Decrease In Global DNA Methylation Research Articles

Vitamin D and K Supplementation Is Associated with Changes in the Methylation Profile of U266-Multiple Myeloma Cells, Influencing the Proliferative Potential and Resistance to Bortezomib.

Multiple myeloma (MM) is a plasma cell malignancy that, despite recent advances in therapy, continues to pose a major challenge to hematologists. Currently, different classes of drugs are applied to treat MM, among others, proteasome inhibitors, immunomodulatory drugs, and monoclonal antibodies. Most of them participate in an interplay with the immune system, hijacking its effector functions and redirecting them to anti-MM activity. Therefore, adjuvant therapies boosting the immune system may be potentially beneficial in MM therapy. Vitamin D (VD) and vitamin K (VK) have multiple so called "non-classical" actions. They exhibit various anti-inflammatory and anti-cancer properties. In this paper, we investigated the influence of VD and VK on epigenetic alterations associated with the proliferative potential of MM cells and the development of BTZ resistance. Our results showed that the development of BTZ resistance is associated with a global decrease in DNA methylation. On the contrary, both control MM cells and BTZ-resistant MM cells exposed to VD alone and to the combination of VD and VK exhibit a global increase in methylation. In conclusion, VD and VK in vitro have the potential to induce epigenetic changes that reduce the proliferative potential of plasma cells and may at least partially prevent the development of resistance to BTZ. However, further ex vivo and in vivo studies are needed to confirm the results and introduce new supplementation recommendations as part of adjuvant therapy.

Mu opioid receptor expressing neurons in the rostral ventromedial medulla are the source of mechanical hypersensitivity induced by repeated restraint stress

Repeated exposure to psychophysical stress often causes an increase in sensitivity and response to pain. This phenomenon is commonly called stress-induced hyperalgesia (SIH). Although psychophysical stress is a well-known risk factor for numerous chronic pain syndromes, the neural mechanism underlying SIH has not yet been elucidated. The rostral ventromedial medulla (RVM) is a key output element of the descending pain modulation system. Descending signals from the RVM have a major impact on spinal nociceptive neurotransmission. In the present study, to clarify changes in the descending pain modulatory system in rats with SIH, we examined the expression of Mu opioid receptor (MOR) mRNA, MeCP2 and global DNA methylation in the RVM after repeated restraint stress for 3 weeks. Additionally, we microinjected neurotoxin dermorphin-SAP into the RVM. The repeated restraint stress for 3 weeks induced mechanical hypersensitivity in the hind paw, a significant increase in the expression of MOR mRNA and MeCP2, and a significant decrease in global DNA methylation in the RVM. The MeCP2 binding to MOR gene promoter in the RVM was significantly decreased in rats with repeated restraint stress. Furthermore, microinjection of dermorphin-SAP into the RVM prevented the mechanical hypersensitivity induced by repeated restraint stress. Although, because of the lack of specific antibody to MOR, we could not show a quantitative analysis in the number of MOR-expressing neurons after the microinjection, these results suggest that MOR-expressing neurons in the RVM induce SIH after repeated restraint stress.

Abstract 4727: RAS and PP2A activities converge on phosphoregulation of epigenetic complexes in cancer

Abstract RAS-mediated human cell transformation requires inhibition of the tumor suppressor Protein Phosphatase 2A (PP2A). Both RAS and PP2A mediate their effects by phosphoregulation, but phosphoprotein targets in which RAS and PP2A activities converge in human cancers have not been systematically analyzed. Here, based on mass spectrometry phosphoproteome data, we discover that phosphosites co-regulated by RAS and PP2A are enriched on proteins involved in epigenetic gene regulation. As examples, RAS and PP2A co-regulate the same phosphorylation sites on HDAC1/2, KDM1A, MTA1/2, RNF168 and TP53BP1. Mechanistically, we validate co-regulation of NuRD chromatin repressor complex by RAS and PP2A. Consistent with their known synergistic effects in cancer, RAS activation and PP2A inhibition resulted in epigenetic reporter de-repression and activation of oncogenic transcription. Notably, transcriptional de-repression by PP2A inhibition was associated with increased euchromatin and decrease in global DNA methylation. In RAS-driven cancers, PP2A activity is inhibited by nuclear PP2A inhibitor proteins PME-1 and SET. We further demonstrate that these PP2A inhibitors drive oncogenic transcription, but that their transcriptional targets are highly diversified. Whereas PME-1 controls RAS and MYC-driven transcription, SET controls TP53 targets and G2/M checkpoint genes. We further provide evidence that these nuclear PP2A inhibitors significantly differ in their roles in regulating DNA methylation. Collectively the results indicate that epigenetic protein complexes involved in oncogenic gene expression constitute a significant point of convergence for RAS hyperactivity and PP2A inhibition in cancer. Further, the results provide a rich source for future understanding of phosphorylation as a previously unappreciated layer of regulation of epigenetic gene regulation in cancer, and in other RAS/PP2A-regulated cellular processes. Citation Format: Mukund Sharma, Anna Aakula, Francesco Tabaro, Henrik Honkanen, Jesse Kamila, Matthieu Schapira, Cheryl Arrowsmith, Matti Nykter, Jukka K. Westermarck. RAS and PP2A activities converge on phosphoregulation of epigenetic complexes in cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4727.

