RESUMO
DNA methylation is an important epigenetic modification involved in transcriptional regulation, nuclear organization, development, aging, and disease. Although DNA methyltransferases have been characterized, the mechanisms for DNA demethylation remain poorly understood. Using a cell-based reporter assay, we performed a functional genomics screen to identify genes involved in DNA demethylation. Here we show that RNF4 (RING finger protein 4), a SUMO-dependent ubiquitin E3-ligase previously implicated in maintaining genome stability, plays a key role in active DNA demethylation. RNF4 reactivates methylation-silenced reporters and promotes global DNA demethylation. Rnf4 deficiency is embryonic lethal with higher levels of methylation in genomic DNA. Mechanistic studies show that RNF4 interacts with and requires the base excision repair enzymes TDG and APE1 for active demethylation. This activity appears to occur by enhancing the enzymatic activities that repair DNA G:T mismatches generated from methylcytosine deamination. Collectively, our study reveals a unique function for RNF4, which may serve as a direct link between epigenetic DNA demethylation and DNA repair in mammalian cells.
Assuntos
Metilação de DNA , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Animais , Sequência de Bases , Linhagem Celular , DNA/química , DNA/genética , DNA/metabolismo , Metilação de DNA/genética , Metilação de DNA/fisiologia , Reparo de Erro de Pareamento de DNA/genética , Reparo de Erro de Pareamento de DNA/fisiologia , Feminino , Genes Letais , Genes Reporter , Genes p16 , Genômica , Humanos , Camundongos , Camundongos Knockout , Proteínas Nucleares/deficiência , Proteínas Nucleares/genética , Gravidez , Regiões Promotoras Genéticas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Fatores de Transcrição/deficiência , Fatores de Transcrição/genética , Ubiquitina-Proteína LigasesRESUMO
OBJECTIVE: GH replacement therapy (GHRT) in adult-onset GH deficiency (AOGHD) reduces carotid intima-media thickness (IMT) and increases myocardial mass, with improvement of systolic and diastolic function. These observations have reinforced the use of GHRT on AOGHD. Conversely, we have previously reported that in adults with lifetime congenital and severe isolated GH deficiency (IGHD) due to a mutation in GHRH receptor gene (GHRHR), a 6-month treatment with depot GH increased carotid IMT, caused the development of atherosclerotic plaques, and an increase in left ventricular mass index (LVMI), posterior wall, and septal thickness and ejection fraction. Such effects persisted 12 months after treatment (12-month washout - 12 mo). METHODS: We have studied the cardiovascular status (by echocardiography and carotid ultrasonography) of these subjects 60 months after completion of therapy (60-month washout - 60 mo). RESULTS: Carotid IMT reduced significantly from 12 to 60 mo, returning to baseline (pre-therapy) value. The number of individuals with plaques was similar at 12 and 60 mo, remaining higher than pre-therapy. LVMI, relative posterior wall thickness, and septum thickness did not change between 12 and 60 mo, but absolute posterior wall increased from 12 to 60 mo. Systolic function, evaluated by ejection fraction and shortening fraction, was reduced at 60 mo in comparison with 12 mo returning to baseline levels. The E/A wave ratio (expression of diastolic function) decreased at 60 mo compared with both 12 mo and baseline. CONCLUSIONS: In adults with lifetime congenital IGHD, the increase in carotid IMT elicited by GHRT was transitory and returned to baseline 5 years after therapy discontinuation. Despite this, the number of subjects with plaques remained stable at 60 mo and higher than at baseline.