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

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

Funder: LightDyNAmicsFunder: Volkswagen Foundation; Id: http://dx.doi.org/10.13039/501100001663Abstract: The four non‐canonical nucleotides in the human genome 5‐methyl‐, 5‐hydroxymethyl‐, 5‐formyl‐ and 5‐carboxydeoxycytidine (mdC, hmdC, fdC and cadC) form a second layer of epigenetic information that contributes to the regulation of gene expression. Formation of the oxidized nucleotides hmdC, fdC and cadC requires oxidation of mdC by ten‐eleven translocation (Tet) enzymes that require oxygen, Fe(II) and α‐ketoglutarate as cosubstrates. Although these oxidized forms of mdC are widespread in mammalian genomes, experimental evidence for their presence in fungi and plants is ambiguous. This vagueness is caused by the fact that these oxidized mdC derivatives are also formed as oxidative lesions, resulting in unclear basal levels that are likely to have no epigenetic function. Here, we report the xdC levels in the fungus Amanita muscaria in comparison to murine embryonic stem cells (mESCs), HEK cells and induced pluripotent stem cells (iPSCs), to obtain information about the basal levels of hmdC, fdC and cadC as DNA lesions in the genome

Proceedings of the 3rd Biennial Conference of the Society for Implementation Research Collaboration (SIRC) 2015: advancing efficient methodologies through community partnerships and team science

It is well documented that the majority of adults, children and families in need of evidence-based behavioral health interventionsi do not receive them [1, 2] and that few robust empirically supported methods for implementing evidence-based practices (EBPs) exist. The Society for Implementation Research Collaboration (SIRC) represents a burgeoning effort to advance the innovation and rigor of implementation research and is uniquely focused on bringing together researchers and stakeholders committed to evaluating the implementation of complex evidence-based behavioral health interventions. Through its diverse activities and membership, SIRC aims to foster the promise of implementation research to better serve the behavioral health needs of the population by identifying rigorous, relevant, and efficient strategies that successfully transfer scientific evidence to clinical knowledge for use in real world settings [3]. SIRC began as a National Institute of Mental Health (NIMH)-funded conference series in 2010 (previously titled the “Seattle Implementation Research Conference”; $150,000 USD for 3 conferences in 2011, 2013, and 2015) with the recognition that there were multiple researchers and stakeholdersi working in parallel on innovative implementation science projects in behavioral health, but that formal channels for communicating and collaborating with one another were relatively unavailable. There was a significant need for a forum within which implementation researchers and stakeholders could learn from one another, refine approaches to science and practice, and develop an implementation research agenda using common measures, methods, and research principles to improve both the frequency and quality with which behavioral health treatment implementation is evaluated. SIRC’s membership growth is a testament to this identified need with more than 1000 members from 2011 to the present.ii SIRC’s primary objectives are to: (1) foster communication and collaboration across diverse groups, including implementation researchers, intermediariesi, as well as community stakeholders (SIRC uses the term “EBP champions” for these groups) – and to do so across multiple career levels (e.g., students, early career faculty, established investigators); and (2) enhance and disseminate rigorous measures and methodologies for implementing EBPs and evaluating EBP implementation efforts. These objectives are well aligned with Glasgow and colleagues’ [4] five core tenets deemed critical for advancing implementation science: collaboration, efficiency and speed, rigor and relevance, improved capacity, and cumulative knowledge. SIRC advances these objectives and tenets through in-person conferences, which bring together multidisciplinary implementation researchers and those implementing evidence-based behavioral health interventions in the community to share their work and create professional connections and collaborations

An integrated study of the affinities of the Aβ16 peptide for Cu(I) and Cu(II): implications for the catalytic production of reactive oxygen species

