Regulation Of DNA Methylation Research Articles

Integrated analysis of DNA methylome and transcriptome revealing epigenetic regulation of CRIR1-promoted cold tolerance

BackgroundDNA methylation contributes to the epigenetic regulation of nuclear gene expression, and is associated with plant growth, development, and stress responses. Compelling evidence has emerged that long non-coding RNA (lncRNA) regulates DNA methylation. Previous genetic and physiological evidence indicates that lncRNA-CRIR1 plays a positive role in the responses of cassava plants to cold stress. However, it is unclear whether global DNA methylation changes with CRIR1-promoted cold tolerance.ResultsIn this study, a comprehensive comparative analysis of DNA methylation and transcriptome profiles was performed to reveal the gene expression and epigenetic dynamics after CRIR1 overexpression. Compared with the wild-type plants, CRIR1-overexpressing plants present gained DNA methylation in over 37,000 genomic regions and lost DNA methylation in about 16,000 genomic regions, indicating a global decrease in DNA methylation after CRIR1 overexpression. Declining DNA methylation is not correlated with decreased/increased expression of the DNA methylase/demethylase genes, but is associated with increased transcripts of a few transcription factors, chlorophyll metabolism and photosynthesis-related genes, which could contribute to the CRIR1-promoted cold tolerance.ConclusionsIn summary, a first set of transcriptome and epigenome data was integrated in this study to reveal the gene expression and epigenetic dynamics after CRIR1 overexpression, with the identification of several TFs, chlorophyll metabolism and photosynthesis-related genes that may be involved in CRIR1-promoted cold tolerance. Therefore, our study has provided valuable data for the systematic study of molecular insights for plant cold stress response.

Microbial Metabolites-Induced Epigenetic Modifications for Inhibition of Colorectal Cancer: Current Status and Future Perspectives.

Globally, one of the most prevalent cancers is colorectal cancer (CRC). Chemotherapy and surgery are two common conventional CRC therapies that are frequently ineffective and have serious adverse effects. Thus, there is a need for complementary and different therapeutic approaches. The use of microbial metabolites to trigger epigenetic alterations as a way of preventing CRC is one newly emerging field of inquiry. Small chemicals called microbial metabolites, which are made by microbes and capable of altering host cell behaviour, are created. Recent research has demonstrated that these metabolites can lead to epigenetic modifications such as histone modifications, DNA methylation, and non-coding RNA regulation, which can control gene expression and affect cellular behaviour. This review highlights the current knowledge on the epigenetic modification for cancer treatment, immunomodulatory and anti-carcinogenic attributes of microbial metabolites, gut epigenetic targeting system, and the role of dietary fibre and gut microbiota in cancer treatment. It also focuses on short-chain fatty acids, especially butyrates (which are generated by microbes), and their cancer treatment perspective, challenges, and limitations, as well as state-of-the-art research on microbial metabolites-induced epigenetic changes for CRC inhibition. In conclusion, the present work highlights the potential of microbial metabolites-induced epigenetic modifications as a novel therapeutic strategy for CRC suppression and guides future research directions in this dynamic field.

TET2 regulates extranodal NK/T cell lymphoma progression through regulation of DNA methylation.

The biological role of Ten-11 translocation 2 (TET2) and the conversion of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) in the development of extra-nodal natural killer/T-cell lymphoma (ENKTL) remains unclear. The level of 5mC and 5hmC was detected in 112 cases of ENKTL tissue specimens by immunohistochemical (IHC) staining. Subsequently, TET2 knockdown and the overexpression cell models were constructed in ENKTL cell lines. Biochemical analyses were used to assess proliferation, apoptosis, cell cycle and monoclonal formation in cells treated or untreated with L-Ascorbic acid sodium salt (LAASS). Dot-Blots were used to detect levels of genome 5mC and 5hmC. Additionally, the ILLUMINA 850k methylation chip was used to analyze the changes of TET2 regulatory genes. RNA-Seq was used to profile differentially expressed genes regulated by TET2. The global level of 5hmC was significantly decreased, while 5mC was highly expressed in ENKTL tissue. TET2 protein expression was negatively correlated with the ratio of 5mC/5hmC (p<0.0001). The 5mC/5hmC status were related to the site of disease, clinical stage, PINK score and Ki-67 index, as well as the 5-year OS. TET2 knockdown prolonged the DNA synthesis period, increased the cloning ability of tumor cells, increased the level of 5mC and decreased the level of 5hmC in ENKTL cells. While overexpression of TET2 presented the opposite effect. Furthermore, treatment of ENKTL cells with LAASS significantly induced ENKTL cell apoptosis. These results suggest that TET2 plays an important role in ENKTL development via regulation of 5mC and 5hmC and may serve as a novel therapeutic target for ENKTL.

