RG108 attenuates acute kidney injury by inhibiting P38 MAPK/FOS and JNK/JUN pathways.

Acute kidney injury (AKI) is an important clinical syndrome characterised by a sudden decline in renal function, often accompanied by renal inflammation and tubular epithelial cell damage. It has been reported that inhibiting DNA methylation significantly suppress the progression of AKI. In the current study, we investigate the effect of the DNA methyltransferase (DNMT) inhibitor RG108 in cisplatin- and hypoxia-reoxygenation-induced AKI. The expression of kidney injury molecules and inflammatory factors was examined by immunofluorescence, Western blotting and Real-time PCR. The results demonstrated that RG108 treatment significantly reduced kidney inflammation and injury. Furthermore, RNA-seq analysis was performed to reveal the regulatory mechanism of RG108 in AKI. The expression of the FOS and JUN genes, which are downstream of the MAPK pathway, were significant increased in AKI. Meanwhile, the expression of FOS and JUN were both inhibited by RG108, which is similar to what we found treatment with a specific JNK inhibitor and a specific p38 MAPK inhibitor, and thus attenuated renal inflammation and injury. In conclusion, we suggest that RG108 inhibits P38 MAPK/FOS and JNK/JUN pathways and attenuates renal injury and inflammatory responses. In these results, RG108 may become a novel MAPK pathway inhibitor and a clinical candidate for the treatment of AKI.

DNA Methyltransferase Inhibition by RG108 Improves Stemness and Multipotential Differentiation of Human Adipose Tissue-derived Stem Cells.

Background: DNA methylation plays important roles in regulating various biological processes, including self-renewal, differentiation and regenerative capacity of stem cells. Previous studies have demonstrated that lineage-specific differentiation of mesenchymal stem cells can be promoted using nontoxic chromatin-modifying drugs. Objectives: Here we evaluated the impact of RG108, a known DNA methyltransferase inhibitor, on the expression of pluripotency genes in human adipose tissue-derived stem cells (hADSCs) and their proliferation and differentiation. Materials and Methods: Human ADSCs were isolated by collagenase treatment and characterized. Then, ADSCs were treated with 5 µM RG108 for four days. The control and RG108-treated cells were analyzed for the cell cycle progression, apoptosis and the expression of pluripotency genes. Also, ADSCs were cultured in adipogenic and osteogenic differentiation media for three weeks and were assessed by Oil Red O and Alizarin Red S staining and qPCR analysis. Results: We showed that RG108 treatment increased proliferation of hADSCs and upregulated the expression of pluripotency-related genes. Additionally, RG108 had a positive impact on the differentiation capability of ADSCs. This was evident through elevated levels of Oil Red O staining in the RG108 treatment group. Also, qPCR analysis showed the upregulation of some adipogenic and osteogenic markers by RG108. Conclusion: These findings indicate that pretreatment with RG108 improves the differentiation potential of ADSCs, probably making these cells more beneficial for cell therapy applications.

DNMT3A dysfunction promotes neuroinflammation and exacerbates acute ischemic stroke.

Somatic mutations related to clonal hematopoiesis of indeterminate potential (CHIP) are risk factors for stroke. The impact of DNMT3A, the most mutated gene in CHIP, on clinical functional outcomes of acute ischemic stroke (AIS) remains unclear. In a well-characterized cohort of 8524 ischemic stroke patients, we demonstrated that DNMT3A-driven CHIP was significantly associated with neurological disability in these patients. With a stroke mouse model of transient middle cerebral artery occlusion (tMCAO), we demonstrated that DNMT3A protein levels in the brain penumbra increased. The DNMT3A inhibitor RG108 administration amplified neutrophil proliferation in the blood, promoted neutrophil infiltration into the brain penumbra, and exaggerated proinflammatory activation in tMCAO male mice. DNMT3A inhibition also significantly increased infarct volume and worsened neurobehavioral function in tMCAO male mice. In conclusion, DNMT3A somatic mutations are associated with worsened neurological disability in some patients with AIS, potentially through increased neutrophil proliferation and infiltration in the ischemic brain region. These findings suggest a possible mechanism for proinflammatory activation and tissue damage in the affected brain tissue, highlighting the need for further research in this area.

DNA Methylation Inhibition Reversibly Impairs the Long-Term Context Memory Maintenance in Helix.

