The regulation of blood vessel formation is of fundamental importance to many physiological processes, and angiogenesis is a major area for novel therapeutic approaches to diseases from ischemia to cancer. A poorly understood clinical manifestation of pathological angiogenesis is angiodysplasia, vascular malformations that cause severe gastrointestinal bleeding. Angiodysplasia can be associated with von Willebrand disease (VWD), the most common bleeding disorder in man. VWD is caused by a defect or deficiency in von Willebrand factor (VWF), a glycoprotein essential for normal hemostasis that is involved in inflammation. We hypothesized that VWF regulates angiogenesis. Inhibition of VWF expression by short interfering RNA (siRNA) in endothelial cells (ECs) caused increased in vitro angiogenesis and increased vascular endothelial growth factor (VEGF) receptor-2 (VEGFR-2)-dependent proliferation and migration, coupled to decreased integrin ␣v␤3 levels and increased angiopoietin (Ang)-2 release. ECs expanded from blood- IntroductionAngiogenesis, the formation of new vessels from pre-existing ones, occurs physiologically in specific circumstances such as wound healing and the menstrual cycle. Dysregulated angiogenesis contributes to the pathogenesis of many disorders, including diabetes, cancer, and macular degeneration (reviewed in Carmeliet 1 ). Angiogenic factors such as vascular endothelial growth factor (VEGF) and the angiopoietins (Ang) orchestrate signaling pathways that promote endothelial cell (EC) migration, proliferation, and ultimately the formation of a new vessel. VEGF-A is a major regulator of angiogenesis (reviewed in Grothey and Galanis 2 ) and acts on ECs mainly through VEGF receptor-2 (VEGFR-2), a tyrosine kinase receptor (reviewed in Olsson 3 ), to promote endothelial proliferation, migration, and sprouting of tip cells in the early stages of angiogenesis (reviewed in Gerhardt 4 ). Ang-1 and Ang-2, which bind to the endothelial Tie-2 receptor, act in the later stages of blood vessel formation and are essential for the maturation of a stable vascular network and for the maintenance of endothelial integrity (reviewed in Thomas and Augustin 5 ). Ang-1 and Ang-2 were originally identified as agonist and antagonist of Tie-2 signaling, respectively, with Ang-1 supporting EC survival and endothelial integrity 6 and Ang-2 promoting blood vessel destabilization and regression. 7 However, recent data suggest a more complex picture that includes cross-talk between the VEGF and the Ang pathways. 8 Growth factor signaling pathways are influenced by surface adhesion molecules that mediate cell-cell or cell-matrix interactions, particularly by members of the integrin superfamily. The integrin that has received most attention in ECs is ␣v␤3 (reviewed in Hodivala-Dilke 9 ), which mediates binding to several extracellular matrix proteins and growth factor receptors including VEGFR-2, thus influencing VEGFR-2 signaling (reviewed in Somanath et al 10 ). ␣v␤3 plays a complex role in angiogenesis. Although the origina...

Genome-wide association studies have identified more than 70 common variants that are associated with breast cancer risk. Most of these variants map to non-protein-coding regions and several map to gene deserts, regions of several hundred kilobases lacking protein-coding genes. We hypothesized that gene deserts harbor long-range regulatory elements that can physically interact with target genes to influence their expression. To test this, we developed Capture Hi-C (CHi-C), which, by incorporating a sequence capture step into a Hi-C protocol, allows high-resolution analysis of targeted regions of the genome. We used CHi-C to investigate long-range interactions at three breast cancer gene deserts mapping to 2q35, 8q24.21, and 9q31.2. We identified interaction peaks between putative regulatory elements (''bait fragments'') within the captured regions and ''targets'' that included both protein-coding genes and long noncoding (lnc) RNAs over distances of 6.6 kb to 2.6 Mb. Target protein-coding genes were IGFBP5, KLF4, NSMCE2, and MYC; and target lncRNAs included DIRC3, PVT1, and CCDC26. For one gene desert, we were able to define two SNPs (rs12613955 and rs4442975) that were highly correlated with the published risk variant and that mapped within the bait end of an interaction peak. In vivo ChIP-qPCR data show that one of these, rs4442975, affects the binding of FOXA1 and implicate this SNP as a putative functional variant.

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IntroductionAngiogenesis, namely the formation of new vessels from preexisting ones, is essential for normal development, as well as for pathologic conditions, including tumor development, diabetic retinopathy, atherosclerosis, and rheumatoid arthritis. In recent years, endothelial apoptosis was shown to play an important role in remodeling the vascular network. Endothelial apoptosis is observed at the initiation of angiogenesis and at the branching and regression of neovessels. [1][2][3] Angiogenesis is controlled by the balance between proangiogenic factors and angiogenesis inhibitors, which also regulate apoptosis of endothelial cells (ECs). Indeed, the mechanism underlying many antiangiogenic therapies appears to be the induction of endothelial cell death. 4 Among the transcription factors involved in the regulation of angiogenesis and vascular development are members of the ETS family. This large family of transcription factors contains approximately 30 members that share a highly conserved DNA-binding domain (ETS domain) and are involved in regulating a wide variety of biologic processes. Many ETS proteins are expressed early in the developing vasculature in several organisms, and loss-of-function studies in mice and zebrafish have shown a critical role for ETS factors in vascular development. 5 In the adult, several endothelial ETS transcription factors were shown to regulate angiogenesis. 6 Among these, the ETS family member Erg is constitutively expressed in ECs, 7 and its expression is apparently restricted to ECs, megakaryocytes, 8 and chondrocytes. 9 Erg drives transcription of genes involved in endothelial homeostasis and angiogenesis, such as eNOS, 10,11 Data from animal models indicate that Erg is involved in endothelial differentiation and vascular development 12 ; for example, Erg overexpression in Xenopus embryos resulted in ectopic endothelial differentiation. 13 We have previously shown that Erg is required for angiogenesis in human ECs in vitro 14 ; however, no data are available on the role of Erg in angiogenesis in the adult in vivo.Endothelial junctions are crucial for the maintenance and regulation of vascular homeostasis and function and mediate a complex signaling network. 15 A major regulator of adherent junctions is vascular endothelial (VE)-cadherin, a Ca 2ϩ -dependent cell-surface adhesion molecule that forms homophilic interactions and is required for the integrity of the endothelial monolayer, endothelial permeability, and the control of cell growth. 16,17 VE-cadherin was clearly shown to regulate vascular development and angiogenesis: genetic inactivation of the VE-cadherin gene leads to early embryonic death because of vascular defects 18,19 and antibodies to VE-cadherin inhibit angiogenesis both in vitro and in vivo (reviewed in Dejana et al 20 ). VE-cadherin regulates a number of signaling events, by intracellular interaction with proteins of the armadillo family, including ␤-catenin and plakoglobin, as well as by clustering signaling molecules and growth factor receptors. 1...

