Disruption Of DNA Methylation Research Articles

Maternal Malnutrition in Mice Impairs Nephrogenesis by Disrupting DNA Methylation of Regulatory Regions.

Maternal caloric restriction during pregnancy significantly impacts kidney development, influencing susceptibility to chronic kidney disease in adulthood. This study explores DNA methylation changes in nephron progenitor cells resulting from caloric restriction and their implications for kidney health. Global DNA hypomethylation is observed in nephron progenitors from caloric-restricted embryos, with specific genomic regions displaying distinct methylation patterns, including hypomethylation and hypermethylation. Differentially methylated regions exhibit enhanced chromatin accessibility, indicating biological relevance. Hypomethylated regions are enriched for genes associated with developmental processes, reflecting changes in gene expression and highlighting their functional relevance in kidney development. The study also reveals that supplementing methionine, an essential amino acid, restores disrupted DNA methylation patterns, particularly in enhancer regions, emphasizing methionine's critical role in regulating nephron progenitor cell epigenetics and ensuring proper kidney development. The intricate relationship between maternal nutrition, dynamic DNA methylation, and kidney development is highlighted, emphasizing the enduring impact of early-life nutritional challenges on kidney function. This research elucidates epigenetic mechanisms as mediators for the lasting effects of maternal caloric restriction on kidney health. The study contributes valuable insights into the origins of chronic kidney diseases during early developmental stages, offering potential interventions to mitigate adverse outcomes.

Clinical and genetic characteristics of ALS patients with variants in genes regulating DNA methylation.

Aberrant DNA methylation alterations are implicated in amyotrophic lateral sclerosis (ALS). Nevertheless, the influence of genetic variants in genes regulating DNA methylation on ALS patients is not well understood. Therefore, we aim to provide a comprehensive variant profile of genes related to DNA methylation (DNMT1, DNMT3A, DNMT3B, DNMT3L) and demethylation (TET1, TET2, TET3, TDG) and to investigate the association of these variants with ALS. Variants were screened in a cohort of 2240 ALS patients from Southwest China, using controls from the Genome Aggregation Database (n = 9976) and the China Metabolic Analytics Project (n = 10,588). The over-representation of rare variants and their association with ALS risk were evaluated using Fisher's exact test with Bonferroni correction at both allele and gene levels. Kaplan-Meier analysis and Cox regression analysis were employed to explore the relationship between variants and survival. A total of 210 variants meeting the criteria were identified. Gene-based burden analysis identified a significant increase in ALS risk associated with rare variants in the TET2 gene (OR = 1.95, 95% CI = 1.29-2.88, P = 0.001). Survival analysis demonstrated that patients carrying variants in demethylation-related genes had a higher risk of death compared to those with methylation-related gene variants (HR = 1.29, 95% CI = 1.03-1.86, P = 0.039). This study provides a genetic variant profile of genes involved in DNA methylation and demethylation regulation, along with the clinical characteristics of ALS patients carrying these variants. The findings offer genetic evidence implicating disrupted DNA methylation dynamics in ALS.

Demethylation Strategies for the Treatment of Acute Myeloid Leukemia: Targeting Protacs That Degrade DNA Methyltransferase

Background DNA methylation is crucial in the pathogenesis of acute myeloid leukemia (AML), characterized by abnormal proliferation of myeloid cells and disrupted DNA methylation patterns. These aberrant patterns contribute to the dysregulation of key genes involved in AML development and progression. Therefore, targeting DNA methyltransferases (DNMTs) to restore proper DNA methylation patterns has emerged as a promising therapeutic strategy. However, traditional demethylation therapies (e.g. azacytidine, decitabine) face challenges in effectively and selectively targeting DNMTs. Methods In this study, we investigated the potential of a novel approach utilizing proteolysis targeting chimeras (PROTACs) for selective degradation of DNMTs in AML cells. A series of PROTAC molecules was designed and synthesized, with GSK3685032 serving as the ligand for DNMTs. The E3 ligase CRBN was employed to mediate the proteasomal degradation process. Results The designed PROTACs efficiently degraded DNMTs in AML cell lines, as demonstrated by immunoblotting and immunofluorescence assays. Genome-wide DNA methylation analysis revealed a significant reduction in global DNA methylation levels following PROTAC-mediated DNMT degradation.In AML PDX models, the PROTACs effectively decreased DNMT protein levels, resulting in substantial reduction of global DNA methylation and significant inhibition of tumor growth. Furthermore, combination studies revealed synergistic effects when the PROTAC-mediated DNMT degradation was combined with standard clinical therapeutic agents.The combination approach showed enhanced therapeutic efficacy compared to either treatment alone, suggesting a potential strategy for improved AML treatment outcomes. Conclusions The study highlights the potential of PROTAC-mediated DNMT degradation as a promising targeted demethylation strategy for AML. This approach holds promise for improving treatment outcomes, particularly in AML patients with DNMT mutations. Further research is warranted to fully evaluate the efficacy of this approach in clinical settings. Keywords: Acute myeloid leukemia, DNA methylation, PROTACs

