Epigenetic effects of single DNA base modifications

点击翻译按钮获取中文摘要

Epigenetic alterations and the resulting deregulation of the affected genes have been implicated in the pathogenesis of numerous diseases of adulthood and advanced age, including cancers, neurological syndromes and immune disorders. Intriguingly, the gene loci demonstrating abnormal epigenetic patterns are often simultaneously affected by genetic mutations, suggesting that both kinds of alterations possibly originate from common DNA damage events earlier in life. However, it has not yet been possible to trace them back to a particular kind of DNA lesion or damaging agent. Using photochemically damaged reporter vectors, my team has provided evidence for a chromatin-mediated gene silencing mechanism that operates on the damaged DNA. In order to get insight into the precise nature of DNA lesions capable to initiate transcriptional silencing, we further refined the experimental system by inventing an efficient method for incorporation of single DNA base modifications of a pre-defined chemical structure into vector DNA. By analyses of numerous synthetic modifications, we found that the induction of transcriptional silencing is a peculiar feature of DNA lesions processed by the base excision DNA repair pathway (BER) and that this silencing is commonly initiated by the lesion-specific DNA glycosylases. Together, these results demonstrate that DNA damage and DNA repair are implicated in an epigenetic mechanism, functionally important for the regulation of gene expression. Further research will proceed in two directions. The first is the investigation of the molecular mechanism of transcriptional silencing initiated by the BER substrates. The aims will be to discover further effectors of the damage-induced gene silencing and to identify the critical biochemical and structural transitions of the template DNA. The second direction is examination of the capacities of DNA repair pathways for the removal of pre-existent DNA modifications with either acknowledged or alleged epigenetic functions. This topic is inspired by recent discovery of several mechanisms for active DNA demethylation, wherein coordinated action of DNA-modifying enzymatic activities and DNA repair pathways results in the oxidation of a stable epigenetic mark 5-methylcytosine to 5-hydroxymethylcytosine, which is successively converted to reparable pyrimidine species. Site-specific incorporation of these DNA modifications into custom designed vector DNA, opens possibilities for monitoring these transitions at a single nucleotide scale. The aims will be to assess the potentials of the pyrimidine oxidation products positioned in a regulatory gene element for the epigenetic regulation of transcription as well as to characterise the impact of DNA repair mechanisms and of collaterally present DNA lesions on the dynamics of these marks.