Profiling RNA at chromatin targets in situ by antibody-targeted tagmentation.

Whereas techniques to map chromatin-bound proteins are well developed, mapping chromatin-associated RNAs remains a challenge. Here, we describe Reverse Transcribe and Tagment (RT&Tag), in which RNAs associated with a chromatin epitope are targeted by an antibody followed by a protein A-Tn5 transposome. Localized reverse transcription generates RNA/cDNA hybrids that are subsequently tagmented by Tn5 transposases for downstream sequencing. We demonstrate the utility of RT&Tag in Drosophila cells for capturing the noncoding RNA roX2 with the dosage compensation complex and maturing transcripts associated with silencing histone modifications. We also show that RT&Tag can detect N6-methyladenosine-modified mRNAs, and show that genes producing methylated transcripts are characterized by extensive promoter pausing of RNA polymerase II. The high efficiency of in situ antibody tethering and tagmentation makes RT&Tag especially suitable for rapid low-cost profiling of chromatin-associated RNAs.

Conserved regulatory logic at accessible and inaccessible chromatin during the acute inflammatory response in mammals.

The regulatory elements controlling gene expression during acute inflammation are not fully elucidated. Here we report the identification of a set of NF-κB-bound elements and common chromatin landscapes underlying the acute inflammatory response across cell-types and mammalian species. Using primary vascular endothelial cells (human/mouse/bovine) treated with the pro-inflammatory cytokine, Tumor Necrosis Factor-α, we identify extensive (~30%) conserved orthologous binding of NF-κB to accessible, as well as nucleosome-occluded chromatin. Regions with the highest NF-κB occupancy pre-stimulation show dramatic increases in NF-κB binding and chromatin accessibility post-stimulation. These 'pre-bound' regions are typically conserved (~56%), contain multiple NF-κB motifs, are utilized by diverse cell types, and overlap rare non-coding mutations and common genetic variation associated with both inflammatory and cardiovascular phenotypes. Genetic ablation of conserved, 'pre-bound' NF-κB regions within the super-enhancer associated with the chemokine-encoding CCL2 gene and elsewhere supports the functional relevance of these elements.

Regulation of CCL2 expression in human vascular endothelial cells by a neighboring divergently transcribed long noncoding RNA.

Atherosclerosis is a chronic inflammatory disease that is driven, in part, by activation of vascular endothelial cells (ECs). In response to inflammatory stimuli, the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling pathway orchestrates the expression of a network of EC genes that contribute to monocyte recruitment and diapedesis across the endothelium. Although many long noncoding RNAs (lncRNAs) are dysregulated in atherosclerosis, they remain poorly characterized, especially in the context of human vascular inflammation. Prior studies have illustrated that lncRNAs can regulate their neighboring protein-coding genes via interaction with protein complexes. We therefore identified and characterized neighboring interleukin-1ß (IL-1ß)-regulated messenger RNA (mRNA)-lncRNA pairs in ECs. We found these pairs to be highly correlated in expression, especially when located within the same chromatin territory. Additionally, these pairs were predominantly divergently transcribed and shared common gene regulatory elements, characterized by active histone marks and NF-κB binding. Further analysis was performed on lncRNA-CCL2, which is transcribed divergently to the gene, CCL2, encoding a proatherosclerotic chemokine. LncRNA-CCL2 and CCL2 showed coordinate up-regulation in response to inflammatory stimuli, and their expression was correlated in unstable symptomatic human atherosclerotic plaques. Knock-down experiments revealed that lncRNA-CCL2 positively regulated CCL2 mRNA levels in multiple primary ECs and EC cell lines. This regulation appeared to involve the interaction of lncRNA-CCL2 with RNA binding proteins, including HNRNPU and IGF2BP2. Hence, our approach has uncovered a network of neighboring mRNA-lncRNA pairs in the setting of inflammation and identified the function of an lncRNA, lncRNA-CCL2, which may contribute to atherogenesis in humans.

c-Myb Exacerbates Atherosclerosis through Regulation of Protective IgM-Producing Antibody-Secreting Cells.