TET2 modulates spatial relocalization of heterochromatin in aged hematopoietic stem and progenitor cells.

DNA methylation deregulation at partially methylated domains (PMDs) represents an epigenetic signature of aging and cancer, yet the underlying molecular basis and resulting biological consequences remain unresolved. We report herein a mechanistic link between disrupted DNA methylation at PMDs and the spatial relocalization of H3K9me3-marked heterochromatin in aged hematopoietic stem and progenitor cells (HSPCs) or those with impaired DNA methylation. We uncover that TET2 modulates the spatial redistribution of H3K9me3-marked heterochromatin to mediate the upregulation of endogenous retroviruses (ERVs) and interferon-stimulated genes (ISGs), hence contributing to functional decline of aged HSPCs. TET2-deficient HSPCs retain perinuclear distribution of heterochromatin and exhibit age-related clonal expansion. Reverse transcriptase inhibitors suppress ERVs and ISGs expression, thereby restoring age-related defects in aged HSPCs. Collectively, our findings deepen the understanding of the functional interplay between DNA methylation and histone modifications, which is vital for maintaining heterochromatin function and safeguarding genome stability in stem cells.

Epigenetic Modulators as Therapeutic Agents in Cancer.

Epigenetics play a crucial role in gene regulation and cellular processes. Most importantly, its dysregulation can contribute to the development of tumors. Epigenetic modifications, such as DNA methylation and histone acetylation, are reversible processes that can be utilized as targets for therapeutic intervention. DNA methylation inhibitors disrupt DNA methylation patterns by inhibiting DNA methyltransferases. Such inhibitors can restore normal gene expression patterns, and they can be effective against various forms of cancer. Histone deacetylase inhibitors increase histone acetylation levels, leading to altered gene expressions. Like DNA methylation inhibitors, histone methyltransferase inhibitors target molecules involved in histone methylation. Bromodomain and extra-terminal domain inhibitors target proteins involved in gene expression. They can be effective by inhibiting oncogene expression and inducing anti-proliferative effects seen in cancer. Understanding epigenetic modifications and utilizing epigenetic inhibitors will offer new possibilities for cancer research.

Placental ischemia disrupts DNA methylation patterns in distal regulatory regions in rats

Preeclampsia (PE) is a leading cause of maternal and fetal morbidity and mortality worldwide. However, the impact of PE on the organization of the functional architecture of the placental methylome remains largely unknown. We performed whole-genome bisulfite sequencing of placental DNA and applied a Hidden Markov Model to investigate epigenome-wide alterations in functional structures, including partially methylated domains (PMDs), low-methylated regions (LMRs), and unmethylated regions (UMRs), in a reduced uterine perfusion pressure (RUPP) rat model of PE. The remarkable similarity we observed between the rat and human placental DNA methylomes suggests that the RUPP rat model is appropriate to elucidate the epigenetic mechanisms underlying human PE. The notable changes in PMDs indicate RUPP-induced perturbation of the stressed placental methylome. This was probably regulated via modulation of the epigenetic modifier expression, including significant downregulation of Dnmt1 and Dnmt3a and upregulation of Tet2. More importantly, changes in RUPP-induced DNA methylation occurred predominately in LMRs (80 %), which represent active enhancers, rather than in canonical UMRs (3 %), which represent promoters, suggesting that placental ischemia disrupts enhancer DNA methylation. Our findings emphasize the role of enhancer methylation in response to PE, corroborating discoveries in human PE studies. We suggest paying more attention to enhancer regions in future studies on PE.

