Availability Of S-adenosylmethionine Research Articles

MAT gene deregulation and liver cancer

Rodent models with low heterogeneity premalignant and malignant lesions provide a valuable contribution to hepatocellular carcinoma (HCC) pathogenesis in the field of research. Studies have been performed in transgenic mice, and rodent strains on signaling pathways deregulation in hepatocarcinogenesis with varying susceptibilities, and human HCC subtypes. Because ethionine, an antagonist of methionine, causes cancer and methyl-deficient diets cause steatohepatitis, followed by the development of HCC, researchers began to look into mechanisms regulating availability of S-adenosylmethionine (SAM) and its role in liver injury, including HCC development. Cirrhotic livers have decreased methionine adenosyltransferase (MATI/III) levels due to oxidation of cysteine residues in the ATP-binding site. MATII upregulation is inhibited by its reaction product, leading to MATI/III downregulation. Decreased MATI/III: MATII activity ratio, along with increased SAM decarboxylation for polyamine synthesis, results in decreased SAM. Some of the molecular pathways associated with specific cancer phenotypes are evolutionarily conserved, according to previous comparative functional genomics research. Cell cycle regulators (WNT/FZD, PI3K/AKT, and MAPK) and key genes (MAPK, IKK/NF-kB) are upregulated in human and rodent HCC progression. Pseudoprognostic markers for HCC include MAT1A/MAT2A switch. Changes in MAT1A expression and SAM levels occur during hepatocarcinogenesis. No evidence of SAM’s therapeutic effect on HCC has been found. The effects of stable MAT1A overexpression or MAT2A/MAT2B inhibition in vivo should be studied first. MAT2A or MAT2B-silenced HepG2 cells proliferate less in leptin. However, intracellular viral vector transduction in vivo has many limitations. This review interprets recent advances in SAM metabolism deregulation in liver injury predisposing to HCC and determining HCC prognosis.

Systematic promoter design for plasmid-encoded S-adenosylmethionine sensing systems.

S-adenosylmethionine (SAM) is an important biomolecule that mainly acts as a methyl donor and plays many roles in a variety of biological functions. SAM is also required for the biosynthesis of valuable methylated compounds, but its supply is a bottleneck for these biosynthetic pathways. To overcome this bottleneck and to reconfigure SAM homeostasis, a high-throughput sensing system for changes in intracellular SAM availability is required. We constructed a plasmid that can detect the factors that can alter SAM availability using minimal components. It does so by placing a fluorescent protein under a promoter controlled by endogenous MetJ, a transcription factor that represses its own regulons upon binding with SAM. Next, to validate SAM-responsive behavior, we systematically reconstructed 10 synthetic promoters with different positions and with different number of metbox sites, sequences of MetJ binding. We found that a position between the -35 box and the -10 box was the most effective for repression and that this setup was suitable for detecting the genetic or environmental factors that can deplete and recover the intracellular SAM availability. Overall, the response patterns of the synthetic MetJ-regulated promoters characterized in this study may be useful for the development of further SAM biosensing systems.

Hematopoietic Stem/Progenitor Cell Intrinsic SAM Synthesis Is Required to Prevent p53 Pathway Activation By Maintaining DNA Stability

