Base editor scanning charts the DNMT3A activity landscape.

DNA methylation is critical for regulating gene expression, necessitating its accurate placement by enzymes such as the DNA methyltransferase DNMT3A. Dysregulation of this process is known to cause aberrant development and oncogenesis, yet how DNMT3A is regulated holistically by its three domains remains challenging to study. Here, we integrate base editing with a DNA methylation reporter to perform in situ mutational scanning of DNMT3A in cells. We identify mutations throughout the protein that perturb function, including ones at an interdomain interface that block allosteric activation. Unexpectedly, we also find mutations in the PWWP domain, a histone reader, that modulate enzyme activity despite preserving histone recognition and protein stability. These effects arise from altered PWWP domain DNA affinity, which we show is a noncanonical function required for full activity in cells. Our findings highlight mechanisms of interdomain crosstalk and demonstrate a generalizable strategy to probe sequence-activity relationships of nonessential chromatin regulators.

Single-nucleus RNA-seq reveals that MBD5, MBD6, and SILENZIO maintain silencing in the vegetative cell of developing pollen.

Silencing of transposable elements (TEs) drives the evolution of numerous redundant mechanisms of transcriptional regulation. Arabidopsis MBD5, MBD6, and SILENZIO act as TE repressors downstream of DNA methylation. Here, we show, via single-nucleus RNA-seq of developing male gametophytes, that these repressors are critical for TE silencing in the pollen vegetative cell, a companion cell important for fertilization that undergoes chromatin decompaction. Instead, other silencing mutants (met1, ddm1, mom1, morc) show loss of silencing in all pollen nucleus types and somatic cells. We show that TEs repressed by MBD5/6 gain chromatin accessibility in wild-type vegetative nuclei despite remaining silent, suggesting that loss of DNA compaction makes them sensitive to loss of MBD5/6. Consistently, crossing mbd5/6 to histone 1 mutants, which have decondensed chromatin in leaves, reveals derepression of MBD5/6-dependent TEs in leaves. MBD5/6 and SILENZIO thus act as a silencing system particularly important when chromatin compaction is compromised.

Epigenetic Effects of Blackberry Extract on Human Colorectal Cancer Cells.

Fruit-derived polyphenolic compounds have been shown to exert anticancer effects via epigenetic mechanisms. In this study, we investigated the effect of blackberry extract on the expression of DNMTs (Dnmt1, Dnmt3a, and Dnmt3b) and HDACs (HDAC1-4 and SIRT1) and its influence on the cellular differentiation and promoter DNA methylation of tumor-related genes using a panel of six human CRC cell lines. Treatment with IC20 and IC50 concentrations of blackberry extract for 72 h significantly reduced Dnmt1 and Dnmt3b transcript levels in HCT116, SW480, HT29/219, SW742, and LS180 cells in a dose-dependent manner. Blackberry also induced promoter DNA demethylation of SFRP2 and p16 genes in four tested CRC cell lines. Berry treatment, however, upregulated Dnmt3a genes in SW480, SW742, and HT29/219 cell lines. A dose-dependent and cell-type-specific reduction of HDAC1, HDAC2, and HDAC4 expressions were observed in CRC-treated cells. Treatment with berry extract induced the expression of SIRT1 gene in HCT116 and HT29/219 cells and increased the expression of two colonic epithelial cell differentiation markers, carcinoembryonic antigen (CEA) and alkaline phosphatase in LS180 cells in a time-dependent manner. This study is the first to report the epigenetic effects of blackberry in cancer cells.

Mindfulness intervention improves cognitive function in older adults by enhancing the level of miRNA-29c in neuron-derived extracellular vesicles.