DNA methylation variation is crucial to restore adventitious rooting ability during in vitro shoot culture-induced rejuvenation in apple rootstock.

In vitro shoot culture has been widely used for restoring adventitious rooting ability in rooting recalcitrant woody perennial species for the past few decades, but its molecular mechanism is largely uncovered. DNA methylation is an essential epigenetic mark that participates in many biological processes. Recent reports suggested a role of DNA methylation in vitro culture in plants. In this study, we characterized the single-base resolution DNA methylome and transcriptome of adult and in vitro shoot culture-induced rejuvenation cuttings of apple rootstock M9T337. We found a global decrease in DNA methylation during rejuvenation, which may be correlated with increased expression of DNA demethylase genes and decreased expression of DNA methyltransferase genes. We additionally documented DNA hypomethylation in 'T337'_R in gene protomer associated with higher transcript levels of several adventitious rooting-related genes. The application of a DNA methylation inhibitor (5-azacytidine) enhanced the adventitious rooting ability and the expression level of adventitious rooting-related genes, such as, MdANT, MdMPK3, MdABCB21, MdCDC48, MdKIN8B, pri-MdMIR156a5 and pri-MdMIR156a12. Together, the DNA hypomethylation is critical for the rejuvenation-dependent adventitious rooting ability in apple rootstock. In addition, increased DNA methylation was also found in thousands of genes in 'T337'_R. We additionally documented that DNA hypermethylation is required for inhibition of adventitious rooting-repressed genes, such as MdGAD5a, encoding glutamate decarboxylase, which can catalyze glutamate decarboxylated to form γ-aminobutyric acid (GABA). Our results revealed that in vitro shoot culture-dependent DNA methylation variation plays important roles in adventitious rooting in apple rootstock.

RAS and PP2A activities converge on epigenetic gene regulation.

RAS-mediated human cell transformation requires inhibition of the tumor suppressor protein phosphatase 2A (PP2A). However, the phosphoprotein targets and cellular processes in which RAS and PP2A activities converge in human cancers have not been systematically analyzed. Here, we discover that phosphosites co-regulated by RAS and PP2A are enriched on proteins involved in epigenetic gene regulation. As examples, RAS and PP2A co-regulate the same phosphorylation sites on HDAC1/2, KDM1A, MTA1/2, RNF168, and TP53BP1. We validate RAS- and PP2A-elicited regulation of HDAC1/2 chromatin recruitment, of RNF168-TP53BP1 interaction, and of gene expression. Consistent with their known synergistic effects in cancer, RAS activation and PP2A inhibition resulted in epigenetic reporter derepression and activation of oncogenic transcription. Transcriptional derepression by PP2A inhibition was associated with an increase in euchromatin and a decrease in global DNA methylation. Collectively, the results indicate that epigenetic protein complexes constitute a significant point of convergence for RAS hyperactivity and PP2A inhibition in cancer. Furthermore, the work provides an important resource for future studies focusing on phosphoregulation of epigenetic gene regulation in cancer and in other RAS/PP2A-regulated cellular processes.

Depletion of tet2 results in age-dependent changes in DNA methylation and gene expression in a zebrafish model of myelodysplastic syndrome.

Myelodysplastic syndrome (MDS) is a heterogeneous group of clonal hematopoietic disorders characterized by ineffective hematopoiesis, cytopenias, and dysplasia. The gene encoding ten-eleven translocation 2 (tet2), a dioxygenase enzyme that catalyzes the conversion of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine, is a recurrently mutated tumor suppressor gene in MDS and other myeloid malignancies. Previously, we reported a stable zebrafish line with a loss-of-function mutation in the tet2 gene. The tet2m/m-mutant zebrafish developed a pre-MDS state with kidney marrow dysplasia, but normal circulating blood counts by 11 months of age and accompanying anemia, signifying the onset of MDS, by 24 months of age. In the current study, we collected progenitor cells from the kidney marrows of the adult tet2m/m and tet2wt/wt fish at 4 and 15 months of age and conducted enhanced reduced representation of bisulfite sequencing (ERRBS) and bulk RNA-seq to measure changes in DNA methylation and gene expression of hematopoietic stem and progenitor cells (HSPCs). A global increase in DNA methylation of gene promoter regions and CpG islands was observed in tet2m/m HSPCs at 4 months of age when compared with the wild type. Furthermore, hypermethylated genes were significantly enriched for targets of SUZ12 and the metal-response-element-binding transcription factor 2 (MTF2)-involved in the polycomb repressive complex 2 (PRC2). However, between 4 and 15 months of age, we observed a paradoxical global decrease in DNA methylation in tet2m/m HSPCs. Gene expression analyses identified upregulation of genes associated with mTORC1 signaling and interferon gamma and alpha responses in tet2m/m HSPCs at 4 months of age when compared with the wild type. Downregulated genes in HSPCs of tet2-mutant fish at 4 months of age were enriched for cell cycle regulation, heme metabolism, and interleukin 2 (IL2)/signal transducer and activator of transcription 5 (STAT5) signaling, possibly related to increased self-renewal and clonal advantage in HSPCs with tet2 loss of function. Finally, there was an overall inverse correlation between overall increased promoter methylation and gene expression.

