Active turnover of genomic methylcytosine in pluripotent cells

Abstract

Epigenetic plasticity underpins cell potency, but the extent to which active turnover of DNA methylation contributes to such plasticity is not known and the underlying pathways are poorly understood. Here we use metabolic labelling with stable isotopes and mass spectrometry to quantitatively address the global turnover of genomic methylcytidine (mdC), hydroxymethylcytidine (hmdC) and formylcytidine (fdC) across mouse pluripotent cell states.High rates of mdC/hmdC oxidation and fdC turnover characterize a formative-like pluripotent state. In primed pluripotent cells the global mdC turnover rate is about 3-6% faster than can be explained by passive dilution through DNA synthesis. While this active component is largely dependent on Tet-mediated mdC oxidation, we unveiled an additional mdC oxidationindependent turnover process based on DNA repair. This process accelerates upon acquisition of primed pluripotency and returns to low levels in lineage committed cells. Thus, in pluripotent cells active mdC turnover involves both mdC oxidation-dependent and -independent processes. .

Epigenetic Regulation of Genomic Stability by Vitamin C

“…TET enzymes have also been shown to oxidize thymine to generate 5hmU directly, leading to 5hmU:A mismatches ( Pfaffeneder et al, 2014 ; Olinski et al, 2016 ). Protein readers of 5hmU:G and 5hmU:A also include chromatin regulators and DNA repair enzymes involved in BER ( Pfaffeneder et al, 2014 ), and the repair of 5hmU:A by long-patch BER or non-canonical mismatch repair could lead to the indirect removal of adjacent 5mC in the genome ( Metivier et al, 2008 ; Santos et al, 2013 ; Grin and Ishchenko, 2016 ; Spada et al, 2020 ). In primed embryonic murine pluripotent stems cells (PSCs) and the developing zygote, other non-oxidative and cell-cycle independent mechanisms of DNA demethylation have also been reported that may utilize deamination of mC by AID and DNA repair processes ( Santos et al, 2013 ; Amouroux et al, 2016 ; Spada et al, 2020 ), however, the majority of active DNA demethylation in mPSCs was shown to be driven by oxidation of 5mC ( Spada et al, 2020 ).…”

5‐Hydroxymethyl‐, 5‐Formyl‐ and 5‐Carboxydeoxycytidines as Oxidative Lesions and Epigenetic Marks

“…The availability of new ultra‐high performance liquid chromatography (UHPLC)‐MS 2 procedures in combination with synthetic isotope standards and reagents enable the quantification of AP and βE‐sites, [9,17] and the parallel determination of the xdC levels and BER repair events. This now allows us to gain information about the ROS induced xdC levels [18–19] . We selected as a model organism the fungus Amanita muscaria , [20] which was collected from its natural environment.…”

“…Our data suggest that, while some of the fdCs have a permanent or semi‐permanent character, because in mESCs even with full BER (Tdg +/+ ) the levels never drop to the ROS level, this is questionable for cadCs. Removing fdC at these (semi)permanent sites then requires Tdg‐independent pathways that were recently investigated in detail [19] …”

“…This now allows us to gain information about the ROS induced xdC levels. [18][19] We selected as a model organism the fungus Amanita muscaria, [20] which was collected from its natural environment. The methylation levels in fungi of the basidiomycota are diverse between 2-5 % per dC.…”

“…Removing fdC at these (semi) permanent sites then requires Tdg-independent pathways that were recently investigated in detail. [19]…”