A combination of 5-azacytidine and nivolumab is a potentially effective rescue therapy in relapsed/refractory AITL.

Angioimmunoblastic T-cell lymphoma (AITL) is a peripheral T-cell lymphoma characterized by a T follicular helper cell phenotype expressing PD-1 (programmed cell death-1). AITL exhibits a poor response to conventional chemotherapy, with a median 5-year overall survival of 44% and a progression-free survival of 32%. Relapse is common, resulting in a median overall survival of 6 months. Recurrent mutations are detected in genes regulating DNA methylation, including TET2, DNMT3A, and IDH2 variants, along with the prevalent RHOA G17V mutation. In this context, patients treated with the hypomethylating agent 5-azacytidine achieved overall response and complete response rates of 75% and 41%, respectively. We hypothesized that targeted therapies combining anti-PD-1 checkpoint blockers with hypomethylating agents could be efficient in AITL patients and less toxic than standard chemotherapy. Here, we report the efficacy of a regimen combining 5-azacytidine and nivolumab in nine relapsed or refractory AITL patients. This regimen was well-tolerated, especially in elderly patients. The overall response rate was 78%, including four partial responses (44%) and three complete responses (33%). Allogeneic hematopoietic stem cell transplantation was performed in two patients who reached complete response. These preliminary favorable results may serve as a basis for further investigation in prospective studies.

MiR-520e and its promoter region DNA methylation as potential biomarkers in atherosclerosis.

In atherosclerosis, DNA methylation plays a key regulatory role in the expression of related genes. However, the molecular mechaism of these processes in HUVECs are unclear. Here, using high-throughput sequencing from the Infinium HumanMethylation450 assay, we manifested that the cg19564375 methylation of miR-520e promoter region in the peripheral blood of acute coronary syndrome (ACS) patients was higher than that of healthy controls. As shown by RQ-MSP, the upstream DNA methylation level of the miR-520e promoter region was considerably increased in ACS patients. miR-520e was markedly down-regulated in ACS patients compared with healthy controls. In the ox-LDL-induced HUVECs injury model, DNA methylation of the upstream region of miR-520e was significantly increased. With increasing concentrations of the methylase inhibitor 5-Aza, miR-520e expression was upregulated. The silence of methyltransferase DNMT1, rather than DNMT3a or DNMT3b, abolished the influence of miR-520e expression by ox-LDL treatment in HUVECs. A dual luciferase reporter assay revealed that miR-520e regulated the TGFBR2 3'-UTR region. After silencing TGFBR2, the promoting effect of miR-520e inhibitor on cell proliferation and migration may be attenuated. In conclusion, the expression of miR-520e is modified by its promoter region DNA methylation, and miR520e and its promoter region DNA methylation may be potential biomarkers in atherosclerosis.

Mediating effects of BMI on the association between DNA methylation regions and 24-h blood pressure in African Americans.

DNA methylation is an important epigenetic mechanism that may influence blood pressure (BP) regulation and hypertension risk. Obesity, a major lifestyle factor associated with hypertension, may interact with DNA methylation to affect BP. However, the indirect effect of DNA methylation on 24-hBP measurements mediated by obesity-related phenotypes such as BMI has not been investigated. Causal mediation analysis was applied to examine the mediating role of BMI in the relation between DNA methylation and 24-h BP phenotypes, including SBP, DBP and mean arterial blood pressure (MAP), in 281 African American participants. Analysis of 38 215 DNA methylation regions, derived from 1,549,368 CpG sites across the genome, identified up to 138 methylation regions that were significantly associated with 24-h BP measurements through BMI mediation. Among them, 38 (19.2%) methylation regions were concurrently associated with SBP, DBP and MAP. Genes associated with BMI-mediated methylation regions are potentially involved in various chronic diseases such as coronary artery disease and renal disease, which are often caused or exacerbated by hypertension. Notably, three genes (CDH4, NOTCH1 and COLGALT1) showed both direct associations with 24-h BP measurements and indirect associations through BMI after adjusting for age and sex covariates. Our findings suggest that DNA methylation may contribute to the regulation of 24-h BP in African Americans both directly and indirectly through BMI mediation.

