Aberrant DNA Methylation Research Articles

Multi-omics integration analysis identifies pathogenic candidate genes of congenital heart defects with vascular malformation

Abstract Background Congenital heart defects (CHD) with vascular malformation harbors numerous genetic and epigenetic aberrations of DNA copy numbers and methylation. Proteomic deregulation by the aberrations plays key driver roles in heterogeneous progression of CHD. However, it still remains largely unknown about integrative analysis of genetic, epigenetic regulation and proteome in CHD. Purpose We employed a multi-omics approach to explore pathogenic candidate genes in CHD with vascular malformation. Methods Previously, we gathered WES data for 216 CHD patients with vascular malformation and 100 healthy controls. Meanwhile, we carried out DNA methylation analysis from 11 patients and 5 controls. Here, we profile proteomics of vascular tissues from 11 patients and 5 controls. Integration analysis of DNA copy-number-correlated (CNVcor) genes, methylation-correlated (METcor) genes and differentially proteins reveal the innovative and crucial pathogenetic genes. We then detected spatiotemporal expression patterns of candidate genes in zebrafish embryos using in situ hybridization and qPCR, and verified the roles in vascular development by morpholino-mediated gene knockdown. Results 90 differentially expressed proteins (DEPs) were obtained using proteomics and were mainly related to cell adhesion and migration, angiogenesis, amino acid metabolism, and HIF-1 signaling pathway through GO and KEGG pathway enrichment analysis. 15 hub proteins were identified by WGCNA analysis (Fig 1). Additionally, we found that the frequencies of the aberrancies of the CNVcor genes were significantly correlated with the METcor genes (Fig 2 C). We obtained 1924 differentially CNVcor genes and 952 differentially METcor genes. Then, we computed the Pearson’s correlation coefficients of all three-omics by pairwise comparison and found that the distribution of correlations was bimodal (Fig 2 D). Meanwhile, 28 overlapped genes were observed among three-omics. We screened out 11 candidate genes (TIMP1, BGN, LTBP1, AKR1C3, LOXL3, MAOA, MAOB, GNA12, IFI30, CNTNAP1, IGF2BP1) according to the node degree of PPI network analysis between 28 genes and known vascular development related genes. We further figured out LOXL3, MAOA, CNTNAP1 and IGF2BP1 are the novel important genes for vascular malformation, of which CNTNAP1 is the joint gene of three-omics. Through expression patterns and gene knockdown in zebrafish embryos, we revealed that LOXL3, MAOA, CNTNAP1 and IGF2BP1 affect vascular development (Fig 2 E-G). Conclusions We identify novel key candidate genes (LOXL3, MAOA, CNTNAP1 and IGF2BP1) potentially related to CHD with vascular malformation and elucidate the possible molecular pathogenesis of CHD. Our multi-omics integrative analysis provides new insights on the multi-layered etiology of CHD, which might be helpful in developing clinical diagnosis and precaution for CHD.Figure 1Figure 2

Effects of Differentially Methylated CpG Sites in Enhancer and Promoter Regions on the Chromatin Structures of Target LncRNAs in Breast Cancer.

Aberrant DNA methylation plays a crucial role in breast cancer progression by regulating gene expression. However, the regulatory pattern of DNA methylation in long noncoding RNAs (lncRNAs) for breast cancer remains unclear. In this study, we integrated gene expression, DNA methylation, and clinical data from breast cancer patients included in The Cancer Genome Atlas (TCGA) database. We examined DNA methylation distribution across various lncRNA categories, revealing distinct methylation characteristics. Through genome-wide correlation analysis, we identified the CpG sites located in lncRNAs and the distally associated CpG sites of lncRNAs. Functional genome enrichment analysis, conducted through the integration of ENCODE ChIP-seq data, revealed that differentially methylated CpG sites (DMCs) in lncRNAs were mostly located in promoter regions, while distally associated DMCs primarily acted on enhancer regions. By integrating Hi-C data, we found that DMCs in enhancer and promoter regions were closely associated with the changes in three-dimensional chromatin structures by affecting the formation of enhancer-promoter loops. Furthermore, through Cox regression analysis and three machine learning models, we identified 11 key methylation-driven lncRNAs (DIO3OS, ELOVL2-AS1, MIAT, LINC00536, C9orf163, AC105398.1, LINC02178, MILIP, HID1-AS1, KCNH1-IT1, and TMEM220-AS1) that were associated with the survival of breast cancer patients and constructed a prognostic risk scoring model, which demonstrated strong prognostic performance. These findings enhance our understanding of DNA methylation's role in lncRNA regulation in breast cancer and provide potential biomarkers for diagnosis.