This work aims to study the epigenetic mechanisms of regulating long-term context memory in the gastropod mollusk: Helix. We have shown that RG108, an inhibitor of DNA methyltransferase (DNMT), impaired long-term context memory in snails, and this impairment can be reversed within a limited time window: no more than 48 h. Research on the mechanisms through which the long-term context memory impaired by DNMT inhibition could be reinstated demonstrated that this effect depends on several biochemical mechanisms: nitric oxide synthesis, protein synthesis, and activity of the serotonergic system. Memory recovery did not occur if at least one of these mechanisms was impaired. The need for the joint synergic activity of several biochemical systems for a successful memory rescue confirms the assumption that the memory recovery process depends on the process of active reconsolidation, and is not simply a passive weakening of the effect of RG108 over time. Finally, we showed that the reactivation of the impaired memory by RG108, followed by administration of histone deacetylase inhibitor sodium butyrate, led to memory recovery only within a narrow time window: no more than 48 h after memory disruption.

Deciphering the potential ability of RG108 in cisplatin-induced HEI-OC1 ototoxicity: a research based on RNA-seq and molecular biology experiment.

BACKGROUND: Drug-induced hearing loss (DIHL) is very common, and seriously affects people's happiness in life. RG108 is a small molecule inhibitor. RG108 is protective against DIHL. Our purpose is to probe the incidence of RG108 on cisplatin-induced ototoxicity. MATERIALS AND METHODS: In our research, the ototoxicity of RG108 was investigated in HEI-OC1. We observed under the microscope whether RG108 had an effect on cisplatin-induced cochlear hair cells. RNA-seq experiments were further performed to explore possible gene ontology (GO) and pathways. ROS assay was applied to supervisory the effect of RG108 on oxidative harm of auditory cells. In auditory cells, RG108 was tested for its effects on apoptosis-related proteins by Western blotting (WB). RESULTS: GO analysis showed that RG108 associated with apoptosis. KEGG analysis shows RG108 may act on PI3K-AKT signaling pathway (PASP) in hearing loss. BIOCARTA analysis showed that RG108 may affect oxidative stress by activating NRF2 pathway. ROS ascerted that RG108 could rescue oxidative harm in HEI-OC1. RG108 rescued cisplatin-induced significant increase in Bax and significant decrease in BCL2. RG108 attenuates cisplatin-induced cochlear apoptosis through upregulated phosphorylated PI3K and phosphorylated AKT and down-regulated caspase3. MTT experiments showed that both PI3K and AKT inhibitors could significantly rescue the damage caused by cisplatin to HEI-OC1. RG108 significantly increases the level of NRF2/HO-1/NQO1 in cisplatin-induced cells. CONCLUSION: Overall, these results provide evidence that NRF2/PI3K-AKT axis may mediate RG108 in the treatment of DIHL, which provide a broader outlook on drug-induced deafness treatment.

Effects of DNA methylase inhibitors in a murine model of severe BPD.

DNA methylation is necessary for developmental gene regulation, but adverse environments result in aberrant methylation and gene silencing. The current pilot study tested the hypothesis that treatment with DNA methylation inhibitors (decitabine; RG108) would improve alveolarization in a newborn murine model of severe bronchopulmonary dysplasia. Newborn mice exposed to maternal inflammation (LPS) and neonatal hyperoxia (85% O2) were treated with decitabine (p3, 0.1 mg/kg; p2, 4, 6, 0.1 mg/kg; or p2, 4, 6, 0.15 mg/kg) or RG108 (p3, 0.0013 mg/kg) delivered intranasally. Modest improvements in alveolarization were observed with decitabine, but no differences were observed with RG108. Attenuated phospho-SMAD2/3 levels and greater surfactant protein C protein levels compared to vehicle were observed with some tested doses. No detrimental side effects were observed with the doses used in this study. In summary, our pilot investigations identified a safe dose for intranasal administration of both methylation inhibitors and provides a foundation for further studies into methylation inhibitors in the context of neonatal lung injury.

Inhibiting DNA methylation alleviates cisplatin-induced hearing loss by decreasing oxidative stress-induced mitochondria-dependent apoptosis via the LRP1-PI3K/AKT pathway.