DNA methylase 1 influences temperature responses and development in the invasive pest Tuta absoluta.

DNA methylase 1 (Dnmt1) is an important regulatory factor associated with biochemical signals required for insect development. It responds to changes in the environment and triggers phenotypic plasticity. Meanwhile, Tuta absolutaMeyrick (Lepidoptera: Gelechiidae)-a destructive invasive pest-can rapidly invade and adapt to different habitats; however, the role of Dnmt1 in this organism has not been elucidated. Accordingly, this study investigates the mechanism(s) underlying the rapid adaptation of Tuta absoluta to temperature stress. Potential regulatory genes were screened via RNAi (RNA interference), and the DNA methylase in Tuta absoluta was cloned by RACE (Rapid amplification of cDNA ends). TaDnmt1 was identified as a potential regulatory gene via bioinformatics; its expression was evaluated in response to temperature stress and during different development stages using real-time polymerase chain reaction. Results revealed that TaDnmt1 participates in hot/cold tolerance, temperature preference and larval development. The full-length cDNA sequence of TaDnmt1 is 3765 bp and encodes a 1254 kDa protein with typical Dnmt1 node-conserved structural features and six conserved DNA-binding active motifs. Moreover, TaDnmt1 expression is significantly altered by temperature stress treatments and within different development stages. Hence, TaDnmt1 likely contributes to temperature responses and organismal development. Furthermore, after treating with double-stranded RNA and exposing Tuta absoluta to 35°C heat shock or -12°C cold shock for 1 h, the survival rate significantly decreases; the preferred temperature is 2°C lower than that of the control group. In addition, the epidermal segments become enlarged and irregularly folded while the surface dries up. This results in a significant increase in larval mortality (57%) and a decrease in pupation (49.3%) and eclosion (50.9%) rates. Hence, TaDnmt1 contributes to temperature stress responses and temperature perception, as well as organismal growth and development, via DNA methylation regulation. These findings suggest that the rapid geographic expansion of T absoluta has been closely associated with TaDnmt1-mediated temperature tolerance. This study advances the research on 'thermos Dnmt' and provides a potential target for RNAi-driven regulation of Tuta absoluta.

Role of the CTCF Binding Site in Human T-Cell Leukemia Virus-1 Pathogenesis.

During HTLV-1 infection, the virus integrates into the host cell genome as a provirus with a single CCCTC binding protein (CTCF) binding site (vCTCF-BS), which acts as an insulator between transcriptionally active and inactive regions. Previous studies have shown that the vCTCF-BS is important for maintenance of chromatin structure, regulation of viral expression, and DNA and histone methylation. Here, we show that the vCTCF-BS also regulates viral infection and pathogenesis in vivo in a humanized (Hu) mouse model of adult T-cell leukemia/lymphoma. Three cell lines were used to initiate infection of the Hu-mice, i) HTLV-1-WT which carries an intact HTLV-1 provirus genome, ii) HTLV-1-CTCF, which contains a provirus with a mutated vCTCF-BS which abolishes CTCF binding, and a stop codon immediate upstream of the mutated vCTCF-BS which deletes the last 23 amino acids of p12, and iii) HTLV-1-p12stop that contains the intact vCTCF-BS, but retains the same stop codon in p12 as in the HTLV-1-CTCF cell line. Hu-mice were infected with mitomycin treated or irradiated HTLV-1 producing cell lines. There was a delay in pathogenicity when Hu-mice were infected with the CTCF virus compared to mice infected with either p12 stop or WT virus. Proviral load (PVL), spleen weights, and CD4 T cell counts were significantly lower in HTLV-1-CTCF infected mice compared to HTLV-1-p12stop infected mice. Furthermore, we found a direct correlation between the PVL in peripheral blood and death of HTLV-1-CTCF infected mice. In cell lines, we found that the vCTCF-BS regulates Tax expression in a time-dependent manner. The scRNAseq analysis of splenocytes from infected mice suggests that the vCTCF-BS plays an important role in activation and expansion of T lymphocytes in vivo. Overall, these findings indicate that the vCTCF-BS regulates Tax expression, proviral load, and HTLV pathogenicity in vivo.