Mechanisms that govern transcriptional regulation of inflammation in atherosclerosis remain largely unknown. Here, we identify the nuclear transcription factor c-Myb as an important mediator of atherosclerotic disease in mice. Atherosclerosis-prone animals fed a diet high in cholesterol exhibit increased levels of c-Myb in the bone marrow. Use of mice that either harbor a c-Myb hypomorphic allele or where c-Myb has been preferentially deleted in B cell lineages revealed that c-Myb potentiates atherosclerosis directly through its effects on B lymphocytes. Reduced c-Myb activity prevents the expansion of atherogenic B2 cells yet associates with increased numbers of IgM-producing antibody-secreting cells (IgM-ASCs) and elevated levels of atheroprotective oxidized low-density lipoprotein (OxLDL)-specific IgM antibodies. Transcriptional profiling revealed that c-Myb has a limited effect on B cell function but is integral in maintaining B cell progenitor populations in the bone marrow. Thus, targeted disruption of c-Myb beneficially modulates the complex biology of B cells in cardiovascular disease.

The transcriptional regulator CCCTC-binding factor limits oxidative stress in endothelial cells.

The CCCTC-binding factor (CTCF) is a versatile transcriptional regulator required for embryogenesis, but its function in vascular development or in diseases with a vascular component is poorly understood. Here, we found that endothelial Ctcf is essential for mouse vascular development and limits accumulation of reactive oxygen species (ROS). Conditional knockout of Ctcf in endothelial progenitors and their descendants affected embryonic growth, and caused lethality at embryonic day 10.5 because of defective yolk sac and placental vascular development. Analysis of global gene expression revealed Frataxin (Fxn), the gene mutated in Friedreich's ataxia (FRDA), as the most strongly down-regulated gene in Ctcf-deficient placental endothelial cells. Moreover, in vitro reporter assays showed that Ctcf activates the Fxn promoter in endothelial cells. ROS are known to accumulate in the endothelium of FRDA patients. Importantly, Ctcf deficiency induced ROS-mediated DNA damage in endothelial cells in vitro, and in placental endothelium in vivo Taken together, our findings indicate that Ctcf promotes vascular development and limits oxidative stress in endothelial cells. These results reveal a function for Ctcf in vascular development, and suggest a potential mechanism for endothelial dysfunction in FRDA.

Somatic Activating KRAS Mutations in Arteriovenous Malformations of the Brain.

BACKGROUND: Sporadic arteriovenous malformations of the brain, which are morphologically abnormal connections between arteries and veins in the brain vasculature, are a leading cause of hemorrhagic stroke in young adults and children. The genetic cause of this rare focal disorder is unknown. METHODS: We analyzed tissue and blood samples from patients with arteriovenous malformations of the brain to detect somatic mutations. We performed exome DNA sequencing of tissue samples of arteriovenous malformations of the brain from 26 patients in the main study group and of paired blood samples from 17 of those patients. To confirm our findings, we performed droplet digital polymerase-chain-reaction (PCR) analysis of tissue samples from 39 patients in the main study group (21 with matching blood samples) and from 33 patients in an independent validation group. We interrogated the downstream signaling pathways, changes in gene expression, and cellular phenotype that were induced by activating KRAS mutations, which we had discovered in tissue samples. RESULTS: We detected somatic activating KRAS mutations in tissue samples from 45 of the 72 patients and in none of the 21 paired blood samples. In endothelial cell-enriched cultures derived from arteriovenous malformations of the brain, we detected KRAS mutations and observed that expression of mutant KRAS (KRASG12V) in endothelial cells in vitro induced increased ERK (extracellular signal-regulated kinase) activity, increased expression of genes related to angiogenesis and Notch signaling, and enhanced migratory behavior. These processes were reversed by inhibition of MAPK (mitogen-activated protein kinase)-ERK signaling. CONCLUSIONS: We identified activating KRAS mutations in the majority of tissue samples of arteriovenous malformations of the brain that we analyzed. We propose that these malformations develop as a result of KRAS-induced activation of the MAPK-ERK signaling pathway in brain endothelial cells. (Funded by the Swiss Cancer League and others.).

Paradoxical Suppression of Atherosclerosis in the Absence of microRNA-146a.