Cisplatin-related ototoxicity is a critical side effect of chemotherapy and can lead to irreversible hearing loss. This study aimed to assess the potential effect of the DNA methyltransferase (DNMT) inhibitor RG108 on cisplatin-induced ototoxicity. Immunohistochemistry, apoptosis assay, and auditory brainstem response (ABR) were employed to determine the impacts of RG108 on cisplatin-induced injury in murine hair cells (HCs) and spiral ganglion neurons (SGNs). Rhodamine 123 and TMRM were utilized for mitochondrial membrane potential (MMP) assessment. Reactive oxygen species (ROS) amounts were evaluated by Cellrox green and Mitosox-red probes. Mitochondrial respiratory function evaluation was performed by determining oxygen consumption rates (OCRs). The results showed that RG108 can markedly reduce cisplatin induced damage in HCs and SGNs, and alleviate apoptotic rate by protecting mitochondrial function through preventing ROS accumulation. Furthermore, RG108 upregulated BCL-2 and downregulated APAF1, BAX, and BAD in HEI-OC1 cells, and triggered the PI3K/AKT pathway. Decreased expression of low-density lipoprotein receptor-related protein 1 (LRP1) and high methylation of the LRP1 promoter were observed after cisplatin treatment. RG108 treatment can increase LRP1 expression and decrease LRP1 promoter methylation. In conclusion, RG108 might represent a new potential agent for preventing hearing loss induced by cisplatin via activating the LRP1-PI3K/AKT pathway.

Epigenetic reprogramming in cloned mouse embryos following treatment with DNA methyltransferase and histone deacetylase inhibitors.

We examined the effects of DNA methyltransferase inhibitor - RG108, and histone deacetylase inhibitor - SAHA, on the reprogramming parameters of cloned mouse embryos produced by somatic cell nuclear transfer into oocytes. The programming parameters studied included dynamics of histone reacetylation, developmental rate, DNA methylation, and transcript levels of genes, all of which are pivotal to lineage specification and blastocyst formation. At the pronuclear stage, somatic nucleus-transplanted oocytes treated with 5 µM SAHA presented higher histone acetylation at H3K9, H3K14, H4K16 and H4K12, compared to untreated clones (p < 0.05). At the morula stage, cloned embryos treated with 5 µM RG108 or 5 µM SAHA presented lower DNA methylation intensity compared to untreated clones (p < 0.05), resembling the intensity levels of fertilized embryos. However, these effects were not observed when RG108 and SAHA were used in combination. The rate of morula formation was significantly higher in cloned embryos treated with 5 µM SAHA than in untreated clones, whereas treatment with RG108 resulted in no obvious effects on morula formation rates. On the other hand, the combined treatment with RG108 and SAHA resulted in inferior rates of cloned morula formation, compared to untreated clones. At the blastocyst stage, the aberrant expression levels of key developmental genes Oct4 and Cdx2, but not Nanog, were corrected in cloned embryos by the treatment with RG108. This is similar to the intensity levels seen in fertilized embryos. The expression of Rpl7l1 gene was significantly higher in embryos treated with both RG108 and SAHA than in untreated and in control groups. In summary, the present study showed that SAHA and RG108, when applied separately, improve the rate and quality of cloned mouse embryos.

DNA methylation inhibitors: Retrospective and perspective view.

DNA methylation is an epigenetic modification that contributes to essential biological processes such as retrotransposon silencing, cell differentiation, genomic imprinting and X-chromosome inactivation. DNA methylation generates a stable epigenetic mark associated with silencing of gene expression. Aberrant DNA methylation is associated with the development of different tumor types. Reversing DNA methylation is a rational strategy to restore gene re-expression and induce cell differentiation in cancer. DNA hypomethylating agents is a class of drugs that demonstrated efficacy in different tumors. In this chapter, the classification of DNA hypomethylating agents, their pharmacodynamics and their potential drawbacks will be discussed.

DNMT1 Inhibitor Restores RUNX2 Expression and Mineralization in Periodontal Ligament Cells.