S-adenosylmethionine (SAM) is a principle methyl group donor to maintain epigenetic status. SAM is synthesized from methionine and ATP via the enzymatic activity of Mat2a (Methionine adenosyltransferase 2a). It has been reported that SAM reduction by methionine restriction or Mat2a inhibition suppresses the proliferation of leukemia cells to attract attentions as a potential therapeutic target for leukemia. To develop the therapy targeting methionine metabolism in leukemia, it is essential to elucidate the significance of methionine metabolism in non-leukemic hematopoiesis. However, still little is known about the importance of SAM synthesis in hematopoiesis, including hematopoietic stem/progenitor cells (HSPCs), in vivo. By using Mx1-Cre mediated Mat2a gene deletion in mice ( Mat2a fl/fl;Mx1-Cre mice), we investigated the roles of Mat2a in hematopoiesis in vivo. Reduction of Mat2a activity in Mat2a knockout (KO) mice in bone marrow (BM) was confirmed by ultra-high performance liquid chromatography tandem mass spectrometry analysis. Mat2a KO mice displayed severe pancytopenia in peripheral blood (PB). Mature cell fractions (Myeloid cells, B cells and T cells) and HSPCs (HSCs, MPP2s, MPP3s and MPP4s) in BM were also significantly decreased in Mat2a KO mice, suggesting that Mat2a is essential for the maintenance of hematopoiesis from HSPCs in vivo. To exclude the possible contributions of the effects induced by Mat2a KO in non-hematopoietic cells, we performed BM transplantation analyses in which hematopoietic cell specific KO or non-hematopoietic cell specific KO were achieved. These analyses clearly confirmed that HSPC intrinsic SAM synthesis is inevitable for the maintenance of hematopoiesis. To reveal the mechanistic insights of the regulatory roles for Mat2a in HSPCs, we performed RNA sequencing analysis ( Mat2a WT vs KO in HSPCs). GSEA (Gene Set Enrichment Analysis) using the Hallmark gene sets showed that p53 pathway was most highly enriched in Mat2a KO. In depth, genes downstream of p53 ( Bax, Apaf1, Bbc3, Casp3, Casp8, Fas, Cdkn1a etc.) were up-regulated by Mat2a KO, causing cell cycle arrest and apoptosis in HSPCs. On the other hand, gene expressions of the upstream of p53 ( Atr, Atm, Chek1, Chek2 etc.) were unchanged, suggesting that p53 activity was not increased at mRNA expression levels. In line with this, mRNA expression levels of Trp53 was unchanged, but total protein amount of p53 was increased in Mat2a KO HSPCs. Since DNA stability is known as a critical regulator of p53 pathway, we analyzed gH2AX levels as DNA damage marker and found that gH2AX levels were increased in Mat2a KO HSPCs. To be noted, expressions of genes involved in Fanc pathway ( Fanca, Fancc, Fancd2, Fance, Fancg, Fancl, Fanci etc), which is responsible for the DNA maintenance, were broadly down-regulated in Mat2a KO HSPCs at transcriptomic levels. Therefore, the Fanc pathway repression by SAM synthesis inhibition could cause DNA damage and p53 pathway dependent loss of maintenance in Mat2a KO HSPCs. To further understand the importance of SAM synthesis under stress hematopoiesis. We analyzed Mat2a expression changes in HSPCs by irradiation (5.5 Gy/mouse). Intriguingly, Mat2a expression was up-regulated by irradiation. We also found that dietary methionine restriction (MR) in mice prevents its hematopoietic recovery from irradiation stress. To be noted, long term MR (24 weeks) did not show any particular compared to control diet. Therefore, methionine metabolism possesses significant roles at least in the situation where acute hematopoietic recovery is required. Collectively, in this study, we found that cell intrinsic SAM synthesis is required for the maintenance (proliferation and survival) of HSPCs in vivo. SAM might be required for the prevention of DNA damage and p53 pathway activation by supporting Fanc pathway gene expressions. Methionine and/or SAM availability might possess critical roles for the regeneration of non-leukemic hematopoiesis. Further investigation about the significance of SAM synthesis in non-leukemic hematopoiesis is required for the proper establishment of SAM synthesis targeting therapy in cancers.

Abstract P3176: One Carbon Metabolism Defect In Failing Heart

Aberrant cardiac metabolism has long been hypothesized to contribute to heart failure (HF). However, understanding of cardiac metabolism in HF remains incomplete. To fill this gap, we performed metabolomic and RNA sequencing analysis of 48 non-failing (NF) and 39 dilated cardiomyopathy (DCM) hearts. Among many differences, metabolites from one-carbon metabolism were significantly reduced, especially methionine (Met) and s-adenosylmethionine (SAM), a universal methyl donor of methylation. We probed the mechanism behind decreased Met/SAM in DCM hearts. Studies in neonatal rat ventricular myocytes (NRVMs), and in vivo steady-state infusions with isotope tracing showed that Met was mostly imported, rather than recycled, suggesting a defective Met transport in DCM hearts. We next explored the impact of low SAM. DNA and histone methylation remained unchanged, but creatine level was significantly decreased. So, we tested potential creatine synthesis in the heart that was previously unclear. To our surprise, NRVMs synthesized ~50% of their creatine pool when external supply is low, and synthesis was dependent on SAM availability. In summary, we find that the majority of Met in cardiomyocyte is imported, with rapid turnover. We also find that cardiomyocytes synthesize creatine and increase creatine production under stress. Together, these findings suggest that defective Met import may contribute to low creatine and SAM levels and ultimately HF.