A new fluorescent probe Aβ16wwa based upon the Aβ16 peptide has been developed with two orders of magnitude greater fluorescence intensity for sensitive detection of interactions with Cu(II). In combination with the Cu(I) probe Ferene S, it is confirmed that the Aβ16 peptide binds either Cu(I) or Cu(II) with comparable affinities at pH 7.4 (log KID = −10.4; log KIID = −10.0). It follows from this property that the Cu–Aβ16 complex is a robust if slow catalyst for the aerial oxidation of ascorbate with H2O2 as primary product (initial rate, ∼0.63 min−1 for Cu–Aβ16 versus >2.5 min−1 for Cuaq2+). An integrated study of variants of this peptide identifies the major ligands and binding modes involved in its copper complexes in solution. The dependence of KID upon pH is consistent with a two-coordinate Cu(I) site in which dynamic processes exchange Cu(I) between the three available pairs of imidazole sidechains provided by His6, His13 and His14. The N-terminal amine is not involved in Cu(I) binding but is a key ligand for Cu(II). Acetylation of the N-terminus alters the redox thermodynamic gradient for the Cu centre and suppresses its catalytic activity considerably. The data indicate the presence of dynamic processes that exchange Cu(II) between the three His ligands and backbone amide at physiological pH. His6 is identified as a key ligand for catalysis as its presence minimises the pre-organisation energy required for interchange of the two copper redox sites. These new thermodynamic data strengthen structural interpretations for the Cu–Aβ complexes and provide valuable insights into the molecular mechanism by which copper chemistry may induce oxidative stress in Alzheimer's disease

G-quadruplex DNA structure is a positive regulator of MYC transcription

DNA structure can regulate genome function. Four-stranded DNA G-quadruplex (G4) structures have been implicated in transcriptional regulation; however, previous studies have not directly addressed the role of an individual G4 within its endogenous cellular context. Using CRISPR to genetically abrogate endogenous G4 structure folding, we directly interrogate the G4 found within the upstream regulatory region of the critical human MYC oncogene. G4 loss leads to suppression of MYC transcription from the P1 promoter that is mediated by the deposition of a de novo nucleosome alongside alterations in RNA polymerase recruitment. We also show that replacement of the endogenous MYC G4 with a different G4 structure from the KRAS oncogene restores G4 folding and MYC transcription. Moreover, we demonstrate that the MYC G4 structure itself, rather than its sequence, recruits transcription factors and histone modifiers. Overall, our work establishes that G4 structures are important features of transcriptional regulation that coordinate recruitment of key chromatin proteins and the transcriptional machinery through interactions with DNA secondary structure, rather than primary sequenceHerchel Smit

5‑Formyl- and 5‑Carboxydeoxycytidines Do Not Cause Accumulation of Harmful Repair Intermediates in Stem Cells

5-Formyl-dC (fdC) and 5-carboxy-dC (cadC) are newly discovered bases in the mammalian genome that are supposed to be substrates for base excision repair (BER) in the framework of active demethylation. The bases are recognized by the monofunctional thymine DNA glycosylase (Tdg), which cleaves the glycosidic bond of the bases to give potentially harmful abasic sites (AP-sites). Because of the turnover of fdC and cadC during cell state transitions, it is an open question to what extent such harmful AP-sites may accumulate during these processes. Here, we report the development of a new reagent that in combination with mass spectrometry (MS) allows us to quantify the levels of AP-sites. This combination also allowed the quantification of β-elimination (βE) products, which are repair intermediates of bifunctional DNA glycosylases. In combination with feeding of isotopically labeled nucleosides, we were able to trace the intermediates back to their original nucleobases. We show that, while the steady-state levels of fdC and cadC are substantially increased in Tdg-deficient cells, those of both AP- and βE-sites are unaltered. The levels of the detected BER intermediates are 1 and 2 orders of magnitude lower than those of cadC and fdC, respectively. Thus, neither the presence of fdC nor that of cadC in stem cells leads to the accumulation of harmful AP- and βE-site intermediates

5-Formylcytosine to Cytosine Conversion by C-C Bond Cleavage in vivo

Tet enzymes oxidise 5-methyl-deoxycytidine (mdC) to 5-hydroxymethyl-dC (hmdC), 5-formyl-dC (fdC) and 5-carboxy-dC (cadC) in DNA. It was proposed that fdC and cadC deformylate and decarboxylate to dC in the course of an active demethylation process. This would re-install canonical dC bases at previously methylated sites. The question whether such direct C-C bond cleavage reactions at fdC and cadC occur in vivo remains an unsolved problem. Here we report the incorporation of synthetic isotope- and (R)-2’-fluorine-labelled dC and fdC-derivatives into the genome of cultured mammalian cells. Following the fate of these probe molecules using UHPLC-MS/MS provided quantitative data about the formed reaction products. The data show that the labelled fdC probe is efficiently converted into the corresponding labelled dC, most likely after its incorporation into the genome. This allows concluding that fdC is undergoing C-C bond cleavage in stem cells that leads to the direct re-installation of unmodified dC