Diagnostic potential of miRNA-135A1 in human papillomavirus associated cervical lesions

Introduction. Human papillomavirus (HPV) infection with high-risk HPVs is an etiological factor in the development of cervical cancer, with HPV type 16 (HPV16) being the most common. The mechanisms leading to disruption of viral oncogene expression and initiation of epithelial cell transformation are poorly understood. Epigenetic regulatory factors, including cellular miRNAs, may play an important role in HPV-induced carcinogenesis, and aberrantly expressed miRNAs may be promising markers for the diagnosis of HPV-associated lesions.Aim. To search for miRNAs involved in the pathogenesis of HPV16-associated cervical cancer and to evaluate their diagnostic potential for the detection of cervical cancer and precancerous lesions.Materials and methods. MiRNA expression in clinical samples was assessed by both next generation sequencing and quantitative stem-loop polymerase chain reaction (sl-qPCR). Plasma miRNAs from patients with precancerous and cancerous lesions and healthy donors were analyzed using sl-qPCR. Loss of heterozygosity in cervical cancer samples was assessed by copy number ratio of MIR135A1 and ACTB genes. A total of 67 patients with cervical cancer, 21 with precancerous cervical lesions and 24 healthy donors were included in the study. The effect of DNA methylation on miRNA-135A1 expression was evaluated after treatment with a demethylating agent of the cervical HPV16-positive SiHa cell line. Changes in the expression of the HPV16 E6 oncogene were analyzed after transfection with synthetic analogues of the mature forms of miRNA-135А1 (miRNA-135a-3p and miRNA-135a-5p).Results. A significant decrease in the expression of miRNA-135A1 and miRNA-135A2 was detected in tumor tissue samples from HPV16-positive cervical cancer, which was confirmed by sl-qPCR in an independent panel of tumor samples. A decrease in miRNA-135A1 expression was shown to result from both loss of heterozygosity of the gene and aberrant DNA methylation. Transfection of mature forms of miRNA-135A1 into SiHa cells resulted in decreased expression of the E6 oncogene of HPV16. Blood plasma samples from patients with cervical cancer and precancerous lesions showed lower levels of miRNA-135a-3p than healthy donors, and ROC analysis indicated its high diagnostic potential.Conclusion. Levels of miRNA-135A1 are significantly reduced in cervical lesions, both in tumor tissue and plasma, and the ability of this miRNA to suppress the expression of the HPV16 E6 oncogene suggests its oncosuppressive properties. Thus, miRNA-135A1 can be used as a promising new marker for the diagnosis of HPV-associated lesions.

Abnormal Promoter Methylation of Nucleotide-Binding Oligomerization Domain Containing 2 (NOD2) Gene in the Pathogenesis of Crohn’s Disease

Background: Changes in the expression of nucleotide-binding oligomerization domain containing 2 (NOD2) play an important role in the pathogenesis of a variety of autoimmune diseases including inflammatory bowel diseases (IBDs). Epigenetic modifications, including DNA methylation, are considered an important mechanism in the suppression of gene activity. In this study, we investigated the relationship between DNA methylation patterns of the promoter region of the NOD2 gene and the pathogenesis of Crohn’s disease (CD). Methods: Colonic mucosa samples were obtained from 15 Iranian patients with IBD and 15 age- and sexmatched healthy controls with no history of autoimmune disease. After the bisulfite conversion of genomic DNA, the DNA methylation status of three CpG sites in the promoter region of the NOD2 gene was determined by the real-time quantitative multiplex methylation-specific PCR (QM-MSP) assay. Results: Using this approach, we identified that IBD patients showed a decreased level of methylation of the NOD2 promoter in the colonic mucosa than did the healthy controls. (Unmethylated DNA in Crohn's disease vs. healthy controls; 0.128±0.093 vs. 0.025±0.016, P<0.000). Conclusion: According to our findings, promoter hypomethylation of the NOD2 gene in the colonic mucosa might contribute to the development and severity of CD. Furthermore, aberrant DNA methylation levels are expected to serve as a clinically useful risk marker.