Although mindfulness-based stress reduction (MBSR) improves cognitive function, the mechanism is not clear. In this study, people aged 65 years and older were recruited from elderly communities in Chitose City, Japan, and assigned to a non-MBSR group or a MBSR group. Before and after the intervention, the Japanese version of the Montreal Cognitive Assessment (MoCA-J) was administered, and blood samples were collected. Then, neuron-derived extracellular vesicles (NDEVs) were isolated from blood samples, and microRNAs, as well as the target mRNAs, were evaluated in NDEVs. A linear mixed model analysis showed significant effects of the MBSR x time interaction on the MoCA-J scores, the expression of miRNA(miR)-29c, DNA methyltransferase 3 alpha (DNMT3A), and DNMT3B in NDEVs. These results indicate that MBSR can improve cognitive function by increasing the expression of miR-29c and decreasing the expression of DNMT3A, as well as DNMT3B, in neurons. It was also found that intracerebroventricular injection of miR-29c mimic into 5xFAD mice prevented cognitive decline, as well as neuronal loss in the subiculum area, by down-regulating Dnmt3a  and Dnmt3b  in the hippocampus. The present study suggests that MBSR can prevent neuronal loss and cognitive impairment by increasing the neuronal expression of miR-29c.

Hairpin-bisulfite sequencing of cells exposed to decitabine documents the process of DNA demethylation.

Although the mechanism of DNA demethylating drugs has been understood for many years, the direct effect of these drugs on methylation of the complementary strands of DNA has not been formally demonstrated. By using hairpin-bisulphite sequencing, we describe the kinetics and pattern of DNA methylation following treatment of cells by the DNA methyltransferase 1 (DNMT1) inhibitor, decitabine. As expected, we demonstrate complete loss of methylation on the daughter strand following S-phase in selected densely methylated genes in synchronized Jurkat cells. Thereafter, cells showed a heterogeneous pattern of methylation reflecting replication of the unmethylated strand and restoration of methylation.

Relationship between redox potential of glutathione and DNA methylation level in liver of newborn guinea pigs.

The metabolism of DNA methylation is reported to be sensitive to oxidant molecules or oxidative stress. Hypothesis: early-life oxidative stress characterized by the redox potential of glutathione influences the DNA methylation level. The in vivo study aimed at the impact of modulating redox potential of glutathione on DNA methylation. Newborn guinea pigs received different nutritive modalities for 4 days: oral nutrition, parenteral nutrition including lipid emulsion Intralipid (PN-IL) or SMOFLipid (PN-SF), protected or not from ambient light. Livers were collected for biochemical determinations. Redox potential (p < 0.001) and DNA methylation (p < 0.01) were higher in PN-infused animals and even higher in PN-SF. Their positive correlation was significant (r2 = 0.51; p < 0.001). Methylation activity was higher in PN groups (p < 0.01). Protein levels of DNA methyltransferase (DNMT)-1 were lower in PN groups (p < 0.01) while those of both DNMT3a isoforms were increased (p < 0.01) and significantly correlated with redox potential (r2 > 0.42; p < 0.001). The ratio of SAM (substrate) to SAH (inhibitor) was positively correlated with the redox potential (r2 = 0.36; p < 0.001). In conclusion, early in life, the redox potential value strongly influences the DNA methylation metabolism, resulting in an increase of DNA methylation as a function of increased oxidative stress. These results support the notion that early-life oxidative stress can reprogram the metabolism epigenetically. This study emphasizes once again the importance of improving the quality of parenteral nutrition solutions administered early in life, especially to newborn infants. Abbreviation of Title: Parenteral nutrition and DNA methylation.

DNA Methylation May be Involved in the Analgesic Effect of Hyperbaric Oxygen via Regulating FUNDC1.

