Reduced DNA Methylation Research Articles

DNMT3A Limits Myeloid Signaling Responses in Committed T Cells during Normal and Leukemic Development

T-cell development and lineage commitment are temporally protracted processes in which the interplay between transcription factors and epigenetic modulators coordinate the sequential exclusion of alternative fates and acquisition of specialized T-cell functions. Alterations during this process can lead to diseases such as T-cell acute lymphoblastic leukemia (T-ALL). While many efforts have focused on the transcription factors which drive normal development and leukemogenesis, the underlying epigenetic constraints remain poorly understood. Further, characterization of the role of particular factors during early stages of human T-cell development is limited by difficulty in obtaining and manipulating primary thymic tissue. Here, we characterize T-cell development as modeled in a chemically defined in vitro differentiation system using primary human CD34+ cord blood (CB) as input material (StemSpan T Cell Generation Kit, Stem Cell Technologies). This culture system yields large numbers of genetically manipulable cells that recapitulate aspects of normal intrathymic development based on immunophenotype, gene expression profiling, T-cell receptor rearrangement, and lineage restriction. DNMT3A is recurrently mutated in human T-ALL with identified variants thought to result in protein loss-of-function (LOF). As well, DNMT3A mutations are enriched in ETP-TALL, a phenotypic subtype with transcriptional signature resembling stem/myeloid-like progenitors. We thus sought to explore the role of DNMT3A in early stages of T cell lineage commitment using the in vitro CB differentiation system in combination with lentiviral shRNA-mediated knockdown (KD) and CRISPR/Cas9-mediated knockout (KO) to mimic DNMT3A LOF mutations. To assess durability of commitment to the T lineage, we transferred cells at the post-commitment CD44- stage from culture conditions supporting T cell differentiation to conditions favoring myeloid differentiation (StemSpan Myeloid Expansion Supplements I and II for granulocytes and monocytes, respectively). We found using clonogenic cell growth assays performed at limiting dilution that T cells with DNMT3A KD/KO were able to grow in myeloid media and expressed an aberrant T/myeloid phenotype at significantly higher frequency as compared to negative controls. Interestingly, this lineage switching activity was nearly exclusively represented in a small subset of cells that expressed myeloid growth factor receptors on the cell surface which were present in both KD and control in vitro derived T cells, as well as in normal thymus at similar frequencies. Minor subsets of myeloid receptor positive cells are also identifiable in established human T-ALL cell lines, and similar to in vitro derived T cells, the receptor positive subset of DNMT3A KD cells showed preferential growth in response to stimulation with their cognate ligands. Phosphoflow assays demonstrated that KD and non-silencing/negative control cells showed similar phosphoprotein signaling responses upon myeloid growth factor stimulation, suggesting that growth responses in KD cells are enacted at the DNA level, presumably by reduced DNA methylation at target gene promoters. These results support a working model in which DNMT3A loss in T-cell progenitors restores their ability to respond positively to myeloid growth factor stimulation both in terms of cell growth and myeloid marker expression. Of note, interrogation of clonal progeny from individual DNMT3A KO cells reveal no evidence of differentiation delay or arrest under T-tropic culture conditions, suggesting that responses to myeloid-tropic conditions are due to reprogramming of T-committed cells rather than increased persistence of uncommitted cells. These processes also appear to be operative in established T-ALL cells, suggesting that DNMT3A LOF mutation may afford T-ALL cells the ability to exploit alternate myeloid factor replete niches in the body.

KCNQ1OT1 promotes genome-wide transposon repression by guiding RNA-DNA triplexes and HP1 binding.

Transposon (de)repression and heterochromatin reorganization are dynamically regulated during cell fate determination and are hallmarks of cellular senescence. However, whether they are sequence specifically regulated remains unknown. Here we uncover that the KCNQ1OT1 lncRNA, by sequence-specific Hoogsteen base pairing with double-stranded genomic DNA via its repeat-rich region and binding to the heterochromatin protein HP1α, guides, induces and maintains epigenetic silencing at specific repetitive DNA elements. Repressing KCNQ1OT1 or deleting its repeat-rich region reduces DNA methylation and H3K9me3 on KCNQ1OT1-targeted transposons. Engineering a fusion KCNQ1OT1 with an ectopically targeting guiding triplex sequence induces de novo DNA methylation at the target site. Phenotypically, repressing KCNQ1OT1 induces senescence-associated heterochromatin foci, transposon activation and retrotransposition as well as cellular senescence, demonstrating an essential role of KCNQ1OT1 to safeguard against genome instability and senescence.