5-Azacytidine treatment inhibits the development of lung cancer models via epigenetic reprogramming and activation of cellular pathways with anti-tumor activity

Background and aims. Non-small cell lung cancer (NSCLC) treatment is challenged by late detection and limited therapeutic options. Aberrant DNA methylation, a common epigenetic alteration in NSCLC, offers new therapeutic avenues. This study aims to evaluate the combined effects of 5-Azacytidine (5- Aza), an epigenetic modifier, and ionizing radiation (IR) on NSCLC, exploring the underlying molecular mechanisms and therapeutic potential. Methods. In this study, we examined the effects of 5-Aza combined with IR in both in vitro and in vivo models of NSCLC. Five human NSCLC cell lines were treated with 5-Aza and IR. Cell viability, colony formation, wound healing, and transwell migration assays were performed to assess treatment effects. Microarray and qPCR analyses were conducted to identify gene expression changes. Additionally, subcutaneous and orthotopic xenograft models were used to evaluate the treatment’s efficacy in vivo. Results. Treatment with 5-Aza and IR resulted in significant reductions in cell viability, colony formation, and migration in NSCLC cell lines. Microarray analysis revealed significant changes in gene expression, including the upregulation of apoptosis-related genes and the downregulation of cell proliferation-related genes. In vivo studies demonstrated a notable reduction in tumor growth and metastasis in both subcutaneous and orthotopic NSCLC models following 5-Aza and IR treatment. Histological and bioluminescent imaging confirmed the therapeutic effects of the combined treatment. Conclusions. The combination of 5-Aza and IR shows promise as an effective treatment for NSCLC, enhancing apoptosis and reducing tumor growth through epigenetic modulation.

Glutaminase inhibition in combination with azacytidine in myelodysplastic syndromes: a phase 1b/2 clinical trial and correlative analyses.

Malignancies are reliant on glutamine as an energy source and a facilitator of aberrant DNA methylation. We demonstrate preclinical synergy of telaglenastat (CB-839), a selective glutaminase inhibitor, combined with azacytidine (AZA), followed by a single-arm, open-label, phase 1b/2 study in persons with advanced myelodysplastic syndrome (MDS). The dual primary endpoints evaluated clinical activity, safety and tolerability; secondary endpoints evaluated pharmacokinetics, pharmacodynamics, overall survival, event-free survival and duration of response. The dose-escalation study included six participants and the dose-expansion study included 24 participants. Therapy was well tolerated and led to an objective response rate of 70% with (marrow) complete remission in 53% of participants and a median overall survival of 11.6 months, with evidence of myeloid differentiation in responders determined by single-cell RNA sequencing. Glutamine transporter solute carrier family 38 member 1 in MDS stem cells was associated with clinical responses and predictive of worse prognosis in a large MDS cohort. These data demonstrate the safety and efficacy of CB-839 and AZA as a combined metabolic and epigenetic approach in MDS. ClinicalTrials.gov identifier: NCT03047993 .

Triphenyl Phosphate Alters Methyltransferase Expression and Induces Genome-Wide Aberrant DNA Methylation in Zebrafish Larvae.