The human malaria parasite Plasmodium falciparum can sense environmental changes and respond by antigenic switching

The primary antigenic and virulence determinant of the human malaria parasite Plasmodium falciparum is a variant surface protein called PfEMP1. Different forms of PfEMP1 are encoded by a multicopy gene family called var, and switching between active genes enables the parasites to evade the antibody response of their human hosts. var gene switching is key for the maintenance of chronic infections; however, what controls switching is unknown, although it has been suggested to occur at a constant frequency with little or no environmental influence. var gene transcription is controlled epigenetically through the activity of histone methyltransferases (HMTs). Studies in model systems have shown that metabolism and epigenetic control of gene expression are linked through the availability of intracellular S-adenosylmethionine (SAM), the principal methyl donor in biological methylation modifications, which can fluctuate based on nutrient availability. To determine whether environmental conditions and changes in metabolism can influence var gene expression, P. falciparum was cultured in media with altered concentrations of nutrients involved in SAM metabolism. We found that conditions that influence lipid metabolism induce var gene switching, indicating that parasites can respond to changes in their environment by altering var gene expression patterns. Genetic modifications that directly modified expression of the enzymes that control SAM levels similarly led to profound changes in var gene expression, confirming that changes in SAM availability modulate var gene switching. These observations directly challenge the paradigm that antigenic variation in P. falciparum follows an intrinsic, programed switching rate, which operates independently of any external stimuli.

Production of S-methyl-methionine using engineered Saccharomyces cerevisiae sake K6.

Introducing a plant-derived MMT gene encoding methionine S-methyltransferase into engineered Saccharomyces cerevisiae sake K6 allowed microbial production of S-methyl-methionine (SMM).

One-carbon metabolism and related pathways in ruminal and small intestinal epithelium of lactating dairy cows.

Physiological and environmental stresses such as the transition into lactation and heat load contribute to gastrointestinal tract (GIT) dysfunction. The nonruminant gastrointestinal tract has mechanisms to cope with pro-oxidant and pro-inflammatory stressors arising from the gut lumen or within intestinal cells. One-carbon metabolism (OCM) contributes to anti-oxidant capacity via the production of glutathione (GSH) and taurine, and the synthesis of phospholipid, creatine, and the osmolyte glycinebetaine among others. A multipronged approach was used to assess the biological relevance of OCM and closely-related pathways on GIT function in dairy cows. Ruminal papillae (Rum) and scrapings from duodenum (Duo), jejunum (Jej), and ileum (Ile) were collected at slaughter from eight multiparous Holstein cows averaging 128 ± 12 d in milk and producing 39 ± 5 kg/d. A MIXED model ANOVA with preplanned orthogonal contrasts was used for statistical analysis. Methionine adenosyl transferase 1 activity (MAT) was ~10-fold greater (P < 0.01) and cystathionine β-synthase activity doubled in Rum vs. small intestine. Total glutathione peroxidase (GPX) activity was greatest (P = 0.03) in Ile, but similar to Rum. Activity and mRNA abundance of betaine-homocysteine S-methyltransferase were undetectable. There was a 2.5-fold greater protein abundance of GPX1 (P < 0.01) and a ~2-fold greater abundance of GPX3 (P < 0.01) in Rum vs. small intestine. Among the various amino acids (AA) with roles in OCM or closely-related pathways (e.g. creatine synthesis), concentrations of arginine, aspartate, glutamine, methionine, and serine were lower (P < 0.01) in Rum vs. small intestine. Unlike AA, concentrations of OCM-related intermediates S-5'-adenosyl-homocysteine (SAH), glycinebetaine, carnitine, creatine (CRE), and cysteinesulfinic acid were greater (P < 0.01) while taurine was lower in Rum vs. small intestine. Intermediates of the folate cycle were undetectable. The fact that S-adenosylmethionine (SAM) was undetectable while MAT activity and SAH were greater in Rum suggested that availability of SAM (a methyl donor) is a key determinant of flux through the folate and methionine cycles in the GIT. Except for adenosine, concentrations of glutamate, glycine, α-ketoglutarate, hypotaurine, and GSH were lowest in Ile. Together, the data underscored unique differences in activity of one-carbon metabolism and related pathways across sections of the GIT.

The Role of the m6A RNA Methyltransferase METTL16 in Gene Expression and SAM Homeostasis.

The RNA methylation of adenosine at the N6-position (m6A) has attracted significant attention because of its abundance and dynamic nature. It accounts for more than 80% of all RNA modifications present in bacteria and eukaryotes and regulates crucial aspects of RNA biology and gene expression in numerous biological processes. The majority of m6A found in mammals is deposited by a multicomponent complex formed between methyltransferase-like (METTL) proteins METTL3 and METTL14. In the last few years, the list of m6A writers has grown, resulting in an expansion of our understanding of the importance of m6A and the methylation machinery. The characterization of the less familiar family member METTL16 has uncovered a new function of the m6A methylation apparatus, namely the fine-tuning of the cellular levels of the major methyl donor S-adenosylmethionine (SAM). METTL16 achieves this by adjusting the levels of the enzyme that synthesizes SAM in direct response to fluctuations in the SAM availability. This review summarizes recent progress made in understanding how METTL16 can sense and relay metabolic information and considers the wider implications. A brief survey highlights similarities and differences between METTL16 and the better-known METTL3/14 complex, followed by a discussion of the target specificity, modes of action and potential roles of METTL16.