PERTURBATION OF DNA METHYLTRANSFERASES IN HUMAN TROPHOBLAST STEM CELLS LEADS TO A FAILURE OF TROPHOBLAST DIFFERENTIATION - A PUTATIVE CAUSE FOR HUMAN MISCARRIAGE

To investigate the role of DNA methyltransferase 1 (DNMT1) in the etiology of miscarriages using a novel human trophoblast stem cell (TSC) model. The DNMTs are an important family of proteins that catalyze the transfer of methyl groups to specific CpG structures in DNA (DNA methylation). DNMT1 has an essential function in development to maintain proper DNA methylation during cell divisions. Prior studies have shown that human chorionic villi from sporadic miscarriages have a 50% reduction of DNMT1 protein with a concomitant decrease in global DNA methylation compared to chorionic villi from control pregnancies (elective terminations).

High Dietary Folic Acid Intake Is Associated with Genomic Instability in Peripheral Lymphocytes of Healthy Adults.

Mandatory fortification of food with synthetic folic acid (FA) was instituted in 1998 to reduce the incidence of neural tube defects. Adequate folate status is correlated with numerous health benefits. However, elevated consumption of FA is controversially associated with deleterious effects on health. We previously reported that excess FA mimicked folate depletion in a lymphoblastoid cell line. To explore the impact of FA intake from fortified food, we conducted an observational human study on 33 healthy participants aged 18–40 not taking any supplements. Food intake, anthropomorphic measurements, and blood samples were collected and analyzed. Our results show that individuals belonging to the highest tertile of folic acid intake, as well as ones with the highest folic acid to total folate intake ratio (FAR), display a significantly greater incidence of lymphocyte genomic damage. A decrease in global DNA methylation is observed in the highest tertile of FAR compared to the lowest (p = 0.055). A downward trend in the overall gene expression of select DNA repair and one carbon cycle genes (MGMT, MLH1, UNG, MTHFR, MTR) is noted with increased folate status and FA intake. These results provide supporting evidence that high consumption of FA from fortified foods can precipitate genomic instability in peripheral lymphocyte in vivo.

Global DNA Methylation Status in Relation to Resistance Training with High vs Low Loads to Failure

ObjectiveOur objective was to determine how global DNA methylation status of skeletal muscle differs with resistance training (RT) to failure in trained participants using either 80% of their estimated one‐repetition maximum (Est. 1‐RM) (80FAIL) or 30% of their Est. 1‐RM (30FAIL).HypothesesWe hypothesize there will be a significant decrease in global DNA methylation with both RT conditions, but that 30FAIL RT will elicit a greater magnitude of hypomethylation than 80FAIL RT.MethodsPreviously trained college‐aged males (n= 11, age 23 ± 4 years, percent fat 11.4 ± 6.4%, training experience 4 ± 3 years, squat strength relative to body weight 1.7 ± 0.3) voluntarily underwent two bouts of RT. Participants completed both the 80FAIL and 30FAIL conditions (separated by one week). For each bout of RT, participants completed 4 sets of back squats and 4 sets leg extensions with either the 80FAIL or 30FAIL training conditions. Muscle biopsies were collected from the vastus lateralis before (PRE), 3 hours (3hPOST), and 6 hours (6hrPOST) after each RT bout. DNA was then batch‐isolated from muscle tissue and submitted for analysis using the Illumina MethylationEPIC array.ResultsTotal number of repetitions performed were significantly higher for 30FAIL training vs 80FAIL training (p< .001), however total training volume (sets x reps x load) was not significantly different between conditions (p= 0.571). Global methylation changes between PRE, 3hPOST, and 6hPOST are presented herein. In addition, significant differences between conditions are emphasized for each post‐exercise time point.ConclusionsWith this study we expand our understanding of how the manipulation of RT variables affect epigenetic modifications in skeletal muscle.