Improve-RRBS: a novel tool to correct the 3’ trimming of reduced representation sequencing reads

Abstract Motivation Reduced Representation Bisulfite Sequencing (RRBS) is a popular approach to determine DNA methylation of the CpG-rich regions of the genome. However, we observed that false positive differentially methylated sites (DMS) are also identified using the standard computational analysis. Results During RRBS library preparation the MspI digested DNA undergo end-repair by a cytosine at the 3’ end of the fragments. After sequencing, Trim Galore cuts these end-repaired nucleotides. However, Trim Galore fails to detect end-repair when it overlaps with the 3’ end of the sequencing reads. We found that these non-trimmed cytosines bias methylation calling, thus can identify DMS erroneously. To circumvent this problem, we developed improve-RRBS, which efficiently identifies and hides these cytosines from methylation calling with a false positive rate of maximum 0.5%. To test improve-RRBS, we investigated four datasets from four laboratories and two different species. We found non-trimmed 3’ cytosines in all datasets analyzed and as much as &amp;gt; 50% of false positive DMS under certain conditions. By applying improve-RRBS, these DMS completely disappeared from all comparisons. Availability improve-RRBS is a freely available python package https://pypi.org/project/iRRBS/ or https://github.com/fothia/improve-RRBS to be implemented in RRBS pipelines. Supplementary information Supplementary data are available at Bioinformatics Advances online.

Oral azacitidine compared with standard therapy in patients with relapsed or refractory follicular helper T-cell lymphoma (ORACLE): an open-label randomised, phase 3 study

Follicular helper T-cell lymphomas (TFHL) harbour frequent alterations in genes that regulate DNA methylation. Preliminary reports suggest that treatment with 5-azacitidine has clinical activity in patients with relapsed or refractory TFHL. We aimed to compare the oral form of azacitidine with investigator's choice standard therapy (ICT; ie, gemcitabine, bendamustine, or romidepsin) in patients with relapsed or refractory TFHL. Patients older than 18 years with relapsed or refractory TFHL (angioimmunoblastic T-cell lymphoma, follicular lymphoma, or nodal T-cell lymphoma with phenotype, ie, positive with two or more markers among CD10, BCL6, CXCL13, PD1, or ICOS) based on the 2017 WHO classification of haematological neoplasms, with an Eastern Cooperative Oncology Group performance status score of 0-3, were recruited in university hospitals from five European countries and from Japan. Patients were randomly assigned 1:1 to treatment with either azacitidine given at a dose of 300 mg once a day (200 mg in Japanese patients) for 14 days in a 28-day cycle or gemcitabine, bendamustine, or romidepsin according to the investigator's choice. Random assignment was stratified by the number of previous lines of therapy and by the presence of previous or concomitant myeloid malignancy. The primary endpoint was investigator-assessed progression-free survival, presented in the intention-to-treat population. This Article is the final analysis of this trial, registered at ClinicalTrials.gov (Europe NCT03593018 and Japan NCT03703375). 86 patients (median age 69 years [IQR 62-76], 50 patients were male, 36 were female) were enrolled between Nov 9, 2018, to Feb 22, 2021; 42 in the azacitidine group and 44 in the ICT group. With a median follow-up of 27·4 months (IQR 20·2-32·9), the median progression-free survival was 5·6 months (95% CI 2·7 -8·1) in the azacitidine group versus 2·8 months (1·9-4·8) in the ICT group (hazard ratio of 0·63 (95% CI 0·38-1·07); 1-sided p=0·042). Grade 3-4 adverse events were reported in 32 (76%) of 42 patients in the azacitidine group versus 42 (98%) of 43 patients in the ICT group. The most adverse grade 3 or worse adverse events were haematological (28 [67%] of 42 patients vs 40 [93%] of 43 patients), infection (8 [19%] and 14 [33%]), and gastrointestinal (5 [12%] vs 1 [2%] for azacitidine and ICT, respectively). There were two treatment-related deaths in the azacitidine group (one endocarditis and one candidiasis) and three in the ICT group (one heart failure, one COVID-19, and one cause unknown). Although the pre-specified primary outcome of the trial was not met, the favourable safety profile suggests that azacitidine could add to the treatment options in these difficult to treat diseases especially in combination with other drugs. Trials with combination are in preparation in a platform trial. Bristol-Myers Squibb. For the French translation of the abstract see Supplementary Materials section.