Periodontal ligament cells (PDLCs) have well documented osteogenic potential; however, this commitment can be highly heterogenous, limiting their applications in tissue regeneration. In this study, we use PDLC populations characterized by high and low osteogenic potential (h-PDLCs and l-PDLCs, respectively) to identify possible sources of such heterogeneity and to investigate whether the osteogenic differentiation can be enhanced by epigenetic modulation. In h-PDLCs, low basal expression levels of pluripotency markers (NANOG, OCT4), DNA methyltransferases (DNMT1, DNMT3B), and enzymes involved in active DNA demethylation (TET1, TET3) were prerequisite to high osteogenic potential. Furthermore, these genes were downregulated upon early osteogenesis, possibly allowing for the increase in expression of the key osteogenic transcription factors, Runt-related transcription factor 2 (RUNX2) and SP7, and ultimately, mineral nodule formation. l-PDLCs appeared locked in the multipotent state and this was further enhanced upon early osteogenic stimulation, correlating with low RUNX2 expression and impaired mineralization. Further upregulation of DNMTs was also evident, while pretreatment with RG108, the DNMTs' inhibitor, enhanced the osteogenic program in l-PDLCs through downregulation of DNMTs, increased RUNX2 expression and nuclear localization, accelerated expression of osteogenic markers, and increased mineralization. These findings point toward the role of DNMTs and Ten Eleven Translocations (TETs) in osteogenic commitment and support application of epigenetic approaches to modulate biomineralization in PDLCs.

The Impact of EGCG and RG108 on SOCS1 Promoter DNA Methylation and Expression in U937 Leukemia Cells.

BACKGROUND: The available evidence has increasingly demonstrated that a combination of genetic and epigenetic factors, such as DNA methylation, could be considered as causing leukemia. Epigenetic changes and methylation of the suppressor of the cytokine signaling 1 promoter (SOCS1) CpG region silence SOCS1 expression in cancer. In the current study, we evaluated the impact of epigallocatechin gallate (EGCG) and RG108 on SOCS1 promoter methylation and expression in U937 cells. METHODS: In the current study, U937 leukemic cells were treated with EGCG and RG108 for 12, 24, 48, and 72 h and SOCS1 promoter methylation and its expression were measured by methylation-specific PCR (MSP) and quantitative real-time PCR, respectively. RESULTS: The outcomes indicated that the SOCS1 promoter is methylated in U937 cells, and treatment of these cells with either EGCG or RG108 reduced its methylation. Moreover, we observed that SOCS1 expression was significantly upregulated in a time-dependent manner by both EGCG and RG108 in U937 cells compared with control cells. In the RG108-treated group at 12, 24, 48, and 72 h, SOCS1 expression was upregulated by 1, 4.2, 16.6, and 32.6 -fold respectively, and in the EGCG-treated group, by 0.5, 3.2, 10.8, and 22.3 -fold, respectively. CONCLUSION: Treatment with either EGCG or RG108 reduced SOCS1 promoter methylation and increased SOCS1 expression in U937 cells in a time-dependent manner, which may play a role in leukemia therapy.

Modulation of DNA Methylation and Gene Expression in Rodent Cortical Neuroplasticity Pathways Exerts Rapid Antidepressant-Like Effects.

BACKGROUND: Stress increases DNA methylation, primarily a suppressive epigenetic mechanism catalyzed by DNA methyltransferases (DNMT), and decreases the expression of genes involved in neuronal plasticity and mood regulation. Despite chronic antidepressant treatment decreases stress-induced DNA methylation, it is not known whether inhibition of DNMT would convey rapid antidepressant-like effects. AIM: This work tested such a hypothesis and evaluated whether a behavioral effect induced by DNMT inhibitors (DNMTi) corresponds with changes in DNA methylation and transcript levels in genes consistently associated with the neurobiology of depression and synaptic plasticity (BDNF, TrkB, 5-HT1A, NMDA, and AMPA). METHODS: Male Wistar rats received intraperitoneal (i.p.) injection of two pharmacologically different DNMTi (5-AzaD 0.2 and 0.6 mg/kg or RG108 0.6 mg/kg) or vehicle (1 ml/kg), 1 h or 7 days before the learned helplessness test (LH). DNA methylation in target genes and the correspondent transcript levels were measured in the hippocampus (HPC) and prefrontal cortex (PFC) using meDIP-qPCR. In parallel separate groups, the antidepressant-like effect of 5-AzaD and RG108 was investigated in the forced swimming test (FST). The involvement of cortical BDNF-TrkB-mTOR pathways was assessed by intra-ventral medial PFC (vmPFC) injections of rapamycin (mTOR inhibitor), K252a (TrkB receptor antagonist), or vehicle (0.2 µl/side). RESULTS: We found that both 5-AzaD and RG108 acutely and 7 days before the test decreased escape failures in the LH. LH stress increased DNA methylation and decreased transcript levels of BDNF IV and TrkB in the PFC, effects that were not significantly attenuated by RG108 treatment. The systemic administration of 5-AzaD (0.2 mg/kg) and RG108 (0.2 mg/kg) induced an antidepressant-like effect in FST, which was, however, attenuated by TrkB and mTOR inhibition into the vmPFC. CONCLUSION: These findings suggest that acute inhibition of stress-induced DNA methylation promotes rapid and sustained antidepressant effects associated with increased BDNF-TrkB-mTOR signaling in the PFC.