The phytochemical brazilin suppress DNMT1 expression by recruiting p53 to its promoter resulting in the epigenetic restoration of p21 in MCF7cells

BackgroundCancer is an outcome of uncontrolled cell division eventually associated with dysregulated epigenetic mechanisms, including DNA methylation. DNA methyltransferase 1 is ubiquitously expressed in the proliferating cells and is essential for the maintenance of DNA methylation. It causes the abnormal silencing of tumor suppressor genes in human cancer which is necessary for proliferation, cell cycle progression, and survival. DNMT1 is involved in tumorigenesis of several cancers, its upregulation potentially upscale the promoter level inactivation of transcription of a tumor inhibitory gene by introducing repressive methylation marks on the CpG islands. This epigenetic perturbation caused by DNMT is targeted for cancer therapeutics. PurposeTo demonstrate the proliferative inhibitory potential of brazilin in human breast cancer cell line (MCF-7) with concurrent mitigation of DNMT1 functional expression and to understand its effect on downstream targets like cell cycle inhibitor p21. Study Design/ MethodsThe impact of brazilin on the growth and proliferation of the MCF-7 cells was determined using the XTT assay. The global DNA 5-methyl cytosine methylation pattern was analyzed upon brazilin treatment. The gene and protein expression of DNMTs were determined with quantitative RTPCR and western blots respectively. The potential binding sites of transcription factors in the human DNMT1 promoter were predicted using the MatInspector tool on the Genomatix software. The chromatin immunoprecipitation (ChIP) assay was performed to demonstrate the transcription factors occupancy at the promoter. Methylation of promoter CpG islands was determined by the methylation-specific PCR (MSP) upon brazilin treatment. The molecular docking of the human DNMT1 with brazilin (ligand) was performed using the Schrödinger suite. ResultsThe heterotetracyclic compound brazilin, present in the wood of Caesalpinia sappan, inhibited the proliferation of the human breast cancer cell line (MCF-7) and reduced the DNMT1 expression with a decrease in global DNA methylation. Brazilin, by activating p38 MAPK and elevating p53 levels within the exposed cells. The elevated level of p53 enriched the occupancy at binding sites within 200 bp upstream to the transcription start site in the DNMT1 promoter, resulting in reduced DNMT1 gene expression. Furthermore, the brazilin restored the p21 levels in the exposed cells as the CpGs in the p21 promoter (-128 bp/+17 bp) were significantly demethylated as observed in the methylation-specific PCR (MSP). ConclusionHighly potential anti-proliferative molecule brazilin can modulate the DNMT1 functional expression and restore the cell cycle inhibitor p21expression. We propose that brazilin can be used in therapeutic interventions to restore the deregulated epigenetic mechanisms in cancer.