Genome-wide DNA methylation analysis in blood identifies differentially methylated regions related to polycystic ovary syndrome

Polycystic ovary syndrome (PCOS) is considered a multifactorial disorder caused due to predisposing alleles of various genes and environmental factors which include lifestyle, dietary habits, and exposure to toxicants. Recently the emphasis has shifted to understanding epigenetic alterations due to the poor correlation between genotypes and the clinical profile of women with PCOS. Investigators have been focusing on differential methylation signatures associated with PCOS, therefore, a pilot study was carried out to investigate PCOS-associated differential DNA methylation regions in the population of north India. A cross-sectional study with sixteen participants comprising eight women with PCOS diagnosed under the Rotterdam criterion and eight age and BMI-matched healthy controls. Subjects were classified into four groups based on BMI and two different groups were compared each time making four comparison types in total. Genome-wide DNA methylation in whole blood was evaluated using an Infinium Human Methylation 850k array which identified PCOS-associated differential sites and differential methylation regions (DMRs) with promoter region, and CpG islands considering (p ≤ 0.01) as statistically significant. Multiple significantly associated hypomethylated and hypermethylated loci of genes were identified. Thirty-four hypomethylated and one hundred twenty-six hypermethylated sites of genes were suggested when all the subjects were of low BMI. But when the groups comprised with high BMI, twenty-seven hypomethylated genes and fifty-nine hypermethylated genes were identified. In silico analysis suggests that most of these genes are involved in estrogen biosynthesis, lipid biosynthetic process, and activation of matrix metalloproteinases (MMP). Seven genes that are found common in both of these comparisons are mostly involved in the inflammasome pathway indicated by BioPlanet and Reactome databases. The study provides information about DMRs in the blood tissue which may be investigated further to understand underlying pathogenesis and as therapeutic targets in the future.

Exploring the epigenetic profile of ID4 in breast cancer: bioinformatic insights into methylation patterns and chromatin accessibility dynamics.

Inhibitor of differentiation 4 (ID4) is a dominant-negative regulator of basic helix-loop-helix (bHLH) transcription factors. The expression of ID4 is dysregulated in various breast cancer subtypes, indicating a potential role for ID4 in subtype-specific breast cancer development. This study aims to elucidate the epigenetic regulation of ID4 within breast cancer subtypes, with a particular focus on DNA methylation and chromatin accessibility. Bioinformatic analyses were conducted to assess DNA methylation and chromatin accessibility in ID4 regulatory regions across breast cancer subtypes. Gene Set Enrichment Analysis (GSEA) was conducted to identify related gene sets. Transcription factor binding within ID4 enhancer and promoter regions was explored. In vitro experiments involved ER+ breast cancer cell lines treated with estradiol (E2) and Tamoxifen. Distinct epigenetic profiles of ID4 were observed, revealing increased methylation and reduced chromatin accessibility in luminal subtypes compared to the basal subtype. Gene Set Enrichment Analysis (GSEA) implicated estrogen-related pathways, suggesting a potential link between estrogen signaling and the regulation of ID4 expression. Transcription factor analysis identified ER and FOXA1 as regulators of ID4 enhancer regions. In vitro experiments confirmed the role of ER, demonstrating reduced ID4 expression and increased methylation with estradiol treatment. Conversely, Tamoxifen treatment increased ID4 expression, indicating the potential involvement of ER signaling through ERα in the epigenetic regulation of ID4 in breast cancer cells. This study shows the intricate epigenetic regulation of ID4 in breast cancer, highlighting subtype-specific differences in DNA methylation and chromatin accessibility.