Integrative Analysis Identifies TCIRG1 as a Potential Prognostic and Immunotherapy-Relevant Biomarker Associated with Malignant Cell Migration in Clear Cell Renal Cell Carcinoma.

Simple SummaryTCIRG1, also known as V-ATPase-a3, is critical for cellular metabolism, membrane transport, and intracellular signaling through its dependent acidification. In earlier research, TCIRG1 was found to be dysregulated in several cancers and to accelerate the growth of various malignancies. The molecular mechanisms behind TCIRG1 and its possible role in the development of clear cell renal cell carcinoma are still poorly understood. Our research is the first to thoroughly examine TCIRG1’s function in clear cell renal cell carcinoma prognosis, immunity, and treatment. The validity that TCIRG1 can accelerate the development of renal clear cell carcinoma was also confirmed in this work by using certain testable experiments. This establishes the theoretical framework for our future investigation into the occurrence and progression of clear cell renal cell carcinoma.Background: TCIRG1, also known as V-ATPase-a3, is critical for cellular life activities through its dependent acidification. Prior to the present research, its relationship with prognostic and tumor immunity in clear cell renal cell carcinoma (ccRCC) had not yet been investigated. Methods: We assessed TCIRG1 expression in normal and tumor tissues using data from TCGA, GEO, GTEX, and IHC. We also analyzed the relationship between TCIRG1 and somatic mutations, TMB, DNA methylation, cancer stemness, and immune infiltration. We evaluated the relevance of TCIRG1 to immunotherapy and potential drugs. Finally, we explored the effect of TCIRG1 knockdown on tumor cells. Results: TCIRG1 was overexpressed in tumor tissue and predicted a significantly unfavorable clinical outcome. High TCIRG1 expression may be associated with fewer PBRM1 and more BAP1 mutations and may reduce DNA methylation, thus leading to a poor prognosis. TCIRG1 was strongly associated with CD8+ T-cell, Treg, and CD4+ T-cell infiltration. Moreover, TCIRG1 was positively correlated with TIDE scores and many drug sensitivities. Finally, experiments showed that the knockdown of TCIRG1 inhibited the migration of ccRCC cells. Conclusions: TCIRG1 may have great potential in identifying prognostic and immunomodulatory mechanisms in tumor patients and may provide a new therapeutic strategy for ccRCC.

Dominant role of DNA methylation over H3K9me3 for IAP silencing in endoderm

Silencing of endogenous retroviruses (ERVs) is largely mediated by repressive chromatin modifications H3K9me3 and DNA methylation. On ERVs, these modifications are mainly deposited by the histone methyltransferase Setdb1 and by the maintenance DNA methyltransferase Dnmt1. Knock-out of either Setdb1 or Dnmt1 leads to ERV de-repression in various cell types. However, it is currently not known if H3K9me3 and DNA methylation depend on each other for ERV silencing. Here we show that conditional knock-out of Setdb1 in mouse embryonic endoderm results in ERV de-repression in visceral endoderm (VE) descendants and does not occur in definitive endoderm (DE). Deletion of Setdb1 in VE progenitors results in loss of H3K9me3 and reduced DNA methylation of Intracisternal A-particle (IAP) elements, consistent with up-regulation of this ERV family. In DE, loss of Setdb1 does not affect H3K9me3 nor DNA methylation, suggesting Setdb1-independent pathways for maintaining these modifications. Importantly, Dnmt1 knock-out results in IAP de-repression in both visceral and definitive endoderm cells, while H3K9me3 is unaltered. Thus, our data suggest a dominant role of DNA methylation over H3K9me3 for IAP silencing in endoderm cells. Our findings suggest that Setdb1-meditated H3K9me3 is not sufficient for IAP silencing, but rather critical for maintaining high DNA methylation.