Knockout of DDM1 in Physcomitrium patens disrupts DNA methylation with a minute effect on transposon regulation and development.

The Snf2 chromatin remodeler, DECREASE IN DNA METHYLATION 1 (DDM1) facilitates DNA methylation. In flowering plants, DDM1 mediates methylation in heterochromatin, which is targeted primarily by MET1 and CMT methylases and is necessary for silencing transposons and for proper development. DNA methylation mechanisms evolved throughout plant evolution, whereas the role of DDM1 in early terrestrial plants remains elusive. Here, we studied the function of DDM1 in the moss, Physcomitrium (Physcomitrella) patens, which has robust DNA methylation that suppresses transposons and is mediated by a MET1, a CMT, and a DNMT3 methylases. To elucidate the role of DDM1 in P. patens, we have generated a knockout mutant and found DNA methylation to be strongly disrupted at any of its sequence contexts. Symmetric CG and CHG sequences were affected stronger than asymmetric CHH sites. Furthermore, despite their separate targeting mechanisms, CG (MET) and CHG (CMT) methylation were similarly depleted by about 75%. CHH (DNMT3) methylation was overall reduced by about 25%, with an evident hyper-methylation activity within lowly-methylated euchromatic transposon sequences. Despite the strong hypomethylation effect, only a minute number of transposons were transcriptionally activated in Ppddm1. Finally, Ppddm1 was found to develop normally throughout the plant life cycle. These results demonstrate that DNA methylation is strongly dependent on DDM1 in a non-flowering plant; that DDM1 is required for plant-DNMT3 (CHH) methylases, though to a lower extent than for MET1 and CMT enzymes; and that distinct and separate methylation pathways (e.g. MET1-CG and CMT-CHG), can be equally regulated by the chromatin and that DDM1 plays a role in it. Finally, our data suggest that the biological significance of DDM1 in terms of transposon regulation and plant development, is species dependent.

Site-specific decreases in DNA methylation in replicating cells following exposure to oxidative stress.

Oxidative stress is a common feature of inflammation-driven cancers, and it promotes genomic instability and aggressive tumour phenotypes. It is known that oxidative stress transiently modulates gene expression through the oxidation of transcription factors and associated regulatory proteins. Neutrophils are our most abundant white blood cells and accumulate at sites of infection and inflammation. Activated neutrophils produce hypochlorous acid and chloramines, which can disrupt DNA methylation by oxidizing methionine. The goal of the current study was to determine whether chloramine exposure results in sequence-specific modifications in DNA methylation that enable long-term alterations in transcriptional output. Proliferating Jurkat T-lymphoma cells were exposed to sublethal doses of glycine chloramine and differential methylation patterns were compared using Illumina EPIC 850K bead chip arrays. There was a substantial genome-wide decrease in methylation 4 h after exposure that correlated with altered RNA expression for 24 and 48h, indicating sustained impacts on exposed cells. A large proportion of the most significant differentially methylated CpG sites were situated towards chromosomal ends, suggesting that these regions are most susceptible to inhibition of maintenance DNA methylation. This may contribute to epigenetic instability of chromosomal ends in rapidly dividing cells, with potential implications for the regulation of telomere length and cellular longevity.

Alcohol and Cancer: Epidemiology and Biological Mechanisms.

Approximately 4% of cancers worldwide are caused by alcohol consumption. Drinking alcohol increases the risk of several cancer types, including cancers of the upper aerodigestive tract, liver, colorectum, and breast. In this review, we summarise the epidemiological evidence on alcohol and cancer risk and the mechanistic evidence of alcohol-mediated carcinogenesis. There are several mechanistic pathways by which the consumption of alcohol, as ethanol, is known to cause cancer, though some are still not fully understood. Ethanol’s metabolite acetaldehyde can cause DNA damage and block DNA synthesis and repair, whilst both ethanol and acetaldehyde can disrupt DNA methylation. Ethanol can also induce inflammation and oxidative stress leading to lipid peroxidation and further DNA damage. One-carbon metabolism and folate levels are also impaired by ethanol. Other known mechanisms are discussed. Further understanding of the carcinogenic properties of alcohol and its metabolites will inform future research, but there is already a need for comprehensive alcohol control and cancer prevention strategies to reduce the burden of cancer attributable to alcohol.

