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

Abstract

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 any systematic comparison. In addition, there is emerging evidence that epigenetic inhibitors cause genomic instability, but the nature of this damage and its repair remain unclear. To bridge these gaps, we compared the effects of 5-azacytidine (AC), 2 0 -deoxy-5-azacytidine (DAC), zebularine and 3-deazaneplanocin A (DZNep) on TGS and DNA damage repair. The most effective inhibitor of TGS 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 us to understand the function of DNA methylation in plants and opens the possibility of using epigenetic inhibitors in biotechnology.

Epigenetic silencing of clustered tRNA genes in Arabidopsis

“…5-azaC treatment significantly released clustered Tyr tDNAs expression whilst no effect was detected for Pro ones (Figure 5A ). Another analog of cytidine, zebularine ( 50 ), confirmed the release of clustered Tyr tDNAs expression (Figure 5A ). This pharmacological approach highlights that DNA methylation represses clustered Tyr tDNAs expression.…”

“…( A ) Northern blots analysis of total tRNAs of untreated (0), 5-azaC or zebularine treated WT Arabidopsis plants. Two conditions were used: direct treatment (DT) or postponed treatment (PT) as described in ( 50 ). ( B and C ) Northern blots analysis of total tRNAs of the indicated mutant genotypes.…”

“…5-Azacytidine (5-azaC) and zebularine assays (0, 5, 25, 50 and 100 μM) were performed using Col0 sterilized seeds and direct (DT) or postponed (PT) treatments as described in ( 50 ).…”

Epigenetic Variation Induced by Gamma Rays, DNA Methyltransferase Inhibitors, and Their Combination in Rice

“…Indices related to epigenetic diversity were increased in all treatment groups compared to the control ( Table 1 ), indicating that GRs as well as DNMTis can affect DNA methylation status. The two types of DNMTi used here, AZA and ZEB, induce epigenetic variation by reducing the methylation rate [ 13 ]. Ionizing irradiation is also known to have a demethylating effect, which was deduced from the activation of TEs by hypomethylation in irradiated plants [ 33 , 45 ].…”

“…Although AZA and ZEB have similar inhibitory effects on DNA methylation, ZEB has a longer half-life and better stability than AZA [ 8 , 9 ]. Many studies have investigated the effects of DNMTis on gene expression and the silencing, growth, development, and induction of DNA damage in plants [ 10 , 11 , 12 , 13 ].…”