RESUMEN
The ability to identify regulatory interactions that mediate gene expression changes through distal elements, such as risk loci, is transforming our understanding of how genomes are spatially organized and regulated. Capture Hi-C (CHi-C) is a powerful tool to delineate such regulatory interactions. However, primary analysis and downstream interpretation of CHi-C profiles remains challenging and relies on disparate tools with ad-hoc input/output formats and specific assumptions for statistical modeling. Here we present a data processing and interaction calling toolkit (CHiCANE), specialized for the analysis and meaningful interpretation of CHi-C assays. In this protocol, we demonstrate applications of CHiCANE to region capture Hi-C (rCHi-C) and promoter capture Hi-C (pCHi-C) libraries, followed by quality assessment of interaction peaks, as well as downstream analysis specific to rCHi-C and pCHi-C to aid functional interpretation. For a typical rCHi-C/pCHi-C dataset this protocol takes up to 3 d for users with a moderate understanding of R programming and statistical concepts, although this is dependent on dataset size and compute power available. CHiCANE is freely available at https://cran.r-project.org/web/packages/chicane .
Asunto(s)
Genómica/métodos , Secuencias Reguladoras de Ácidos Nucleicos/genética , Elementos de Facilitación Genéticos/genética , Epigenoma , Genoma , Código de Histonas , Modelos Genéticos , Anotación de Secuencia Molecular , Mutación/genética , Polimorfismo de Nucleótido Simple/genética , Regiones Promotoras Genéticas , Sitios de Carácter Cuantitativo/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Estadística como AsuntoRESUMEN
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.
Asunto(s)
Neoplasias de la Mama/genética , Predisposición Genética a la Enfermedad , Epistasis Genética , Femenino , Estudio de Asociación del Genoma Completo , Genotipo , Humanos , Mutación , Polimorfismo de Nucleótido Simple , Sitios de Carácter CuantitativoRESUMEN
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.
Asunto(s)
Neoplasias de la Mama/genética , Predisposición Genética a la Enfermedad/genética , Estudio de Asociación del Genoma Completo/métodos , Polimorfismo de Nucleótido Simple , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Línea Celular Tumoral , Inmunoprecipitación de Cromatina , Mapeo Cromosómico , Cromosomas Humanos Par 2/genética , Cromosomas Humanos Par 8/genética , Cromosomas Humanos Par 9/genética , Genoma Humano/genética , Factor Nuclear 3-alfa del Hepatocito/genética , Factor Nuclear 3-alfa del Hepatocito/metabolismo , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Factor 4 Similar a Kruppel , Células MCF-7 , Análisis de Secuencia por Matrices de Oligonucleótidos , Unión Proteica , Mapeo de Interacción de Proteínas , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Secuencias Reguladoras de Ácidos Nucleicos/genética , Reproducibilidad de los Resultados , Análisis de Secuencia de ADNRESUMEN
BACKGROUND: 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.
Asunto(s)
Antígenos CD/metabolismo , Cadherinas/metabolismo , Permeabilidad Capilar , Moléculas de Adhesión Celular/metabolismo , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Proteína de Unión al GTP rac1/metabolismo , Animales , Antígenos CD/genética , Sitios de Unión , Moléculas de Adhesión Celular/química , Moléculas de Adhesión Celular/genética , Línea Celular Tumoral , Proteínas del Citoesqueleto/metabolismo , Uniones Comunicantes/metabolismo , Humanos , Proteínas de la Membrana/metabolismo , Ratones , Proteínas de Microfilamentos/metabolismo , Unión Proteica , Transporte de Proteínas , Transducción de SeñalRESUMEN
The interaction of transcription factors with specific DNA sequences is critical for activation of gene expression programs. In endothelial cells (EC), the transcription factor NF-κB is important in the switch from quiescence to activation, and is tightly controlled to avoid excessive inflammation and organ damage. Here we describe a novel mechanism that controls the activation of NF-κB in EC. The transcription factor Erg, the most highly expressed ETS member in resting EC, controls quiescence by repressing proinflammatory gene expression. Focusing on intercellular adhesion molecule 1(ICAM)-1 as a model, we identify two ETS binding sites (EBS -118 and -181) within the ICAM-1 promoter required for Erg-mediated repression. We show that Erg binds to both EBS -118 and EBS -181, the latter located within the NF-κB binding site. Interestingly, inhibition of Erg expression in quiescent EC results in increased NF-κB-dependent ICAM-1 expression, indicating that Erg represses basal NF-κB activity. Erg prevents NF-κB p65 from binding to the ICAM-1 promoter, suggesting a direct mechanism of interference. Gene set enrichment analysis of transcriptome profiles of Erg and NF-κB-dependent genes, together with chromatin immunoprecipitation (ChIP) studies, reveals that this mechanism is common to other proinflammatory genes, including cIAP-2 and IL-8. These results identify a role for Erg as a gatekeeper controlling vascular inflammation, thus providing an important barrier to protect against inappropriate endothelial activation.