The DNA methylation landscape of multiple myeloma shows extensive inter- and intrapatient heterogeneity that fuels transcriptomic variability

BackgroundCancer evolution depends on epigenetic and genetic diversity. Historically, in multiple myeloma (MM), subclonal diversity and tumor evolution have been investigated mostly from a genetic perspective.MethodsHere, we performed an analysis of 42 MM samples from 21 patients by using enhanced reduced representation bisulfite sequencing (eRRBS). We combined several metrics of epigenetic heterogeneity to analyze DNA methylation heterogeneity in MM patients.ResultsWe show that MM is characterized by the continuous accumulation of stochastic methylation at the promoters of development-related genes. High combinatorial entropy change is associated with poor outcomes in our pilot study and depends predominantly on partially methylated domains (PMDs). These PMDs, which represent the major source of inter- and intrapatient DNA methylation heterogeneity in MM, are linked to other key epigenetic aberrations, such as CpG island (CGI)/transcription start site (TSS) hypermethylation and H3K27me3 redistribution as well as 3D organization alterations. In addition, transcriptome analysis revealed that intratumor methylation heterogeneity was associated with low-level expression and high variability.ConclusionsWe propose that disrupted DNA methylation in MM is responsible for high epigenetic and transcriptomic instability allowing tumor cells to adapt to environmental changes by tapping into a pool of evolutionary trajectories.

Pan-cancer characterization of long non-coding RNA and DNA methylation mediated transcriptional dysregulation.

BackgroundDisruption of DNA methylation (DNAm) is one of the key signatures of cancer, however, detailed mechanisms that alter the DNA methylome in cancer remain to be elucidated.MethodsHere we present a novel integrative analysis framework, called MeLncTRN (Methylation mediated LncRNA Transcriptional Regulatory Network), that integrates genome-wide transcriptome, DNA methylome and copy number variation profiles, to systematically identify the epigenetically-driven lncRNA-gene regulation circuits across 18 cancer types.FindingWe show that a significant fraction of the aberrant DNAm and gene expression landscape in cancer is associated with long noncoding RNAs (lncRNAs). We reveal distinct types of regulation between lncRNA modulators and target genes that are operative in either only specific cancers or across cancers. Functional studies identified a common theme of cancer hallmarks that lncRNA modulators may participate in. The coupled lncRNA gene interactions via DNAm also serve as markers for classifications of cancer subtypes with different prognoses.InterpretationOur study reveals a vital layer of DNAm and associated expression regulation for many cancer-related genes and we also provide a valuable database resource for interrogating epigenetically mediated lncRNA-gene interactions in cancer.FundingNational Natural Science Foundation of China [91959106, 31871255].

Cadmium disturbs epigenetic modification and induces DNA damage in mouse preimplantation embryos

Cadmium is an environmental pollutant that has extensive deleterious effects on the reproductive system. However, the mechanisms underlying the effects of cadmium on preimplantation embryos are unclear. Here, we used a mouse model to investigate the effects of maternal cadmium (32 mg/l) exposure in drinking water for 2 days on early embryonic development, and studied the mechanisms associated with epigenetic modifications and DNA damage induced by oxidative stress. We observed that maternal cadmium exposure impaired preimplantation embryo development by inducing embryo death, fragmentation, or developmental blockade. After cadmium exposure, the most survived embryos were at the 8-cell stage, which were used for all measurements. Histone acetylation, not methylation, was disturbed by increasing histone deacetylase 1 (HDAC1) levels after cadmium exposure. Cadmium also disrupted DNA methylation of H19; however genomic DNA methylation can be normally reprogrammed in embryos. Furthermore, cadmium increased reactive oxygen species (ROS) levels and DNA damage, and partly inhibited gene expression related to DNA repair. The distribution and activity of mitochondria was increased; therefore, embryos maintain intracellular homeostasis for survival. Collectively, our findings revealed that maternal cadmium exposure impairs preimplantation embryo development by disturbing the epigenetic modification and inducing DNA damage.

TET2: A cornerstone in normal and malignant hematopoiesis.

