|dc.description.abstract||Chemical modification of DNA bases is a key strategy employed by cells in epigenetic regulation of gene expression. 5-methylcytosine (5mC) is an important epigenetic mark and transcription repressor in the human genome which its occurrence in promoter regions leads to gene silencing. However, the mechanism is reversible by Ten-eleven translocation (TET) dioxygenases that can initiate DNA demethylation pathways by hydroxylating these epigenetic marks. Oxidation of 5mC by TET proteins causes a modification resembling the oxidative base damages that DNA suffers daily due to unstable levels of reactive oxidative species in cells. Several lines of evidence suggest that the base excision repair (BER) machinery is triggered at this point. Then, DNA glycosylases are recruited to restore the cytosine base to its non-methylated state resulting in transcription activation. However, it is still unclear how and in which manner BER enzymes are contributing to the demethylation of hypermethylated promoters of silenced genes.
In this study, we aimed at performing a quantitative basic research study to investigate the impact of NEIL2 and other BER enzymes associated with oxidative DNA damage response on TET1-mediated DNA demethylation. The experiments were performed using the CRISPR-based methylation editing platform (Casilio-ME) to target the hypermethylated promoter of the MutL Homolog 1 (MLH1) gene in human embryonic kidney cells (HEK293T). The contribution of each BER enzyme on DNA demethylation was then evaluated quantitatively by measuring the mRNA levels of MLH1 using real-time quantitative PCR with TaqMan® probes.
In line with previous findings, we were able to verify that the enzymatic coupling of TET1 and NEIL2 enhances demethylation considerably. Additionally, our results suggest that the downregulation of endogenous NEIL2 diminishes the demethylation rate but does not inhibit the process completely. This indicates that DNA demethylation might only be partially dependent on NEIL2 expression. Moreover, further overexpression analyses with TET1 and other BER enzymes revealed that NEIL2, NEIL1 and APE1, stimulate the process positively by comparable levels, while OGG1 was not observed to have a large impact on the process. On the other hand, we witnessed a tremendous stimulation effect by NTHL1 which has not been reported before. TET1- mediated DNA demethylation coupled with NTHL1 was significantly more powerful compared to the originally published version of Casilio-ME. Thus, we have potentially discovered an improved and more powerful version of the Casilio-ME genome editing tool that can benefit a wide range of clinical studies on human pathologies associated with abnormal DNA methylation.||