Ficus dubia latex extract prevent DMH-induced rat early colorectal carcinogenesis through the regulation of xenobiotic metabolism, inflammation, cell proliferation and apoptosis

Ficus dubia latex is recognized as a remedy in Asian traditional medicine with various therapeutic effects. The present study aimed to determine the preventive action of Ficus dubia latex extract (FDLE) on 1,2-dimethylhydrazine (DMH)-induced rat colorectal carcinogenesis and its mechanisms. The experiment included an initiation model in which rats were orally administered with FDLE daily for 1 week before DMH injection until the end of the experiment, while only after DMH injection until the end in the post-initiation model. The results firstly indicated that FDLE treatment could reduce the level of methylazoxymethanol (MAM) in rat colonic lumen by inhibition of the activities of both phase I xenobiotic metabolizing enzymes in the liver and β-glucuronidase in the colon, leading to reduced DNA methylation in colonic mucosal cells, related to the number of ACF in the initiation stage. Besides, FDLE modulated the inflammation which could suppress the growth and induce apoptosis of aberrant colonic mucosal cells, leading to retardation of ACF multiplicity. Therefore, FDLE showed the ability to suppress the DMH-induced rat ACF formation and inflammation promoted growth of ACF. In conclusion, FDLE had the potential to prevent carcinogens-induced rat colorectal carcinogenesis in the initiation stage.

Exercise for osteoporosis: A literature review of pathology and mechanism.

Osteoporosis (OP) is a disease that weakens bones and has a high morbidity rate worldwide, which is prevalent among the elderly, particularly, women of postmenopausal age. The dynamic balance between bone formation and resorption is necessary for normal bone metabolism. Many factors, including aging, estrogen deficiency, and prolonged immobilization, disrupt normal apoptosis, autophagy, and inflammation, leading to abnormal activation of osteoclasts, which gradually overwhelm bone formation by bone resorption. Moderate exercise as an effective non-drug treatment helps increase bone formation and helps relieve OP. The possible mechanisms are that exercise affects apoptosis and autophagy through the release of exercise-stimulated myohormone and the secretion of anti-inflammatory cytokines via mechanical force. In addition, exercise may also have an impact on the epigenetic processes involved in bone metabolism. Mechanical stimulation promotes bone marrow mesenchymal stem cells (BMSCs) to osteogenic differentiation by altering the expression of non-coding RNAs. Besides, by reducing DNA methylation, the mechanical stimulus can also alter the epigenetic status of osteogenic genes and show associated increased expression. In this review, we reviewed the possible pathological mechanisms of OP and summarized the effects of exercise on bone metabolism, and the mechanisms by which exercise alleviates the progression of OP, to provide a reference for the prevention and treatment of OP.

OS08.1.A Integrative molecular analysis of matched primary and recurrent IDH-mutant astrocytoma; an update from the GLASS-NL consortium

Abstract Background The evolutionary processes that drive tumor progression in patients with IDH-mutant astrocytoma remain largely unclear. The GLASS-NL consortium was initiated to gain insight into the molecular mechanisms underlying glioma evolution and to identify markers of progression in IDH-mutant astrocytomas. Ultimately, such markers can assist clinical decision making. Here, we present the DNA methylation profiling, RNA-sequencing and shallow whole-genome sequencing (sWGS) of samples included in the GLASS-NL study. Methods Eligible were patients with an IDH-mutant, 1p19q non-codeleted, astrocytoma at first diagnosis who underwent surgical resection of the tumor at least twice separated by >6 months, and of whom paired tumor samples were available for analyses. Overall survival (OS) was measured from date of initial surgery. DNA methylation profiling was performed with the 850kEPIC array, and transcriptome and sWGS by NGS. After quality control, DNA methylation data of 103, expression data of 91, and sWGS data of 92 patients was available for further analysis. Methylation classes were determined according to Capper et al. and copy number alterations (CNAs) were extracted from both sWGS and DNA-methylation data. Results 110 patients were identified from various medical centers in the Netherlands. The median time between surgical resections was 41.9 months (IQR:26.5-65.9) and after initial surgery, 63% and 22% of the patients were treated with radiotherapy or chemotherapy respectively. The proportion of samples assigned to the high grade methylation class increased ~three-fold at recurrence. 83% of patients that progressed from low to high grade, received treatment prior to recurrent surgery, as compared to 53% of the patients that remained low grade. Genome wide DNA methylation levels of recurrent samples were lower than that of initial samples. This difference is explained by the increased number of high grades at recurrence, since near identical DNA methylation levels were observed in samples that remained low grade. Analysis of CNAs revealed chromosomal arms that were more frequently altered in high grade samples. Univariate analysis showed that losses in 3p, 9p, 10q, 13q and 14q were associated with poor OS. More than 800 differentially expressed genes between initial and recurrent tumor samples were found. Chromosomal enrichment analysis revealed a locus on chromosome 6 enriched with histone genes, to be significantly up-regulated over time. Conclusion Longitudinal methylation profiling of IDH-mutant astrocytoma reveals a shift towards a higher grade at tumor recurrence coinciding with reduced DNA methylation levels, and increased frequency of CNAs. Longitudinal expression analysis showed changes in expression of >800 genes, including an up-regulated locus enriched with histone genes. Further integrative analyses are ongoing and will be reported at the meeting.