Emerging environmental contaminants, organophosphate flame retardants (OPFRs), pose significant threats to ecosystems and human health. Despite numerous studies reporting the toxic effects of OPFRs, research on their epigenetic alterations remains limited. In this study, we investigated the effects of exposure to 2-ethylhexyl diphenyl phosphate (EHDPP), tricresyl phosphate (TMPP), and triphenyl phosphate (TPHP) on DNA methylation patterns during zebrafish embryonic development. We assessed general toxicity and morphological changes, measured global DNA methylation and hydroxymethylation levels, and evaluated DNA methyltransferase (DNMT) enzyme activity, as well as mRNA expression of DNMTs and ten-eleven translocation (TET) methylcytosine dioxygenase genes. Additionally, we analyzed genome-wide methylation patterns in zebrafish larvae using reduced-representation bisulfite sequencing. Our morphological assessment revealed no general toxicity, but a statistically significant yet subtle decrease in body length following exposure to TMPP and EHDPP, along with a reduction in head height after TPHP exposure, was observed. Eye diameter and head width were unaffected by any of the OPFRs. There were no significant changes in global DNA methylation levels in any exposure group, and TMPP showed no clear effect on DNMT expression. However, EHDPP significantly decreased only DNMT1 expression, while TPHP exposure reduced the expression of several DNMT orthologues and TETs in zebrafish larvae, leading to genome-wide aberrant DNA methylation. Differential methylation occurred primarily in introns (43%) and intergenic regions (37%), with 9% and 10% occurring in exons and promoter regions, respectively. Pathway enrichment analysis of differentially methylated region-associated genes indicated that TPHP exposure enhanced several biological and molecular functions corresponding to metabolism and neurological development. KEGG enrichment analysis further revealed TPHP-mediated potential effects on several signaling pathways including TGFβ, cytokine, and insulin signaling. This study identifies specific changes in DNA methylation in zebrafish larvae after TPHP exposure and brings novel insights into the epigenetic mode of action of TPHP.

Perturbing TET2 condensation promotes aberrant genome-wide DNA methylation and curtails leukaemia cell growth.

The ten-eleven translocation (TET) family of dioxygenases maintain stable local DNA demethylation during cell division and lineage specification. As the major catalytic product of TET enzymes, 5-hydroxymethylcytosine is selectively enriched at specific genomic regions, such as enhancers, in a tissue-dependent manner. However, the mechanisms underlying this selectivity remain unresolved. Here we unveil a low-complexity insert domain within TET2 that facilitates its biomolecular condensation with epigenetic modulators, such as UTX and MLL4. This co-condensation fosters a permissive chromatin environment for precise DNA demethylation. Disrupting low-complexity insert-mediated condensation alters the genomic binding of TET2 to cause promiscuous DNA demethylation and genome reorganization. These changes influence the expression of key genes implicated in leukaemogenesis to curtail leukaemia cell proliferation. Collectively, this study establishes the pivotal role of TET2 condensation in orchestrating precise DNA demethylation and gene transcription to support tumour cell growth.

MIR124-3 and NKX6-1 hypermethylation profiles accurately predict metachronous gastric lesions in a Caucasian population

BackgroundEarly gastric cancer is treated endoscopically, but patients require surveillance due to the risk of metachronous gastric lesions (MGLs). Epigenetic alterations, particularly aberrant DNA methylation in genes, such as MIR124-3, MIR34b/c, NKX6-1, EMX1, MOS and CDO1, have been identified as promising biomarkers for MGL in Asian populations. We aimed to determine whether these changes could predict MGL risk in intermediate-risk Caucasian patients.MethodsThis case–cohort study included 36 patients who developed MGL matched to 48 patients without evidence of MGL in the same time frame (controls). Multiplex quantitative methylation-specific PCR was performed using DNA extracted from the normal mucosa adjacent to the primary lesion. The overall risk of progression to MGL was assessed using Kaplan–Meier and Cox proportional hazards model analyses.ResultsMIR124-3, MIR34b/c and NKX6-1 were successfully analyzed in 77 samples. MIR124-3 hypermethylation was detected in individuals who developed MGL (relative quantification 78.8 vs 50.5 in controls, p = 0.014), particularly in females and Helicobacter pylori-negative patients (p = 0.021 and p = 0.0079, respectively). This finding was further associated with a significantly greater risk for MGL development (aHR = 2.31, 95% CI 1.03–5.17, p = 0.042). Similarly, NKX6-1 was found to be hypermethylated in patients with synchronous lesions (relative quantification 7.9 vs 0.0 in controls, p = 0.0026). A molecular-based methylation model incorporating both genes was significantly associated with a threefold increased risk for MGL development (aHR = 3.10, 95% CI 1.07–8.95, p = 0.037).ConclusionsThis preliminary study revealed an association between MIR124-3 and NKX6-1 hypermethylation and the development of MGL in a Western population. These findings may represent a burden reduction and a greener approach to patient care.

