Francesco Spallotta1, Chiara Cencioni2, Sandra Atlante2, Davide Garella2, Mattia Cocco2, Mattia Mori2, Raffaella Mastrocola2, Carsten Kuenne2, Stefan Guenther2, Simona Nanni2, Valerio Azzimato2, Sven Zukunft2, Angela Kornberger2, Duran Sürün2, Frank Schnütgen2, Harald von Melchner2, Antonella Di Stilo2, Manuela Aragno2, Maarten Braspenning2, Wim van Criekinge2, Miles J De Blasio2, Rebecca H Ritchie2, Germana Zaccagnini2, Fabio Martelli2, Antonella Farsetti2, Ingrid Fleming2, Thomas Braun2, Andres Beiras-Fernandez2, Bruno Botta2, Massimo Collino2, Massimo Bertinaria2, Andreas M Zeiher2, Carlo Gaetano1. 1. From the Goethe University, Frankfurt am Main, Germany (F. Spallotta, C.C., S.A., S.Z., D.S., F. Schnütgen, H.v.M., A.F., I.F., A.M.Z., C.G.); University of Turin, Torino, Italy (D.G., M. Cocco, R.M., A.D.S., M.A., M. Collino, M. Bertinaria); Istituto Italiano di Tecnologia CLNS@Sapienza Rome, Italy (M.M.); Max Planck Institute for Heart and Lung Research, Bad Nauheim, Germany (C.K., S.G., T.B.); Università Cattolica del Sacro Cuore, Rome, Italy (S.N.); Karolinska Institutet, Huddinge, Sweden (V.A.); University of Mainz, Germany (A.K., A.B.-F.); NXT-Dx, Ghent, Belgium (M. Braspenning); Ghent University, Belgium (W.v.C.); Baker IDI Heart and Diabetes Institute, Melbourne VIC, Australia (M.J.D.B., R.H.R.); IRCCS Policlinico San Donato, Milan, Italy (G.Z., F.M.); National Research Council, Rome, Italy (A.F., C.C.); and Sapienza University, Rome, Italy (B.B.). gaetano@em.uni-frankfurt.de fspallotta@gmail.com. 2. From the Goethe University, Frankfurt am Main, Germany (F. Spallotta, C.C., S.A., S.Z., D.S., F. Schnütgen, H.v.M., A.F., I.F., A.M.Z., C.G.); University of Turin, Torino, Italy (D.G., M. Cocco, R.M., A.D.S., M.A., M. Collino, M. Bertinaria); Istituto Italiano di Tecnologia CLNS@Sapienza Rome, Italy (M.M.); Max Planck Institute for Heart and Lung Research, Bad Nauheim, Germany (C.K., S.G., T.B.); Università Cattolica del Sacro Cuore, Rome, Italy (S.N.); Karolinska Institutet, Huddinge, Sweden (V.A.); University of Mainz, Germany (A.K., A.B.-F.); NXT-Dx, Ghent, Belgium (M. Braspenning); Ghent University, Belgium (W.v.C.); Baker IDI Heart and Diabetes Institute, Melbourne VIC, Australia (M.J.D.B., R.H.R.); IRCCS Policlinico San Donato, Milan, Italy (G.Z., F.M.); National Research Council, Rome, Italy (A.F., C.C.); and Sapienza University, Rome, Italy (B.B.).
Abstract
RATIONALE: Human cardiac mesenchymal cells (CMSCs) are a therapeutically relevant primary cell population. Diabetes mellitus compromises CMSC function as consequence of metabolic alterations and incorporation of stable epigenetic changes. OBJECTIVE: To investigate the role of α-ketoglutarate (αKG) in the epimetabolic control of DNA demethylation in CMSCs. METHODS AND RESULTS: Quantitative global analysis, methylated and hydroxymethylated DNA sequencing, and gene-specific GC methylation detection revealed an accumulation of 5-methylcytosine, 5-hydroxymethylcytosine, and 5-formylcytosine in the genomic DNA of human CMSCs isolated from diabetic donors. Whole heart genomic DNA analysis revealed iterative oxidative cytosine modification accumulation in mice exposed to high-fat diet (HFD), injected with streptozotocin, or both in combination (streptozotocin/HFD). In this context, untargeted and targeted metabolomics indicated an intracellular reduction of αKG synthesis in diabetic CMSCs and in the whole heart of HFD mice. This observation was paralleled by a compromised TDG (thymine DNA glycosylase) and TET1 (ten-eleven translocation protein 1) association and function with TET1 relocating out of the nucleus. Molecular dynamics and mutational analyses showed that αKG binds TDG on Arg275 providing an enzymatic allosteric activation. As a consequence, the enzyme significantly increased its capacity to remove G/T nucleotide mismatches or 5-formylcytosine. Accordingly, an exogenous source of αKG restored the DNA demethylation cycle by promoting TDG function, TET1 nuclear localization, and TET/TDG association. TDG inactivation by CRISPR/Cas9 knockout or TET/TDG siRNA knockdown induced 5-formylcytosine accumulation, thus partially mimicking the diabetic epigenetic landscape in cells of nondiabetic origin. The novel compound (S)-2-[(2,6-dichlorobenzoyl)amino]succinic acid (AA6), identified as an inhibitor of αKG dehydrogenase, increased the αKG level in diabetic CMSCs and in the heart of HFD and streptozotocin mice eliciting, in HFD, DNA demethylation, glucose uptake, and insulin response. CONCLUSIONS: Restoring the epimetabolic control of DNA demethylation cycle promises beneficial effects on cells compromised by environmental metabolic changes.
RATIONALE: Human cardiac mesenchymal cells (CMSCs) are a therapeutically relevant primary cell population. Diabetes mellitus compromises CMSC function as consequence of metabolic alterations and incorporation of stable epigenetic changes. OBJECTIVE: To investigate the role of α-ketoglutarate (αKG) in the epimetabolic control of DNA demethylation in CMSCs. METHODS AND RESULTS: Quantitative global analysis, methylated and hydroxymethylated DNA sequencing, and gene-specific GC methylation detection revealed an accumulation of 5-methylcytosine, 5-hydroxymethylcytosine, and 5-formylcytosine in the genomic DNA of human CMSCs isolated from diabetic donors. Whole heart genomic DNA analysis revealed iterative oxidative cytosine modification accumulation in mice exposed to high-fat diet (HFD), injected with streptozotocin, or both in combination (streptozotocin/HFD). In this context, untargeted and targeted metabolomics indicated an intracellular reduction of αKG synthesis in diabetic CMSCs and in the whole heart of HFD mice. This observation was paralleled by a compromised TDG (thymine DNA glycosylase) and TET1 (ten-eleven translocation protein 1) association and function with TET1 relocating out of the nucleus. Molecular dynamics and mutational analyses showed that αKG binds TDG on Arg275 providing an enzymatic allosteric activation. As a consequence, the enzyme significantly increased its capacity to remove G/T nucleotide mismatches or 5-formylcytosine. Accordingly, an exogenous source of αKG restored the DNA demethylation cycle by promoting TDG function, TET1 nuclear localization, and TET/TDG association. TDG inactivation by CRISPR/Cas9 knockout or TET/TDG siRNA knockdown induced 5-formylcytosine accumulation, thus partially mimicking the diabetic epigenetic landscape in cells of nondiabetic origin. The novel compound (S)-2-[(2,6-dichlorobenzoyl)amino]succinic acid (AA6), identified as an inhibitor of αKG dehydrogenase, increased the αKG level in diabetic CMSCs and in the heart of HFD and streptozotocinmice eliciting, in HFD, DNA demethylation, glucose uptake, and insulin response. CONCLUSIONS: Restoring the epimetabolic control of DNA demethylation cycle promises beneficial effects on cells compromised by environmental metabolic changes.
Authors: Jinhua Li; Yu Bo Yang Sun; Weiyi Chen; Jinjin Fan; Songhui Li; Xinli Qu; Qikang Chen; Riling Chen; Dajian Zhu; Jinfeng Zhang; Zhuguo Wu; Honggang Chi; Simon Crawford; Viola Oorschot; Victor G Puelles; Peter G Kerr; Yi Ren; Susan K Nilsson; Mark Christian; Huanwen Tang; Wei Chen; John F Bertram; David J Nikolic-Paterson; Xueqing Yu Journal: EMBO Rep Date: 2020-01-09 Impact factor: 8.807
Authors: Chiara Cencioni; Francesco Spallotta; Matteo Savoia; Carsten Kuenne; Stefan Guenther; Agnese Re; Susanne Wingert; Maike Rehage; Duran Sürün; Mauro Siragusa; Jacob G Smith; Frank Schnütgen; Harald von Melchner; Michael A Rieger; Fabio Martelli; Antonella Riccio; Ingrid Fleming; Thomas Braun; Andreas M Zeiher; Antonella Farsetti; Carlo Gaetano Journal: Nat Commun Date: 2018-03-29 Impact factor: 14.919