Regulation of genome‐wide DNA methylation is fundamental for a variety of biological processes such as mammalian development, stem cell function, cellular proliferation/differentiation, and oncogenesis. Among the regulators of DNA methylation, ten‐eleven translocation 2 (TET2) is one of the most frequently mutated genes in clonal hematopoiesis of indeterminate potential and in various hematological malignancies, underscoring a pivotal role for TET2 in blood homeostasis and hematopoietic transformation. TET2 oxidizes methylated cytosines to further modify cytosines, which behave as intermediates in active/passive DNA demethylation processes. TET2 itself associates with histone modifiers, thereby regulating histone modifications and expression of target genes. A number of studies have reported pleiotropic effects of TET2 on hematopoietic stem cell self‐renewal, hematopoietic differentiation, genome instability and inflammatory response. Recent single‐cell genomics studies have identified gene promoters as well as transcription factor binding sites as TET2‐targeted genetic loci in which disruption of DNA methylation can fundamentally modify hematopoietic differentiation and promote leukemogenesis. TET2 mutations show convergent cooperativity with other disease alleles in signaling molecules, epigenetic modifiers, and spliceosome factors in hematopoietic transformation. Future studies focusing on the molecular basis of stem cell and immune regulation by TET2 loss will further deepen our understanding of the entire landscape of pathophysiology and molecular vulnerabilities of TET2‐mutated hematological malignancies.

Autocrine IL6-Mediated Activation of the STAT3-DNMT Axis Silences the TNFα-RIP1 Necroptosis Pathway to Sustain Survival and Accumulation of Myeloid-Derived Suppressor Cells.

Although accumulation of myeloid-derived suppressor cells (MDSC) is a hallmark of cancer, the underlying mechanism of this accumulation within the tumor microenvironment remains incompletely understood. We report here that TNFα-RIP1-mediated necroptosis regulates accumulation of MDSCs. In tumor-bearing mice, pharmacologic inhibition of DNMT with the DNA methyltransferease inhibitor decitabine (DAC) decreased MDSC accumulation and increased activation of antigen-specific cytotoxic T lymphocytes. DAC-induced decreases in MDSC accumulation correlated with increased expression of the myeloid cell lineage-specific transcription factor IRF8 in MDSCs. However, DAC also suppressed MDSC-like cell accumulation in IRF8-deficient mice, indicating that DNA methylation may regulate MDSC survival through an IRF8-independent mechanism. Instead, DAC decreased MDSC accumulation by increasing cell death via disrupting DNA methylation of RIP1-dependent targets of necroptosis. Genome-wide DNA bisulfite sequencing revealed that the Tnf promoter was hypermethylated in tumor-induced MDSCs in vivo. DAC treatment dramatically increased TNFα levels in MDSC in vitro, and neutralizing TNFα significantly increased MDSC accumulation and tumor growth in tumor-bearing mice in vivo. Recombinant TNFα induced MDSC cell death in a dose- and RIP1-dependent manner. IL6 was abundantly expressed in MDSCs in tumor-bearing mice and patients with human colorectal cancer. In vitro, IL6 treatment of MDSC-like cells activated STAT3, increased expression of DNMT1 and DNMT3b, and enhanced survival. Overall, our findings reveal that MDSCs establish a STAT3-DNMT epigenetic axis, regulated by autocrine IL6, to silence TNFα expression. This results in decreased TNFα-induced and RIP1-dependent necroptosis to sustain survival and accumulation. SIGNIFICANCE: These findings demonstrate that targeting IL6 expression or function represent potentially effective approaches to suppress MDSC survival and accumulation in the tumor microenvironment.

Hypomethylation of LINE-1 retrotransposons is associated with cadmium-induced testicular injury.

Retrotransposons, as vital regulator of male fertility, are essential for spermatogenesis. Cadmium (Cd) is an environmental toxicant and endocrine disruptor, targeting the reproductive system. Growing evidence shows that Cd exposure can induce male infertility in mammals. In this study, we generated a male C57BL/6J mice model with consecutive 35days cadmium chloride (CdCl2) in different concentrations of 0, 0.25, 0.5, 1.0, and 2.0mg/kg. The results indicated that 1.0 and 2.0mg/kg CdCl2 significantly affected the body weight. Meanwhile, the highest dose group with 2.0mg/kg CdCl2 presented low fertility. Furthermore, the expression of retrotransposon mRNA was markedly increased in the higher doses group. We examined methylcytosine (mC) levels of the three active LINE-1 subfamilies TfI, A, and GfII in testis. Conclusively, Cd exposure probably undermines the male mice fertility by disrupting DNA methylation to regulate the retrotransposons. Further studies are required for identifying whether retrotransposon activation has any significant impacts on genome structure, stability, and expression in Cd-induced testicular injury, laying foundation for the treatment for male infertility.

