Comparative analysis of epigenetic inhibitors reveals different degrees of interference with transcriptional gene silencing and induction of DNA damage.

Repetitive DNA sequences and some genes are epigenetically repressed by transcriptional gene silencing (TGS). When genetic mutants are not available or problematic to use, TGS can be suppressed by chemical inhibitors. However, informed use of epigenetic inhibitors is partially hampered by the absence of their systematic comparison. In addition, there is emerging evidence that epigenetic inhibitors cause genome instability, but the nature of this damage and its repair remain unclear. To bridge these gaps, we compared effects of 5-azacytidine (AC), 2´-deoxy-5-azacytidine (DAC), zebularine and 3-deazaneplanocin A (DZNep) on TGS and DNA damage repair. The most effective TGS inhibitor was DAC, followed by DZNep, zebularine, and AC. We confirmed that all inhibitors induce DNA damage and suggest that this damage is repaired by multiple pathways with a critical role of homologous recombination and of the SMC5/6 complex. A strong positive link between the degree of cytidine analog-induced DNA demethylation and the amount of DNA damage suggests that DNA damage is an integral part of cytidine analog-induced DNA demethylation. This helps understanding function of DNA methylation in plants and opens the possibilities towards the use of epigenetic inhibitors in biotechnology.