MLL (KMT2A)-rearranged Acute Lymphoblastic Leukemias Are Addicted to S-Adenosyl Methionine (SAM): Implications for Therapy

MLL (KMT2A)-rearranged acute lymphoblastic leukemia (MLLr ALL) is an aggressive subset associated with poor survival and relapse representing 5% of childhood ALL cases and 75% of infant leukemias. Though current intensive chemotherapy regimens have improved survival in childhood ALL overall, they fail to cure many of these high-risk patient subgroups and inflict significant long-term side effects, demonstrating the urgent need for novel therapies. While exploring the effect of different amino acid depletion strategies, we observed rapid induction of apoptosis upon methionine restriction (MR) in MLLr cells. The histone methyltransferase function of the MLL fusion protein complex requires increased amounts of the methionine metabolite s-adenosylmethionine (SAM) to maintain its hypermethylated state, creating a selective sensitivity of MLLr ALL for perturbations in methionine availability. To explore how MR impinges on MLLr cells' metabolic state we performed global metabolomics on SEM cells and observed rapid effects on metabolism, with 416 metabolites out of the 650 measured differing significantly upon 12 hours of MR. We performed in parallel mRNA sequencing on SEM cells to understand the effect of MR on gene expression and identified 1351 differentially expressed genes. Integrated transcriptomics and metabolomics analysis revealed several key pathways interlinked with SAM, including the folate cycle, polyamine synthesis, arginine/redox metabolism, nucleotide synthesis, and the creatine/urea cycle, with several SAM-dependent enzymes and key byproducts in these pathways being significantly reduced upon MR. Moreover, addition of SAM completely rescued MLLr cell lines from methionine depletion induced apoptosis, an effect not observed in non-MLLr cells, confirming the critical role of SAM in this leukemia subset. To better understand the effect of methionine and SAM restriction on epigenetic modifications, we looked at histone methylation in a panel of MLLr and non-MLLr B-ALL cell lines and observed a global suppression of methylation upon MR specifically in the pediatric MLLr cells. Additionally, gene expression analysis revealed downregulation of key MLL fusion target genes associated with proliferation and maintenance of the oncogenic phenotype in response to MR, including ZMYND8, ERG, BCL2, PROM1, HMX3, HOXA10, HMGA2, and PBX3. Several of these gene targets are known to be transcriptionally upregulated in MLLr leukemia via aberrant H3K79 methylation. Consistent with this notion, knockdown of the H3K79 demethylase, KDM2B, displayed a more MR-resistant phenotype than its wildtype counterparts. Taken together, MR has a direct effect on global histone methylation in MLLr ALL cells, which in turn disturbs the MLL gene signature critical for their survival. Lastly, immunocompromised mice were engrafted with MLLr SEM cells and treated with a 95% MR diet while leukemia progression was monitored by flow cytometry. The MR diet was well tolerated with minimal weight loss, methionine levels in plasma were roughly 50% reduced compared to controls, and that alone was enough to significantly delay leukemic growth (p = 0.015). While several compounds targeting MAT2A, the enzyme that produces SAM from methionine, are currently in Phase 1 clinical trials, the availability of an FDA approved methionine-free formula for infants allows for rapid translation to the clinic. As a follow-up, we also performed a drug screen using 214 compounds to identify drugs that interact with MR, showing that vinca-alkaloids, including vincristine, strongly synergize. Validation experiments demonstrate that MR triggers a stress induced G2 arrest, priming cells for mitotic spindle destruction with vincristine. In vivo experiments are currently underway using patient-derived xenografts exploring synergistic potential of vincristine with targeting methionine metabolism. In summary, we found that MLLr leukemic cells are selectively vulnerable to perturbations of the methionine cycle due to their increased need for SAM to maintain hypermethylation and aberrant gene activation. Limiting methionine and/or SAM availability provides an attractive adjuvant therapy potentiating the effects of current or novel (targeted) therapies for MLLr leukemia.