Integrated Methylome and Transcriptome Analysis between the CMS-D2 Line ZBA and Its Maintainer Line ZB in Upland Cotton.

DNA methylation is an important epigenetic modification involved in multiple biological processes. Altered methylation patterns have been reported to be associated with male sterility in some plants, but their role in cotton cytoplasmic male sterility (CMS) remains unclear. Here, integrated methylome and transcriptome analyses were conducted between the CMS-D2 line ZBA and its near-isogenic maintainer line ZB in upland cotton. More methylated cytosine sites (mCs) and higher methylation levels (MLs) were found among the three sequence contexts in ZB compared to ZBA. A total of 4568 differentially methylated regions (DMRs) and 2096 differentially methylated genes (DMGs) were identified. Among the differentially expressed genes (DEGs) associated with DMRs (DMEGs), 396 genes were upregulated and 281 genes were downregulated. A bioinformatics analysis of these DMEGs showed that hyper-DEGs were significantly enriched in the “oxidative phosphorylation” pathway. Further qRT-PCR validation indicated that these hypermethylated genes (encoding the subunits of mitochondrial electron transport chain (ETC) complexes I and V) were all significantly upregulated in ZB. Our biochemical data revealed a higher extent of H2O2 production but a lower level of adenosine triphosphate (ATP) synthesis in CMS-D2 line ZBA. On the basis of the above results, we propose that disrupted DNA methylation in ZBA may disrupt the homeostasis of reactive oxygen species (ROS) production and ATP synthesis in mitochondria, triggering a burst of ROS that is transferred to the nucleus to initiate programmed cell death (PCD) prematurely, ultimately leading to microspore abortion. This study illustrates the important role of DNA methylation in cotton CMS.

Dynamic DNA methylation of ovaries during pubertal transition in gilts

BackgroundIn female mammals, the initiation of puberty, coupling with the dramatically morphological changes in ovaries, indicates the sexual and follicular maturation. Previous studies have suggested that the disrupted DNA methylation results in the delayed puberty. However, to date, the changes in ovarian methylomes during pubertal transition have not been investigated. In this study, using gilts as a pubertal model, the genome-wide DNA methylation were profiled to explore their dynamics during pubertal transition across Pre-, In- and Post-puberty.ResultsDuring pubertal transition, the follicles underwent maturation and luteinization, coupled with the significant changes in the mRNA expression of DNMT1 and DNMT3a. DNA methylation levels of In-puberty were higher than that of Pre- and Post-puberty at the locations of genes and CpG islands (CGIs). Analysis of the DNA methylation changes identified 12,313, 20,960 and 17,694 differentially methylated CpGs (DMCs) for the comparisons of Pre- vs. In-, In vs. Post-, and Pre- vs. Post-puberty, respectively. Moreover, the CGIs, upstream and exonic regions showed a significant underrepresentation of DMCs, but the CGI shores, CGI shelves, intronic, downstream and intergenic regions showed a significant overrepresentation of DMCs. Furthermore, biological functions of these methylation changes enriched in PI3K-Akt signaling pathway, GnRH signaling pathway, and Insulin secretion, and the mRNA expressions of several genes of these signaling pathway, including MMP2, ESR1, GSK3B, FGF21, IGF1R, and TAC3, were significantly changed across Pre-, In- and Post-puberty in ovaries.ConclusionsDuring pubertal transition in gilts, the DNA methylation changes of ovaries were likely to affect the transcription of genes related to PI3K-Akt signaling pathway, GnRH signaling pathway, and Insulin secretion. These observations can provide new insight into the epigenetic mechanism of follicular and sexual maturation during pubertal transition in mammals.

Examining the Impact of Imputation Errors on Fine-Mapping Using DNA Methylation QTL as a Model Trait.