Novel Therapeutic Targets for DNMT3A Mutant Myeloid Neoplasms

The DNMT3A mutations (DNMT3AMT) that occur in acute myeloid leukemia (AML) and Myelodysplastic syndromes (MDS) are either hypomorphic or inactivating, consistent with bone fide tumor suppression function of DNMT3A . The most recurrent mutation involves the canonical R882, resulting in a hypomorphic protein most likely also acting in a dominant-negative manner by affecting the methyltransferase activity of the remaining wild type DNMT3A allele (DNMT3AWT).To assess the clinical context of DNMT3A alterations, 1059 samples from patients with myeloid neoplasia (422 MDS, 240 MDS/myeloproliferative neoplasms (MPN), 163 secondary (s) AML, 234 primary (p) AML were examined. DNMT3AMT were found in 21% of patients, of which 71% were missense mutations [52% R882; 48% non-canonical mutation (NCM)] and 29% were truncating mutations or frameshift mutations. DNMT3AR882 was significantly overrepresented in pAML (P=.04) and underrepresented in MDS (P=.03). In contrast, truncating mutations were more common in MDS and sAML vs. pAML (55% vs. 29%; P<.01). DNMT3AMT were associated with ASXL1 (P=.05) and SF3B1 (P<.01) mutations in MDS but not in pAML. In contrast, DNMT3AMT associated with NPM1 (P<.01), FLT3ITD/TKD (P=.04) and IDH1 mutations in pAML but not sAML. Clonal architecture analyses showed that 49% of R882 were ancestral events with TET2, FLT3ITD/TKD and NPM1 as the most common secondary hits. In contrast, only 33% of non-R882 variants (39% truncating; 33% NCM) were an ancestral event (P=.04). Gene expression analyses of AML [n=167 (26% DNMT3AMT)] and MDS [n=175 (8% DNMT3AMT)] showed a decrease in DNMT3A mRNA levels in DNMT3AMTvs. DNMT3AWT AML (mean ± SD= 1079 ± 582 vs. 1376 ± 556), though not in MDS, probably due to the high percentage of truncating mutations and resulting nonsense mediated decay of mRNA.Exclusively heterozygous DNMT3AMT configurations imply that biallelic DNMT3A loss may be lethal and thus that mutant cells may be amenable to synthetic lethal therapeutic approaches. DNMT3A inhibitors may however, also phenocopy DNMT3A loss in normal DNMT3AWT cells. Although such concerns are not huge, as they would likely be reversed by withdrawing the inhibitor, there is also rationale to instead overcome haploinsufficiency by increasing the throughput of the remaining DNMT3AWT allele, i.e. to revert malignant cells toward normalcy rather than attempt to kill them. We achieve this by supplementation of the universal methyl donor S-adenosyl- L -methionine (SAM), i.e. by increasing DNMT3A substrate availability.To test this principle we quantified the differential sensitivity of DNMT3AMTvs. DNMT3AWT and primary cells to SAM (disulfate tosylate) supplementation. As in vitro models, we selected a myeloid derived cell line, OCI-AML3, which harbors R882 and shows a 9% decrease in global DNA-methylation. As controls we used a panel of myeloid cell lines with DNMT3AWT (K562, THP1, U937, MOLM13). We observed that OCI-AML3R882 exhibited higher sensitivity to 24h SAM supplementation than DNMT3AWT cell lines using an MTT assay (IC50 of ~2mM). This dose did not affect healthy human bone marrow cells as shown by no change in growth upon SAM treatment.We then assessed DNMT3A enzymatic activity using an ELISA-like colorimetric assay and found that 2mM SAM exposures increased DNMT3A activity by 20% vs. baseline (P=.04) in OCI-AML3R882 cells, suggesting that SAM supplementation was able to increase DNMT3AWT throughput. DNMT3AWT cells did not show increases in DNMT3A activity with SAM supplementation.We also generated a cellular model of DNMT3A insufficiency using lentiviral shRNA in K562 (55% reduction by qPCR) (K562-shRNA DNMT3A). SAM treatment (500µM, 1mM, 2mM, 3mM) induced a decrease in cell growth in K562-shRNA DNMT3A vs. not (30% vs. 60%; P<.01).In sum, we present the first novel DNMT3AMT therapeutic strategy based on the principle of overcoming the functional deficiency of a monoallelic mutants through up-modulation of DNMT3AWT activity. SAM showed a promising increase in DNMT3AWT activity in mutant cells at safe/feasible doses, opening the possibility of combinational strategies with existing/ approved agents. SAM could be beneficial in healthy older adults with clonal hematopoiesis of indeterminate potential and DNMT3AMT. We are in the process of determining if SAM treatment will enhance DNMT3AWT in murine models of DNMT3A (Dnmt3aMT, Flt3ITD, Npm1C) resulting in a fully penetrant leukemic phenotype. DisclosuresSekeres:Celgene: Membership on an entity's Board of Directors or advisory committees.

Genome-wide analysis of DNA methylation in Hirschsprung enteric precursor cells: unraveling the epigenetic landscape of enteric nervous system development

BackgroundHirschsprung disease (HSCR, OMIM 142623) is a rare congenital disorder that results from a failure to fully colonize the gut by enteric precursor cells (EPCs) derived from the neural crest. Such incomplete gut colonization is due to alterations in EPCs proliferation, survival, migration and/or differentiation during enteric nervous system (ENS) development. This complex process is regulated by a network of signaling pathways that is orchestrated by genetic and epigenetic factors, and therefore alterations at these levels can lead to the onset of neurocristopathies such as HSCR. The goal of this study is to broaden our knowledge of the role of epigenetic mechanisms in the disease context, specifically in DNA methylation. Therefore, with this aim, a Whole-Genome Bisulfite Sequencing assay has been performed using EPCs from HSCR patients and human controls.ResultsThis is the first study to present a whole genome DNA methylation profile in HSCR and reveal a decrease of global DNA methylation in CpG context in HSCR patients compared with controls, which correlates with a greater hypomethylation of the differentially methylated regions (DMRs) identified. These results agree with the de novo Methyltransferase 3b downregulation in EPCs from HSCR patients compared to controls, and with the decrease in the global DNA methylation level previously described by our group. Through the comparative analysis of DMRs between HSCR patients and controls, a set of new genes has been identified as potential susceptibility genes for HSCR at an epigenetic level. Moreover, previous differentially methylated genes related to HSCR have been found, which validates our approach.ConclusionsThis study highlights the relevance of an adequate methylation pattern for a proper ENS development. This is a research area that provides a novel approach to deepen our understanding of the etiopathogenesis of HSCR.Graphic abstract

Quantitative determination of histone methylation via fluorescence resonance energy transfer (FRET) technology in immortalized bovine mammary alveolar epithelial cells supplemented with methionine.