Mapping of pituitary stress-induced gene regulation connects Nrcam to negative emotions

SummaryEnvironmental stressors such as repeated social defeat may initiate powerful activation of subconscious parts of the brain. Here, we examine the consequences of such stress (induced by resident-intruder paradigm) on the pituitary gland. In male stressed vs. control rats, by RNA- and bisulfite DNA sequencing, we found regulation of genes involved in neuron morphogenesis and communication. Among these, Neuronal cell adhesion molecule (Nrcam) showed reduced transcription and reduced DNA methylation in a region corresponding to intron 1 in human NRCAM. Also, genetic variability in this area was associated with altered stress response in male humans exposed to repeated social defeat in the form of abusive supervision. Thus, our data show that the pituitary gene expression may be affected by social stress and that genetic variability in NRCAM intron 1 region influences stress-induced negative emotions. We hope our shared datasets will facilitate further exploration of the motions triggered by social stressors.

5-Aza-dC promotes T-cell acute lymphoblastic leukemia cell invasion via downregulation of DNMT1 and upregulation of MMP-2 and MMP-9

5-Aza-2'-deoxycytidine (5-Aza-dC) is a demethylation agent known to deplete DNA methyltransferases (DNMTs) in leukemia cancer cells, and can restore the expression of their target genes in Jurkat cells. The goal of this study was to discern the potential effect of 5-Aza-dC on the invasion of T-ALL cells in acute lymphoblastic leukemia (ALL). The role of matrix metallopeptidase (MMP)-2, MMP-9, and DNMT1 in cell invasion was determined using loss- and gain-of-function investigations in Jurkat- and Sup-T1-R cells. A nude mouse model of ALL was established for further exploration of their roles in vivo. MMP-2 and MMP-9 exhibited high expression and low DNA methylation levels in 5-Aza-dC-resistant T-ALL cells. DNMT1 was poorly expressed in 5-Aza-dC-resistant T-ALL cells and exhibited decreased enrichment in the promoter region of MMP-2 and MMP-9. Silencing of MMP-2 and MMP-9 or DNMT1 overexpression reduced T-ALL cell invasion. After treatment of Sup-T1 cells with 5-Aza-dC, MMP-2 and MMP-9 presented with reduced DNA methylation levels but increased expression, and DNMT1 expression was identified to be suppressed. Further, in vivo assays revealed that DNMT1 alleviated T-ALL by reducing the expression of MMP-2 and MMP-9 in vivo. All in all, 5-Aza-dC activates MMP-2 and MMP-9 expression by reducing DNMT1-dependent DNA methylation levels and, hence, promotes the invasion of T-ALL cells.

Bisphenol-A reduces DNA methylation after metabolic activation

Bisphenol-A (BPA) is an important environmental contaminant with adverse health effects suspected to be mediated through epigenetic mechanisms. We had reported that the FLO1-dependent flocculation of transgenic yeast expressing human DNA methyltransferase (DNMT yeast) is a useful tool in epigenotoxicology studies. In this report, we have investigated the effects of BPA in the presence of metabolic activation (S-9 mix) on the transcription level of the FLO1 gene in the DNMT yeast. In the presence of metabolic activation, BPA inhibited the intensity of green fluorescence reporter protein (GFP) driven by the FLO1 promoter. A metabolite of BPA, 4-methyl-2,4-bis(p-hydroxyphenyl) pent-1-ene (MBP), also exhibited similar inhibitory effect. Furthermore, BPA in the presence of S-9 mix had only a weak while MBP had no inhibitory effects on the expression of modified GFP reporter gene under the control of FLO1 promoter with reduced CpG motifs. Aforementioned behavior was confirmed by the inhibition of flocculation as well as FLO1 gene mRNA expression. In addition, the global DNA methylation level in the human HEK293 cells was also reduced by MBP. These results indicate that BPA metabolites have inhibitory effect on DNA methylation. Our approach offers a novel in vitro method for screening for chemicals that can alter the epigenome by a mechanism dependent on their metabolic activation.