Multiple regulatory elements distant from their targets on the linear genome can influence the expression of a single gene through chromatin looping. Chromosome conformation capture implemented in Hi-C allows for genome-wide agnostic characterization of chromatin contacts. However, detection of functional enhancer–promoter interactions is precluded by its effective resolution that is determined by both restriction fragmentation and sensitivity of the experiment. Here we develop a capture Hi-C (cHi-C) approach to allow an agnostic characterization of these physical interactions on a genome-wide scale. Single-nucleotide polymorphisms associated with complex diseases often reside within regulatory elements and exert effects through long-range regulation of gene expression. Applying this cHi-C approach to 14 colorectal cancer risk loci allows us to identify key long-range chromatin interactions in cis and trans involving these loci.

We have identified a novel physiological anti-inflammatory pathway under the control of the transcription factor Erg; this pathway inhibits NF-κB-dependent transcription and TNF-α-induced inflammation in vivo. These results suggest a novel approach to anti-inflammatory therapies.

Genome-wide association studies (GWAS) have identified approximately 100 breast cancer risk loci. Translating these findings into a greater understanding of the mechanisms that influence disease risk requires identification of the genes or non-coding RNAs that mediate these associations. Here, we use Capture Hi-C (CHi-C) to annotate 63 loci; we identify 110 putative target genes at 33 loci. To assess the support for these target genes in other data sources we test for associations between levels of expression and SNP genotype (eQTLs), disease-specific survival (DSS), and compare them with somatically mutated cancer genes. 22 putative target genes are eQTLs, 32 are associated with DSS and 14 are somatically mutated in breast, or other, cancers. Identifying the target genes at GWAS risk loci will lead to a greater understanding of the mechanisms that influence breast cancer risk and prognosis.

The endothelial ETS transcription factor Erg plays an important role in homeostasis and angiogenesis by regulating many endothelial functions including survival and junction stability. Here we show that Erg regulates endothelial cell (EC) migration. Transcriptome profiling of Ergdeficient ECs identified ϳ 80 genes involved in cell migration as candidate Erg targets, including many regulators of RhoGTPases. Inhibition of Erg expression in HUVECs resulted in decreased migration in vitro, while Erg overexpression using adenovirus caused increased migration. Live-cell imaging of Erg-deficient HUVECs showed a reduction in lamellipodia, in line with decreased motility. Both actin and tubulin cytoskeletons were disrupted in Erg-deficient ECs, with a dramatic increase in tubulin acetylation. Among the most significant microarray hits was the cytosolic histone deacetylase 6 (HDAC6), a regulator of cell migration. Chromatin immunoprecipitation (ChIP) and transactivation studies demonstrated that Erg regulates HDAC6 expres- IntroductionAngiogenesis is a tightly controlled process that involves a cascade of events. A breakthrough in understanding the cellular and molecular mechanisms underlying angiogenesis has been the identification of highly motile, polarized endothelial cells (ECs), named tip cells, which lead the growth of the developing sprout. 1 Dynamic reorganization of the cytoskeleton in tip cells supports the projection of lamellipodia and filopodia, which allow the cell to sense the microenvironment for migration cues and guide the sprouting vessel toward an angiogenic stimulus. The functional cross-talk between the actin and tubulin cytoskeletal systems is essential for cell motility. 2 Several families of intracellular regulators control these pathways. Among the most studied are the small GTPases of the Rho family, with Rac as the main regulator of lamellipodia and CDC42 as the regulator of filopodia; both of these GTPases have been shown to be involved in angiogenesis. 3 The class II histone deacetylase 6 (HDAC6), a known regulator of cell motility, 4 has been recently shown to be involved in EC migration and to support angiogenesis. 5 HDAC6 is expressed in many cells (including ECs) and localizes predominantly to the cytoplasm, binding to microtubules and deacetylating several cytosolic proteins, including ␣-tubulin 4,6 and cortactin. 7 Acetylation of tubulin, characteristic of stabilized microtubules, is thought to contribute to regulating microtubule dynamics, 8 while acetylation of cortactin is thought to modulate actin dynamics by regulating the interaction of cortactin with F-actin. 7 HDAC6 modulation of both cytoskeletons mediates its role in regulating endothelial migration, cytoskeletal dynamics, and angiogenesis. 4,8 We have been interested in the transcriptional regulation of endothelial gene expression and angiogenesis, and have focused on the ETS transcription factor Erg (ETS-related gene). Erg is the most abundantly expressed ETS factor in resting ECs and its expression, although not uniquely ...