Metabolic control of DNA methylation in naive pluripotent cells.

Naive epiblast and embryonic stem cells (ESCs) give rise to all cells of adults. Such developmental plasticity is associated with genome hypomethylation. Here, we show that LIF-Stat3 signaling induces genomic hypomethylation via metabolic reconfiguration. Stat3-/- ESCs show decreased α-ketoglutarate production from glutamine, leading to increased Dnmt3a and Dnmt3b expression and DNA methylation. Notably, genome methylation is dynamically controlled through modulation of α-ketoglutarate availability or Stat3 activation in mitochondria. Alpha-ketoglutarate links metabolism to the epigenome by reducing the expression of Otx2 and its targets Dnmt3a and Dnmt3b. Genetic inactivation of Otx2 or Dnmt3a and Dnmt3b results in genomic hypomethylation even in the absence of active LIF-Stat3. Stat3-/- ESCs show increased methylation at imprinting control regions and altered expression of cognate transcripts. Single-cell analyses of Stat3-/- embryos confirmed the dysregulated expression of Otx2, Dnmt3a and Dnmt3b as well as imprinted genes. Several cancers display Stat3 overactivation and abnormal DNA methylation; therefore, the molecular module that we describe might be exploited under pathological conditions

Metabolic control of DNA methylation in naive pluripotent cells.

Naive epiblast and embryonic stem cells (ESCs) give rise to all cells of adults. Such developmental plasticity is associated with genome hypomethylation. Here, we show that LIF-Stat3 signaling induces genomic hypomethylation via metabolic reconfiguration. Stat3-/- ESCs show decreased α-ketoglutarate production from glutamine, leading to increased Dnmt3a and Dnmt3b expression and DNA methylation. Notably, genome methylation is dynamically controlled through modulation of α-ketoglutarate availability or Stat3 activation in mitochondria. Alpha-ketoglutarate links metabolism to the epigenome by reducing the expression of Otx2 and its targets Dnmt3a and Dnmt3b. Genetic inactivation of Otx2 or Dnmt3a and Dnmt3b results in genomic hypomethylation even in the absence of active LIF-Stat3. Stat3-/- ESCs show increased methylation at imprinting control regions and altered expression of cognate transcripts. Single-cell analyses of Stat3-/- embryos confirmed the dysregulated expression of Otx2, Dnmt3a and Dnmt3b as well as imprinted genes. Several cancers display Stat3 overactivation and abnormal DNA methylation; therefore, the molecular module that we describe might be exploited under pathological conditions

Effect of angiotensin-converting enzyme inhibitor and angiotensin receptor blocker initiation on organ support-free days in patients hospitalized with COVID-19

IMPORTANCE Overactivation of the renin-angiotensin system (RAS) may contribute to poor clinical outcomes in patients with COVID-19. Objective To determine whether angiotensin-converting enzyme (ACE) inhibitor or angiotensin receptor blocker (ARB) initiation improves outcomes in patients hospitalized for COVID-19. DESIGN, SETTING, AND PARTICIPANTS In an ongoing, adaptive platform randomized clinical trial, 721 critically ill and 58 non–critically ill hospitalized adults were randomized to receive an RAS inhibitor or control between March 16, 2021, and February 25, 2022, at 69 sites in 7 countries (final follow-up on June 1, 2022). INTERVENTIONS Patients were randomized to receive open-label initiation of an ACE inhibitor (n = 257), ARB (n = 248), ARB in combination with DMX-200 (a chemokine receptor-2 inhibitor; n = 10), or no RAS inhibitor (control; n = 264) for up to 10 days. MAIN OUTCOMES AND MEASURES The primary outcome was organ support–free days, a composite of hospital survival and days alive without cardiovascular or respiratory organ support through 21 days. The primary analysis was a bayesian cumulative logistic model. Odds ratios (ORs) greater than 1 represent improved outcomes. RESULTS On February 25, 2022, enrollment was discontinued due to safety concerns. Among 679 critically ill patients with available primary outcome data, the median age was 56 years and 239 participants (35.2%) were women. Median (IQR) organ support–free days among critically ill patients was 10 (–1 to 16) in the ACE inhibitor group (n = 231), 8 (–1 to 17) in the ARB group (n = 217), and 12 (0 to 17) in the control group (n = 231) (median adjusted odds ratios of 0.77 [95% bayesian credible interval, 0.58-1.06] for improvement for ACE inhibitor and 0.76 [95% credible interval, 0.56-1.05] for ARB compared with control). The posterior probabilities that ACE inhibitors and ARBs worsened organ support–free days compared with control were 94.9% and 95.4%, respectively. Hospital survival occurred in 166 of 231 critically ill participants (71.9%) in the ACE inhibitor group, 152 of 217 (70.0%) in the ARB group, and 182 of 231 (78.8%) in the control group (posterior probabilities that ACE inhibitor and ARB worsened hospital survival compared with control were 95.3% and 98.1%, respectively). CONCLUSIONS AND RELEVANCE In this trial, among critically ill adults with COVID-19, initiation of an ACE inhibitor or ARB did not improve, and likely worsened, clinical outcomes. TRIAL REGISTRATION ClinicalTrials.gov Identifier: NCT0273570

Gas-phase stabilization of Cu(II) reveals phosphatidylcholine isomeric features by ESI and MALDI-MS