Multiomics and machine learning-based analysis of pancancer pseudouridine modifications

Pseudouridine widely affects the stability and function of RNA. However, our knowledge of pseudouridine properties in tumors is incomplete. We systematically analyzed pseudouridine synthases (PUSs) expression, genomic aberrations, and prognostic features in 10907 samples from 33 tumors. We found that the pseudouridine-associated pathway was abnormal in tumors and affected patient prognosis. Dysregulation of the PUSs expression pattern may arise from copy number variation (CNV) mutations and aberrant DNA methylation. Functional enrichment analyses determined that the PUSs expression was closely associated with the MYC, E2F, and MTORC1 signaling pathways. In addition, PUSs are involved in the remodeling of the tumor microenvironment (TME) in solid tumors, such as kidney and lung cancers. Particularly in lung cancer, increased expression of PUSs is accompanied by increased immune checkpoint expression and Treg infiltration. The best signature model based on more than 112 machine learning combinations had good prognostic ability in ACC, DLBC, GBM, KICH, MESO, THYM, TGCT, and PRAD tumors, and is expected to guide immunotherapy for 19 tumor types. The model was also effective in identifying patients with tumors amenable to etoposide, camptothecin, cisplatin, or bexarotene treatment. In conclusion, our work highlights the dysregulated features of PUSs and their role in the TME and patient prognosis, providing an initial molecular basis for future exploration of pseudouridine. Studies targeting pseudouridine are expected to lead to the development of potential diagnostic strategies and the evaluation and improvement of antitumor therapies.

Determination of global DNA methylation level by methylation-sensitive comet assay in patients with urinary bladder cancer.

DNA methylation is an important mechanism in the regulation of gene expression and maintenance of genomic integrity. Aberrant DNA methylation is an early event in carcinogenesis. DNA methyltransferase inhibitors are used to restore aberrant DNA methylation and inhibit tumor growth. Evaluation of DNA methylation level is important for an effective anti-cancer therapy. In the present study, the determination of global DNA methylation levels in patients with urinary bladder cancer was proposed. The methylation-sensitive comet assay determined the global DNA methylation level at the level of single cells. McrBC enzyme, a methylation-sensitive restriction endonuclease was used for enzymatic digestion to generate additional breaks at methylated sites. % DNA methylation level was significantly higher in patients with bladder cancer compared to the control group. The clinical performance of % DNA methylation analysis by methylation-sensitive comet assay was evaluated by ROC curve. Using the cut-off value of 6,5% DNA methylation, 92% sensitivity, and 42% specificity were obtained. In conclusion, global DNA methylation measured by methylation-sensitive comet assay may be a promising non-invasive biomarker that reduces interventional tests required in the diagnosis and follow-up of urinary bladder cancer.

Methyltransferase DNMT3B promotes colorectal cancer cell proliferation by inhibiting PLCG2.

Aberrant DNA methylation patterns in the promoter region of PLCG2 are associated with dysregulated signaling pathways and cellular functions. Its role in colorectal cancer cells is still unknown. In this study, qRT-PCR is used to measure DNMT3B expression in colorectal cancer. Western blot analysis and immunohistochemistry are used to analyze DNMT3B and PLCG2 protein levels in colorectal tissues and cell lines. Cell Counting Kit-8 (CCK-8) and colony formation assays are used to assess the proliferation of colorectal cancer cells. Methylation-specific PCR (MSP) and bisulfite-sequencing PCR (BSP) are used to measure DNA methylation level. Our results show that DNMT3B is overexpressed in colorectal cells in the TCGA datasets according to Kaplan-Meier plots. DNMT3B is significantly overexpressed in tumor tissues compared to that in adjacent nontumor tissues. Western blot analysis results demonstrate high expression of DNMT3B in tumor tissues. Compared to normal colonic epithelial cells, colorectal cancer cell lines exhibit elevated level of PLCG2 methylation. Overexpression of PLCG2 effectively prevents the growth of colorectal cancer xenograft tumors in vivo. PLCG2 is identified as a key downstream regulatory protein of DNMT3B in colorectal cancer. DNMT3B inhibits PLCG2 transcription through methylation of the PLCG2 promoter region. DNMT3B controls colorectal cancer cell proliferation through PLCG2, which is useful for developing therapeutic approaches that target PLCG2 expression for the treatment of colorectal cancer.

Single-molecule epiallelic profiling of DNA derived from routinely collected Pap specimens for noninvasive detection of ovarian cancer.