Structure of DNMT3B homo-oligomer reveals vulnerability to impairment by ICF mutations

DNA methyltransferase DNMT3B plays an essential role in establishment of DNA methylation during embryogenesis. Mutations of DNMT3B are associated with human diseases, notably the immunodeficiency, centromeric instability and facial anomalies (ICF) syndrome. How ICF mutations affect DNMT3B activity is not fully understood. Here we report the homo-oligomeric structure of DNMT3B methyltransferase domain, providing insight into DNMT3B-mediated DNA methylation in embryonic stem cells where the functional regulator DNMT3L is dispensable. The interplay between one of the oligomer interfaces (FF interface) and the catalytic loop renders DNMT3B homo-oligomer a conformation and activity distinct from the DNMT3B-DNMT3L heterotetramer, and a greater vulnerability to certain ICF mutations. Biochemical and cellular analyses further reveal that the ICF mutations of FF interface impair the DNA binding and heterochromatin targeting of DNMT3B, leading to reduced DNA methylation in cells. Together, this study provides a mechanistic understanding of DNMT3B-mediated DNA methylation and its dysregulation in disease.

A Novel Cartesian Plot Analysis for Fixed Monolayers That Relates Cell Phenotype to Transfer of Contents between Fibroblasts and Cancer Cells by Cell-Projection Pumping.

We recently described cell-projection pumping as a mechanism transferring cytoplasm between cells. The uptake of fibroblast cytoplasm by co-cultured SAOS-2 osteosarcoma cells changes SAOS-2 morphology and increases cell migration and proliferation, as seen by single-cell tracking and in FACS separated SAOS-2 from co-cultures. Morphological changes in SAOS-2 seen by single cell tracking are consistent with previous observations in fixed monolayers of SAOS-2 co-cultures. Notably, earlier studies with fixed co-cultures were limited by the absence of a quantitative method for identifying sub-populations of co-cultured cells, or for quantitating transfer relative to control populations of SAOS-2 or fibroblasts cultured alone. We now overcome that limitation by a novel Cartesian plot analysis that identifies individual co-cultured cells as belonging to one of five distinct cell populations, and also gives numerical measure of similarity to control cell populations. We verified the utility of the method by first confirming the previously established relationship between SAOS-2 morphology and uptake of fibroblast contents, and also demonstrated similar effects in other cancer cell lines including from melanomas, and cancers of the ovary and colon. The method was extended to examine global DNA methylation, and while there was no clear effect on SAOS-2 DNA methylation, co-cultured fibroblasts had greatly reduced DNA methylation, similar to cancer associated fibroblasts.

Modification of Gene Expression, DNA Methylation and Small RNAs Expression in Rice Plants under In Vitro Culture

Tissue culture is an important experimental technique widely used for plant transformation and can induce somaclonal variation that is shown to be associated with genetic and epigenetic changes. However, the molecular basis of somaclonal variation and plant cell response to tissue culture has yet to be fully understood. In this study, we investigated gene expression, DNA methylation, and small RNA changes in regenerated lines (RL) compared with the wild-type progenitor plants (WT) of rice cv. Hitomebore. Using microarray, we identified many genes that were differentially expressed in the shoot-tip tissue and showed that TEs were generally activated in RL. Methylation Sensitive Amplification Polymorphism (MSAP) analysis of 5′CCGG sites combined with bisulfite sequencing detected a generally reduced DNA methylation in the RL lines. Small RNA sequencing analysis detected widespread changes in small RNA accumulation between RL and WT. In particular, repeat and TE-associated 24-nt size class of small RNAs, the inducer of RNA-directed DNA methylation, was in general down-regulated in RL, consistent with reduced CHG and CHH methylation at some of the differentially methylated TE loci. A large number of differentially expressed miRNAs were identified in RL and WT lines, including known and novel miRNAs. The expression of some of these miRNAs exhibited inverse correlation with the predicted target genes, suggesting a regulatory function. The RL plants looked similar to WT plants under normal conditions but showed significant phenotypic alterations under abiotic stress conditions. The widespread changes in DNA methylation, small RNA accumulation and gene expression in regenerated plants supports the role of epigenetic changes in tissue culture-induced somaclonal variation.