Methionine (Met) is an essential precursor of S-adenosylmethionine (SAM), which is the primary methyl donor required for biological processes such as DNA and histone methylation, which alter gene expression. In dairy cows, dietary Met has been observed to exert transcriptional alterations with beneficial effects on milk biosynthesis; however, the extent of these effects via SAM remains unknown. Therefore, we evaluated the effect of Met supply on histone methylation in lysine residues K9 and K27 in the histone tail H3 via a fluorescence resonance energy transfer (FRET) system in immortalized bovine mammary alveolar epithelial cells (MACT) incubated varying concentration of Met. The histone methylation data was complemented with global DNA methylation, cellular protein synthesis, and RT-qPCR analysis of genes related to Met cycle, DNA and histone methylation, AA transporters, and protein synthesis. The histone methylation data was performed on MACT cells seeded at 30,000 cells/well in 96-well plates 24 h prior to transfection. The transfections of FRET gene reporter plasmids H3K9 and H3K27 was performed with 0.3 μL/well of Lipofectamine® 3000 and 50 ng of plasmid DNA per well. At 24 h post-transfection, cells were treated with 0, 125, 250, and 500 μM of Met, and quantification of histone methylation was performed at 0, 12, and 24 h post-treatment as well as cell viability at 24 h using CellProfiler software. An inverted microscope for live imagining (EVOS® FL Auto) equipped with a motorized scanning stage, and an environment-controlled chamber at 37˚C and 5.0% of CO2 was used to take 4 pictures/well at 4x magnification. A more defined response on histone methylation was observed in H3K9 than H3K27 to Met supply, where maximal histone methylation in H3K9 was observed with 125 μM of Met. This greater histone methylation in H3K9 at 125 μM was accompanied by greater cellular protein concentration. The linear increase in Met supply causes a linear decrease in global DNA methylation, while linearly upregulating genes related to the Met cycle (i.e., MAT1A, PEMT, SAHH, and MTR). The histone methylation data suggest that, to some extent, methyl-donors such as Met may affect the methylation sites, H3K9 and H3K27, and consequently causing a different epigenetic alteration. In the context of the dairy cow, further refinement to this FRET assay to study histone methylation could lead to establishing novel potential mechanisms of how dietary methyl donors may control the structural conformation of the bovine genome and, by extension, gene expression.

Genome-wide investigation of DNA methylation dynamics reveals a critical role of DNA demethylation during the early somatic embryogenesis of Dimocarpus longan Lour.

DNA methylation plays essential roles in gene regulation, chromatin structure stability, gene imprinting, X chromosome inactivation and embryonic development. However, the dynamics and functions of DNA methylation during the early stage of longan (Dimocarpus longan) somatic embryogenesis (SE) are still unclear. In this study, we carried out whole genome bisulphite sequencing and transcriptome sequencing analyses for embryogenic callus (EC), incomplete compact pro-embryogenic cultures (ICpEC) and globular embryos (GE) in an early SE system. At a global level, the DNA 5-methylcytosine content in EC, ICpEC and GE was 24.59, 19.65 and 19.74%, respectively, suggesting a global decrease in DNA methylation from EC to ICpEC and then a slight increase from ICpEC to GE. Differentially methylated region (DMR) analysis showed that hypomethylation mainly occurred in CHH contexts. Gene ontology and Kyoto encyclopedia of genes and genomes analysis of hypomethylated-CHH-DMR-associated genes revealed that zein biosynthesis, fatty acid biosynthesis, circadian rhythm and mitophagy pathways were involved in longan early SE. Expression patterns of DNA methyltransferase and demethylase genes during longan early SE suggested that the decrease in DNA methylation was probably regulated by DNA methyltransferase genes and the DNA demethylase gene REPRESSOR OF SILENCING 1 (ROS1). The correlation between DNA hypomethylation and gene expression revealed that decreased DNA methylation did not cause extensive changes in gene expression during early longan SE and that gene expression may be affected by methylation changes in gene and downstream regions. Inhibiting DNA methylation with 5-azacytidine treatment in EC promoted the formation of GE and enhanced the capability of longan SE. Our results suggest that DNA demethylation has important roles in longan SE development.

A valepotriate-enriched fraction from Valeriana glechomifolia decreases DNA methylation and up-regulate TrkB receptors in the hippocampus of mice.

DNA methylation, an epigenetic modification that mediates gene silencing, has been shown to play a role in the neurobiology of major depression. Studies suggested that terpenes inhibit DNA methylation and increase gene expression. The present study investigated the involvement of DNA methylation in the antidepressant-like activity of diene valepotriates, non-glicosilated carbocyclic iridoids that comprise a family of terpenes obtained from Valeriana glechomifolia. The antidepressant-like effect of diene valepotriates acute administration (5 mg/kg, p.o.) in mice submitted to the forced swimming test was followed by a decrease in global DNA methylation in animals' hippocampus (but not in the pre-frontal cortex). Mice pretreatment with anysomicin (a protein synthesis inhibitor) and K252a (an inhibitor of Trk receptors) attenuated diene valepotriates-induced antidepressant-like effect in the forced swimming test. Diene valepotriates elicited an upregulation in the TrkB receptor and a tendency to increase BDNF levels in mice hippocampus. These results demonstrate that DNA methylation could be an in vivo molecular target of diene valepotriates. The diene valepotriates-triggered reduction in hippocampal DNA methylation is accompanied by increased protein synthesis, which is involved in its antidepressant-like activity. Furthermore, BDNF-mediated TrkB signaling may contribute for diene valepotriates antidepressant-like effect.