Inhibition of DNA Methyltransferase by RG108 Promotes Pluripotency-Related Character of Porcine Bone Marrow Mesenchymal Stem Cells.

Mesenchymal stem/stromal cells (MSCs) have been identified in almost all adult human tissues and been used in numerous clinical trials for a variety of diseases. Studies have shown that MSCs would undergo cellular senescence when cultured over a long term, which is brought on by increased epigenetic modifications, including DNA methylation. However, the mechanism of MSCs senescence is not well studied. In this study, the effects of RG108, a DNA methyltransferase inhibitor (DNMTi), on senescence, apoptosis, and pluripotency gene expressions in porcine bone marrow (pBM)-MSCs were investigated. First, we determined the optimized dose and time of RG108 treatment in pBM-MSCs to be 10 µM for 48 hours, respectively. Under these conditions, the pluripotency genes (NANOG, POU5F1), the anti-senescence genes (TERT, bFGF), and the anti-apoptosis gene (BCL2) were increased, whereas the apoptotic gene (BAX) was decreased. RG108 protected against apoptosis when pBM-MSC induces apoptosis with H2O2 for 1.5 hours. We also found that RG108 significantly induced the expression of NANOG and POU5F1 by decreasing DNA methylation in gene promoter regions. These results indicate that an optimized dose of RG108 may promote the pluripotency-related character of pBM-MSCs through improving cellular anti-senescence, anti-apoptosis, and pluripotency, which provide a better cell origin for stem cell therapy.

The role of DNA methyltransferase activity in cocaine treatment and withdrawal in the nucleus accumbens of mice.

An increasing number of reports have provided crucial evidence that epigenetic modifications, such as DNA methylation, may be involved in initiating and establishing psychostimulant-induced stable changes at the cellular level by coordinating the expression of gene networks, which then manifests as long-term behavioral changes. In this study, we evaluated the enzyme activity of DNA methyltransferases (DNMTs) after cocaine treatment and during withdrawal. Furthermore, we studied how genetic or pharmacological inhibition of DNMTs in mouse nucleus accumbens (NAc) affects the induction and expression of cocaine-induced behavioral sensitization. Our results showed that after silencing Dnmt3a in the NAc during the induction phase of cocaine-induced sensitization, overall DNMT activity decreases, correlating negatively with behavioral sensitization. Reduced Dnmt3a mRNA during this phase was the largest contributing factor for decreased DNMT activity. Cocaine withdrawal and a challenge dose increased DNMT activity in the NAc, which was associated with the expression of behavioral sensitization. Long-term selective Dnmt3a transcription silencing in the NAc did not alter DNMT activity or the expression of cocaine-induced behavioral sensitization. However, bilateral intra-NAc injection of a non-specific inhibitor of DNMT (RG108) during withdrawal from cocaine decreased DNMT activity in the NAc and had a small effect on the expression of cocaine-induced behavioral sensitization. Thus, cocaine treatment and withdrawal is associated with biphasic changes in DNMT activity in the NAc, and the expression of behavioral sensitization decreases with non-selective inhibition of DNMT but not with selective silencing of Dnmt3a.

RG108 affects the proliferation and apoptosis of NSCLC cells by regulating TFPI-2 methylation and TMPRSS4 expression / 中国肿瘤生物治疗杂志