Recent advances in molecular analyses of ovarian cancer have revealed a wealth of promising tumour-specific biomarkers, including protein, DNA mutations and methylation; however, reliably detecting such alterations at satisfactorily high sensitivity and specificity through low-cost methods remains challenging, especially in early-stage diseases. Here we present PapDREAM, a new approach that enables detection of rare, ovarian-cancer-specific aberrations of DNA methylation from routinely-collected cervical Pap specimens. The PapDREAM approach employs a microfluidic platform that performs highly parallelized digital high-resolution melt to analyze locus-specific DNA methylation patterns on a molecule-by-molecule basis at or near single CpG-site resolution at a fraction (<1/10th) of the cost of next-generation sequencing techniques. We demonstrate the feasibility of the platform by assessing intermolecular heterogeneity of DNA methylation in a panel of methylation biomarker loci using DNA derived from Pap specimens obtained from a cohort of 43 women, including 18 cases with ovarian cancer and 25 cancer-free controls. PapDREAM leverages systematic multidimensional bioinformatic analyses of locus-specific methylation heterogeneity to improve upon Pap-specimen-based detection of ovarian cancer, demonstrating a clinical sensitivity of 50% at 99% specificity in detecting ovarian cancer cases with an area under the receiver operator curve of 0.90. We then establish a logistic regression model that could be used to identify high-risk patients for subsequent clinical follow-up and monitoring. The results of this study support the utility of PapDREAM as a simple, low-cost screening method with the potential to integrate with existing clinical workflows for early detection of ovarian cancer. KEY POINTS: We present a microfluidic platform for detection and analysis of rare, heterogeneously methylated DNA within Pap specimens towards detection of ovarian cancer. The platform achieves high sensitivity (fractions <0.00005%) at a suitably low cost (∼$25) for routine screening applications. Furthermore, it provides molecule-by-molecule quantitative analysis to facilitate further study on the effect of heterogeneous methylation on cancer development.

Epigenetic Landscapes of Pain: DNA Methylation Dynamics in Chronic Pain.

Chronic pain is a prevalent condition with a multifaceted pathogenesis, where epigenetic modifications, particularly DNA methylation, might play an important role. This review delves into the intricate mechanisms by which DNA methylation and demethylation regulate genes associated with nociception and pain perception in nociceptive pathways. We explore the dynamic nature of these epigenetic processes, mediated by DNA methyltransferases (DNMTs) and ten-eleven translocation (TET) enzymes, which modulate the expression of pro- and anti-nociceptive genes. Aberrant DNA methylation profiles have been observed in patients with various chronic pain syndromes, correlating with hypersensitivity to painful stimuli, neuronal hyperexcitability, and inflammatory responses. Genome-wide analyses shed light on differentially methylated regions and genes that could serve as potential biomarkers for chronic pain in the epigenetic landscape. The transition from acute to chronic pain is marked by rapid DNA methylation reprogramming, suggesting its potential role in pain chronicity. This review highlights the importance of understanding the temporal dynamics of DNA methylation during this transition to develop targeted therapeutic interventions. Reversing pathological DNA methylation patterns through epigenetic therapies emerges as a promising strategy for pain management.

Aberrant DNA methylation associated with the development of metabolic dysfunction-associated fatty liver disease

The literature review deals with DNA methylation, a key epigenetic mechanism that controls the activity of gene transcription, plays a decisive role in the formation of genomic imprinting, gene silencing, X-chromosome inactivation, RNA splicing, DNA repair, cell differentiation and cell reprogramming, and also determines the occurrence and development of liver steatotic lesions and metabolic disorders. Methylation of DNA cytosine dinucleotide (CpG) can be represented in two types: de novo CpG methylation, which is carried out by 5mC DNA writers — DNA-(cytosine-5)-methyltransferase (DNMT) 3a and 3b, and suppor­ting DNA methylation, which is performed by DNMT1 during DNA replication. It has been found that the maintenance DNA methylation allows the preservation of the methylation pattern characteristic of progenitor cells in the cells of the new generation, and the DNA methylation of the gene body is associated with its increased expression. Active demethylation of 5mC is carried out by TET dioxygenases, including three enzymatic representatives: TET1, TET2 and TET3. It has been demonstrated that aberrant methylation of DNA nucleotides is directly related to the activity of lipid synthesis, the degree of oxidative stress, the development of liver steatosis, low-grade inflammation, insulin resistance, and the progression of liver fibrosis. The authors presented in detail the functions and features of DNA methyltransferases, erasers, and readers of 5mC sites; possible violations of the balance of activity of writers and erasers of 5mC DNA; DNA methylation landscape and patterns; clinical significance of DNA methylation signatures in metabolic dysfunction-associated fatty liver disease. Global hypomethylation of genome, at least 55 genes, is observed in patients with metabolic dysfunction-associated fatty liver disease. The authors emphasize that the use of DNA methylation signatures is a promising direction for early diagnosis and prognosis of the course of metabolic dysfunction-associated fatty liver disease, while the study of molecular components of DNA methylation mechanisms involved in the regulation of gene expression, the dependence of their activity on exposure to the exposome will allow to persona­lize and improve recommendations for lifestyle and diet modification in patients with metabolic dysfunction-associated fatty liver disease.