Background: Neuropathic pain (NP) is a type of chronic pain which lacks predictable, effective, and safe therapeutic options. We investigated the role of hyperbaric oxygen (HBO) in expression of FUN14 domain-containing 1 (FUNDC1), which is associated with DNA methylation. Methods: We randomly divided rats into four groups: sham operation (S), S + HBO, chronic constriction injury (CCI), and CCI + HBO. Lumbar (L)4 and L5 dorsal root ganglia (DRGs) were used to assess expression of DNA methyltransferase (DNMT)1, DNMT3a, and DNMT3b by western blotting and RT-PCR. Pain-related behaviors were evaluated using mechanical withdrawal threshold and thermal withdrawal latency analysis. Western blotting was also used to assess expression of FUNDC1, BCL2, and adenovirus E1B19 kDa-interacting protein 3-like (NIX) and BCL2 and adenovirus E1B19 kDa-interacting protein3 (BNIP3). And we also examined the changes of FUNDC1 with immunofluorescence. Nonnucleoside DNA methyltransferase inhibitor RG108 was administered prior to CCI. The pain-related behavior and western blotting changes were examined in all groups. Results: DNMT3a expression was higher on day 14 after CCI. HBO downregulated DNMT3a mRNA and protein expression, but not those of DNMT1 and DNMT3b. HBO increased pain-related behavior significantly, while it was down-regulated by RG108. In HBO groups, FUNDC1, NIX, and BNIP3 expression was upregulated more significantly than in the CCI group. In addition, FUNDC1 protein colocalized with NeuN and rarely with glutamine synthetase. However, expression was reduced when RG108 was administered. Immunofluorescence showed that FUNDC1 was upregulated after HBO treatment. Conclusion: Our findings suggest that DNA methylation is involved in the analgesic effect of HBO via the regulation of FUNDC1.

HDAC inhibitors reverse mania-like behavior and modulate epigenetic regulatory enzymes in an animal model of mania induced by Ouabain.

BACKGROUND: The etiology of bipolar disorder (BD) is multifactorial, involving both environmental and genetic factors. Current pharmacological treatment is associated with several side effects, which are the main reason patients discontinue treatment. Epigenetic alterations have been studied for their role in the pathophysiology of BD, as they bridge the gap between gene and environment. OBJECTIVE: Evaluate the effects of histone deacetylase inhibitors on behavior and epigenetic enzymes activity in a rat model of mania induced by ouabain. METHODS: Adult male rats were subjected to a single intracerebroventricular injection of ouabain (10-3 M) followed by 7 days of valproate (200 mg/kg) or sodium butyrate (600 mg/kg) administration. Locomotor and exploratory activities were evaluated in the open-field test. Histone deacetylase, DNA methyltransferase, and histone acetyltransferase activity were assessed in the frontal cortex, hippocampus, and striatum. RESULTS: Ouabain induced hyperactivity in rats, which was reversed by valproate and sodium butyrate treatment. Ouabain did not alter the activity of any of the enzymes evaluated. However, valproate and sodium butyrate decreased the activity of histone deacetylase and DNA methyltransferase. Moreover, there was a positive correlation between these two enzymes. CONCLUSION: These results suggest that targeting epigenetic mechanisms may play an important role in mania-like behavior management.

Bacosides Encapsulated in Lactoferrin Conjugated PEG-PLA-PCL-OH Based Polymersomes Act as Epigenetic Modulator in Chemically Induced Amnesia.

The present study demonstrates the epigenetic mechanisms underlying the effect of Bacoside rich extract of Bacopa monniera-a nootropic herb, on scopolamine treated amnesic mice conferred via chromatin modifying enzymes. The focus of the work was to elucidate the modulation of the chromatin modifying enzymes: DNMT1, DNMT3a, DNMT3b, HDAC2, HDAC5 and CPB in scopolamine induced amnesic mice after treatment with bacoside rich extract of Bacopa monniera (BA) and BA encapsulated in lactoferrin conjugated PEG-PLA-PCL-OH based polymersomes (BAN). We observed remarkable difference between the results obtained after the treatment with BA and BAN. Interestingly BAN was found to be more efficient in downregulating DNA methylation and histone chain deacetylation. Scopolamine treatment showed up-regulation of DNMT1 expression in qRT-PCR by 3.14-fold as compared to the control, which was considerably decreased by 1.5-fold after treatment with BA and remarkably decreased 0.11-fold by BAN treatment. Scopolamine treatment up-regulated the expression of DNMT3a by 1.6-fold while for DNMT3b by 3.13-fold. In DNMT3a and DNMT3b the fold change decreased to 0.64 and 0.76 after BA treatment, whereas the BAN treatment further down-regulated to 0.32- and 0.63-fold, respectively. Similarly scopolamine up-regulated HDAC2 and HDAC5 by 3.12 fold and 3.64-fold, respectively. BA treatment reversed the changes by reducing HDAC2 mRNA to 0.89-fold and HDAC5 mRNA 0.83-fold. BAN further reduced expression of HDAC2 further to 0.39-fold and HDAC5 to 0.31-fold. On the other hand scopolamine down-regulated CBP mRNA expression by 0.28-fold and increased by 1.09 after BA treatment. BAN significantly increased the CPB expression by 1.65-fold as compared to BA treatment. These findings were consolidated by DNMT and HDAC enzyme activity assay, methylation in the promoter region of the memory related genes: ARC and BDNF and Dot blot assay for DNA methylation. The percent activity increase of DNMT and HDAC after scopolamine administration was 375.74 and 240.90 respectively. After treatment with BA the downfall in percent activity was observed as 167.99 in DMNT and 130.57 in HDAC. BAN treatment further decreased the percent enzyme activity of DNMT and HDAC significantly by 30.0 and 61.81 respectively. The potency of BAN in reversing the epigenetic changes of scopolamine induced amnesic mouse brain, can be attributed to the brain specific delivery of BA through polymersomes which are able to cross the blood brain barrier (BBB) via receptor mediated endocytosis.