@#[Abstract] Objective: To investigate the effect of RG108 on the proliferation and apoptosis of human non-small cell lung cancer (NSCLC) cell lines (A549, H1299) and explore its molecular mechanism. Methods: A549 and H1299 cells were cultured in vitro and treated with different concentrations of RG108. The cell proliferation, cell cycle and apoptosis were detected by MTT assay and Flow cytometry, respectively. qPCR and Western blotting (WB) were used to detect the TFPI-2 mRNA and protein expressions as well as the expression of TMPRSS4 in cells. Meanwhile, the methylation status and degree of TFPI-2 promoter in cells were detected with Methylation-specific PCR (MSP) and colorimetry. Finally, siRNA-TFPI-2 and pcDNA3.0-TMPRSS4 plasmids were used to silence TFPI-2 or overexpress TMPRSS4, and then the changes in cell proliferation and apoptosis were detected. Results: After treatment with RG108, the proliferation rate of A549 and H1299 cells were significantly decreased (all P<0.05), while the apoptosis rate were significantly increased(P<0.05), the cell cycle were arrested in G1/S phase (P<0.05), and the intracellular mRNA and protein expressions of TFPI-2 were significantly increased (P<0.01 or P<0.05). Meanwhile, the methylase degree in TFPI-2 promoter region and the expression of TMPRSS4 in cells were all significanly decreased ( all P<0.05). After TFPI-2 silence, the proliferation levels of A549 and H1299 cells were significantly increased(all P<0.05); however, the apoptosis rate of A549 and H1299 cells were significantly reduced after transfection with pcDNA3.0-TMPRSS4(all P<0.05). Conclusion: RG108 can inhibit proliferation of A549 and H1299 cells and promote apoptosis by inhibiting the methylation of TFPI-2 and negatively regulates the expression of TMPRSS4.

Optimizing treatment of DNA methyltransferase inhibitor RG108 on porcine fibroblasts for somatic cell nuclear transfer.

Aberration in DNA methylation is believed to be one of the major causes of abnormal gene expression and inefficiency of somatic cell nuclear transfer (SCNT). RG108, a non-nucleoside DNA methyltransferase (DNMT) inhibitor, has been reported to facilitate somatic nuclear reprogramming and improved blastocyst formation. The aim of this study was to investigate interaction effect of RG108 treatment time (24-72 hr) and concentrations (0.05-50 µM) on donor cells, and further to optimize the treatment for porcine SCNT. Our results showed that RG108 treatment resulted in time-dependent decrease of genome-wide DNA methylation on foetal fibroblasts, which only happened after 72-hr treatment in our experiments, and no interaction effect between treatment time and concentration. Remarkable decrease of methylation in imprinted gene H19 and increased apoptosis was observed in 5 and 50 µM RG108-treated cells. Furthermore, the blastocyst rates of SCNT embryos were increased as the fibroblasts treated with RG108 at 5 and 50 µM, and additional treatment during cultivation of SCNT embryos would not provide any advantage for blastocyst formation. In conclusion, the RG108 treatment of 72 hr and 5 µM would be optimized time and concentration for porcine foetal fibroblasts to improve the SCNT embryonic development. In addition, combined treatment of RG108 on donor cells and SCNT embryos would not be beneficial for embryonic development.

DNA methylation inhibitor attenuates polyglutamine-induced neurodegeneration by regulating Hes5.

Spinal and bulbar muscular atrophy (SBMA) is a polyglutamine-mediated neuromuscular disease caused by a CAG repeat expansion in the androgen receptor (AR) gene. While transcriptional dysregulation is known to play a critical role in the pathogenesis of SBMA, the underlying molecular pathomechanisms remain unclear. DNA methylation is a fundamental epigenetic modification that silences the transcription of various genes that have a CpG-rich promoter. Here, we showed that DNA methyltransferase 1 (Dnmt1) is highly expressed in the spinal motor neurons of an SBMA mouse model and in patients with SBMA. Both genetic Dnmt1 depletion and treatment with RG108, a DNA methylation inhibitor, ameliorated the viability of SBMA model cells. Furthermore, a continuous intracerebroventricular injection of RG108 mitigated the phenotype of SBMA mice. DNA methylation array analysis identified hairy and enhancer of split 5 (Hes5) as having a CpG island with hyper-methylation in the promoter region, and the Hes5 expression was strongly silenced in SBMA. Moreover, Hes5 over-expression rescued the SBMA cells possibly by inducing Smad2 phosphorylation. Our findings suggest DNA hyper-methylation underlies the neurodegeneration in SBMA.

Dynamic Methylation Changes of DNA and H3K4 by RG108 Improve Epigenetic Reprogramming of Somatic Cell Nuclear Transfer Embryos in Pigs.