International audienceGlycerophospholipids (GPLs) are amphiphilic metabolites that make up most of the lipidbilayer in mammalian cells. literature has pointed out the significant implications of doublebond (DB) positions and linkage positions on the glycerol moiety in the chemical, biochemical,and biophysical roles of lipids.1 Currently, derivatization or instrumental modifications are re-quested to highlight these isomeric features in GPLs.Copper-adducted fatty acid proved to be efficient in localizing DB in aliphatic chains. However,the driving force of this fragmentation mechanism was attributed to the presence of the freecarboxylic acid function that is lacking in GPLs. We recently demonstrated that radical-drivenfragmentation could be obtained from quaternary copper complexes of GPLs. The CID MS/MSfragmentation of such species yields both DB and stereo-numbering (sn) locations in a singleexperiment.In this work, we will present our mechanistic hypothesis for phosphatidylcholine analogs basedon MS/MS and MSn experiments. Different isomers and classes of GPLs were used to confirmthe unambiguous location of stereoisomers and the role of the polar head. In addition, thismethod was applied to generate Cu(II) quaternary PC complexes by MALDI-MS.We tried different MALDI matrixes to characterize the quaternary Cu(II)-based complex. Forthis purpose, 1,5 DAN, 9-AA, DCTB, DHB, and THAP were tested to ionize this complex.DCTB was the only matrix that allowed to keep the +II oxidation state of Cu, suggesting highcomplex stability.3 MALDI-TOF/TOF experiments were performed on these complexes and al-lowed to locate the double bonds

Gas-phase stabilization of Cu(II) reveals phosphatidylcholine isomeric features by ESI and MALDI-MS

Glycerophospholipids (GPLs) are amphiphilic metabolites that make up most of the lipid bilayer in mammalian cells. Literature has pointed out the significant implications of double bond (DB) positions and linkage positions on the glycerol moiety in the chemical, biochemical, and biophysical roles of lipids.1 Currently, derivatization or instrumental modifications are requested to highlight these isomeric features in GPLs.2 Copper-adducted fatty acid dissociation spectra proved to be efficient in locating DB in aliphatic chains. However, the driving force of the diagnostic fragmentation mechanism was attributed to the presence of the free carboxylic acid function that is lacking in GPLs. We have recently demonstrated that radical-driven fragmentations could be obtained from quaternary copper complexes of GPLs. The CID MS/MS fragmentation of such species yields both DB and stereo-numbering (sn) locations in a single experiment.In this work, we will present our mechanistic hypothesis for phosphatidylcholine (PC) analogs, based on MS/MS and MSn experiments. Different isomers and classes of GPLs were used to confirm the unambiguous location of stereoisomers and the role of the polar head. In addition, this method was applied to generate Cu(II) quaternary PC complexes by MALDI-MS.We tried different MALDI matrices such as 1,5 DAN, 9-AA, DCTB, DHB, and THAP ionize and characterize the quaternary Cu(II)-based complex, and it turned out that DCTB was the only matrix that allowed the +II oxidation state of Cu to be preserved, suggesting high complex stability.3 MALDI-TOF/TOF experiments were performed on these complexes and allowed locating the double bonds.[1] X. Ma and Y. Xia, Angew. Chem. Int. Ed. Engl., 2014, 53, 2592–2596. [2] A. Bednařík, et al. Anal. Chem. 2022, 94, 4889–4900.[3] J. Zhang, et al. J. Am. Soc. Mass Spectrom. 2003, 14, 42–50

MadID, a Versatile Approach to Map Protein-DNA Interactions, Highlights Telomere-Nuclear Envelope Contact Sites in Human Cells

Summary: Mapping the binding sites of DNA- or chromatin-interacting proteins is essential to understanding biological processes. DNA adenine methyltransferase identification (DamID) has emerged as a comprehensive method to map genome-wide occupancy of proteins of interest. A caveat of DamID is the specificity of Dam methyltransferase for GATC motifs that are not homogenously distributed in the genome. Here, we developed an optimized method named MadID, using proximity labeling of DNA by the methyltransferase M.EcoGII. M.EcoGII mediates N6-adenosine methylation in any DNA sequence context, resulting in deeper and unbiased coverage of the genome. We demonstrate, using m6A-specific immunoprecipitation and deep sequencing, that MadID is a robust method to identify protein-DNA interactions at the whole-genome level. Using MadID, we revealed contact sites between human telomeres, repetitive sequences devoid of GATC sites, and the nuclear envelope. Overall, MadID opens the way to identification of binding sites in genomic regions that were largely inaccessible. : Mapping the binding sites of DNA- or chromatin-interacting proteins is essential to understanding biological processes. Sobecki et al. developed an optimized method named MadID based on proximity labeling of DNA by the bacterial methyltransferase M.EcoGII. MadID results in deep and unbiased coverage for genome-wide mapping studies. Keywords: MadID, M.EcoGII, m6A, LADs, telomeres, nuclear envelope, proximity labeling, methylatio

Enzyme Activation with a Synthetic Catalytic Co-enzyme Intermediate: Nucleotide Methylation by Flavoenzymes

International audienceTo facilitate production of functional enzymes and to study their mechanisms, especially in the complex cases of coenzyme-dependent systems, activation of an inactive apoenzyme preparation with a catalytically competent coenzyme intermediate is an attractive strategy. This is illustrated with the simple chemical synthesis of a flavin-methylene iminium compound previously proposed as a key intermediate in the catalytic cycle of several important flavoenzymes involved in nucleic acid metabolism. Reconstitution of both flavin-dependent RNA methyltransferase and thymidylate synthase apoproteins with this synthetic compound led to active enzymes for the C5-uracil methylation within their respective transfer RNA and dUMP substrate. This strategy is expected to be of general application in enzymology