Developmental changes and sex differences in DNA methylation and demethylation in hypothalamic regions of the mouse brain.

DNA methylation is dynamically modulated during postnatal brain development, and plays a key role in neuronal lineage commitment. This epigenetic mark has also recently been implicated in the development of neural sex differences, many of which are found in the hypothalamus. The level of DNA methylation depends on a balance between the placement of methyl marks by DNA methyltransferases (Dnmts) and their removal, which is catalyzed by ten-eleven translocation (Tet) methylcytosine dioxygenases. Here, we examined developmental changes and sex differences in the expression of Tet and Dnmt enzymes from birth to adulthood in two hypothalamic regions (the preoptic area and ventromedial nucleus) and the hippocampus of mice. We found highest expression of all Tet enzymes (Tet1, Tet2, Tet3) and Dnmts (Dnmt1, Dnmt3a, Dnmt3b) in newborns, despite the fact that global methylation and hydroxymethylation were at their lowest levels at birth. Expression of the Dnmt co-activator, Dnmt3l, followed a pattern opposite to that of the canonical Dnmts (i.e., was very low in newborns and increased with age). Tet enzyme activity was much higher at birth than at weaning in both the hypothalamus and hippocampus, mirroring developmental changes in gene expression. Sex differences in Tet enzyme expression were seen in all brain regions examined during the first week of life, whereas Dnmt expression was more balanced between the sexes. Neonatal testosterone treatment of females only partially masculinized enzyme expression. Thus, Tet expression and activity are elevated during neonatal brain development, and may play important roles in sexual differentiation of the brain.

Danhong Injection Alleviates Cardiac Fibrosis via Preventing the Hypermethylation of Rasal1 and Rassf1 in TAC Mice.