Methylation and transcriptome analyses construct a prognostic model and reveal the suppressor role of VMO1 in lung adenocarcinoma

BackgroundDNA methylation is an important epigenetic mechanism of gene regulation. The aberrant DNA methylation has been found to play an important role in the initiation and progression of tumors. ResultsTranscriptome and DNA methylation data of lung adenocarcinoma (LUAD) patients were co-analyzed and 95 methylation-driven genes (MDGs) was found in relation to LUAD. A prognostic model based on 3 MDGs (GMNN, SPINK2 and VMO1) was constructed by Univariate and Multivariate cox regression analyses. The risk score generated from the prognostic model could be used to classify LUAD patients into high and low risk groups. Furthermore, it was found that the risk score was associated with tumor microenvironment (TME) and clinical characteristics (survival status and T stage) of patients. Interestingly, we identified and validated that the patients in the low-risk group responded better to immunotherapy treatment. Then, a nomogram model based on the risk score and clinical characteristics was established which showed significant prediction value. The down-regulation and hypermethylation levels of vitelline membrane outer layer protein 1 homolog (VMO1) were verified in paired LUAD tumor and non-tumor tissues by pyrosequencing assay and RT-qPCR. Furthermore, MTT, migration and wound healing assays were performed with lentivector-mediated ectopic over-expression and 5-Aza-dC demethylation followed by siRNA rescue experiments to investigate the role of VMO1 in LUAD cells. Our results indicated that VMO1 could inhibit proliferation and migration of A549 and NCI-H1299 cells. ConclusionsIn summary, our experiments constructed a prognostic model with high capacity for risk prediction in LUAD patients. VMO1 had a malignant suppressor role in LUAD cells. The correlation between risk score and TME might elucidate a potential mechanism of oncogenesis and provide an avenue for further therapeutic targets.

Structural basis for the H2AK119ub1-specific DNMT3A-nucleosome interaction

Isoform 1 of DNA methyltransferase DNMT3A (DNMT3A1) specifically recognizes nucleosome monoubiquitylated at histone H2A lysine-119 (H2AK119ub1) for establishment of DNA methylation. Mis-regulation of this process may cause aberrant DNA methylation and pathogenesis. However, the molecular basis underlying DNMT3A1−nucleosome interaction remains elusive. Here we report the cryo-EM structure of DNMT3A1’s ubiquitin-dependent recruitment (UDR) fragment complexed with H2AK119ub1-modified nucleosome. DNMT3A1 UDR occupies an extensive nucleosome surface, involving the H2A-H2B acidic patch, a surface groove formed by H2A and H3, nucleosomal DNA, and H2AK119ub1. The DNMT3A1 UDR’s interaction with H2AK119ub1 affects the functionality of DNMT3A1 in cells in a context-dependent manner. Our structural and biochemical analysis also reveals competition between DNMT3A1 and JARID2, a cofactor of polycomb repression complex 2 (PRC2), for nucleosome binding, suggesting the interplay between different epigenetic pathways. Together, this study reports a molecular basis for H2AK119ub1-dependent DNMT3A1−nucleosome association, with important implications in DNMT3A1-mediated DNA methylation in development.