Rice melatonin deficiency causes premature leaf senescence via DNA methylation regulation

In a study of DNA methylation changes in melatonin-deficient rice mutants, mutant plants showed premature leaf senescence during grain-filling and reduced grain yield. Melatonin deficiency led to transcriptional reprogramming, especially of genes involved in chlorophyll and carbon metabolism, redox regulation, and transcriptional regulation, during dark-induced leaf senescence. Hypomethylation of mCG and mCHG in the melatonin-deficient rice mutants was associated with the expression change of both protein-coding genes and transposable element-related genes. Changes in gene expression and DNA methylation in the melatonin-deficient mutants were compensated by exogenous application of melatonin. A decreased S-adenosyl-L-methionine level may have contributed to the DNA methylation variations in rice mutants of melatonin deficiency under dark conditions.

Elevation of miR-150-5p in Brain Microvascular Endothelial Cells as a Result of Prenatal Alcohol Exposure Is Mediated by Transcriptional Mechanisms

Fetal Alcohol Spectrum Disorders (FASD) encompass a variety of conditions impacting development in children exposed to alcohol in utero. Vascular alterations such as defective angiogenesis, blood brain barrier permeability, vessel reactivity, and blood flow have been found in various animal models of prenatal alcohol exposure (PAE). The underlying mechanisms of these vascular defects are not fully understood. Our lab previously showed that miR-150-5p was upregulated in brain microvascular endothelial cells (BMVECs) isolated from embryonic mouse cortices following moderate PAE. We identified downstream vascular targets of miR-150-5p in BMVECs and demonstrated that miR-150-5p inhibition could counter alcohol-mediated effects on BMVECS and the cortical microvasculature in vitro and in vivo, respectively. Here, we investigate upstream mechanisms that contribute to miR-150-5p elevation in BMVECs following PAE. We hypothesized that multiple mechanisms, including pathways at the transcriptional level, contribute to increased miR-150-5p abundance in BMVECs following ethanol (EtOH) exposure. Overall transcriptional activity at the miR-150 promoter was increased in BMVECs with EtOH treatment, and pri-miR-150, the primary transcript for miR-150-5p, was elevated in EtOH-treated BMVECs. Additionally, miR-150 promoter methylation was decreased in PAE cortices compared to saccharine-exposed controls (SAC). Current work aims to confirm the role of DNA methylation in transcriptional regulation of the miR-150 promoter in BMVECs and characterize DNA methyltransferase activity in BMVECs during PAE. We also assessed transcription factor binding at the miR-150 promoter and found that miR-150 promoter activation by several transcription factors was blunted with EtOH treatment. Current work involves the investigation of global transcription factor activation in BMVECs in PAE versus SAC. In summary, our research sheds light on multiple mechanisms of miR-150-5p regulation that may contribute to brain microvascular defects as a result of PAE. American Physiological Society Porter Physiology Development Fellowship University of New Mexico Health School of Medicine Department of Cell Biology and Physiology. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Coordinated Regulation of Myonuclear DNA Methylation, mRNA, and miRNA Levels Associates with Mitochondrial Function During an Acute Skeletal Muscle Growth Stimulus

The central dogma of molecular biology dictates the general flow of molecular information from DNA that leads to a functional cellular outcome. In skeletal muscle fibers, the extent to which global myonuclear transcriptional alterations, accounting for epigenetic and post-transcriptional influences, contribute to an adaptive stress response is not clearly defined. In this investigation, we leveraged an integrated analysis of the myonucleus-specific DNA methylome and transcriptome, as well as myonuclear small RNA profiling to molecularly define the early phase of skeletal muscle fiber hypertrophy. The analysis of myonucleus-specific mature miRNA and other small RNA species provides new directions for exploring muscle adaptation and complemented the methylation and transcriptional information. Our multi-omics interrogation revealed a coordinated myonuclear molecular landscape during muscle loading that coincides with an acute and rapid reduction of oxidative metabolism. This response may favor a biosynthesis-oriented metabolic program that supports rapid hypertrophic growth. Y.W. was supported by the National Institutes of Health (K99 AR081367) K.A.M. was supported by the National Institutes of Health (R00 AG063994 and R01 AG080047). F.v.W. was supported by the Swedish research council (2022-01392), AFM-Telethon (23137), SMDF, Åke Wiberg, Swedish Medical Association, and the Swedish Research Council for Sport Science (2022/10). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Single-molecule long-read methylation profiling reveals regional DNA methylation regulated by Elongator Complex Subunit 2 in Arabidopsis roots experiencing spaceflight

