RESUMO
Processes that regulate gene transcription are directly under the influence of the genome organization. The epigenome contains additional information that is not brought by DNA sequence, and generates spatial and functional constraints that complement genetic instructions. DNA methylation on CpGs constitutes an epigenetic mark generally correlated with transcriptionally silent condensed chromatin. Replication of methylation patterns by DNA methyltransferases maintains genome stability through cell division. Here we present evidence of an unanticipated dynamic role for DNA methylation in gene regulation in human cells. Periodic, strand-specific methylation/demethylation occurs during transcriptional cycling of the pS2/TFF1 gene promoter on activation by oestrogens. DNA methyltransferases exhibit dual actions during these cycles, being involved in CpG methylation and active demethylation of 5mCpGs through deamination. Inhibition of this process precludes demethylation of the pS2 gene promoter and its subsequent transcriptional activation. Cyclical changes in the methylation status of promoter CpGs may thus represent a critical event in transcriptional achievement.
Assuntos
Metilação de DNA , Regulação da Expressão Gênica , Regiões Promotoras Genéticas/genética , Transcrição Gênica/genética , Ativação Transcricional/genética , Proteínas Supressoras de Tumor/genética , Linhagem Celular , Imunoprecipitação da Cromatina , Ilhas de CpG/genética , DNA (Citosina-5-)-Metiltransferases/antagonistas & inibidores , DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilação de DNA/efeitos dos fármacos , Reparo do DNA , Desaminação , Estrogênios/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Cinética , Timina DNA Glicosilase/metabolismo , Transcrição Gênica/efeitos dos fármacos , Ativação Transcricional/efeitos dos fármacos , Fator Trefoil-1RESUMO
Unliganded (apo-) estrogen receptor alpha (ERalpha, NR3A1) is classically considered as transcriptionally unproductive. Reassessing this paradigm demonstrated that apo-human ERalpha (ERalpha66) and its N-terminally truncated isoform (ERalpha46) are both predominantly nuclear transcription factors that cycle on the endogenous estrogen-responsive pS2 gene promoter in vivo. Importantly, isoform-specific consequences occur in terms of poising the promoter for transcription, as evaluated by determining (i) the engagement of several cofactors and the resulting nucleosomal organization; and (ii) the CpG methylation state of the pS2 promoter. Although transcriptionally unproductive, cycling of apo-ERalpha66 prepares the promoter to respond to ligand, through sequentially targeting chromatin remodeling complexes and general transcription factors. Additionally, apo-ERalpha46 recruits corepressors, following engagement of cofactors identical to those recruited by apo-ERalpha66. Together, these data describe differential activities of ERalpha isoforms. Furthermore, they depict the maintenance of a promoter in a repressed state as a cyclical process that is intrinsically dependent on initial poising of the promoter.