BACKGROUND/AIM: Danhong injection (DHI) is a Chinese patent drug used for relieving cardiovascular diseases. Recent studies have suggested that DNA methylation plays a pivotal role in the maintenance of cardiac fibrosis (CF) in cardiovascular diseases. This study was aimed at identifying the effect and the underlying mechanism of DHI on CF, especially the DNA methylation. METHODS: A CF murine model was established by thoracic aortic constriction (TAC). A 28-day daily treatment with or without DHI via intraperitoneal injection was carried out immediately following TAC surgery. The changes in cardiac function, pathology, and fibrosis following TAC were measured by echocardiography and immunostaining. We used methyl-seq analysis to assess the DNA methylation changes in whole genes and identified the methylation changes of two Ras signaling-related genes in TAC mice, including Ras protein activator like-1 (Rasal1) and Ras-association domain family 1 (Rassf1). Next, the methylation status and expression levels of Rasal1 and Rassf1 genes were consolidated by bisulfite sequencing, quantitative reverse transcription polymerase chain reaction (RT-qPCR), and Western blotting, respectively. To determine the underlying molecular mechanism, the expressions of DNA methyltransferases (DNMTs), Tet methylcytosine dioxygenase 3 (TET3), fibrosis-related genes, and the activity of Ras/ERK were measured by RT-qPCR and Western blotting. RESULTS: DHI treatment alleviated CF and significantly improved cardiac function on day 28 of TAC. The methyl-seq analysis identified 42,606 differential methylated sites (DMSs), including 19,618 hypermethylated DMSs and 22,988 hypomethylated DMSs between TAC and sham-operated mice. The enrichment analysis of these DMSs suggested that the methylated regulation of Ras signal transduction and focal adhesion-related genes would be involved in the TAC-induced CF development. The results of bisulfite sequencing revealed that the TAC-induced methylation affected the CpG site in both of Rasal1 and Rassf1 genes, and DHI treatment remarkably downregulated the promoter methylation of Rasal1 and Rassf1 in CF hearts. Furthermore, DHI treatment upregulated the expressions of Rasal1 and Rassf1, inhibited the hyperactivity of Ras/ERK, and decreased the expressions of fibrosis-related genes. Notably, we found that DHI treatment markedly downregulated the expression of DNMT3B in CF hearts, while it did not affect the expressions of DNMT1, DNMT3A, and TET3. CONCLUSION: Aberrant DNA methylation of Rasal1 and Rassf1 genes was involved in the CF development. DHI treatment alleviated CF, prevented the hypermethylation of Rasal1 and Rassf1, and downregulated DNMT3B expression in CF hearts.

DNA methylation in AgRP neurons regulates voluntary exercise behavior in mice.

DNA methylation regulates cell type-specific gene expression. Here, in a transgenic mouse model, we show that deletion of the gene encoding DNA methyltransferase Dnmt3a in hypothalamic AgRP neurons causes a sedentary phenotype characterized by reduced voluntary exercise and increased adiposity. Whole-genome bisulfite sequencing (WGBS) and transcriptional profiling in neuronal nuclei from the arcuate nucleus of the hypothalamus (ARH) reveal differentially methylated genomic regions and reduced expression of AgRP neuron-associated genes in knockout mice. We use read-level analysis of WGBS data to infer putative ARH neural cell types affected by the knockout, and to localize promoter hypomethylation and increased expression of the growth factor Bmp7 to AgRP neurons, suggesting a role for aberrant TGF-ß signaling in the development of this phenotype. Together, these data demonstrate that DNA methylation in AgRP neurons is required for their normal epigenetic development and neuron-specific gene expression profiles, and regulates voluntary exercise behavior.

Dietary-phytochemical mediated reversion of cancer-specific splicing inhibits Warburg effect in head and neck cancer.

BACKGROUND: The deregulated alternative splicing of key glycolytic enzyme, Pyruvate Kinase muscle isoenzyme (PKM) is implicated in metabolic adaptation of cancer cells. The splicing switch from normal PKM1 to cancer-specific PKM2 isoform allows the cancer cells to meet their energy and biosynthetic demands, thereby facilitating the cancer cells growth. We have investigated the largely unexplored epigenetic mechanism of PKM splicing switch in head and neck cancer (HNC) cells. Considering the reversible nature of epigenetic marks, we have also examined the utility of dietary-phytochemical in reverting the splicing switch from PKM2 to PKM1 isoform and thereby inhibition of HNC tumorigenesis. METHODS: We present HNC-patients samples, showing the splicing-switch from PKM1-isoform to PKM2-isoform analyzed via immunoblotting and qRT-PCR. We performed methylated-DNA-immunoprecipitation to examine the DNA methylation level and chromatin-immunoprecipitation to assess the BORIS (Brother of Regulator of Imprinted Sites) recruitment and polII enrichment. The effect of dietary-phytochemical on the activity of denovo-DNA-methyltransferase-3b (DNMT3B) was detected by DNA-methyltransferase-activity assay. We also analyzed the Warburg effect and growth inhibition using lactate, glucose uptake assay, invasion assay, cell proliferation, and apoptosis assay. The global change in transcriptome upon dietary-phytochemical treatment was assayed using Human Transcriptome Array 2.0 (HTA2.0). RESULTS: Here, we report the role of DNA-methylation mediated recruitment of the BORIS at exon-10 of PKM-gene regulating the alternative-splicing to generate the PKM2-splice-isoform in HNC. Notably, the reversal of Warburg effect was achieved by employing a dietary-phytochemical, which inhibits the DNMT3B, resulting in the reduced DNA-methylation at exon-10 and hence, PKM-splicing switch from cancer-specific PKM2 to normal PKM1. Global-transcriptome-analysis of dietary-phytochemical-treated cells revealed its effect on alternative splicing of various genes involved in HNC. CONCLUSION: This study identifies the epigenetic mechanism of PKM-splicing switch in HNC and reports the role of dietary-phytochemical in reverting the splicing switch from cancer-specific PKM2 to normal PKM1-isoform and hence the reduced Warburg effect and growth inhibition of HNC. We envisage that this approach can provide an effective way to modulate cancer-specific-splicing and thereby aid in the treatment of HNC.