BackgroundThe Advanced Plant Experiment-04 - Epigenetic Expression (APEX-04-EpEx) experiment onboard the International Space Station examined the spaceflight-altered cytosine methylation in two genetic lines of Arabidopsis thaliana, wild-type Col-0 and the mutant elp2-5, which is deficient in an epigenetic regulator Elongator Complex Subunit 2 (ELP2). Whole-genome bisulfite sequencing (WGBS) revealed distinct spaceflight associated methylation differences, presenting the need to explore specific space-altered methylation at single-molecule resolution to associate specific changes over large regions of spaceflight related genes. To date, tools of multiplexed targeted DNA methylation sequencing remain limited for plant genomes.ResultsTo provide methylation data at single-molecule resolution, Flap-enabled next-generation capture (FENGC), a novel targeted multiplexed DNA capture and enrichment technique allowing cleavage at any specified sites, was applied to survey spaceflight-altered DNA methylation in genic regions of interest. The FENGC capture panel contained 108 targets ranging from 509 to 704 nt within the promoter or gene body regions of gene targets derived from spaceflight whole-genome data sets. In addition to genes with significant changes in expression and average methylation levels between spaceflight and ground control, targets with space-altered distributions of the proportion of methylated cytosines per molecule were identified. Moreover, trends of co-methylation of different cytosine contexts were exhibited in the same DNA molecules. We further identified significant DNA methylation changes in three previously biological process-unknown genes, and loss-of-function mutants of two of these genes (named as EMO1 and EMO2 for ELP2-regulated Methylation in Orbit 1 and 2) showed enhanced root growth rate.ConclusionsFENGC simplifies and reduces the cost of multiplexed, targeted, single-molecule profiling of methylation in plants, providing additional resolution along each DNA molecule that is not seen in population-based short-read data such as WGBS. This case study has revealed spaceflight-altered regional modification of cytosine methylation occurring within single DNA molecules of cell subpopulations, which were not identified by WGBS. The single-molecule survey by FENGC can lead to identification of novel functional genes. The newly identified EMO1 and EMO2 are root growth regulators which may be epigenetically involved in plant adaptation to spaceflight.

EPIGENETIC MODIFICATIONS OF STRESS-RELATED DISORDERS: MECHANISMS, IMPLICATIONS AND THERAPEUTIC PERSPECTIVES

Stress-related disorders, encompassing conditions such as anxiety, depression, and post-traumatic stress disorder (PTSD), pose significant challenges to global mental health. While the etiology of these disorders involves a complex interplay of genetic, environmental, and psychosocial factors, emerging evidence suggests a pivotal role of epigenetic modifications in their pathogenesis. Epigenetic mechanisms, including DNA methylation, histone modifications, and non-coding RNA-mediated regulation, dynamically regulate gene expression in response to stressors, thereby influencing brain function and behaviour. Alterations in these epigenetic processes contribute to aberrant stress responses, increased susceptibility to stress-related disorders, and individual differences in stress vulnerability and resilience. Moreover, epigenetic modifications can be transmitted across generations, highlighting the enduring impact of stress on mental health outcomes. Understanding the mechanisms underlying epigenetic regulation in stress-related disorders provides insights into disease pathogenesis and potential biomarkers for diagnosis and prognosis. Additionally, targeting epigenetic pathways represents a promising therapeutic approach for these disorders. Epigenetic drugs, such as DNA methyltransferase inhibitors and histone deacetylase inhibitors, hold potential for mitigating the adverse effects of stress on mental health. However, challenges such as off-target effects and lack of specificity necessitate further refinement of epigenetic-based therapies.

DNA methyltransferases-associated long non-coding RNA PRKCQ-AS1 regulate DNA methylation in myelodysplastic syndrome.

