The endothelial transcription factor ERG promotes vascular stability and growth through Wnt/β-catenin signaling.

Blood vessel stability is essential for embryonic development; in the adult, many diseases are associated with loss of vascular integrity. The ETS transcription factor ERG drives expression of VE-cadherin and controls junctional integrity. We show that constitutive endothelial deletion of ERG (Erg(cEC-KO)) in mice causes embryonic lethality with vascular defects. Inducible endothelial deletion of ERG (Erg(iEC-KO)) results in defective physiological and pathological angiogenesis in the postnatal retina and tumors, with decreased vascular stability. ERG controls the Wnt/β-catenin pathway by promoting β-catenin stability, through signals mediated by VE-cadherin and the Wnt receptor Frizzled-4. Wnt signaling is decreased in ERG-deficient endothelial cells; activation of Wnt signaling with lithium chloride, which stabilizes β-catenin levels, corrects vascular defects in Erg(cEC-KO) embryos. Finally, overexpression of ERG in vivo reduces permeability and increases stability of VEGF-induced blood vessels. These data demonstrate that ERG is an essential regulator of angiogenesis and vascular stability through Wnt signaling.This work was funded by grants from the British Heart Foundation (PG/09/096 and RG/11/17/29256). A.V.S. is a recipient of a National Lung and Heart Institute Foundation Studentship. I.M.A. is a recipient of a DOC-fFORTE fellowship of the Austrian Academy of Sciences at the London Research Institute.This paper was published by Cell Press in Developmental Cell (GM Birdsey, AV Shah, N Dufton, LE Reynolds, LO Almagro, Y Yang, IM Aspalter, ST Khan, JC Mason, E Dejana, B Göttgens, K Hodivala-Dilke, Gerhardt, RH Adams, AM Randi, Developmental Cell 2015, 32, 82-96

The Transcription Factor ERG Regulates Super-Enhancers Associated with an Endothelial-Specific Gene Expression Program

Rationale: The ETS transcription factor (TF) ERG is essential for endothelial homeostasis, driving expression of lineage genes and repressing pro-inflammatory genes. Loss of ERG expression is associated with diseases including atherosclerosis. ERG’s homeostatic function is lineage-specific, since aberrant ERG expression in cancer is oncogenic. The molecular basis for ERG lineage-specific activity is unknown. Transcriptional regulation of lineage specificity is linked to enhancer clusters (super-enhancers). Objective: To investigate whether ERG regulates endothelial-specific gene expression via super-enhancers. Methods and Results: Chromatin immunoprecipitation with high-throughput sequencing (ChIP-seq) in human umbilical vein endothelial cells (HUVEC) showed that ERG binds 93% of super-enhancers ranked according to H3K27ac, a mark of active chromatin. These were associated with endothelial genes such as DLL4, CLDN5, VWF and CDH5. Comparison between HUVEC and prostate cancer TMPRSS2:ERG fusion-positive VCaP cells revealed distinctive lineage-specific transcriptome and super-enhancer profiles. At a subset of endothelial super-enhancers (including DLL4 and CLDN5), loss of ERG results in significant reduction in gene expression which correlates with decreased enrichment of H3K27ac and Mediator subunit MED1, and reduced recruitment of acetyltransferase p300. At these super-enhancers, co-occupancy of GATA2 and AP-1 is significantly lower compared to super-enhancers that remained constant following ERG inhibition. These data suggest distinct mechanisms of super-enhancer regulation in EC and highlight the unique role of ERG in controlling a core subset of super-enhancers. Most disease-associated single nucleotide polymorphisms (SNPs) from genome-wide association studies (GWAS) lie within noncoding regions and perturb TF recognition sequences in relevant cell types. Analysis of GWAS data shows significant enrichment of risk variants for CVD and other diseases, at ERG endothelial enhancers and superenhancers. Conclusions: The TF ERG promotes endothelial homeostasis via regulation of lineage-specific enhancers and super-enhancers. Enrichment of CVD-associated SNPs at ERG super-enhancers suggests that ERGdependent transcription modulates disease risk.This work was funded by grants from the British Heart Foundation (RG/11/17/29256; RG/17/4/32662; FS/15/65/32036; PG/17/33/32990) and Cancer Research U

ICAM-2 regulates vascular permeability and N-cadherin localization through ezrin-radixin-moesin (ERM) proteins and Rac-1 signalling.

