Dataset Information

Endothelial cell-specific expression of roundabout 4 is regulated by differential DNA methylation of the proximal promoter.

ABSTRACT: The molecular basis of endothelial cell (EC)-specific gene expression is poorly understood. Roundabout 4 (Robo4) is expressed exclusively in ECs. We previously reported that the 3-kb 5'-flanking region of the human Robo4 gene contains information for lineage-specific expression in the ECs. Our studies implicated a critical role for GA-binding protein and specificity protein 1 (SP1) in mediating overall expression levels. However, these transcription factors are also expressed in non-ECs. In this study, we tested the hypothesis that epigenetic mechanisms contribute to EC-specific Robo4 gene expression.Bisulfite sequencing analysis indicated that the proximal promoter of Robo4 is methylated in non-ECs but not in ECs. Treatment with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine increased Robo4 gene expression in non-ECs but not in ECs. Proximal promoter methylation significantly decreased the promoter activity in ECs. Electrophoretic mobility shift assays showed that DNA methylation of the proximal promoter inhibited SP1 binding to the -42 SP1 site. In DNase hypersensitivity assays, chromatin condensation of the Robo4 promoter was observed in some but not all nonexpressing cell types. In Hprt (hypoxanthine phosphoribosyltransferase)-targeted mice, a 0.3-kb proximal promoter directed cell-type-specific expression in the endothelium. Bisulfite sequencing analysis using embryonic stem cell-derived mesodermal cells and ECs indicated that the EC-specific methylation pattern of the promoter is determined by demethylation during differentiation and that binding of GA-binding protein and SP1 to the proximal promoter is not essential for demethylation.The EC-specific DNA methylation pattern of the Robo4 proximal promoter is determined during cell differentiation and contributes to regulation of EC-specific Robo4 gene expression.

SUBMITTER: Okada Y

PROVIDER: S-EPMC5378492 | biostudies-literature | 2014 Jul

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Endothelial cell-specific expression of roundabout 4 is regulated by differential DNA methylation of the proximal promoter.

Okada Yoshiaki Y Funahashi Nobuaki N Tanaka Toru T Nishiyama Yuji Y Yuan Lei L Shirakura Keisuke K Turjman Alexis S AS Kano Yoshihiro Y Naruse Hiroki H Suzuki Ayano A Sakai Miki M Zhixia Jiang J Kitajima Kenji K Ishimoto Kenji K Hino Nobumasa N Kondoh Masuo M Mukai Yohei Y Nakagawa Shinsaku S García-Cardeña Guillermo G Aird William C WC Doi Takefumi T

Arteriosclerosis, thrombosis, and vascular biology 20140522 7

<h4>Objective</h4>The molecular basis of endothelial cell (EC)-specific gene expression is poorly understood. Roundabout 4 (Robo4) is expressed exclusively in ECs. We previously reported that the 3-kb 5'-flanking region of the human Robo4 gene contains information for lineage-specific expression in the ECs. Our studies implicated a critical role for GA-binding protein and specificity protein 1 (SP1) in mediating overall expression levels. However, these transcription factors are also expressed i ...[more]

PMID: 24855053

Similar Datasets

Project description:RationaleHemodynamic disturbed flow (DF) is associated with susceptibility to atherosclerosis. Endothelial Kruppel-Like Factor 4 (KLF4) is an important anti-inflammatory atheroprotective transcription factor that is suppressed in regions of DF.ObjectiveThe plasticity of epigenomic KLF4 transcriptional regulation by flow-mediated DNA methylation was investigated in vitro and in arterial tissue.Methods and resultsTo recapitulate dominant flow characteristics of atheroprotected and atherosusceptible arteries, human aortic endothelial cells were subjected to pulsatile undisturbed flow or oscillatory DF containing a flow-reversing phase. Differential CpG site methylation was measured by methylation-specific polymerase chain reaction, bisulfite pyrosequencing, and restriction enzyme-polymerase chain reaction. The methylation profiles of endothelium from disturbed and undisturbed flow sites of adult swine aortas were also investigated. In vitro, DF increased DNA methylation of CpG islands within the KLF4 promoter that significantly contributed to suppression of KLF4 transcription; the effects were mitigated by DNA methyltransferase (DNMT) inhibitors and knockdown of DNMT3A. Contributory mechanisms included DF-induced increase of DNMT3A protein (1.7-fold), DNMT3A enrichment (11-fold) on the KLF4 promoter, and competitive blocking of a myocyte enhancer factor-2 binding site in the KLF4 promoter near the transcription start site. DF also induced DNMT-sensitive propathological expression of downstream KLF4 transcription targets nitric oxide synthase 3, thrombomodulin, and monocyte chemoattractant protein-1. In support of the in vitro findings, swine aortic endothelium isolated from DF regions expressed significantly lower KLF4 and nitric oxide synthase 3, and bisulfite sequencing of KLF4 promoter identified a hypermethylated myocyte enhancer factor-2 binding site.ConclusionsHemodynamics influence endothelial KLF4 expression through DNMT enrichment/myocyte enhancer factor-2 inhibition mechanisms of KLF4 promoter CpG methylation with regional consequences for atherosusceptibility.

Dataset Information

Endothelial cell-specific expression of roundabout 4 is regulated by differential DNA methylation of the proximal promoter.

Publications

Endothelial cell-specific expression of roundabout 4 is regulated by differential DNA methylation of the proximal promoter.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets