Epimutations in both the TESK2 and MMACHC promoters in the Epi-cblC inherited disorder of intracellular metabolism of vitamin B12

Background: epi-cblC is a recently discovered inherited disorder of intracellular vitamin B12 metabolism associating hematological, neurological, and cardiometabolic outcomes. It is produced by an epimutation at the promoter common to CCDC163P and MMACHC, which results from an aberrant antisense transcription due to splicing mutations in the antisense PRDX1 gene neighboring MMACHC. We studied whether the aberrant transcription produced a second epimutation by encompassing the CpG island of the TESK2 gene neighboring CCDC163P. Methods: We unraveled the methylome architecture of the CCDC163P-MMACHC CpG island (CpG:33) and the TESK2 CpG island (CpG:51) of 17 epi-cblC cases. We performed an integrative analysis of the DNA methylome profiling, transcriptome reconstruction of RNA-sequencing (RNA-seq), chromatin immunoprecipitation sequencing (ChIP-Seq) of histone H3, and transcription expression of MMACHC and TESK2. Results: The PRDX1 splice mutations and activation of numerous cryptic splice sites produced antisense readthrough transcripts encompassing the bidirectional MMACHC/CCDC163P promoter and the TESK2 promoter, resulting in the silencing of both the MMACHC and TESK2 genes through the deposition of SETD2-dependent H3K36me3 marks and the generation of epimutations in the CpG islands of the two promoters. Conclusions: The antisense readthrough transcription of the mutated PRDX1 produces an epigenetic silencing of MMACHC and TESK2. We propose using the term 'epi-digenism' to define this epigenetic disorder that affects two genes. Epi-cblC is an entity that differs from cblC. Indeed, the PRDX1 and TESK2 altered expressions are observed in epi-cblC but not in cblC, suggesting further evaluating the potential consequences on cancer risk and spermatogenesis. Keywords: Epi-cblC; MMACHC; Methylmalonic aciduria and homocystinuria, cblC type; Promoter hypermethylation; Secondary epimutation; TESK2

Genome-wide and MMACHC gene promoter epivariations induced by cellular stress factors and aberrant antisense transcription in normal, pluripotent, and cancer cells

La méthionine-dépendance désigne l'incapacité des cellules à proliférer en l'absence de méthionine, et ce, malgré la présence de son précurseur métabolique, l'homocystéine. Deux modèles cellulaires connus pour leur méthionine-dépendance sont les cellules dérivées de patients atteints d'épi-cblC et la lignée cellulaire de mélanome, MeWo-LC1. La méthionine-dépendance de ces deux modèles s'explique par la méthylation du promoteur du gène MMACHC, un gène majeur du métabolisme des monocarbones, induisant l'inhibition de son expression. Cette méthylation est associée à la transcription aberrante de PRDX1, un gène adjacent impliqué dans la réponse au stress cellulaire. De plus, des études récentes ont montré un nouveau type de transcrit induit par le stress pouvant générer une transcription anti-sens aberrante dont l’effet sur la méthylation de l’ADN reste encore à déchiffrer. L'objectif de cette étude est d'évaluer l'influence des facteurs de stress cellulaire sur l'épigénome humain et sur la méthylation du promoteur MMACHC induite ou non par des transcrits anti-sens aberrants. Dans un premier temps, nous avons exposé trois modèles cancéreux de carcinome hépatocellulaire, de mélanome (deux lignées cellulaires présentant des phénotypes opposés de méthionine-dépendance) et de glioblastome à des rayonnements ionisants (RI). Nous avons trouvé des épivariations sur de nombreuses sondes CpG avec des voies biologiques partagées entre les différentes lignées cellulaires testées, particulièrement celles associées au cycle et à la division cellulaires, aux réponses aux RI, à l'angiogenèse soutenue, à l'invasion tissulaire et aux métastases. Dans un deuxième temps, nous avons exposé des cellules normales, pluripotentes ou cancéreuses à trois types de facteurs de stress cellulaires connus pour leur induction de l'expression de PRDX1 ou des transcrits aberrants : arséniate, hypoxie/hypoxie-réoxygénation et stress hyperosmotique (sucrose). Nous avons trouvé une augmentation modeste de la méthylation au niveau du promoteur MMACHC et une diminution de son expression liée à la transcription anti-sens aberrante de PRDX1 après une exposition à l'arsenate et au sucrose. En outre, les analyses d'enrichissement des épivariations à l'échelle du génome ont montré des mécanismes d'adaptation spécifiques au stress et des mécanismes partagés hautement corrélés entre le traitement hypoxie-réoxygénation/arséniate et entre le traitement répétitif au sucrose à court/long terme. En conclusion, notre étude a mis en évidence des épivariations au niveau du promoteur du gène MMACHC avec son expression réduite liée à la transcription anti-sens de PRDX1. De plus, nos résultats ont fourni un aperçu des mécanismes adaptatifs partagés de l'épigénome par le biais d'épivariations à la résolution des dinucléotides CpG. Les analyses de RNA-seq complémenteront celles du méthylome et nous permettront d'adopter une approche multi-omique pour disséquer le lien entre la transcription anti-sens aberrante et les épivariations générées à l'échelle du génome.Methionine-dependence refers to the inability of cells to proliferate in the absence of methionine despite the presence of its metabolic precursor, homocysteine. Two cellular models known for their methionine-dependence are cells derived from patients with epi-cblC and the melanoma cell line, MeWo-LC1. The methionine dependence of these two models is explained by the methylation of MMACHC promoter, a major one carbon metabolism gene, leading to inhibition of its expression. This methylation is associated with aberrant transcription of PRDX1, an adjacent gene involved in the cellular stress response. Moreover, recent studies have shown a new type of stress-induced transcript that can generate an aberrant antisense transcript whose effect on DNA methylation remains to be deciphered. The objective of this study was to evaluate the influence of cellular stressors on the human epigenome and on MMACHC promoter methylation induced or not by aberrant antisense transcripts. First, we exposed three cancer models of hepatocellular carcinoma, melanoma (two cell lines with opposite methionine-dependent phenotypes) and glioblastoma to ionizing radiation (IR). We found epivariations on many CpG probes with shared biological pathways between the different cell lines tested, notably those associated with cell cycle and division, responses to IR, sustained angiogenesis, tissue invasion, and metastasis. Second, we exposed normal, pluripotent or cancer cells to three types of cellular stressors known to induce PRDX1 expression or aberrant transcripts: arsenate, hypoxia/hypoxia-reoxygenation and hyperosmotic stress (sucrose). We found a modest increase in methylation at the MMACHC promoter and a decrease in its expression related to the aberrant antisense transcript of PRDX1 after arsenate and sucrose exposure. In addition, genome-wide epivariations enrichment analyses showed stress-specific annotation enrichment and highly correlated shared adaptation mechanisms between hypoxia-reoxygenation/arsenate treatment and between short/long term repetitive sucrose treatment. In conclusion, our study revealed epivariations at the promoter level of the MMACHC gene with its reduced expression linked to the antisense transcription of PRDX1. Furthermore, our results provided insight into shared adaptive mechanisms of the epigenome at CpG dinucleotide resolution. RNA-seq analyses will complement methylome analyses and allow us to take a multi-omics approach to dissect the link between aberrant antisense transcription and genome-wide generated epivariations

Épivariations du génome entier et du promoteur du gène MMACHC induites par les facteurs de stress cellulaire et la transcription anti-sens aberrante dans les cellules normales, pluripotentes et cancéreuses

