Analysis of an Active Deformylation Mechanism of 5‐Formyl‐deoxycytidine (fdC) in Stem Cells

The removal of 5‐methyl‐deoxycytidine (mdC) from promoter elements is associated with reactivation of the silenced corresponding genes. It takes place through an active demethylation process involving the oxidation of mdC to 5‐hydroxymethyl‐deoxycytidine (hmdC) and further on to 5‐formyl‐deoxycytidine (fdC) and 5‐carboxy‐deoxycytidine (cadC) with the help of α‐ketoglutarate‐dependent Tet oxygenases. The next step can occur through the action of a glycosylase (TDG), which cleaves fdC out of the genome for replacement by dC. A second pathway is proposed to involve C−C bond cleavage that converts fdC directly into dC. A 6‐aza‐5‐formyl‐deoxycytidine (a‐fdC) probe molecule was synthesized and fed to various somatic cell lines and induced mouse embryonic stem cells, together with a 2′‐fluorinated fdC analogue (F‐fdC). While deformylation of F‐fdC was clearly observed in vivo, it did not occur with a‐fdC, thus suggesting that the C−C bond‐cleaving deformylation is initiated by nucleophilic activation

Active turnover of genomic methylcytosine in pluripotent cells

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 unveil additional oxidation-independent mdC turnover, possibly through 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

Chromatin-dependent allosteric regulation of DNMT3A activity by MeCP2

Despite their central importance in mammalian development, the mechanisms that regulate the DNA methylation machinery and thereby the generation of genomic methylation patterns are still poorly understood. Here, we identify the 5mC-binding protein MeCP2 as a direct and strong interactor of DNA methyltransferase 3( DNMT3) proteins. We mapped the interaction interface to the transcriptional repression domain of MeCP2 and the ADD domain of DNMT3A and find that binding of MeCP2 strongly inhibits the activity of DNMT3A in vitro. This effect was reinforced by cellular studies where a global reduction of DNA methylation levels was observed after overexpression of MeCP2 in human cells. By engineering conformationally locked DNMT3A variants as novel tools to study the allosteric regulation of this enzyme, we show that MeCP2 stabilizes the closed, autoinhibitory conformation of DNMT3A. Interestingly, the interaction with MeCP2 and its resulting inhibition were relieved by the binding of K4 unmodified histone H3 N-terminal tail to the DNMT3A-ADD domain. Taken together, our data indicate that the localization and activity of DNMT3A are under the combined control of MeCP2 and H3 tailmodifications where, depending on the modification status of the H3 tail at the binding sites, MeCP2 can act as either a repressor or activator of DNA methylation

Influencing epigenetic information with a hydrolytically stable carbocyclic 5-aza-2’-deoxycytidine

5-Aza-2’-deoxycytidine (AzadC) is an antimetabolite in clinical use, which reduces the level of the epigenetic modification 5-methyl-2’-deoxycytidine (mdC). AzadC is incorporated into the genome of proliferating cells, where it inhibits the DNA methyltransferases (DNMTs) in a suicide process leading to a reduction of mdC. The loss of mdC, which is a transcriptional silencer in promoters, leads to the reactivation of genes including tumor suppressor genes, which elicits a beneficial effect. The problem associated with AzadC is that the compound is hydrolytically unstable. It decomposes during treatment to a variety of poorly characterized hydrolysis products. After its incorporation into the genome, this hydrolytic instability generates abasic sites. It is consequently difficult to dissect if the activity of the compound is caused by DNMT inhibition or more generally by DNA lesion formation. We now discovered that a disarmed version of AzadC, in which the ribose oxygen was replaced by a CH2-group, is surprisingly stable under a variety of pH values while keeping the epigenetic activity against the DNMTs

Associations of long-term exposure to air pollution and noise with body composition in children and adults: Results from the LEAD general population study

Background: While long-term air pollution and noise exposure has been linked to increasing cardiometabolic disease risk, potential effects on body composition remains unclear. This study aimed to investigate the associations of long-term air pollution, noise and body composition. Methods: We used repeated data from the LEAD (Lung, hEart, sociAl, boDy) study conducted in Vienna, Austria. Body mass index (BMI; kg/m2), fat mass index (FMI; z-score), and lean mass index (LMI; z-score) were measured using dual-energy x-ray absorptiometry at the first (t0; 2011-ongoing) and second (t1; 2017-ongoing) examinations. Annual particulate matter (PM10) and nitrogen dioxide (NO2) concentrations were estimated with the GRAMM/GRAL model (2015–2021). Day-evening-night (Lden) and night-time (Lnight) noise levels from transportation were modeled for 2017 following the European Union Directive 2002/49/EC. Exposures were assigned to residential addresses. We performed analyses separately in children/adolescents and adults, using linear mixed-effects models with random participant intercepts and linear regression models for cross-sectional and longitudinal associations, respectively. Models were adjusted for co-exposure, lifestyle and sociodemographics. Results: A total of 19,202 observations (nt0 = 12,717, nt1 = 6,485) from participants aged 6–86 years (mean age at t0 = 41.0 years; 52.9 % female; mean PM10 = 21 µg/m3; mean follow-up time = 4.1 years) were analyzed. Among children and adolescents (age ≤ 18 years at first visit), higher PM10 exposure was cross-sectionally associated with higher FMI z-scores (0.09 [95 % Confidence Interval (CI): 0.03, 0.16]) and lower LMI z-scores (−0.05 [95 % CI: −0.10, −0.002]) per 1.8 µg/m3. Adults showed similar trends in cross-sectional associations as children, though not reaching statistical significance. We observed no associations for noise exposures. Longitudinal analyses on body composition changes over time yielded positive associations for PM10, but not for other exposures. Conclusion: Air pollution exposure, mainly PM10, was cross-sectionally and longitudinally associated with body composition in children/adolescents and adults. Railway/road-traffic noise exposures showed no associations in both cross-sectional and longitudinal analyses