Dietary Methionine Restriction Reverses Liver Cancer Driving Metabotype in GNMT KO Mice

ObjectivesGlycine N-methyltransferase (GNMT) is a major liver enzyme that catalyzes the S-adenosyl-methionine (SAM) dependent synthesis of sarcosine from glycine and regulates the availability of SAM for cellular methylation reactions, as well as for detoxification pathways in liver cells. The whole-body enzyme knockout in mice results in dysregulated methionine metabolism, including massive elevations of SAM. As a result, Gnmt–/– mice develop fatty livers and subsequently, hepatocellular carcinomas (HCC). We hypothesize that since the HCC development in Gnmt–/– mice is associated with the deregulation of metabolic processes, alteration of nutrients supply may delay/prevent cancer development in animals with genetic deficiency of GNMT. MethodsWild type and Gnmt–/– littermates of both sexes were randomized into dietary treatment groups. Experimental diets contained defined mixtures of purified amino acids instead of protein, and identical amounts and sources of carbohydrates, fat and vitamins. A standard amino acid defined diet (AAD, Envigo) was used as a control. Methionine adjusted diet (MA, same levels of amino acids as standard diet, except methionine was 8-fold lower) was tested for the effect on liver metabolome. Mice were kept on respective diets for 8 months after weaning, and blood and tissue samples were collected at endpoint. Aliquots of frozen liver samples (∼60–70 mg) were subjected to untargeted metabolomic analysis through Metabolon®. ResultsMetabolomic data underscore dramatic elevation of polyamines, TCA metabolites, bile acids and glycogen degradation products in Gnmt–/- livers on standard diet compared to WT controls. On the MA diet however, metabotypes of Gnmt–/- mice were similar to those of their wildtype littermates, with reversals in elevations of SAM and almost all the aforementioned metabolites. There were no appreciable differences between diets in WT mice metabotypes. ConclusionsOur data demonstrate that diet alteration can reverse the deregulation of metabolic processes associated with HCC development in Gnmt–/- mice. These results also suggest that diet modification should be investigated for the potential of improving liver cancer treatments. Funding SourcesUNC NRI 2018 Pilot Project.

SAMS-1 coordinates HLH-30/TFEB and PHA-4/FOXA activities through histone methylation to mediate dietary restriction-induced autophagy and longevity

ABSTRACT Dietary restriction (DR) is known to promote autophagy to exert its longevity effect. While SAMS-1 (S-adenosyl methionine synthetase-1) has been shown to be a key mediator of the DR response, little is known about the roles of S-adenosyl methionine (SAM) and SAM-dependent methyltransferase in autophagy and DR-induced longevity. In this study, we show that DR and SAMS-1 repress the activity of SET-2, a histone H3K4 methyltransferase, by limiting the availability of SAM. Consequently, the reduced H3K4me3 levels promote the expression and activity of two transcription factors, HLH-30/TFEB and PHA-4/FOXA, which both regulate the transcription of autophagy-related genes. We then find that HLH-30/TFEB and PHA-4/FOXA act collaboratively on their common target genes to mediate the transcriptional response of autophagy-related genes and consequently the lifespan of the animals. Our study thus shows that the SAMS-1-SET-2 axis serves as a nutrient-sensing module to epigenetically coordinate the activation of HLH-30/TFEB and PHA-4/FOXA transcription factors to control macroautophagy/autophagy and longevity in response to DR. Abbreviations: ChIP: chromatin immunoprecipitation; ChIP-seq: chromatin immuno precipitation-sequencing; COMPASS: complex of proteins associated with Set1; DR: dietary restriction; GO: gene ontology; SAM: S-adenosyl methionine; SAMS-1: S-adenosyl methionine synthetase-1; TSS: transcription start site; WT: wild-type.

Disrupted liver oxidative metabolism in glycine N-methyltransferase-deficient mice is mitigated by dietary methionine restriction

ObjectiveOne-carbon metabolism is routinely dysregulated in nonalcoholic fatty liver disease. This includes decreased glycine N-methyltransferase (GNMT), a critical regulator of s-adenosylmethionine (SAM). Deletion of GNMT in mice increases SAM and promotes liver steatosis. Lower liver oxidative metabolism, as indicated by a decline in gluconeogenesis, citric acid cycle flux, and oxidative phosphorylation contributes to liver steatosis in GNMT-null mice; however, the extent to which higher SAM mediates this phenotype remains unclear. Here, we determined the SAM-dependent impairment in liver oxidative metabolism by loss of GNMT. MethodsGNMT knockout (KO) mice were fed a methionine-restricted diet to prevent increased SAM. 2H/13C metabolic flux analysis was performed in conscious, unrestrained mice to quantify liver nutrient fluxes. Metabolomics and high-resolution respirometry were used to quantify liver nutrient pool sizes and mitochondrial oxidative phosphorylation, respectively. Folic acid-supplemented and serine/glycine-deficient diets were used independently to further define the metabolic implications of perturbed one-carbon donor availability. ResultsDietary methionine restriction prevented a 75-fold increase in SAM and a 53% rise in triacylglycerides in livers of KO mice. Dietary methionine restriction increased gluconeogenesis, independent of genotype, and restored cytochrome c oxidase respiratory function in KO mice. Citric acid cycle fluxes remained lower in KO mice irrespective of diet. Restricting dietary methionine abrogated markers of increased lipogenesis and folate cycle dysfunction in KO mice. ConclusionsThe impaired liver oxidative metabolism following loss of GNMT is both dependent and independent of greater SAM availability. Lower in vivo citric acid cycle flux is independent of increased SAM. In contrast, gluconeogenesis and oxidative phosphorylation are negatively regulated by excess SAM. Lipid accumulation in livers of mice lacking GNMT is also linked to higher SAM.