Methionine-dependence refers to the inability of cells to proliferate in the absence of methionine despite the presence of its metabolic precursor, homocysteine. Two cellular models known for their methionine-dependence are cells derived from patients with epi-cblC and the melanoma cell line, MeWo-LC1. The methionine dependence of these two models is explained by the methylation of MMACHC promoter, a major one carbon metabolism gene, leading to inhibition of its expression. This methylation is associated with aberrant transcription of PRDX1, an adjacent gene involved in the cellular stress response. Moreover, recent studies have shown a new type of stress-induced transcript that can generate an aberrant antisense transcript whose effect on DNA methylation remains to be deciphered. The objective of this study was to evaluate the influence of cellular stressors on the human epigenome and on MMACHC promoter methylation induced or not by aberrant antisense transcripts. First, we exposed three cancer models of hepatocellular carcinoma, melanoma (two cell lines with opposite methionine-dependent phenotypes) and glioblastoma to ionizing radiation (IR). We found epivariations on many CpG probes with shared biological pathways between the different cell lines tested, notably those associated with cell cycle and division, responses to IR, sustained angiogenesis, tissue invasion, and metastasis. Second, we exposed normal, pluripotent or cancer cells to three types of cellular stressors known to induce PRDX1 expression or aberrant transcripts: arsenate, hypoxia/hypoxia-reoxygenation and hyperosmotic stress (sucrose). We found a modest increase in methylation at the MMACHC promoter and a decrease in its expression related to the aberrant antisense transcript of PRDX1 after arsenate and sucrose exposure. In addition, genome-wide epivariations enrichment analyses showed stress-specific annotation enrichment and highly correlated shared adaptation mechanisms between hypoxia-reoxygenation/arsenate treatment and between short/long term repetitive sucrose treatment. In conclusion, our study revealed epivariations at the promoter level of the MMACHC gene with its reduced expression linked to the antisense transcription of PRDX1. Furthermore, our results provided insight into shared adaptive mechanisms of the epigenome at CpG dinucleotide resolution. RNA-seq analyses will complement methylome analyses and allow us to take a multi-omics approach to dissect the link between aberrant antisense transcription and genome-wide generated epivariations.La méthionine-dépendance désigne l'incapacité des cellules à proliférer en l'absence de méthionine, et ce, malgré la présence de son précurseur métabolique, l'homocystéine. Deux modèles cellulaires connus pour leur méthionine-dépendance sont les cellules dérivées de patients atteints d'épi-cblC et la lignée cellulaire de mélanome, MeWo-LC1. La méthionine-dépendance de ces deux modèles s'explique par la méthylation du promoteur du gène MMACHC, un gène majeur du métabolisme des monocarbones, induisant l'inhibition de son expression. Cette méthylation est associée à la transcription aberrante de PRDX1, un gène adjacent impliqué dans la réponse au stress cellulaire. De plus, des études récentes ont montré un nouveau type de transcrit induit par le stress pouvant générer une transcription anti-sens aberrante dont l’effet sur la méthylation de l’ADN reste encore à déchiffrer. L'objectif de cette étude est d'évaluer l'influence des facteurs de stress cellulaire sur l'épigénome humain et sur la méthylation du promoteur MMACHC induite ou non par des transcrits anti-sens aberrants. Dans un premier temps, nous avons exposé trois modèles cancéreux de carcinome hépatocellulaire, de mélanome (deux lignées cellulaires présentant des phénotypes opposés de méthionine-dépendance) et de glioblastome à des rayonnements ionisants (RI). Nous avons trouvé des épivariations sur de nombreuses sondes CpG avec des voies biologiques partagées entre les différentes lignées cellulaires testées, particulièrement celles associées au cycle et à la division cellulaires, aux réponses aux RI, à l'angiogenèse soutenue, à l'invasion tissulaire et aux métastases. Dans un deuxième temps, nous avons exposé des cellules normales, pluripotentes ou cancéreuses à trois types de facteurs de stress cellulaires connus pour leur induction de l'expression de PRDX1 ou des transcrits aberrants : arséniate, hypoxie/hypoxie-réoxygénation et stress hyperosmotique (sucrose). Nous avons trouvé une augmentation modeste de la méthylation au niveau du promoteur MMACHC et une diminution de son expression liée à la transcription anti-sens aberrante de PRDX1 après une exposition à l'arsenate et au sucrose. En outre, les analyses d'enrichissement des épivariations à l'échelle du génome ont montré des mécanismes d'adaptation spécifiques au stress et des mécanismes partagés hautement corrélés entre le traitement hypoxie-réoxygénation/arséniate et entre le traitement répétitif au sucrose à court/long terme. En conclusion, notre étude a mis en évidence des épivariations au niveau du promoteur du gène MMACHC avec son expression réduite liée à la transcription anti-sens de PRDX1. De plus, nos résultats ont fourni un aperçu des mécanismes adaptatifs partagés de l'épigénome par le biais d'épivariations à la résolution des dinucléotides CpG. Les analyses de RNA-seq complémenteront celles du méthylome et nous permettront d'adopter une approche multi-omique pour disséquer le lien entre la transcription anti-sens aberrante et les épivariations générées à l'échelle du génome

