Minimal role of base excision repair in TET-induced global DNA demethylation in HEK293T cells

Abstract

Oxidation of 5-methylcytosine by TET family proteins can induce DNA replication-dependent (passive) DNA demethylation and base excision repair (BER)-based (active) DNA demethylation. The balance of active vs. passive TET-induced demethylation remains incompletely determined. In the context of large scale DNA demethylation, active demethylation may require massive induction of the DNA repair machinery and thus compromise genome stability. To study this issue, we constructed a tetracycline-controlled TET-induced global DNA demethylation system in HEK293T cells. Upon TET overexpression, we observed induction of DNA damage and activation of a DNA damage response; however, BER genes are not upregulated to promote DNA repair. Depletion of TDG (thymine DNA glycosylase) or APEX1 (apurinic/apyrimidinic endonuclease 1), two key BER enzymes, enhances rather than impairs global DNA demethylation, which can be explained by stimulated proliferation. By contrast, growth arrest dramatically blocks TET-induced global DNA demethylation. Thus, in the context of TET-induction in HEK293T cells, the DNA replication-dependent passive mechanism functions as the predominant pathway for global DNA demethylation. In the same context, BER-based active demethylation is markedly restricted by limited BER upregulation, thus potentially preventing a disastrous DNA damage response to extensive active DNA demethylation.

TET2-mediated 5-hydroxymethylcytosine induces genetic instability and mutagenesis

“…Therefore, these data suggest that TET2-mediated active demethylation may be a threat to genome stability and should not be used to entirely demethylate the genome, but only specific loci. In accordance with our results, Jin et al have recently shown that the BER pathway plays only a minor role in the TET1-mediated global DNA demethylation [35].…”

“…This cell cycle delay could be linked to the generation of 5-hmC, 5-fC or 5-caC residues which might either (i) perturb DNA replication by themselves or by generating genotoxic DNA repair intermediates such as AP sites and DNA strand breaks by TDG-BER [8,10,13] or (ii) activate intra-S checkpoints as suggested by P53 activation, possibly through ATM/ATR and CHK1/CHK2 pathways. Increase in ␥-H2AX foci formation and activation of CHK2 and P53 have also been observed with TET1 catalytic domain, suggesting a TET1-mediated role in increased DNA damages or decreased DNA repair [35]. Moreover, TET2-mediated 5-hmC has recently been found to colocalize with ␥-H2AX and to participate to DNA damage foci [36].…”

Genome-wide analysis reveals a role for TDG in estrogen receptor-mediated enhancer RNA transcription and 3-dimensional reorganization

“…Genome-wide studies using ChIP-based technologies have shown that the majority of ERα binding sites in breast cancer cells are found distally from gene promoters, and a significant component is found within gene-specific “enhancer” regions in response to the E2 [ 2 ]. Enhancers are essential regulatory regions found in noncoding regions that control temporal and tissue-specific gene expression.…”

DNA demethylation pathways: Additional players and regulators

“…Instead, the base (or a nearby base) is always first modified, either by deamination, oxidation, or -according to models that now appear less likely -a combination of both, followed by replacement of the modified nucleotide (possibly together with surrounding nucleotides [18,19]). Most evidence points to the base excision repair (BER) pathway [20][21][22][23][24], but its role in global DNA demethylation has been challenged [25]. Alternative pathways include nucleotide excision [26,27] and non-canonical mismatch repair [28].…”