Chronic‐Low Dose Cadmiun Exposure Alters Sperm DNA Methylation in Mice

Millions of people are exposed to cadmium and chronic‐low dose exposure has been linked to increased risks of breast and endometrial cancer, osteoporosis, renal dysfunction, cardiovascular disease and reproductive defects. One possible mechanism is dysregulation of gene expression through oxidative stress. Since cadmium is not a mutagen, we hypothesize that it could also exert its effects through epigenetic reprogramming, modulating regulatory targets with consequences in gene expression. Studies have reported that these dysregulations in gene expression is transgenerational; however these studies have focused heavily on maternal exposure while the impact on sperm and fertility has received very little attention. This discrepancy is due to the long‐held belief that, DNA methylation, one of the most stable epigenetic marks, is erased during fertilization. Recent evidence now suggests that at least some methylation marks are retained and can be passed on to the next generation. Here, we investigate the impact of chronic‐low dose cadmium exposure on DNA methylation patterns in sperm using a mouse model and an environmentally relevant cadmium dose. We hypothesize that chronic‐low dose exposure to cadmium modifies DNA methylation patterns in sperm leading to developmental effects. Our preliminary data shows that exposure to cadmium did not cause any histological changes to the testes of cadmium‐treated compared to control animals. However, using the 5‐mC DNA ELISA method we demonstrated a global decrease in DNA methylation in our treatment group. Additionally, Reduced Representation Bisulfite Sequencing (RRBS) which measures DNA methylation changes at specific genomic loci, further validated the impact of cadmium exposure on DNA methylation. A stringent statistical cut‐off (q&lt;0.01 with at least 25% difference between the groups and at least 10X reads), showed that chronic‐low dose cadmium exposure leads to 1,788 differentially methylated sites. Importantly, some of these sites, which we are now validating, were at promoters and are likely to have functional consequences in gene expression. Our results show for the first time that mice sperm methylome is altered in response to cadmium exposure while the testes integrity remain intact. Our data highlight a foundational framework to study gene expression patterns that may affect offspring in a paternal inheritance context.Support or Funding InformationUK‐CARES Grant P30 ES026529

HP1s modulate the S-Adenosyl Methionine synthesis pathway in liver cancer cells.

Hepatocellular carcinoma (HCC) is the most frequent primary liver cancer in adults. Among the altered pathways leading to HCC, an increasing role is attributed to abnormal epigenetic regulation. Members of the Heterochromatin Protein (HP1) 1 family are key players in chromatin organisation, acting as docking sites for chromatin modifiers. Here, we inactivated HP1α in HepG2 human liver carcinoma cells and showed that HP1α participated in cell proliferation. HP1α-depleted cells have a global decrease in DNA methylation and consequently a perturbed chromatin organisation, as exemplified by the reactivation of transcription at centromeric and pericentromeric regions, eventhough the protein levels of chromatin writers depositing methylation marks, such as EZH2, SETDB1, SUV39H1, G9A and DNMT3A remained unaltered. This decrease was attributed mainly to a low S-Adenosyl Methionine (SAM) level, a cofactor involved in methylation processes. Furthermore, we showed that this decrease was due to a modification in the Methionine adenosyl transferase 2A RNA (MAT2A) level, which modifies the ratio of MAT1A/MAT2A, two enzymes that generate SAM. Importantly, HP1α reintroduction into HP1α-depleted cells restored the MAT2A protein to its initial level. Finally, we demonstrated that this transcriptional deregulation of MAT2A in HP1α-depleted cells relied on a lack of recruitment of HP1β and HP1γ to MAT2A promoter where an improper non-CpG methylation site was promoted in the vicinity of the transcription start site where HP1β and HP1γ bound. Altogether, these results highlight an unanticipated link between HP1 and the SAM synthesis pathway, and emphasise emerging functions of HP1s as sensors of some aspects of liver cell metabolism.