Stemness of Normal and Cancer Cells: The Influence of Methionine Needs and SIRT1/PGC-1α/PPAR-α Players

Stem cells are a population of undifferentiated cells with self-renewal and differentiation capacities. Normal and cancer stem cells share similar characteristics in relation to their stemness properties. One-carbon metabolism (OCM), a network of interconnected reactions, plays an important role in this dependence through its role in the endogenous synthesis of methionine and S-adenosylmethionine (SAM), the universal donor of methyl groups in eukaryotic cells. OCM genes are differentially expressed in stem cells, compared to their differentiated counterparts. Furthermore, cultivating stem cells in methionine-restricted conditions hinders their stemness capacities through decreased SAM levels with a subsequent decrease in histone methylation, notably H3K4me3, with a decrease in stem cell markers. Stem cells’ reliance on methionine is linked to several mechanisms, including high methionine flux or low endogenous methionine biosynthesis. In this review, we provide an overview of the recent discoveries concerning this metabolic dependence and we discuss the mechanisms behind them. We highlight the influence of SIRT1 on SAM synthesis and suggest a role of PGC-1α/PPAR-α in impaired stemness produced by methionine deprivation. In addition, we discuss the potential interest of methionine restriction in regenerative medicine and cancer treatment

Ionizing radiations induce shared epigenomic signatures unraveling adaptive mechanisms of cancerous cell lines with or without methionine dependency

International audienceBackground: Although radiation therapy represents a core cancer treatment modality, its efficacy is hampered by radioresistance. The effect of ionizing radiations (IRs) is well known regarding their ability to induce genetic alterations; however, their impact on the epigenome landscape in cancer, notably at the CpG dinucleotide resolution, remains to be further deciphered. In addition, no evidence is available regarding the effect of IRs on the DNA methylome profile according to the methionine dependency phenotype, which represents a hallmark of metabolic adaptation in cancer. Methods: We used a case-control study design with a fractionated irradiation regimen on four cancerous cell lines representative of HCC (HepG2), melanoma (MeWo and MeWo-LC1, which exhibit opposed methionine dependency phenotypes), and glioblastoma (U251). We performed high-resolution genome-wide DNA methylome profiling using the MethylationEPIC BeadChip on baseline conditions, irradiated cell lines (cumulative dose of 10 Gy), and nonirradiated counterparts. We performed epigenome-wide association studies to assess the effect of IRs and methionine-dependency-oriented analysis by carrying out epigenome-wide conditional logistic regression. We looked for epigenome signatures at the locus and single-probe (CpG dinucleotide) levels and through enrichment analyses of gene ontologies (GO). The EpiMet project was registered under the ID#AAP-BMS_003_211. Results: EWASs revealed shared GO annotation pathways associated with increased methylation signatures for several biological processes in response to IRs, including blood circulation, plasma membrane-bounded cell projection organization, cell projection organization, multicellular organismal process, developmental process, and animal organ morphogenesis. Epigenome-wide conditional logistic regression analysis on the methionine dependency phenotype highlighted several epigenome signatures related to cell cycle and division and responses to IR and ultraviolet light. Conclusions: IRs generated a variation in the methylation level of a high number of CpG probes with shared biological pathways, including those associated with cell cycle and division, responses to IRs, sustained angiogenesis, tissue invasion, and metastasis. These results provide insight on shared adaptive mechanisms of the epigenome in cancerous cell lines in response to IR. Future experiments should focus on the tryptic association between IRs, the initiation of a radioresistance phenotype, and their interaction with methionine dependency as a hallmark of metabolic adapta‑tion in cancer

A transgenic mice model of retinopathy of cblG-type inherited disorder of one-carbon metabolism highlights epigenome-wide alterations related to cone photoreceptor cells development and retinal metabolism