DNMT3b/OCT4 expression confers sorafenib resistance and poor prognosis of hepatocellular carcinoma through IL-6/STAT3 regulation.

BACKGROUND: The inflammatory cytokine interleukin-6 (IL-6) is critical for the expression of octamer-binding transcription factor 4 (OCT4), which is highly associated with early tumor recurrence and poor prognosis of hepatocellular carcinomas (HCC). DNA methyltransferase (DNMT) family is closely linked with OCT4 expression and drug resistance. However, the underlying mechanism regarding the interplay between DNMTs and IL-6-induced OCT4 expression and the sorafenib resistance of HCC remains largely unclear. METHODS: HCC tissue samples were used to examine the association between DNMTs/OCT4 expression levels and clinical prognosis. Serum levels of IL-6 were detected using ELISA assays (n = 144). Gain- and loss-of-function experiments were performed in cell lines and mouse xenograft models to determine the underlying mechanism in vitro and in vivo. RESULTS: We demonstrate that levels of DNA methyltransferase 3 beta (DNMT3b) are significantly correlated with the OCT4 levels in HCC tissues (n = 144), and the OCT4 expression levels are positively associated with the serum IL-6 levels. Higher levels of IL-6, DNMT3b, or OCT4 predicted early HCC recurrence and poor prognosis. We show that IL-6/STAT3 activation increases DNMT3b/1 and OCT4 in HCC. Activated phospho-STAT3 (STAT-Y640F) significantly increased DNMT3b/OCT4, while dominant negative phospho-STAT3 (STAT-Y705F) was suppressive. Inhibiting DNMT3b with RNA interference or nanaomycin A (a selective DNMT3b inhibitor) effectively suppressed the IL-6 or STAT-Y640F-induced increase of DNMT3b-OCT4 and ALDH activity in vitro and in vivo. The fact that OCT4 regulates the DNMT1 expressions were further demonstrated either by OCT4 forced expression or DNMT1 silence. Additionally, the DNMT3b silencing reduced the OCT4 expression in sorafenib-resistant Hep3B cells with or without IL-6 treatment. Notably, targeting DNMT3b with nanaomycin A significantly increased the cell sensitivity to sorafenib, with a synergistic combination index (CI) in sorafenib-resistant Hep3B cells. CONCLUSIONS: The DNMT3b plays a critical role in the IL-6-mediated OCT4 expression and the drug sensitivity of sorafenib-resistant HCC. The p-STAT3 activation increases the DNMT3b/OCT4 which confers the tumor early recurrence and poor prognosis of HCC patients. Findings from this study highlight the significance of IL-6-DNMT3b-mediated OCT4 expressions in future therapeutic target for patients expressing cancer stemness-related properties or sorafenib resistance in HCC.

Extracellular Spermine Activates DNA Methyltransferase 3A and 3B.