RATIONALE: Inflammation is a key contributor to atherosclerosis. MicroRNA-146a (miR-146a) has been identified as a critical brake on proinflammatory nuclear factor κ light chain enhancer of activated B cells signaling in several cell types, including endothelial cells and bone marrow (BM)-derived cells. Importantly, miR-146a expression is elevated in human atherosclerotic plaques, and polymorphisms in the miR-146a precursor have been associated with risk of coronary artery disease. OBJECTIVE: To define the role of endogenous miR-146a during atherogenesis. METHODS AND RESULTS: Paradoxically, Ldlr-/- (low-density lipoprotein receptor null) mice deficient in miR-146a develop less atherosclerosis, despite having highly elevated levels of circulating proinflammatory cytokines. In contrast, cytokine levels are normalized in Ldlr-/-;miR-146a-/- mice receiving wild-type BM transplantation, and these mice have enhanced endothelial cell activation and elevated atherosclerotic plaque burden compared with Ldlr-/- mice receiving wild-type BM, demonstrating the atheroprotective role of miR-146a in the endothelium. We find that deficiency of miR-146a in BM-derived cells precipitates defects in hematopoietic stem cell function, contributing to extramedullary hematopoiesis, splenomegaly, BM failure, and decreased levels of circulating proatherogenic cells in mice fed an atherogenic diet. These hematopoietic phenotypes seem to be driven by unrestrained inflammatory signaling that leads to the expansion and eventual exhaustion of hematopoietic cells, and this occurs in the face of lower levels of circulating low-density lipoprotein cholesterol in mice lacking miR-146a in BM-derived cells. Furthermore, we identify sortilin-1(Sort1), a known regulator of circulating low-density lipoprotein levels in humans, as a novel target of miR-146a. CONCLUSIONS: Our study reveals that miR-146a regulates cholesterol metabolism and tempers chronic inflammatory responses to atherogenic diet by restraining proinflammatory signaling in endothelial cells and BM-derived cells.

Fingerprint of long non-coding RNA regulated by cyclic mechanical stretch in human aortic smooth muscle cells: implications for hypertension.

Emerging evidence suggests that long non-coding RNAs (lncRNAs) represent a cellular hub coordinating various cellular processes that are critical in health and disease. Mechanical stress triggers changes in vascular smooth muscle cells (VSMCs) that in turn contribute to pathophysiological changes within the vasculature. We sought to evaluate the role that lncRNAs play in mechanical stretch-induced alterations of human aortic smooth muscle cells (HASMCs). RNA (lncRNA and mRNA) samples isolated from HASMCs that had been subjected to 10 or 20% elongation (1 Hz) for 24 h were profiled with the Arraystar Human LncRNA Microarray V3.0. LncRNA expression was quantified in parallel via qRT-PCR. Of the 30,586 human lncRNAs screened, 580 were differentially expressed (DE, P < 0.05) in stretched HASMCs. Amongst the 26,109 protein-coding transcripts evaluated, 25 of those DE were associated with 25 of the aforementioned DE lncRNAs (P < 0.05). Subsequent Kyoto Encyclopedia of Genes and Genomes analysis revealed that the DE mRNAs were largely associated with the tumor necrosis factor signaling pathway and inflammation. Gene Ontology analysis indicated that the DE mRNAs were associated with cell differentiation, stress response, and response to external stimuli. We describe the first transcriptome profile of stretch-induced changes in HASMCs and provide novel insights into the regulatory switches that may be fundamental in governing aberrant VSMC remodeling.

Dynamic regulation of VEGF-inducible genes by an ERK/ERG/p300 transcriptional network.

The transcriptional pathways activated downstream of vascular endothelial growth factor (VEGF) signaling during angiogenesis remain incompletely characterized. By assessing the signals responsible for induction of the Notch ligand delta-like 4 (DLL4) in endothelial cells, we find that activation of the MAPK/ERK pathway mirrors the rapid and dynamic induction of DLL4 transcription and that this pathway is required for DLL4 expression. Furthermore, VEGF/ERK signaling induces phosphorylation and activation of the ETS transcription factor ERG, a prerequisite for DLL4 induction. Transcription of DLL4 coincides with dynamic ERG-dependent recruitment of the transcriptional co-activator p300. Genome-wide gene expression profiling identified a network of VEGF-responsive and ERG-dependent genes, and ERG chromatin immunoprecipitation (ChIP)-seq revealed the presence of conserved ERG-bound putative enhancer elements near these target genes. Functional experiments performed in vitro and in vivo confirm that this network of genes requires ERK, ERG and p300 activity. Finally, genome-editing and transgenic approaches demonstrate that a highly conserved ERG-bound enhancer located upstream of HLX (which encodes a transcription factor implicated in sprouting angiogenesis) is required for its VEGF-mediated induction. Collectively, these findings elucidate a novel transcriptional pathway contributing to VEGF-dependent angiogenesis.