International audienceBACKGROUND: Endothelial junctions control functions such as permeability, angiogenesis and contact inhibition. VE-Cadherin (VECad) is essential for the maintenance of intercellular contacts. In confluent endothelial monolayers, N-Cadherin (NCad) is mostly expressed on the apical and basal membrane, but in the absence of VECad it localizes at junctions. Both cadherins are required for vascular development. The intercellular adhesion molecule (ICAM)-2, also localized at endothelial junctions, is involved in leukocyte recruitment and angiogenesis. RESULTS: In human umbilical vein endothelial cells (HUVEC), both VECad and NCad were found at nascent cell contacts of sub-confluent monolayers, but only VECad localized at the mature junctions of confluent monolayers. Inhibition of ICAM-2 expression by siRNA caused the appearance of small gaps at the junctions and a decrease in NCad junctional staining in sub-confluent monolayers. Endothelioma lines derived from WT or ICAM-2-deficient mice (IC2neg) lacked VECad and failed to form junctions, with loss of contact inhibition. Re-expression of full-length ICAM-2 (IC2 FL) in IC2neg cells restored contact inhibition through recruitment of NCad at the junctions. Mutant ICAM-2 lacking the binding site for ERM proteins (IC2 ΔERM) or the cytoplasmic tail (IC2 ΔTAIL) failed to restore junctions. ICAM-2-dependent Rac-1 activation was also decreased in these mutant cell lines. Barrier function, measured in vitro via transendothelial electrical resistance, was decreased in IC2neg cells, both in resting conditions and after thrombin stimulation. This was dependent on ICAM-2 signalling to the small GTPase Rac-1, since transendothelial electrical resistance of IC2neg cells was restored by constitutively active Rac-1. In vivo, thrombin-induced extravasation of FITC-labeled albumin measured by intravital fluorescence microscopy in the mouse cremaster muscle showed that permeability was increased in ICAM-2-deficient mice compared to controls. CONCLUSIONS: These results indicate that ICAM-2 regulates endothelial barrier function and permeability through a pathway involving N-Cadherin, ERMs and Rac-1

The Transcription Factor ERG Regulates Super-Enhancers Associated with an Endothelial-Specific Gene Expression Program

Rationale: The ETS transcription factor (TF) ERG is essential for endothelial homeostasis, driving expression of lineage genes and repressing pro-inflammatory genes. Loss of ERG expression is associated with diseases including atherosclerosis. ERG’s homeostatic function is lineage-specific, since aberrant ERG expression in cancer is oncogenic. The molecular basis for ERG lineage-specific activity is unknown. Transcriptional regulation of lineage specificity is linked to enhancer clusters (super-enhancers). Objective: To investigate whether ERG regulates endothelial-specific gene expression via super-enhancers. Methods and Results: Chromatin immunoprecipitation with high-throughput sequencing (ChIP-seq) in human umbilical vein endothelial cells (HUVEC) showed that ERG binds 93% of super-enhancers ranked according to H3K27ac, a mark of active chromatin. These were associated with endothelial genes such as DLL4, CLDN5, VWF and CDH5. Comparison between HUVEC and prostate cancer TMPRSS2:ERG fusion-positive VCaP cells revealed distinctive lineage-specific transcriptome and super-enhancer profiles. At a subset of endothelial super-enhancers (including DLL4 and CLDN5), loss of ERG results in significant reduction in gene expression which correlates with decreased enrichment of H3K27ac and Mediator subunit MED1, and reduced recruitment of acetyltransferase p300. At these super-enhancers, co-occupancy of GATA2 and AP-1 is significantly lower compared to super-enhancers that remained constant following ERG inhibition. These data suggest distinct mechanisms of super-enhancer regulation in EC and highlight the unique role of ERG in controlling a core subset of super-enhancers. Most disease-associated single nucleotide polymorphisms (SNPs) from genome-wide association studies (GWAS) lie within noncoding regions and perturb TF recognition sequences in relevant cell types. Analysis of GWAS data shows significant enrichment of risk variants for CVD and other diseases, at ERG endothelial enhancers and superenhancers. Conclusions: The TF ERG promotes endothelial homeostasis via regulation of lineage-specific enhancers and super-enhancers. Enrichment of CVD-associated SNPs at ERG super-enhancers suggests that ERGdependent transcription modulates disease risk.This work was funded by grants from the British Heart Foundation (RG/11/17/29256; RG/17/4/32662; FS/15/65/32036; PG/17/33/32990) and Cancer Research U

ZO-1 controls endothelial adherens junctions, cell-cell tension, angiogenesis, and barrier formation

Intercellular junctions are crucial for mechanotransduction, but whether tight junctions contribute to the regulation of cell–cell tension and adherens junctions is unknown. Here, we demonstrate that the tight junction protein ZO-1 regulates tension acting on VE-cadherin–based adherens junctions, cell migration, and barrier formation of primary endothelial cells, as well as angiogenesis in vitro and in vivo. ZO-1 depletion led to tight junction disruption, redistribution of active myosin II from junctions to stress fibers, reduced tension on VE-cadherin and loss of junctional mechanotransducers such as vinculin and PAK2, and induced vinculin dissociation from the α-catenin–VE-cadherin complex. Claudin-5 depletion only mimicked ZO-1 effects on barrier formation, whereas the effects on mechanotransducers were rescued by inhibition of ROCK and phenocopied by JAM-A, JACOP, or p114RhoGEF down-regulation. ZO-1 was required for junctional recruitment of JACOP, which, in turn, recruited p114RhoGEF. ZO-1 is thus a central regulator of VE-cadherin–dependent endothelial junctions that orchestrates the spatial actomyosin organization, tuning cell–cell tension, migration, angiogenesis, and barrier formation