RESUMO
Proximal promoter DNA methylation has been shown to be important for regulating gene expression. However, its relative contribution to the cell-specific expression of endothelial cell (EC)-enriched genes has not been defined. We used methyl-DNA immunoprecipitation and bisulfite conversion to analyze the DNA methylation profile of EC-enriched genes in ECs vs nonexpressing cell types, both in vitro and in vivo. We show that prototypic EC-enriched genes exhibit functional differential patterns of DNA methylation in proximal promoter regions of most (eg, CD31, von Willebrand factor [vWF], VE-cadherin, and intercellular adhesion molecule-2), but not all (eg, VEGFR-1 and VEGFR-2), EC-enriched genes. Comparable findings were evident in cultured ECs, human blood origin ECs, and murine aortic ECs. Promoter-reporter episomal transfection assays for endothelial nitric oxide synthase, VE-cadherin, and vWF indicated functional promoter activity in cell types where the native gene was not active. Inhibition of DNA methyltransferase activity indicated important functional relevance. Importantly, profiling DNA replication timing patterns indicated that EC-enriched gene promoters with differentially methylated regions replicate early in S-phase in both expressing and nonexpressing cell types. Collectively, these studies highlight the functional importance of promoter DNA methylation in controlling vascular EC gene expression.
Assuntos
Metilação de DNA , Período de Replicação do DNA , Endotélio Vascular/citologia , Regulação da Expressão Gênica , Regiões Promotoras Genéticas/genética , Fase S/fisiologia , Animais , Antígenos CD/genética , Aorta/citologia , Aorta/metabolismo , Caderinas/genética , Bovinos , Moléculas de Adesão Celular/genética , Células Cultivadas , Imunoprecipitação da Cromatina , Derme/citologia , Derme/metabolismo , Endotélio Vascular/metabolismo , Hepatócitos/citologia , Hepatócitos/metabolismo , Células Endoteliais da Veia Umbilical Humana/citologia , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Queratinócitos/citologia , Queratinócitos/metabolismo , Camundongos , Músculo Liso Vascular/citologia , Músculo Liso Vascular/metabolismo , Óxido Nítrico Sintase Tipo III/genética , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Fator de von Willebrand/genéticaRESUMO
A commonly-assumed paradigm holds that the primary genetic determinant of cardiovascular disease resides within the DNA sequence of our genes. This paradigm can be challenged. For example, how do sequence changes in the non-coding region of the genome influence phenotype? Why are all diseases not shared between identical twins? Part of the answer lies in the fact that the environment or exogenous stimuli clearly influence disease susceptibility, but it was unclear in the past how these effects were signalled to the static DNA code. Epigenetics is providing a newer perspective on these issues. Epigenetics refers to chromatin-based mechanisms important in the regulation of gene expression that do not involve changes to the DNA sequence per se. The field can be broadly categorized into three areas: DNA base modifications (including cytosine methylation and cytosine hydroxymethylation), post-translational modifications of histone proteins, and RNA-based mechanisms that operate in the nucleus. Cardiovascular disease pathways are now being approached from the epigenetic perspective, including those associated with atherosclerosis, angiogenesis, ischemia-reperfusion damage, and the cardiovascular response to hypoxia and shear stress, among many others. With increasing interest and expanding partnerships in the field, we can expect new insights to emerge from epigenetic perspectives of cardiovascular health. This paper reviews the principles governing epigenetic regulation, discusses their presently-understood importance in cardiovascular disease, and considers the growing significance we are likely to attribute to epigenetic contributions in the future, as they provide new mechanistic insights and a host of novel clinical applications.
Assuntos
Doenças Cardiovasculares/genética , Epigênese Genética , Predisposição Genética para Doença , Humanos , FenótipoRESUMO
Classical models of transcription in vascular endothelial cells, specifically the cis/trans paradigm, have limitations. For instance, how does the environment have chronic effects on gene expression in endothelial cells after weeks or years? When an endothelial cell divides, how is this information transmitted to daughter cells? Epigenetics refers to chromatin-based pathways important in the regulation of gene expression and includes three distinct, but highly interrelated, mechanisms: DNA methylation, histone density and posttranslational modifications, and RNA-based mechanisms. Together they offer a newer perspective on transcriptional control paradigms in vascular endothelial cells and provide a molecular basis for understanding how the environment impacts the genome to modify disease susceptibility. This alternative viewpoint for transcriptional regulation allows a reassessment of the cis/trans model and even helps explain some of its limitations. This review provides an introduction to epigenetic concepts for vascular biologists and uses topical examples in cell biology to provide insight into how cell types or even whole organisms, such as monozygotic human twins with the same DNA sequence, can exhibit heterogeneous patterns of gene expression, phenotype, or diseases prevalence. Using endothelial nitric oxide synthase (NOS3) as an example, we examine the growing body of evidence implicating epigenetic pathways in the control of vascular endothelial gene expression in health and disease.
Assuntos
Doenças Cardiovasculares/genética , Endotélio Vascular/metabolismo , Epigênese Genética , Animais , Doenças Cardiovasculares/metabolismo , Doenças Cardiovasculares/fisiopatologia , Diferenciação Celular/genética , Montagem e Desmontagem da Cromatina , Metilação de DNA , Endotélio Vascular/fisiopatologia , Regulação da Expressão Gênica , Histonas/metabolismo , Humanos , Óxido Nítrico Sintase Tipo III/genética , Óxido Nítrico Sintase Tipo III/metabolismo , Processamento de Proteína Pós-Traducional , RNA não Traduzido/metabolismo , Transcrição GênicaRESUMO
We describe a PFIC2 patient with a good response to ursodeoxycholic acid for 9 years. We found two novel ABCB11 gene mutations in the patient, i.e. I498T and 2098delA. The correlation of the patient's genotypes with the clinical course supports the existence of a phenotypic continuum between BRIC2 and PFIC2.