Asunto(s)
Regulación de la Expresión Génica , Células Endoteliales de la Vena Umbilical Humana/fisiología , Transactivadores/fisiología , Factor de Transcripción ReIA/metabolismo , Sitios de Unión , Unión Competitiva , Células Cultivadas , ADN/química , Ensayo de Cambio de Movilidad Electroforética , Perfilación de la Expresión Génica , Genes Reporteros , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Molécula 1 de Adhesión Intercelular/genética , Molécula 1 de Adhesión Intercelular/metabolismo , Luciferasas de Renilla/biosíntesis , Luciferasas de Renilla/genética , Regiones Promotoras Genéticas , Unión Proteica , Fase de Descanso del Ciclo Celular , Transactivadores/química , Transactivadores/metabolismo , Sitio de Iniciación de la Transcripción , Transcripción Genética , Regulador Transcripcional ERGRESUMEN
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 Erg-deficient ECs identified â¼ 80 genes involved in cell migration as candidate Erg targets, including many regulators of Rho- GTPases. 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 expression. Rescue experiments confirmed that HDAC6 mediates the Erg-dependent regulation of tubulin acetylation and actin localization. In vivo, inhibition of Erg expression in angiogenic ECs resulted in decreased HDAC6 expression with increased tubulin acetylation. Thus, we have identified a novel function for the transcription factor Erg in regulating HDAC6 and multiple pathways essential for EC migration and angiogenesis.
Asunto(s)
Biomarcadores/metabolismo , Movimiento Celular , Endotelio Vascular/metabolismo , Regulación de la Expresión Génica , Histona Desacetilasas/genética , Neovascularización Fisiológica , Transducción de Señal , Transactivadores/metabolismo , Acetilación , Actinas/metabolismo , Western Blotting , Células Cultivadas , Inmunoprecipitación de Cromatina , Endotelio Vascular/citología , Perfilación de la Expresión Génica , Histona Desacetilasa 6 , Histona Desacetilasas/metabolismo , Humanos , Análisis de Secuencia por Matrices de Oligonucleótidos , Regiones Promotoras Genéticas/genética , ARN Mensajero/genética , ARN Interferente Pequeño/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transactivadores/antagonistas & inhibidores , Transactivadores/genética , Regulador Transcripcional ERG , Venas Umbilicales/citología , Venas Umbilicales/metabolismoRESUMEN
OBJECTIVE: To test whether ETS-related gene (Erg) inhibits tumor necrosis factor (TNF)-α-dependent endothelial activation and inflammation. METHODS AND RESULTS: Endothelial activation underlies many vascular diseases, including atherosclerosis. Endothelial activation by proinflammatory cytokines decreases expression of the ETS transcription factor Erg. By using human umbilical vein endothelial cells (HUVECs), we showed that Erg overexpression by adenovirus (AdErg) repressed basal and TNF-α-induced expression of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule (VCAM), and interleukin 8 (IL-8). Erg inhibited TNF-α-dependent activation of the ICAM-1 promoter, nuclear factor (NF)-κB activity, and NF-κB p65 phosphorylation. Basal NF-κB activity was also inhibited by Erg overexpression. Chromatin immunoprecipitation showed that Erg binds to the ICAM-1 proximal promoter region, which contains 7 putative ETS binding sites. To test the anti-inflammatory role of Erg in vivo, we used a murine model of TNF-α-dependent acute inflammation. The injection of AdErg into the paw decreased TNF-α-induced inflammation compared with control. Finally, staining of human coronary plaques showed loss of Erg expression from the endothelium overlaying active plaque shoulders. CONCLUSIONS: 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.