Myelodysplastic syndrome (MDS) is a group of clonal hematopoietic stem cell disorders. DNA hypermethylation is considered to be the key mechanism of pathogenesis for MDS. Studies have demonstrated that DNA methylation can be regulated by the co-effect between long non-coding RNAs (lncRNAs) and DNA methyltransferases (DNMTs). The aim of this study was to identify DNMTs-associated differentially expressed (DE) lncRNAs, which may be a novel diagnostic and therapeutic target for MDS. Two gene expression profile datasets (GSE4619 and GSE19429) were downloaded from the Gene Expression Omnibus (GEO) database. Systematic bioinformatics analysis was conducted. Then we verified the expression of PRKCQ-AS1 in MDS patients and features in SKM-1 cells. Bioinformatics analysis revealed that the DNMT-associated DE-lncRNA PRKCQ-AS1 was functionally related to DNA methylation. The target genes of PRKCQ-AS1 associated with DNA methylation are mainly methionine synthetase (MTR) and ten-eleven-translocation 1 (TET1). Moreover, the high expression of PRKCQ-AS1 was verified in real MDS cases. Further cellular analysis in SKM-1 cells revealed that overexpressed PRKCQ-AS1 promoted methylation levels of long interspersed nuclear element 1 (LINE-1) and cell proliferation, and apparently elevated both mRNA and protein levels of MTR and TET1, while knockdown of PRKCQ-AS1 showed opposite trend in SKM-1 cells. DNMT-associated DE-lncRNA PRKCQ-AS1 may affects DNA methylation levels by regulating MTR and TET1.

DNA methylation regulator-based molecular subtyping and tumor microenvironment characterization in hepatocellular carcinoma.

Although recent studies have reported the regulation of the immune response in hepatocellular carcinoma (HCC) through DNA methylation, the comprehensive impact methylation modifications on tumor microenvironment characteristics and immunotherapy efficacy has not been fully elucidated. In this research, we conducted a comprehensive assessment of the patterns of DNA methylation regulators and the profiles of the tumor microenvironment (TME) in HCC, focusing on 21 specific DNA methylation regulators. We subsequently developed a unique scoring system, a DNA methylation score (DMscore), to assess the individual DNA methylation modifications among the three distinct methylation patterns for differentially expressed genes (DEGs). Three distinct methylation modification patterns were identified with distinct TME infiltration characteristics. We demonstrated that the DMscore could predict patient subtype, TME infiltration, and patient prognosis. A low DMscore, characterized by an elevated tumor mutation burden (TMB), hepatitis B virus (HBV)/hepatitis C virus (HCV) infection, and immune activation, indicates an inflamed tumor microenvironment phenotype with a 5-year survival rate of 7.8%. Moreover, a low DMscore appeared to increase the efficacy of immunotherapy in the anti-CTLA-4/PD-1/PD-L1 cohort. In brief, this research has enhanced our understanding of the correlation between modifications in DNA methylation patterns and the profile of the tumor microenvironment in individuals diagnosed with HCC. The DMscore may serve as an alternative biomarker for survival and efficacy of immunotherapy in patients with HCC.

DNA methylation of imprint control regions associated with Alzheimer’s disease in non-Hispanic Blacks and non-Hispanic Whites

Alzheimer’s disease (AD) prevalence is twice as high in non-Hispanic Blacks (NHBs) as in non-Hispanic Whites (NHWs). The objective of this study was to determine whether aberrant methylation at imprint control regions (ICRs) is associated with AD. Differentially methylated regions (DMRs) were bioinformatically identified from whole-genome bisulfite sequenced DNA derived from brain tissue of 9 AD (5 NHBs and 4 NHWs) and 8 controls (4 NHBs and 4 NHWs). We identified DMRs located within 120 regions defined as candidate ICRs in the human imprintome (https://genome.ucsc.edu/s/imprintome/hg38.AD.Brain_track). Eighty-one ICRs were differentially methylated in NHB-AD, and 27 ICRs were differentially methylated in NHW-AD, with two regions common to both populations that are proximal to the inflammasome gene, NLRP1, and a known imprinted gene, MEST/MESTIT1. These findings indicate that early developmental alterations in DNA methylation of regions regulating genomic imprinting may contribute to AD risk and that this epigenetic risk differs between NHBs and NHWs.