BACKGROUND/AIMS: DNA methylation and histone modifications are essential epigenetic marks that can significantly affect the mammalian somatic cell nuclear transfer (SCNT) embryo development. However, the mechanisms by which the DNA methylation affects the epigenetic reprogramming have not been fully elucidated. METHODS: In our study, we used quantitative polymerase chain reaction (qPCR), Western blotting, immunofluorescence staining (IF) and sodium bisulfite genomic sequencing to examine the effects of RG108, a DNA methyltransferase inhibitor (DNMTi), on the dynamic pattern of DNA methylation and histone modifications in porcine SCNT embryos and investigate the mechanism by which the epigenome status of donor cells' affects SCNT embryos development and the crosstalk between epigenetic signals. RESULTS: Our results showed that active DNA demethylation was enhanced by the significantly improving expression levels of TET1, TET2, TET3 and 5hmC, and passive DNA demethylation was promoted by the remarkably inhibitory expression levels of DNMT1, DNMT3A and 5mC in embryos constructed from the fetal fibroblasts (FFs) treated with RG108 (RG-SCNT embryos) compared to the levels in embryos from control FFs (FF-SCNT embryos). The signal intensity of histone H3 lysine 4 trimethylation (H3K4me3) and histone H3 lysine 9 acetylation (H3K9Ac) was significantly increased and the expression levels of H3K4 methyltransferases were more than 2-fold higher expression in RG-SCNT embryos. RG-SCNT embryos had significantly higher cleavage and blastocyst rates (69.3±1.4%, and 24.72±2.3%, respectively) than FF-SCNT embryos (60.1±2.4% and 18.38±1.9%, respectively). CONCLUSION: Dynamic changes in DNA methylation caused by RG108 result in dynamic alterations in the patterns of H3K4me3, H3K9Ac and histone H3 lysine 9 trimethylation (H3K9me3), which leads to the activation of embryonic genome and epigenetic modification enzymes associated with H3K4 methylation, and contributes to reconstructing normal epigenetic modifications and improving the developmental efficiency of porcine SCNT embryos.

RASgrf1, a Potential Methylatic Mediator of Anti-epileptogenesis?

Epileptogenesis, induced by status epilepticus (SE), is a chronic process, and intervention in this progress may prevent chronic epilepsy. It has been proposed that DNA methylation might be related with epileptogenesis. RASgrf1 has a differentially methylated region at the promoter which can silence gene expression. We have previously observed the down-regulation of RASgrf1 in epilepsy patients and proved that hypermethylation of RASgrf1 reaches maximal level at the latent period in mice after kainate-induced SE (KA mice), with corresponding alteration of RASgrf1 expression. In the present study, N-phthalyl-L-tryptophan (RG108), a DNA methyltransferase inhibitor, was applied in KA mice at latent phase and the behavior, electroencephalogram and pathological changes were observed in chronic phase. Methylation and expression of RASgrf1 were determined by polymerase chain reaction (PCR), western blotting, and bisulfite sequencing PCR. The results showed that the incidence of spontaneous recurrent seizures (SRS) was significantly lower in the RG108 group than the normal saline (NS) group. Subgroup analysis showed significant hypermethylation and lower expression of RASgrf1 in the RG108-SRS subgroup and the NS-SRS subgroup but not in the RG108-NSRS (no SRS) subgroup and the NS-NSRS subgroup compared with the control group. No significant difference was found between the RG108-SRS and NS-SRS subgroups. Meanwhile, hippocampal neuronal loss was observed in RG108-SRS and NS-SRS subgroups. We thus demonstrated that RG108 could modify the progression of epileptogenesis after KA induced SE and prevent chronic epilepsy. Meanwhile, hypermethylation of RASgrf1 after KA induced SE could be reversed with corresponding changes of RASgrf1 expression. Additionally, we speculated that RASgrf1 might be a potential epigenetic mediator in epileptogenesis and chronic epilepsy.

Epigenetic modulation by small molecule compounds for neurodegenerative disorders.

The accumulation of somatic and genetic mutations which altered the structure and coding information of the DNA are the major cause of neurological disorders. However, our recent understanding of molecular mechanisms of 'epigenetic' phenomenon reveals that the modifications of chromatin play a significant role in the development and severity of neurological disorders. These epigenetic processes are dynamic and reversible as compared to genetic ablations which are stable and irreversible. Therefore, targeting these epigenetic processes through small molecule modulators are of great therapeutic potential. To date, large number of small molecule modulators have been discovered which are capable of altering the brain pathology by targeting epigenetic enzymes. In this review, we shall put forward the key studies supporting the role of altered epigenetic processes in neurological disorders with especial emphasis on neurodegenerative disorders. A few small molecule modulators which have been shown to possess promising results in the animal model system of neurological disorders will also be discussed with future perspectives.