Tumor detection by analysis of both symmetric- and hemi-methylation of plasma cell-free DNA

Aberrant DNA methylation patterns have been used for cancer detection. However, DNA hemi-methylation, present at about 10% CpG dinucleotides, has been less well studied. Here we show that a majority of differentially hemi-methylated regions (DHMRs) in liver tumor DNA or plasma cells free (cf) DNA do not overlap with differentially methylated regions (DMRs) of the same samples, indicating that DHMRs could serve as independent biomarkers. Furthermore, we analyzed the cfDNA methylomes of 215 samples from individuals with liver or brain cancer and individuals without cancer (controls), and trained machine learning models using DMRs, DHMRs or both. The models incorporated with both DMRs and DHMRs show a superior performance compared to models trained with DMRs or DHMRs, with AUROC being 0.978, 0.990, and 0.983 in distinguishing control, liver and brain cancer, respectively, in a validation cohort. This study supports the potential of utilizing both DMRs and DHMRs for multi-cancer detection.

Multiomics analysis of IgM monoclonal gammopathies reveals epigenetic influence on oncogenesis via DNA methylation

AbstractCurrently, the role of DNA methylation in the immunoglobulin M (IgM) monoclonal gammopathy disease spectrum remains poorly understood. In the present study, a multiomics prospective analysis was conducted integrating DNA methylation, RNA sequencing (RNA-seq), and whole-exome sequencing data in 34 subjects (23 with Waldenström macroglobulinemia [WM], 6 with IgM monoclonal gammopathy of undetermined significance [MGUS], and 5 normal controls). Analysis was focused on defining differences between IgM gammopathies (WM/IgM-MGUS) compared with controls, and specifically between WM and IgM-MGUS. Between groups, genome-wide DNA methylation analysis demonstrated a significant number of differentially methylated regions that were annotated according to genomic region. Next, integration of RNA-seq data was performed to identify potentially epigenetically deregulated pathways. We found that pathways involved in cell cycle, metabolism, cytokine/immune signaling, cytoskeleton, tumor microenvironment, and intracellular signaling were differentially activated and potentially epigenetically regulated. Importantly, there was a positive enrichment of the CXCR4 signaling pathway along with several interleukin (interleukin 6 [IL-6], IL-8, and IL-15) signaling pathways in WM compared with IgM-MGUS. Further assessment of known tumor suppressor genes and oncogenes uncovered differential promoter methylation of several targets with concordant change in gene expression, including CCND1 and CD79B. Overall, this report defines how aberrant DNA methylation in IgM gammopathies may play a critical role in the epigenetic control of oncogenesis and key cellular functions.

A novel iPSC-based model of ICF syndrome subtype 2 recapitulates the molecular phenotype of ZBTB24 deficiency.

Immunodeficiency, Centromeric instability and Facial anomalies (ICF) syndrome is a rare genetic disorder characterized by variable immunodeficiency. More than half of the affected individuals show mild to severe intellectual disability at early onset. This disorder is genetically heterogeneous and ZBTB24 is the causative gene of the subtype 2, accounting for about 30% of the ICF cases. ZBTB24 is a multifaceted transcription factor belonging to the Zinc-finger and BTB domain-containing protein family, which are key regulators of developmental processes. Aberrant DNA methylation is the main molecular hallmark of ICF syndrome. The functional link between ZBTB24 deficiency and DNA methylation errors is still elusive. Here, we generated a novel ICF2 disease model by deriving induced pluripotent stem cells (iPSCs) from peripheral CD34+-blood cells of a patient homozygous for the p.Cys408Gly mutation, the most frequent missense mutation in ICF2 patients and which is associated with a broad clinical spectrum. The mutation affects a conserved cysteine of the ZBTB24 zinc-finger domain, perturbing its function as transcriptional activator. ICF2-iPSCs recapitulate the methylation defects associated with ZBTB24 deficiency, including centromeric hypomethylation. We validated that the mutated ZBTB24 protein loses its ability to directly activate expression of CDCA7 and other target genes in the patient-derived iPSCs. Upon hematopoietic differentiation, ICF2-iPSCs showed decreased vitality and a lower percentage of CD34+/CD43+/CD45+ progenitors. Overall, the ICF2-iPSC model is highly relevant to explore the role of ZBTB24 in DNA methylation homeostasis and provides a tool to investigate the early molecular events linking ZBTB24 deficiency to the ICF2 clinical phenotype.