Taurine Antagonizes Macrophages M1 Polarization by Mitophagy-Glycolysis Switch Blockage via Dragging SAM-PP2Ac Transmethylation.

The excessive M1 polarization of macrophages drives the occurrence and development of inflammatory diseases. The reprogramming of macrophages from M1 to M2 can be achieved by targeting metabolic events. Taurine promotes for the balance of energy metabolism and the repair of inflammatory injury, preventing chronic diseases and complications. However, little is known about the mechanisms underlying the action of taurine modulating the macrophage polarization phenotype. In this study, we constructed a low-dose LPS/IFN-γ-induced M1 polarization model to simulate a low-grade pro-inflammatory process. Our results indicate that the taurine transporter TauT/SlC6A6 is upregulated at the transcriptional level during M1 macrophage polarization. The nutrient uptake signal on the membrane supports the high abundance of taurine in macrophages after taurine supplementation, which weakens the status of methionine metabolism, resulting in insufficient S-adenosylmethionine (SAM). The low availability of SAM is directly sensed by LCMT-1 and PME-1, hindering PP2Ac methylation. PP2Ac methylation was found to be necessary for M1 polarization, including the positive regulation of VDAC1 and PINK1. Furthermore, its activation was found to promote the elimination of mitochondria by macrophages via the mitophagy pathway for metabolic adaptation. Mechanistically, taurine inhibits SAM-dependent PP2Ac methylation to block PINK1-mediated mitophagy flux, thereby maintaining a high mitochondrial density, which ultimately hinders the conversion of energy metabolism to glycolysis required for M1. Our findings reveal a novel mechanism of taurine-coupled M1 macrophage energy metabolism, providing novel insights into the occurrence and prevention of low-grade inflammation, and propose that the sensing of taurine and SAM availability may allow communication to inflammatory response in macrophages.

Histone H3 lysine 4 trimethylation in sperm is transmitted to the embryo and associated with diet-induced phenotypes in the offspring.

A father's lifestyle impacts offspring health; yet, the underlying molecular mechanisms remain elusive. We hypothesized that a diet that changes methyl donor availability will alter the sperm and embryo epigenomes to impact embryonic gene expression and development. Here, we demonstrate that a folate-deficient (FD) dietalters histone H3 lysine 4 trimethylation (H3K4me3) in sperm at developmental genes and putative enhancers. A subset of H3K4me3 alterations in sperm are retained in the pre-implantation embryo and associated with deregulated embryonic gene expression. Using a genetic mouse model in which sires have pre-existing altered H3K4me2/3 in sperm, we show that a FD diet exacerbates alterations in sperm H3K4me3 and embryonic gene expression, leading to an increase in developmental defect severity. These findings imply that paternal H3K4me3 is transmitted to the embryo and influences gene expression and development. It further suggests that epigenetic errors can accumulate in sperm to worsen offspring developmental outcomes.

Cellular stress signaling activates type-I IFN response through FOXO3-regulated lamin posttranslational modification

Neural stem/progenitor cells (NSPCs) persist over the lifespan while encountering constant challenges from age or injury related brain environmental changes like elevated oxidative stress. But how oxidative stress regulates NSPC and its neurogenic differentiation is less clear. Here we report that acutely elevated cellular oxidative stress in NSPCs modulates neurogenic differentiation through induction of Forkhead box protein O3 (FOXO3)-mediated cGAS/STING and type I interferon (IFN-I) responses. We show that oxidative stress activates FOXO3 and its transcriptional target glycine-N-methyltransferase (GNMT) whose upregulation triggers depletion of s-adenosylmethionine (SAM), a key co-substrate involved in methyl group transfer reactions. Mechanistically, we demonstrate that reduced intracellular SAM availability disrupts carboxymethylation and maturation of nuclear lamin, which induce cytosolic release of chromatin fragments and subsequent activation of the cGAS/STING-IFN-I cascade to suppress neurogenic differentiation. Together, our findings suggest the FOXO3-GNMT/SAM-lamin-cGAS/STING-IFN-I signaling cascade as a critical stress response program that regulates long-term regenerative potential.