MiR-26a-5p Suppresses Wnt/β-Catenin Signaling Pathway by Inhibiting DNMT3A-Mediated SFRP1 Methylation and Inhibits Cancer Stem Cell-Like Properties of NSCLC

Background Lung cancer is a malignant cancer which results in the most cancer incidence and mortality worldwide. There is increasing evidence that the pattern of DNA methylation affects tumorigenesis and progression. However, the molecules and mechanisms regulating DNA methylation remain unclear. Methods The expression of miR-26a-5p in NSCLC cell lines was detected by qPCR and verified in NSCLC tissues from TCGA using Limma R package. CCK-8 assay, plate clone formation assay, flow cytometry, and sphere formation assay were used to detect the cell proliferation, colony formation, cell cycle, and cancer stem cell- (CSC-) like property in NSCLC cell lines. The immunoblotting was used to detect the protein levels of DNMT3A, SFRP1, and Ki67. Global DNA methylation levels and DNA methylation levels of SFRP1 promoter were examined using ELISA and MSP-PCR assay, respectively. The distribution of β-catenin was examined using immunofluorescence (IF). Besides, xenograft mouse model was used to investigate the antitumor effects of miR-26a-5p in vivo. The pathology and protein levels were, respectively, detected by hematoxylin and eosin (H&E) and immunocytochemistry (IHC). Results The expression of miR-26a-5p was downregulated in the tumor tissues comparted to adjacent normal tissues as well as NSCLC cell lines compared to normal lung epithelial cell (BEAS2B). The overexpression of miR-26a-5p inhibited cell proliferation, colony formation, CSC-like property, and arrested cell cycle at G1 phase. DNMT3A was a target of miR-26a-5p and upregulated DNA methylation on SFRP1 promoter. Mechanistically, miR-26a-5p repressed cell proliferation, colony formation, CSC-like property, and arrested cell cycle at G1 phase by binding DNMT3A to reduce DNA methylation levels of SFRP1 then upregulated SFRP1 expression. Moreover, miR-26a-5p exerted antitumor effects in vivo. Conclusion Our results revealed that miR-26a-5p acted as a tumor suppressor through targeting DNMT3A to upregulate SFRP1 via reducing DNMT3A-dependent DNA methylation.

The Activation of Protamine 1 Using Epigenome Editing Decreases the Proliferation of Tumorigenic Cells.

DNA methyltransferases (DNMT) and histone deacetylases (HDAC) inhibitors are used as cancer epigenome drugs. However, these epigenetic drugs lack targeting specificity and could risk inducing genome instability and the expression of oncogenes. Therefore, there is a need to develop new therapeutic strategies where specific cancer genes can be targeted for silencing or activation. The CRISPR/dCas9 system represents a promising, powerful therapeutic tool because of its simplicity and specificity. Protamine 1 (PRM1) is exclusively expressed in sperm and has a vital role in the tight packaging of DNA, thus inducing transcriptional silencing in sperm cells. We hypothesized that the activation of the PRM1 gene in tumorigenic cells would lead to DNA condensation and reduce the proliferation of these cells. To test our hypothesis, we transfected human embryonic kidney cells 293T with a dCas9-P300 plasmid that adds acetyl groups to the promoter region of PRM1 via specific gRNAs plasmids. RNA-Seq analysis of transfected cells revealed high specificity of targeted gene activation. PRM1 expression resulted in a significant decrease in cell proliferation as measured by the BrdU ELISA assay. To confirm that the activation of PRM1 was due to acetyl groups deposited to H3K27, a ChIP-qPCR was performed. The acetylation of the PRM1 promoter region targeted by dCas9-p300 in transfected cells was higher than that of the control cells. Interestingly, the targeted promoter region for acetylation showed reduced DNA methylation. These findings demonstrate the efficacy of epigenome editing in activating PRM1 in non-expressing tumorigenic cells, which could be used as a promising therapeutic strategy in cancer treatment.

Abstract 295: Methyl Transferase Inhibitor 5-azacytidine Suppresses Experimental Abdominal Aortic Aneurysms

Background: Increased vascular DNA methylation is present in disease states including abdominal aortic aneurysms (AAA). The impact of dysregulated DNA methylation on aneurysm pathogenesis, however, remains uncertain. We evaluated the influence of 5-azacytine (AZA), a methyl transferase inhibitor, on experimental AAA initiation and progression. Methods and Results: AAAs were induced in male C57BL/6J mice via intra-aortic porcine pancreatic elastase infusion. AZA or vehicle were administered intraperitoneally starting one day prior to (prophylactic, 2 mg/kg), or 4 days following (therapeutic, 1 or 2 mg/kg), elastase infusion and continuing daily through 14 days. AAA development and progression were evaluated by serial ultrasonographic aortic diameter measurements and histopathology at sacrifice.Time dependent post-elastase aortic diameter enlargement was markedly reduced in mice receiving prophylactic AZA treatment. A substantial delay in AAA onset was noted as well. While therapeutic AZA tended to mitigate further aneurysmal enlargement at either dose, a significant reduction was noted only in the 2 mg/kg group at 14 days. On histopathological assessment, AZA was associated with marked attenuation of characteristic aneurysmal pathologic features including medial elastin and smooth muscle destruction, leukocyte accumulation and neoangiogenesis in all groups. Conclusion: AZA treatment attenuates aneurysm development and progression in the intra-aortic porcine pancreatic elastase infusion AAA model. Pharmacologic strategies effective at limiting or reducing DNA methylation may have translational applications in the medical management of AAA disease.

Epigenetic regulation of Anoctamin1 in pulmonary arterial hypertension

Introduction/HypothesisPulmonary arterial hypertension (PAH) is associated with substantial pulmonary vascular remodeling wherein endothelial cells (ECs) within the lumen hyper‐proliferate leading to occlusive plexiform lesions. We previously reported that Anoctamin‐1 (ANO1) expression is increased in lung ECs from patients with PAH and is associated with increased EC proliferation. However, the mechanism of ANO1 expression regulation in PAH remains unclear. DNA methylation has been recognized as an important mechanism in gene regulation. In this study, we test the hypothesis that ANO1 expression in PAH is epigenetically mediated via DNA methylation.Methods/ResultsDNA was extracted from lung ECs of patients with idiopathic PAH (n=7) and controls (n=6), followed by Bisulfite Next‐Generation Sequencing (EpigenDx, MA) to determine DNA methylation profile in human ANO1 gene (Ensembl Gene ID: ENSG00000131620). We used 19 assays to cover 97 CpG sites in the human ANO1 gene, including regions of 5’‐upstream, 5’‐UTR, and different introns. Our data show that the methylation of 29 of 97 (30%) CpG sites in human ANO1 gene is significantly altered in lung ECs from PAH patients than that from control subjects. Among the significantly altered 29 CpG sites, the methylation at 22 CpG sites (76%) is significantly decreased. To determine whether decreased DNA methylation changes human ANO1 gene expression, we then exposed normal human pulmonary artery ECs to RG108, which is a DNA methyltransferase inhibitor that broadly reduces DNA methylation. Our data show that cells treated with RG108 for 72 hours have a significant 2‐fold increase in ANO‐1 mRNA expression in comparison to control cells, suggesting that decreased DNA methylation is sufficient to upregulate human ANO1 expression. We are developing CRISPR‐based approaches to target these identified CpG sites to determine the specific contribution of their methylation in regulation of human ANO1 gene.ConclusionsOur results demonstrate that DNA methylation in human ANO1 gene is significantly altered in PAH and reduction in DNA methylation may serve as a regulatory mechanism for ANO1 upregulation in settings of PAH.

H3K36 dimethylation shapes the epigenetic interaction landscape by directing repressive chromatin modifications in embryonic stem cells.

Epigenetic modifications on the chromatin do not occur in isolation. Chromatin-associated proteins and their modification products form a highly interconnected network, and disturbing one component may rearrange the entire system. We see this increasingly clearly in epigenetically dysregulated cancers. It is important to understand the rules governing epigenetic interactions. Here, we use the mouse embryonic stem cell (mESC) model to describe in detail the relationships within the H3K27-H3K36-DNA methylation subnetwork. In particular, we focus on the major epigenetic reorganization caused by deletion of the histone 3 lysine 36 methyltransferase NSD1, which in mESCs deposits nearly all of the intergenic H3K36me2. Although disturbing the H3K27 and DNA methylation (DNAme) components also affects this network to a certain extent, the removal of H3K36me2 has the most drastic effect on the epigenetic landscape, resulting in full intergenic spread of H3K27me3 and a substantial decrease in DNAme. By profiling DNMT3A and CHH methylation (mCHH), we show that H3K36me2 loss upon Nsd1-KO leads to a massive redistribution of DNMT3A and mCHH away from intergenic regions and toward active gene bodies, suggesting that DNAme reduction is at least in part caused by redistribution of de novo methylation. Additionally, we show that pervasive acetylation of H3K27 is regulated by the interplay of H3K36 and H3K27 methylation. Our analysis highlights the importance of H3K36me2 as a major determinant of the developmental epigenome and provides a framework for further consolidating our knowledge of epigenetic networks.

Selenium Supplementation Improved Cardiac Functions by Suppressing DNMT2-Mediated GPX1 Promoter DNA Methylation in AGE-Induced Heart Failure.

Objective Advanced glycation end products (AGEs) are featured metabolites associated with diabetic cardiomyopathy which is characterized by heart failure caused by myocyte apoptosis. Selenium was proved cardioprotective. This study was aimed at investigating the therapeutic effects and underlying mechanisms of selenium supplementation on AGE-induced heart failure. Methods Rats and primary myocytes were exposed to AGEs. Selenium supplementation was administrated. Cardiac functions and myocyte apoptosis were evaluated. Oxidative stress was assessed by total antioxidant capacity (TAC), reactive oxygen species (ROS) generation, and GPX activity. Expression levels of DNA methyltransferases (DNMTs) and glutathione peroxidase 1 (GPX1) were evaluated. DNA methylation of the GPX1 promoter was analyzed. Results AGE exposure elevated intracellular ROS generation, induced myocyte apoptosis, and impaired cardiac functions. AGE exposure increased DNMT1 and DNMT2 expression, leading to the reduction of GPX1 expression and activity in the heart. Selenium supplementation decreased DNMT2 expression, recovered GPX1 expression and activity, and alleviated intracellular ROS generation and myocyte apoptosis, resulting in cardiac function recovery. DNA methylation analysis in primary myocytes indicated that selenium supplementation or DNMT inhibitor AZA treatment reduced DNA methylation of the GPX1 gene promoter. Selenium supplementation and AZA administration showed synergic inhibitory effect on GPX1 gene promoter methylation. Conclusions Selenium supplementation showed cardioprotective effects on AGE-induced heart failure by suppressing ROS-mediated myocyte apoptosis. Selenium supplementation suppressed ROS generation by increasing GPX1 expression via inhibiting DNMT2-induced GPX1 gene promoter DNA methylation in myocytes exposed to AGEs.

Developmental arsenic exposure impairs cognition, directly targets DNMT3A, and reduces DNA methylation.

Developmental arsenic exposure has been associated with cognitive deficits in epidemiological studies, but the underlying mechanisms remain poorly understood. Here, we establish a mouse model of developmental arsenic exposure exhibiting deficits of recognition and spatial memory in the offspring. These deficits are associated with genome-wide DNA hypomethylation and abnormal expression of cognition-related genes in the hippocampus. Arsenic atoms directly bind to the cysteine-rich ADD domain of DNA methyltransferase 3A (DNMT3A), triggering ubiquitin- and proteasome-mediated degradation of DNMT3A in different cellular contexts. DNMT3A degradation leads to genome-wide DNA hypomethylation in mouse embryonic fibroblasts but not in non-embryonic cell lines. Treatment with metformin, a first-line antidiabetic agent reported to increase DNA methylation, ameliorates the behavioral deficits and normalizes the aberrant expression of cognition-related genes and DNA methylation in the hippocampus of arsenic-exposed offspring. Our study establishes a DNA hypomethylation effect of developmental arsenic exposure and proposes a potential treatment against cognitive deficits in the offspring of pregnant women in arsenic-contaminated areas.