Asunto(s)
Células Endoteliales/inmunología , Mediadores de Inflamación/metabolismo , Inflamación/prevención & control , FN-kappa B/metabolismo , Transactivadores/metabolismo , Animales , Secuencia de Bases , Sitios de Unión , Células Cultivadas , Enfermedad de la Arteria Coronaria/metabolismo , Modelos Animales de Enfermedad , Regulación hacia Abajo , Humanos , Inflamación/genética , Inflamación/inmunología , Molécula 1 de Adhesión Intercelular/genética , Molécula 1 de Adhesión Intercelular/metabolismo , Interleucina-8/genética , Interleucina-8/metabolismo , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Fosforilación , Regiones Promotoras Genéticas , Interferencia de ARN , Factores de Tiempo , Transactivadores/genética , Factor de Transcripción ReIA/metabolismo , Regulador Transcripcional ERG , Transfección , Factor de Necrosis Tumoral alfa/metabolismo , Molécula 1 de Adhesión Celular Vascular/genética , Molécula 1 de Adhesión Celular Vascular/metabolismoRESUMEN
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 αvß3 levels and increased angiopoietin (Ang)-2 release. ECs expanded from blood-derived endothelial progenitor cells of VWD patients confirmed these results. Finally, 2 different approaches, in situ and in vivo, showed increased vascularization in VWF-deficient mice. We therefore identify a new function of VWF in ECs, which confirms VWF as a protein with multiple vascular roles and defines a novel link between hemostasis and angiogenesis. These results may have important consequences for the management of VWD, with potential therapeutic implications for vascular diseases.
Asunto(s)
Células Endoteliales/metabolismo , Neovascularización Fisiológica , Factor de von Willebrand/metabolismo , Adulto , Anciano de 80 o más Años , Angiopoyetina 2/genética , Angiopoyetina 2/metabolismo , Animales , Línea Celular , Movimiento Celular , Proliferación Celular , Células Endoteliales/citología , Femenino , Hemostasis , Humanos , Immunoblotting , Integrina alfaVbeta3/genética , Integrina alfaVbeta3/metabolismo , Masculino , Ratones , Ratones Noqueados , Persona de Mediana Edad , Neovascularización Patológica , Interferencia de ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Receptor 2 de Factores de Crecimiento Endotelial Vascular/genética , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo , Enfermedades de von Willebrand/genética , Enfermedades de von Willebrand/metabolismo , Enfermedades de von Willebrand/patología , Factor de von Willebrand/genéticaRESUMEN
Transcription factors of the ETS family are important regulators of endothelial gene expression. Here, we review the evidence that ETS factors regulate angiogenesis and briefly discuss the target genes and pathways involved. Finally, we discuss novel evidence that shows how these transcription factors act in a combinatorial fashion with others, through composite sites that may be crucial in determining endothelial specificity in gene transcription.
Asunto(s)
Neovascularización Fisiológica/fisiología , Proteínas Proto-Oncogénicas c-ets/metabolismo , Animales , Endotelio Vascular/fisiología , Regulación del Desarrollo de la Expresión Génica , Humanos , Filogenia , Unión Proteica , Proteínas Proto-Oncogénicas c-ets/clasificación , Proteínas Proto-Oncogénicas c-ets/genéticaRESUMEN
Tight regulation of the balance between apoptosis and survival is essential in angiogenesis. The ETS transcription factor Erg is required for endothelial tube formation in vitro. Inhibition of Erg expression in human umbilical vein endothelial cells (HUVECs), using antisense oligonucleotides, resulted in detachment of cell-cell contacts and increased cell death. Inhibition of Erg expression by antisense in HUVECs also lowered expression of the adhesion molecule vascular endothelial (VE)-cadherin, a key regulator of endothelial intercellular junctions and survival. Using chromatin immunoprecipitation, we showed that Erg binds to the VE-cadherin promoter. Furthermore, Erg was found to enhance VE-cadherin promoter activity in a transactivation assay. Apoptosis induced by inhibition of Erg was partly rescued by overexpression of VE-cadherin-GFP, suggesting that VE-cadherin is involved in the Erg-dependent survival signals. To show the role of Erg in angiogenesis in vivo, we used siRNA against Erg in a Matrigel plug model. Erg inhibition resulted in a significant decrease in vascularization, with increase in caspase-positive endothelial cells (ECs). These results identify a new pathway regulating angiogenesis and endothelial survival, via the transcription factor Erg and the adhesion molecule VE-cadherin.