We first demonstrated that long-term increased polyamine (spermine, spermidine, putrescine) intake elevated blood spermine levels in mice and humans, and lifelong consumption of polyamine-rich chow inhibited aging-associated increase in aberrant DNA methylation, inhibited aging-associated pathological changes, and extend lifespan of mouse. Because gene methylation status is closely associated with aging-associated conditions and polyamine metabolism is closely associated with regulation of gene methylation, we investigated the effects of extracellular spermine supplementation on substrate concentrations and enzyme activities involved in gene methylation. Jurkat cells and human mammary epithelial cells were cultured with spermine and/or D,L-alpha-difluoromethylornithine (DFMO), an inhibitor of ornithine decarboxylase. Spermine supplementation inhibited enzymatic activities of adenosylmethionine decarboxylase in both cells. The ratio of decarboxylated S-adenosylmethionine to S-adenosyl-L-methionine increased by DFMO and decreased by spermine. In Jurkat cells cultured with DFMO, the protein levels of DNA methyltransferases (DNMTs) 1, 3A and 3B were not changed, however the activity of the three enzymes markedly decreased. The protein levels of these enzymes were not changed by addition of spermine, DNMT 3A and especially 3B were activated. We show that changes in polyamine metabolism dramatically affect substrate concentrations and activities of enzymes involved in gene methylation.

The Effects of Low Selenium on DNA Methylation in the Tissues of Chickens.

DNA methylation is involved in epigenetic mechanisms associated with gene suppression, and its abnormalities lead to gene instability and disease development. As an essential trace element in humans and animals, selenium (Se) is also associated with abnormal changes in DNA methylation. However, the effect of low Se on DNA methylation in avian tissues has not been reported. In the current study, chickens were fed a low-Se diet (0.033 mg Se/kg) or supplemented with 0.15 mg Se/kg as selenite for up to 55 days. DNA methylation levels were examined by high-performance liquid chromatography (HPLC). DNA methyltransferases (DNMTs) and methyl-DpG-binding domain protein 2 (MBD2) mRNA levels were examined through the applications of RT-PCR. The experiment aims to explore the relationship between low Se and DNA methylation. The results showed that total DNA methylation levels in the muscle tissues, brain, immune tissues, and liver of the low-selenium diet group were decreased compared with the control group. The degree of DNA methylation reduction in different tissues from largest to smallest was liver > cerebellum > thymus > brain > spleen ≥ leg muscles > pectoral muscles > bursa of Fabricius > thalamus > wing muscles. DNMT1, DNMT3A, and DNMT3B mRNA expression levels of the low-selenium diet group were decreased compared with those in the control group. The mRNA expression of the MBD2 gene was increased. The results indicate that low Se can reduce the DNA methylation levels of tissues, especially within the liver. These conclusions provide a basis for exploring the pathogenesis of selenium deficiency from the perspective of DNA methylation and create a new basis for comparative medicine.

Regulation of Brain DNA Methylation Factors and of the Orexinergic System by Cocaine and Food Self-Administration.

Inhibitors of DNA methylation and orexin type-1 receptor antagonists modulate the neurobiological effects driving drugs of abuse and natural reinforcers by activating common brain structures of the mesolimbic reward system. In this study, we applied a self-administration paradigm to assess the involvement of factors regulating DNA methylation processes and satiety or appetite signals. These factors include Dnmts and Tets, miR-212/132, orexins, and orx-R1 genes. The study focused on dopamine projection areas such as the prefrontal cortex (PFCx) and caudate putamen (CPu) and in the hypothalamus (HP) that is interconnected with the reward system. Striking changes were observed in response to both reinforcers, but differed depending on contingent and non-contingent delivery. Expression also differed in the PFCx and the CPu. Cocaine and food induced opposite effects on Dnmt3a expression in both brain structures, whereas they repressed both miRs to a different extent, without affecting their primary transcript in the CPu. Unexpectedly, orexin mRNAs were found in the CPu, suggesting a transport from their transcription site in the HP. The orexin receptor1 gene was found to be induced by cocaine in the PFCx, consistent with a regulation by DNA methylation. Global levels of 5-methylcytosines in the PFCx were not significantly altered by cocaine, suggesting that it is rather their distribution that contributes to long-lasting behaviors. Together, our data demonstrate that DNA methylation regulating factors are differentially altered by cocaine and food. At the molecular level, they support the idea that neural circuits activated by both reinforcers do not completely overlap.

