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Arterioscler Thromb Vasc Biol ; 34(7): 1531-8, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24855053

RESUMO

OBJECTIVE: 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. METHODS AND RESULTS: 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. CONCLUSIONS: 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.


Assuntos
Metilação de DNA , Células Endoteliais/metabolismo , Epigênese Genética , Regiões Promotoras Genéticas , Receptores de Superfície Celular/metabolismo , Animais , Sítios de Ligação , Diferenciação Celular , Linhagem da Célula , Montagem e Desmontagem da Cromatina , Metilação de DNA/efeitos dos fármacos , Metilases de Modificação do DNA/antagonistas & inibidores , Metilases de Modificação do DNA/metabolismo , Células-Tronco Embrionárias/metabolismo , Células Endoteliais/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Epigênese Genética/efeitos dos fármacos , Fibroblastos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Células HEK293 , Humanos , Hipoxantina Fosforribosiltransferase/genética , Hipoxantina Fosforribosiltransferase/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Miócitos de Músculo Liso/metabolismo , Regiões Promotoras Genéticas/efeitos dos fármacos , Receptores de Superfície Celular/genética , Fator de Transcrição Sp1/metabolismo , Transfecção
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