Asunto(s)
Antígenos CD/biosíntesis , Apoptosis/fisiología , Cadherinas/biosíntesis , Proteínas de Unión al ADN/metabolismo , Células Endoteliales/metabolismo , Regulación de la Expresión Génica/fisiología , Neovascularización Fisiológica/fisiología , Transactivadores/metabolismo , Antígenos CD/genética , Apoptosis/efectos de los fármacos , Cadherinas/genética , Caspasas/genética , Caspasas/metabolismo , Adhesión Celular/efectos de los fármacos , Adhesión Celular/fisiología , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Células Cultivadas , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/genética , Células Endoteliales/citología , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Uniones Intercelulares/efectos de los fármacos , Uniones Intercelulares/genética , Uniones Intercelulares/metabolismo , Neovascularización Fisiológica/efectos de los fármacos , Oligonucleótidos Antisentido/genética , Oligonucleótidos Antisentido/farmacología , Regiones Promotoras Genéticas/fisiología , Unión Proteica/efectos de los fármacos , Unión Proteica/fisiología , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/farmacología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Transactivadores/antagonistas & inhibidores , Transactivadores/genética , Regulador Transcripcional ERGRESUMEN
Orf virus infection causes a contagious pustular dermatitis characterized by extensive vascular changes that have been linked to a virally encoded vascular endothelial growth factor (VEGF). The VEGF genes of different strains of orf virus can vary extensively in amino acid sequence. Functional analyses of two major variant VEGF proteins derived from orf virus strains, NZ2 and NZ7, have revealed quantitative differences in biological activities and receptor binding specificities suggesting that these viral VEGFs could have different roles in the pathology of orf virus infection. In this study, we show that both orf virus strains express equivalent levels of the viral VEGF variants and during infection of sheep skin induce comparable levels of vascularization, edema, epidermal rete ridge and scab formation. Recombinants of orf virus NZ2 and NZ7 strains in which the variant VEGF genes were disrupted showed markedly reduced vascular changes and evidence of partially attenuated viral growth. These results demonstrate that despite substantial differences in sequence and biological activity in vitro, these virally expressed virulence factors are functionally equivalent in their natural host, contributing equally to orf virus pathology.
Asunto(s)
Secuencia de Aminoácidos , Ectima Contagioso/patología , Variación Genética , Virus del Orf/patogenicidad , Factores de Crecimiento Endotelial Vascular/genética , Proteínas Virales/genética , Animales , Células Cultivadas , Ectima Contagioso/virología , Eliminación de Gen , Masculino , Virus del Orf/clasificación , Virus del Orf/genética , Ovinos/virología , Piel/patología , Testículo/citología , Testículo/virología , Factores de Crecimiento Endotelial Vascular/química , Factores de Crecimiento Endotelial Vascular/metabolismo , Proteínas Virales/química , Proteínas Virales/metabolismoRESUMEN
Infections of humans and ungulates by parapoxviruses result in skin lesions characterized by extensive vascular changes that have been linked to viral-encoded homologues of vascular endothelial growth factor (VEGF). VEGF acts via a family of receptors (VEGFRs) to mediate endothelial cell proliferation, vascular permeability, and angiogenesis. The VEGF genes from independent parapoxvirus isolates show an extraordinary degree of inter-strain sequence variation. We conducted functional comparisons of five representatives of the divergent viral VEGFs. These revealed that despite the sequence divergence, all were equally active mitogens, stimulating proliferation of human endothelial cells in vitro and vascularization of sheep skin in vivo with potencies equivalent to VEGF. This was achieved even though the viral VEGFs bound VEGFR-2 less avidly than did VEGF. Surprisingly the viral VEGFs varied in their ability to cross-link VEGFR-2, induce vascular permeability and bind neuropilin-1. Correlations between these three activities were detected. In addition it was possible to correlate these functional variations with certain sequence and structural motifs specific to the viral VEGFs. In contrast to the conserved ability to bind human VEGFR-2, the viral growth factors did not bind either VEGFR-1 or VEGFR-3. We propose that the extensive sequence divergence seen in the viral VEGFs was generated primarily by selection against VEGFR-1 binding.