Effect of titanium dioxide nanoparticles on DNA methylation in multiple human cell lines

Nanoscale titanium dioxide (TiO2) is manufactured in wide scale, with a range of applications in consumer products. Significant toxicity of TiO2 nanoparticles has, however, been recognized, suggesting considerable risk to human health. To evaluate fully their toxicity, assessment of the epigenetic action of these nanoparticles is critical. However, only few studies are available examining capability of nanoparticles to alter epigenetic integrity. In the present study, the effect of TiO2 nanoparticles exposure on DNA methylation, a major epigenetic mechanism, was investigated in in vitro cellular model systems. A panel of cells relevant to portals of human exposure (Caco-2 (colorectal), HepG2 (liver), NL20 (lung), and A-431 (skin)) was exposed to TiO2 nanoparticles to assess effects on global methylation, gene-specific methylation, and expression levels of DNA methyltransferases, MBD2, and UHRF1. Global methylation was determined by enzyme-linked immunosorbent assay-based immunochemical analysis. Degree of promoter methylation across a defined panel of genes was evaluated using EpiTect Methyl II Signature PCR System Array technology. Expression of DNMT1, DNMT3a, DNMT3b, MBD2, and URHF1 was quantified by qRT-PCR. Decrease in global DNA methylation in cell lines Caco-2, HepG2, and A-431 exposed to TiO2 nanoparticles was shown. Across four cell lines, eight genes (CDKN1A, DNAJC15, GADD45A, GDF15, INSIG1, SCARA3, TP53, and BNIP3) were identified in which promotors were methylated after exposure. Altered expression of these genes is associated with disease etiology. The results also revealed aberrant expression of epigenetic regulatory genes involved in DNA methylation (DNMT1, DNMT3a, DNMT3b, MBD2, and UHRF1) in TiO2 exposed cells, which was cell type dependent. Findings from this study clearly demonstrate the impact of TiO2 nanoparticles exposure on DNA methylation in multiple cell types, supporting potential involvement of this epigenetic mechanism in the toxicity of TiO2 nanoparticles. Hence for complete assessment of potential risk from nanoparticle exposure, epigenetic studies are critical.

Neurological Effects in the Offspring After Switching From Tobacco Cigarettes to E-Cigarettes During Pregnancy in a Mouse Model.

Maternal smoking is currently a public health concern and has been associated with a number of complications in the offspring. E-cigarettes are gaining popularity as a "safer" alternative to tobacco cigarettes during pregnancy, however, there are a limited number of studies to suggest that it is actually "safe." Balb/C female mice were exposed to ambient air (n = 8; Sham), or tobacco cigarette smoke (n = 8; SE) before gestation, during gestation and lactation. A third group was exposed to cigarette smoke before gestation followed by e-cigarette aerosols during gestation and lactation (n = 8; Switch). Male offspring (12-week old, n = 10-14/group) underwent behavioral assessments to investigate short-term memory, anxiety, and activity using the novel object recognition and elevated plus maze tests. Brains were collected at postnatal day (P)1, P20, and Week 13 for global DNA methylation, epigenetic gene expression, and neuronal cell counts. The offspring from mothers switching to e-cigarettes exhibited no change in exploration/activity but showed a decrease in global DNA methylation, Aurora Kinase (Aurk) A and AurkB gene expression and a reduction in neuronal cell numbers in the cornu ammonis 1 region of the dorsal hippocampus compared with the SE group. Continuous tobacco cigarette smoke exposure during pregnancy resulted in marked neurological deficits in the offspring. Switching to e-cigarettes during pregnancy reduced these neurological deficits compared with cigarette smoke exposure. However, neurological changes were still observed, so we therefore conclude that e-cigarette use during pregnancy is not advised.

Epigenetic, transcriptional and phenotypic responses in two generations of Daphnia magna exposed to the DNA methylation inhibitor 5-azacytidine.

The water flea Daphnia magna is a keystone species in freshwater ecosystems and has been widely used as a model organism in environmental ecotoxicology. This aquatic crustacean is sensitive to environmental stressors and displays considerable plasticity in adapting to changing environmental conditions. Part of this plasticity may be due to epigenetic regulation of gene expression, including changes to DNA methylation and histone modifications. Because of the generally hypomethylated genome of this species, we hypothesized that the histone code may have an essential role in the epigenetic control and that histone modifications might be an early marker for stress. This study aims to characterize the epigenetic, transcriptional and phenotypic responses and their causal linkages in directly exposed adult (F0) Daphnia and peritoneal exposed neonates (F1) after a chronic (7-day) exposure to a sublethal concentration (10 mg/l) of 5-azacytidine, a well-studied vertebrate DNA methylation inhibitor. Exposure of the F0 generation significantly reduced the cumulative fecundity, accompanied with differential expression of genes in the one-carbon-cycle metabolic pathway. In the epigenome of the F0 generation, a decrease in global DNA methylation, but no significant changes on H3K4me3 or H3K27me3, were observed. In the F1 offspring generation, changes in gene expression, a significant reduction in global DNA methylation and changes in histone modifications were identified. The results indicate that exposure during adulthood may result in more pronounced effects on early development in the offspring generation, though interpretation of the data should be carefully done since both the exposure regime and developmental period is different in the two generations examined. The obtained results improve our understanding of crustacean epigenetics and the tools developed may promote use of epigenetic markers in hazard assessment of environmental stressors.