Epigenetic control of hypersensitivity in chronic inflammatory pain by the de novo DNA methyltransferase Dnmt3a2.

Chronic pain is a pathological manifestation of neuronal plasticity supported by altered gene transcription in spinal cord neurons that results in long-lasting hypersensitivity. Recently, the concept that epigenetic regulators might be important in pathological pain has emerged, but a clear understanding of the molecular players involved in the process is still lacking. In this study, we linked Dnmt3a2, a synaptic activity-regulated de novo DNA methyltransferase, to chronic inflammatory pain. We observed that Dnmt3a2 levels are increased in the spinal cord of adult mice following plantar injection of Complete Freund's Adjuvant, an in vivo model of chronic inflammatory pain. In vivo knockdown of Dnmt3a2 expression in dorsal horn neurons blunted the induction of genes triggered by Complete Freund's Adjuvant injection. Among the genes whose transcription was found to be influenced by Dnmt3a2 expression in the spinal cord is Ptgs2, encoding for Cox-2, a prime mediator of pain processing. Lowering the levels of Dnmt3a2 prevented the establishment of long-lasting inflammatory hypersensitivity. These results identify Dnmt3a2 as an important epigenetic regulator needed for the establishment of central sensitization. Targeting expression or function of Dnmt3a2 may be suitable for the treatment of chronic pain.

yylncT Defines a Class of Divergently Transcribed lncRNAs and Safeguards the T-mediated Mesodermal Commitment of Human PSCs.

Human protein-coding genes are often accompanied by divergently transcribed non-coding RNAs whose functions, especially in cell fate decisions, are poorly understood. Using an hESC-based cardiac differentiation model, we define a class of divergent lncRNAs, termed yin yang lncRNAs (yylncRNAs), that mirror the cell-type-specific expression pattern of their protein-coding counterparts. yylncRNAs are preferentially encoded from the genomic loci of key developmental cell fate regulators. Most yylncRNAs are spliced polyadenylated transcripts showing comparable expression patterns in vivo in mouse and in human embryos. Signifying their developmental function, the key mesoderm specifier BRACHYURY (T) is accompanied by yylncT, which localizes to the active T locus during mesoderm commitment. yylncT binds the de novo DNA methyltransferase DNMT3B, and its transcript is required for activation of the T locus, with yylncT depletion specifically abolishing mesodermal commitment. Collectively, we report a lncRNA-mediated regulatory layer safeguarding embryonic cell fate transitions.

Counteraction of Oxidative Stress by Vitamin E Affects Epigenetic Regulation by Increasing Global Methylation and Gene Expression of MLH1 and DNMT1 Dose Dependently in Caco-2 Cells.

Obesity- or diabetes-induced oxidative stress is discussed as a major risk factor for DNA damage. Vitamin E and many polyphenols exhibit antioxidative activities with consequences on epigenetic regulation of inflammation and DNA repair. The present study investigated the counteraction of oxidative stress by vitamin E in the colorectal cancer cell line Caco-2 under normal (1 g/l) and high (4.5 g/l) glucose cell culture condition. Malondialdehyde (MDA) as a surrogate marker of lipid peroxidation and reactive oxygen species (ROS) was analyzed. Gene expression and promoter methylation of the DNA repair gene MutL homolog 1 (MLH1) and the DNA methyltransferase 1 (DNMT1) as well as global methylation by LINE-1 were investigated. Results revealed a dose-dependent counteracting effect of vitamin E on H2O2-induced oxidative stress. Thereby, 10 µM vitamin E proved to be more efficient than did 50 µM in reducing MDA. Further, an induction of MLH1 and DNMT1 gene expression was noticed, accompanied by an increase in global methylation. Whether LINE-1 hypomethylation is a cause or effect of oxidative stress is still unclear. In conclusion, supplementation of exogenous antioxidants like vitamin E in vitro exhibits beneficial effects concerning oxidative stress as well as epigenetic regulation involved in DNA repair.