kb from their target genes, VEGFC, FGD6, and KIF26B. Regulatory SNPs identified were enriched in coronary artery disease (CAD) loci, and this result has specific implications for PECAM-1, FES, and AXL. We also found significant roles for EC regulatory variants in modifying the traits pulse pressure, blood protein levels, and monocyte count. Lastly, we present two unlinked SNPs in the promoter of MFAP2 that exhibit pleiotropic effects on human disease traits. Together, this supports the possibility that genetic predisposition for complex disease is manifested through the endothelium.
Functional consequences of genetic variation in the noncoding human genome are difficult to ascertain despite demonstrated associations to common, complex disease traits. To elucidate properties of ...functional noncoding SNPs with effects in human endothelial cells (ECs), we utilized our previous molecular quantitative trait locus (molQTL) analysis for transcription factor binding, chromatin accessibility, and H3K27 acetylation to nominate a set of likely functional noncoding SNPs. Together with information from genome-wide association studies (GWASs) for vascular disease traits, we tested the ability of 34,344 variants to perturb enhancer function in ECs using the highly multiplexed STARR-seq assay. Of these, 5711 variants validated, whose enriched attributes included: (1) mutations to TF binding motifs for ETS or AP-1 that are regulators of the EC state; (2) location in accessible and H3K27ac-marked EC chromatin; and (3) molQTL associations whereby alleles associate with differences in chromatin accessibility and TF binding across genetically diverse ECs. Next, using pro-inflammatory IL1B as an activator of cell state, we observed robust evidence (>50%) of context-specific SNP effects, underscoring the prevalence of noncoding gene-by-environment (GxE) effects. Lastly, using these cumulative data, we fine-mapped vascular disease loci and highlighted evidence suggesting mechanisms by which noncoding SNPs at two loci affect risk for pulse pressure/large artery stroke and abdominal aortic aneurysm through respective effects on transcriptional regulation of
and
Together, we highlight the attributes and context dependence of functional noncoding SNPs and provide new mechanisms underlying vascular disease risk.
CALCRL (calcitonin receptor-like) protein is an important mediator of the endothelial fluid shear stress response, which is associated with the genetic risk of coronary artery disease. In this study, ...we functionally characterized the noncoding regulatory elements carrying coronary artery disease that risks single-nucleotide polymorphisms and studied their role in the regulation of
expression in endothelial cells.
To functionally characterize the coronary artery disease single-nucleotide polymorphisms harbored around the gene
, we applied an integrative approach encompassing statistical, transcriptional (RNA-seq), and epigenetic (ATAC-seq transposase-accessible chromatin with sequencing, chromatin immunoprecipitation assay-quantitative polymerase chain reaction, and electromobility shift assay) analyses, alongside luciferase reporter assays, and targeted gene and enhancer perturbations (siRNA and clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeat-associated 9) in human aortic endothelial cells.
We demonstrate that the regulatory element harboring rs880890 exhibits high enhancer activity and shows significant allelic bias. The A allele was favored over the G allele, particularly under shear stress conditions, mediated through alterations in the HSF1 (heat shock factor 1) motif and binding. CRISPR deletion of rs880890 enhancer resulted in downregulation of
expression, whereas HSF1 knockdown resulted in a significant decrease in rs880890-enhancer activity and
expression. A significant decrease in HSF1 binding to the enhancer region in endothelial cells was observed under disturbed flow compared with unidirectional flow.
knockdown and variant perturbation experiments indicated the role of CALCRL in mediating eNOS (endothelial nitric oxide synthase), APLN (apelin), angiopoietin, prostaglandins, and EDN1 (endothelin-1) signaling pathways leading to a decrease in cell proliferation, tube formation, and NO production.
Overall, our results demonstrate the existence of an endothelial-specific HSF (heat shock factor)-regulated transcriptional enhancer that mediates
expression. A better understanding of
gene regulation and the role of single-nucleotide polymorphisms in the modulation of
expression could provide important steps toward understanding the genetic regulation of shear stress signaling responses.
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