RESUMO
The ten-eleven translocation family of proteins (Tet1/2/3, Tets) converts 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), which can be further oxidized and repaired by thymine DNA glycosylase (TDG), to influence gene transcription in embryonic and adult tissues. However the mechanisms of how Tets and TDG levels are regulated are unknown. We show that miR-29 can directly regulate Tet1-3 and TDG mRNA levels through binding to their 3'UTRs. miR-29 mimic decreases global 5hmC levels, a hallmark of Tet activity. Moreover, the mRNA levels for Tet3 and TDG are inversely correlated with the levels of miR-29 in aged mouse aorta implying that aging may affect methylation patterns via miRNA. In summary, our data show that Tets and TDG are direct targets of miR-29 and unravel a novel regulatory role for this miRNA in epigenetic DNA demethylation pathways.
Assuntos
Metilação de DNA/genética , Proteínas de Ligação a DNA/metabolismo , Dioxigenases/metabolismo , Marcação de Genes/métodos , MicroRNAs/fisiologia , Proteínas Proto-Oncogênicas/metabolismo , Transdução de Sinais/genética , Timina DNA Glicosilase/biossíntese , Regiões 3' não Traduzidas/genética , Animais , Células Cultivadas , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/genética , Dioxigenases/antagonistas & inibidores , Dioxigenases/genética , Repressão Epigenética/genética , Células HEK293 , Humanos , Camundongos , MicroRNAs/antagonistas & inibidores , Oxigenases de Função Mista , Mimetismo Molecular/fisiologia , Oxirredução , Ligação Proteica/genética , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas/genética , RNA Mensageiro/antagonistas & inibidores , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Timina DNA Glicosilase/antagonistas & inibidores , Timina DNA Glicosilase/genéticaRESUMO
Previous studies have demonstrated that the base excision repair enzyme thymine DNA glycosylase (TDG) mediates recruitment of histone acetyltransferases CREB-binding protein (CBP) and p300 to DNA, suggesting a plausible role for these factors in TDG-mediated repair. Furthermore, TDG was found to potentiate CBP/p300-dependent transcription and serve as a substrate for CBP/p300 acetylation. Here, we show that the small ubiquitin-like modifier 1 (SUMO-1) protein binding activity of TDG is essential for activation of CBP and localization to promyelocytic leukemia protein oncogenic domains (PODs). SUMO-1 binding is mediated by two distinct amino- and carboxy-terminal motifs (residues 144 to 148 and 319 to 322) that are negatively regulated by DNA binding via an amino-terminal hydrophilic region (residues 1 to 121). TDG is also posttranslationally modified by covalent conjugation of SUMO-1 (sumoylation) to lysine 341. Interestingly, we found that sumoylation of TDG blocks interaction with CBP and prevents TDG acetylation in vitro. Furthermore, sumoylation effectively abrogates intermolecular SUMO-1 binding and a sumoylation-deficient mutant accumulates in PODs, suggesting that sumoylation negatively regulates translocation to these nuclear structures. These findings suggest that TDG sumoylation promotes intramolecular interactions with amino- and carboxy-terminal SUMO-1 binding motifs that dramatically alter the biochemical properties and subcellular localization of TDG.