Epigenetics of Atherosclerosis: Emerging Mechanisms and Methods.

Atherosclerosis is a vascular pathology characterized by inflammation and plaque build-up within arterial vessel walls. Vessel occlusion, often occurring after plaque rupture, can result in myocardial and cerebral infarction. Epigenetic changes are increasingly being associated with atherosclerosis and are of interest from both therapeutic and biomarker perspectives. Emerging genomic approaches that profile DNA methylation, chromatin accessibility, post-translational histone modifications, transcription factor binding, and RNA expression in low or single cell populations are poised to enhance our spatiotemporal understanding of atherogenesis. Here, we review recent therapeutically relevant epigenetic discoveries and emerging technologies that may generate new opportunities for atherosclerosis research.

Noncoding RNAs regulate NF-κB signaling to modulate blood vessel inflammation.

Cardiovascular diseases such as atherosclerosis are one of the leading causes of morbidity and mortality worldwide. The clinical manifestations of atherosclerosis, which include heart attack and stroke, occur several decades after initiation of the disease and become more severe with age. Inflammation of blood vessels plays a prominent role in atherogenesis. Activation of the endothelium by inflammatory mediators leads to the recruitment of circulating inflammatory cells, which drives atherosclerotic plaque formation and progression. Inflammatory signaling within the endothelium is driven predominantly by the pro-inflammatory transcription factor, NF-κB. Interestingly, activation of NF-κB is enhanced during the normal aging process and this may contribute to the development of cardiovascular disease. Importantly, studies utilizing mouse models of vascular inflammation and atherosclerosis are uncovering a network of noncoding RNAs, particularly microRNAs, which impinge on the NF-κB signaling pathway. Here we summarize the literature regarding the control of vascular inflammation by microRNAs, and provide insight into how these microRNA-based pathways might be harnessed for therapeutic treatment of disease. We also discuss emerging areas of endothelial cell biology, including the involvement of long noncoding RNAs and circulating microRNAs in the control of vascular inflammation.

Ezh2-mediated repression of a transcriptional pathway upstream of Mmp9 maintains integrity of the developing vasculature.

Maintenance of vascular integrity is required for embryogenesis and organ homeostasis. However, the gene expression programs that stabilize blood vessels are poorly understood. Here, we show that the histone methyltransferase Ezh2 maintains integrity of the developing vasculature by repressing a transcriptional program that activates expression of Mmp9. Inactivation of Ezh2 in developing mouse endothelium caused embryonic lethality with compromised vascular integrity and increased extracellular matrix degradation. Genome-wide approaches showed that Ezh2 targets Mmp9 and its activators Fosl1 and Klf5. In addition, we uncovered Creb3l1 as an Ezh2 target that directly activates Mmp9 gene expression in the endothelium. Furthermore, genetic inactivation of Mmp9 rescued vascular integrity defects in Ezh2-deficient embryos. Thus, epigenetic repression of Creb3l1, Fosl1, Klf5 and Mmp9 by Ezh2 in endothelial cells maintains the integrity of the developing vasculature, potentially linking this transcriptional network to diseases with compromised vascular integrity.

Nonribosomal assembly of natural lipocyclocarbamate lipoprotein-associated phospholipase inhibitors.

EXPANDING OUR KNOWLEDGE: Natural lipocyclocarbamate natural products have provided the inspiration for the first-in-class synthetic phospholipase inhibitor darapladib, currently in phase III clinical trials for the treatment of atherosclerosis. Here, we discuss their biosynthesis by a nonribosomal peptide synthetase.