Methionine-Mediated Protein Phosphatase 2A Catalytic Subunit (PP2Ac) Methylation Ameliorates the Tauopathy Induced by Manganese in Cell and Animal Models.

The molecular mechanism of Alzheimer-like cognitive impairment induced by manganese (Mn) exposure has not yet been fully clarified, and there are currently no effective interventions to treat neurodegenerative lesions related to manganism. Protein phosphatase 2 A (PP2A) is a major tau phosphatase and was recently identified as a potential therapeutic target molecule for neurodegenerative diseases; its activity is directed by the methylation status of the catalytic C subunit. Methionine is an essential amino acid, and its downstream metabolite S-adenosylmethionine (SAM) participates in transmethylation pathways as a methyl donor. In this study, the neurotoxic mechanism of Mn and the protective effect of methionine were evaluated in Mn-exposed cell and rat models. We show that Mn-induced neurotoxicity is characterized by PP2Ac demethylation accompanied by abnormally decreased LCMT-1 and increased PME-1, which are associated with tau hyperphosphorylation and spatial learning and memory deficits, and that the poor availability of SAM in the hippocampus is likely to determine the loss of PP2Ac methylation. Importantly, maintenance of local SAM levels through continuous supplementation with exogenous methionine, or through specific inhibition of PP2Ac demethylation by ABL127 administration in vitro, can effectively prevent tau hyperphosphorylation to reduce cellular oxidative stress, apoptosis, damage to cell viability, and rat memory deficits in cell or animal Mn exposure models. In conclusion, our data suggest that SAM and PP2Ac methylation may be novel targets for the treatment of Mn poisoning and neurotoxic mechanism-related tauopathies.

Abstract 155: Global DNA hypomethylation can be caused by decreased methyl-donor content in tissue and liquid biopsy samples in colorectal cancer progression

Abstract Backgrounds: Global DNA hypomethylation is characteristic in various cancer types including colorectal cancer. Alterations of DNA methylation related enzymes expression and decreased level of methyl-donor molecules (folic acid (FA), S-adenosylmethionine (SAM)) can lead to aberrant DNA methylation pattern and elevated homocysteine level. Aims: Our aim was to examine global DNA methylation changes during aging and colorectal normal-adenoma-carcinoma sequence and in inflammatory bowel disease in tissue and liquid biopsy samples for diagnostic purposes. Moreover, we aimed to explore the reasons of global hypomethylation on gene expression level and methyl-donor molecule content. Methods: Bisulfite treatment was performed on DNA isolated from 30 normal (N), 10 adenoma (Ad), 10 colorectal carcinoma (CRC), 10 colitis ulcerosa (UC) tissue samples and on 11 N, 10 Ad, 15 CRC, 12 UC plasma specimens. 30 N samples contained different age groups derived from under 20 to 70 years old healthy controls for examination of aging process. LINE-1 PCR product was generated and pyrosequenced. Whole genome expression level of 60 biopsy samples was evaluated by HTA 2.0 RNA microarraychip (Affymetrix). In situ tissue appearance of 5-methylcytosine, FA, SAM, homocysteine, and expression of DNA methyltransferases (DNMTs) were analyzed by immunohistochemistry staining (IHC). Results: According to LINE-1 bisulfite sequencing results, DNA methylation was 72.6±1% in samples of healthy controls under 50 years old and 71.6±1.8% in specimens of patients over 50 years old. Significant DNA hypomethylation was found in CRC (62.9±8.7%; p&amp;lt;0.001, early stages: 67.4±7.8%, late stages: 58.3±0.1%), Ad (66.7±5.1%; p&amp;lt;0.001) tissue samples in comparison with N samples (72±1.4%). Significant decrease of DNA methylation was observed in CRC (78.8±1.7%; p&amp;lt;0.02), and Ad (80.1±1.7%; p&amp;lt;0.02) plasma samples compared to N specimens (82.2±1.8%). Global DNA hypomethylation was not detected in UC samples. Significantly elevated RNA expression of enzymes connected to nucleotide synthesis was observed in Ad and CRC samples compared to N (p&amp;lt;0.05), while no changes were detected in the RNA levels of DNA methylation-related proteins. The intensity of 5-mC labeling of CRC and Ad samples was lower than in N tissue samples. Decreased FA, SAM, and increased homocysteine levels were noticed in CRC compared to N specimens; however no expression changes of DNMT enzymes were observed. Conclusion: Significant decrease in DNA methylation level was found in tissue and liquid biopsy samples of colorectal normal-adenoma-carcinoma sequence, but not in UC specimens. Our results suggest that determination of global DNA hypomethylation could have prognostic and diagnostic value as well, and reduction of DNA methylation level could be linked to decreased FA and SAM availability and not to methylation related ezymes activity. Citation Format: Krisztina A. Szigeti, Orsolya Galamb, Alexandra Kalmár, Gábor Valcz, Sára Zsigrai, Barbara K. Barták, Zsófia B. Nagy, Zsolt Tulassay, Péter Igaz, Béla Molnár. Global DNA hypomethylation can be caused by decreased methyl-donor content in tissue and liquid biopsy samples in colorectal cancer progression [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 155.