Genetic variants disrupting DNA methylation at CpG dinucleotides (CpG-SNP) provide a set of known causal variants to serve as models to test fine-mapping methodology. We use 1716 CpG-SNPs to test three fine-mapping approaches (Bayesian imputation-based association mapping, Bayesian sparse linear mixed model, and the J-test), assessing the impact of imputation errors and the choice of reference panel by using both whole-genome sequence (WGS), and genotype array data on the same individuals (n = 1166). The choice of imputation reference panel had a strong effect on imputation accuracy, with the 1000 Genomes Project Phase 3 (1000G) reference panel (n = 2504 from 26 populations) giving a mean nonreference discordance rate between imputed and sequenced genotypes of 3.2% compared to 1.6% when using the Haplotype Reference Consortium (HRC) reference panel (n = 32,470 Europeans). These imputation errors had an impact on whether the CpG-SNP was included in the 95% credible set, with a difference of ∼23% and ∼7% between the WGS and the 1000G and HRC imputed datasets, respectively. All of the fine-mapping methods failed to reach the expected 95% coverage of the CpG-SNP. This is attributed to secondary cis genetic effects that are unable to be statistically separated from the CpG-SNP, and through a masking mechanism where the effect of the methylation disrupting allele at the CpG-SNP is hidden by the effect of a nearby SNP that has strong linkage disequilibrium with the CpG-SNP. The reduced accuracy in fine-mapping a known causal variant in a low-level biological trait with imputed genetic data has implications for the study of higher-order complex traits and disease.

DNA methylation regulates the neonatal CD4+ T-cell response to pneumonia in mice

Pediatric acute lung injury, usually because of pneumonia, has a mortality rate of more than 20% and an incidence that rivals that of all childhood cancers combined. CD4+ T-cells coordinate the immune response to pneumonia but fail to function robustly among the very young, who have poor outcomes from lung infection. We hypothesized that DNA methylation represses a mature CD4+ T-cell transcriptional program in neonates with pneumonia. Here, we found that neonatal mice (3-4 days old) aspirated with Escherichia coli bacteria had a higher mortality rate than juvenile mice (11-14 days old). Transcriptional profiling with an unsupervised RNA-Seq approach revealed that neonates displayed an attenuated lung CD4+ T-cell transcriptional response to pneumonia compared with juveniles. Unlike neonates, juveniles up-regulated a robust set of canonical T-cell immune response genes. DNA methylation profiling with modified reduced representation bisulfite sequencing revealed 44,119 differentially methylated CpGs, which preferentially clustered around transcriptional start sites and CpG islands. A methylation difference-filtering algorithm detected genes with a high likelihood of differential promoter methylation regulating their expression; these 731 loci encoded important immune response and tissue-protective T-cell pathway components. Disruption of DNA methylation with the hypomethylating agent decitabine induced plasticity in the lung CD4+ T-cell marker phenotype. Altogether, multidimensional profiling suggested that DNA methylation within the promoters of a core set of CD4+ T-cell pathway genes contributes to the hyporesponsive neonatal immune response to pneumonia. These findings also suggest that DNA methylation could serve as a mechanistic target for disease-modifying therapies in pediatric lung infection and injury.

Disrupted Genome Methylation in Response to High Temperature Has Distinct Affects on Microspore Abortion and Anther Indehiscence.

High-temperature (HT) stress induces male sterility, leading to yield reductions in crops. DNA methylation regulates a range of processes involved in plant development and stress responses, but its role in male sterility under HT remains unknown. Here, we investigated DNA methylation levels in cotton (Gossypium hirsutum) anthers under HT and normal temperature (NT) conditions by performing whole-genome bisulfite sequencing to investigate the regulatory roles of DNA methylation in male fertility under HT. Global disruption of DNA methylation, especially CHH methylation (where H = A, C, or T), was detected in an HT-sensitive line. Changes in the levels of 24-nucleotide small-interfering RNAs were significantly associated with DNA methylation levels. Experimental suppression of DNA methylation led to pollen sterility in the HT-sensitive line under NT conditions but did not affect the normal dehiscence of anther walls. Further transcriptome analysis showed that the expression of genes in sugar and reactive oxygen species (ROS) metabolic pathways were significantly modulated in anthers under HT, but auxin biosynthesis and signaling pathways were only slightly altered, indicating that HT disturbs sugar and ROS metabolism via disrupting DNA methylation, leading to microspore sterility. This study opens up a pathway for creating HT-tolerant cultivars using epigenetic techniques.