Abstract Background MTR gene encodes the cytoplasmic enzyme methionine synthase, which plays a pivotal role in the methionine cycle of one-carbon metabolism. This cycle holds a significant importance in generating S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH), the respective universal methyl donor and end-product of epigenetic transmethylation reactions. cblG type of inherited disorders of vitamin B12 metabolism due to mutations in MTR gene exhibits a wide spectrum of symptoms, including a retinopathy unresponsive to conventional therapies. Methods To unveil the underlying epigenetic pathological mechanisms, we conducted a comprehensive study of epigenomic-wide alterations of DNA methylation by NGS of bisulfited retinal DNA in an original murine model with conditional Mtr deletion in retinal tissue. Our focus was on postnatal day 21, a critical developmental juncture for ocular structure refinement and functional maturation. Results We observed delayed eye opening and impaired visual acuity and alterations in the one-carbon metabolomic profile, with a notable dramatic decline in SAM/SAH ratio predicted to impair DNA methylation. This metabolic disruption led to epigenome-wide changes in genes involved in eye development, synaptic plasticity, and retinoid metabolism, including promoter hypermethylation of Rarα, a regulator of Lrat expression. Consistently, we observed a decline in cone photoreceptor cells and reduced expression of Lrat, Rpe65, and Rdh5, three pivotal genes of eye retinoid metabolism. Conclusion We introduced an original in vivo model for studying cblG retinopathy, which highlighted the pivotal role of altered DNA methylation in eye development, cone differentiation, and retinoid metabolism. This model can be used for preclinical studies of novel therapeutic targets

Additional file 1 of A transgenic mice model of retinopathy of cblG-type inherited disorder of one-carbon metabolism highlights epigenome-wide alterations related to cone photoreceptor cells development and retinal metabolism

Additional file 1: Table S1 Conversion rate of the methylated spike-in controls of RRBS Diagenode kit. Table S2 Primers used for RT-qPCR analysis. Fig. S1 RBPMS and Rhodopsin protein expression in the retina of Mtr-cKO vs Wild-type mice. Fig. S2 Enhanced Genome Browser View panel revealing the Rara gene with ENCODE’s annotations

Epimutations in both the TESK2 and MMACHC promoters in the Epi-cblC inherited disorder of intracellular metabolism of vitamin B12

BACKGROUND: epi-cblC is a recently discovered inherited disorder of intracellular vitamin B12 metabolism associating hematological, neurological, and cardiometabolic outcomes. It is produced by an epimutation at the promoter common to CCDC163P and MMACHC, which results from an aberrant antisense transcription due to splicing mutations in the antisense PRDX1 gene neighboring MMACHC. We studied whether the aberrant transcription produced a second epimutation by encompassing the CpG island of the TESK2 gene neighboring CCDC163P. METHODS: We unraveled the methylome architecture of the CCDC163P-MMACHC CpG island (CpG:33) and the TESK2 CpG island (CpG:51) of 17 epi-cblC cases. We performed an integrative analysis of the DNA methylome profiling, transcriptome reconstruction of RNA-sequencing (RNA-seq), chromatin immunoprecipitation sequencing (ChIP-Seq) of histone H3, and transcription expression of MMACHC and TESK2. RESULTS: The PRDX1 splice mutations and activation of numerous cryptic splice sites produced antisense readthrough transcripts encompassing the bidirectional MMACHC/CCDC163P promoter and the TESK2 promoter, resulting in the silencing of both the MMACHC and TESK2 genes through the deposition of SETD2-dependent H3K36me3 marks and the generation of epimutations in the CpG islands of the two promoters. CONCLUSIONS: The antisense readthrough transcription of the mutated PRDX1 produces an epigenetic silencing of MMACHC and TESK2. We propose using the term 'epi-digenism' to define this epigenetic disorder that affects two genes. Epi-cblC is an entity that differs from cblC. Indeed, the PRDX1 and TESK2 altered expressions are observed in epi-cblC but not in cblC, suggesting further evaluating the potential consequences on cancer risk and spermatogenesis.Isite LU