Genetic variants in S-adenosyl-methionine synthesis pathway and nonsyndromic cleft lip with or without cleft palate in Chile

The S-adenosyl-methionine (SAM) availability is crucial for DNA methylation, an epigenetic mechanism involved in nonsyndromic cleft lip with or without cleft palate (NSCL/P) expression. The aim of this study was to assess the association between single-nucleotide polymorphisms (SNPs) of genes involved in SAM synthesis and NSCL/P in a Chilean population. In 234 cases and 309 controls, 18 SNPs in AHCY, MTR, MTRR, and MAT2A were genotyped, and the association between them and the phenotype was evaluated based on additive (allele), dominant, recessive and haplotype models, by odds ratio (OR) computing. Three deep intronic SNPs of MTR showed a protective effect on NSCL/P expression: rs10925239 (OR 0.68; p = 0.0032; q = 0.0192), rs10925254 (OR 0.66; p = 0.0018; q = 0.0162), and rs3768142 (OR 0.66; p = 0.0015; q = 0.0162). Annotations in expression database demonstrate that the protective allele of the three SNPs is associated with a reduction of MTR expression summed to the prediction by bioinformatic tools of its potentiality to modify splicing sites. The protective effect against NSCL/P of these intronic MTR SNPs seems to be related to a decrease in MTR enzyme expression, modulating the SAM availability for proper substrate methylation. However, functional analyses are necessary to confirm our findings. SAM synthesis pathway genetic variants are factors associated to NSCL/P. This article adds new evidence for folate related genes in NSCL/P in Chile. Its impact is to contribute with potential new markers for genetic counseling.

Methyl-Metabolite Depletion Elicits Adaptive Responses to Support Heterochromatin Stability and Epigenetic Persistence

S-adenosylmethionine (SAM) is the methyl-donor substrate for DNA and histone methyltransferases that regulate epigenetic states and subsequent gene expression. This metabolism-epigenome link sensitizes chromatin methylation to altered SAM abundance, yet the mechanisms that allow organisms to adapt and protect epigenetic information during life-experienced fluctuations in SAM availability are unknown. We identified a robust response to SAM depletion that is highlighted by preferential cytoplasmic and nuclear mono-methylation of H3 Lys 9 (H3K9) at the expense of broad losses in histone di- and tri-methylation. Under SAM-depleted conditions, H3K9 mono-methylation preserves heterochromatin stability and supports global epigenetic persistence upon metabolic recovery. This unique chromatin response was robust across the mouse lifespan and correlated with improved metabolic health, supporting a significant role for epigenetic adaptation to SAM depletion invivo. Together, these studies provide evidence for an adaptive response that enables epigenetic persistence to metabolic stress.

Epigenetic engineering of yeast reveals dynamic molecular adaptation to methylation stress and genetic modulators of specific DNMT3 family members.

Cytosine methylation is a ubiquitous modification in mammalian DNA generated and maintained by several DNA methyltransferases (DNMTs) with partially overlapping functions and genomic targets. To systematically dissect the factors specifying each DNMT’s activity, we engineered combinatorial knock-in of human DNMT genes in Komagataella phaffii, a yeast species lacking endogenous DNA methylation. Time-course expression measurements captured dynamic network-level adaptation of cells to DNMT3B1-induced DNA methylation stress and showed that coordinately modulating the availability of S-adenosyl methionine (SAM), the essential metabolite for DNMT-catalyzed methylation, is an evolutionarily conserved epigenetic stress response, also implicated in several human diseases. Convolutional neural networks trained on genome-wide CpG-methylation data learned distinct sequence preferences of DNMT3 family members. A simulated annealing interpretation method resolved these preferences into individual flanking nucleotides and periodic poly(A) tracts that rotationally position highly methylated cytosines relative to phased nucleosomes. Furthermore, the nucleosome repeat length defined the spatial unit of methylation spreading. Gene methylation patterns were similar to those in mammals, and hypo- and hypermethylation were predictive of increased and decreased transcription relative to control, respectively, in the absence of mammalian readers of DNA methylation. Introducing controlled epigenetic perturbations in yeast thus enabled characterization of fundamental genomic features directing specific DNMT3 proteins.