Loss Of DNA Methylation Research Articles

Dnmt3a Inactivation Leads to Slow DNA Methylation Loss in Murine Hematopoietic Cells In Vivo

IntroductionSomatic mutations in the gene encoding DNA methyltransferase 3A (DNMT3A) are the most common cause of clonal hematopoiesis, and among the most common initiating events for acute myeloid leukemia (AML). Constitutional DNMT3A mutations cause DNMT3A Overgrowth Syndrome, associated with obesity, overgrowth, behavioral problems, and increased risk of hematopoietic malignancies after years of latency [1]. Studies of germline and somatic Dnmt3a knockout mice have identified focal, canonical hypomethylation phenotypes in hematopoietic cells. This hypomethylation phenotype is thought to contribute to the epigenetic “fitness” of hematopoietic cells to acquire cooperating mutations that cause clonal progression and AML. However, the kinetics of methylation loss following mutational inactivation of DNMT3A in hematopoietic cells is essentially unknown.MethodsBone marrow from C57Bl/6-CD45.2 Dnmt3a fl/fl -ERT2-cre mice was transplanted into lethally irradiated C57Bl/6-CD45.1 recipients. Engraftment frequencies of >80% were detected in virtually all mice. Inactivation of Dnmt3a was induced with 9 doses of Tamoxifen (tam) given by gavage on MWF for three consecutive weeks. Bone marrow samples were serially collected for whole-genome bisulfite sequencing (WGBS) and analysis as previously described [1-4]. We compared these data to WGBS data from previously published doxycycline inducible DNMT3A transgene addback data, described in [3].ResultsCre-mediated deletion of exon 19 (flanked by loxP sites) leads to out-of-frame splicing of the mRNAs encoding all active isoforms of Dnmt3a. To limit Dnmt3a inactivation to hematopoietic cells, we used a transplantation model, which allowed us to eliminate the effects Dnmt3a loss in non-hematopoietic tissues. Floxing efficiency in the bone marrow was >90% 1 week after the last dose of Tam, and these levels persisted until the last mice in the cohort were analyzed 35 weeks later. We defined differentially methylated regions (DMRs) using WGBS data derived from WT vs Dnmt3a knockout (KO) bone marrow samples, using previously described criteria [4]. We identified 33,519 focal, highly canonical DMRs in the KO samples; 33,504 (99.96%) of these were hypomethylated, compared to WT marrow. Passively plotting the methylation values of the Dnmt3afl/fl + tam (“takeaway”) samples for these DMRs over time (Figure 1a) revealed a slow, progressive, ordered loss of methylation, although the degree of methylation loss did not reach KO levels by 36 weeks. In contrast, remethylation of most of these DMRs with transgene-driven DNMT3A “addback” in KOmice revealed methylation ‘correction’ that started rapidly (within 1-2 weeks), and that approached wild-type levels by 24 weeks (Figure 1b). Combined, these data show that methylation loss after Dnmt3a inactivation occurs much more slowly than remethylation of Dnmt3a-dependent sites following restoration of DNMT3A activity. This trend was true across all functionally annotated regions of the genome, including gene bodies, promoters, enhancers, transcriptional start sites (TSS) and CpG islands. Importantly, DMRs that lost methylation rapidly in the takeaway setting, also gained methylation rapidly with addback, suggesting that there is a canonical set of genomic regions where DNA methylation is actively “remodeled”. These “Dynamic DMRs” are significantly smaller (and had fewer CpGs) than DMRs associated with slower rates of remodeling.ConclusionsThese data describe a very slow rate of methylation loss following Dnmt3a inactivation in hematopoietic cells. We also describe regions of the genome (Dynamic DMRs) where DNA methylation is actively remodeled in hematopoietic cells, presumably by the collaborative action of de novo DNA methylases (Dnmt3a and Dnmt3b) vs. the DNA demethylases (i.e. the TET family of enzymes). Since clonal hematopoiesis patients with DNMT3A mutations can have very long latent periods before developing hematopoietic malignancies, these data suggest that long latency may be (at least in part) related to the very slow loss of DNA methylation at relevant regions of the genome after a DNMT3A allele acquires an inactivating somatic mutation. [Display omitted] DisclosuresLeight: Amgen: Current Employment.

DNA Methylation and Recurrent Pregnancy Loss: A Mysterious Compass?

Recurrent pregnancy loss (RPL) is a common and severe pathological pregnancy, whose pathogenesis is not fully understood. With the development of epigenetics, the study of DNA methylation, provides a new perspective on the pathogenesis and therapy of RPL. The abnormal DNA methylation of imprinted genes, placenta-specific genes, immune-related genes and sperm DNA may, directly or indirectly, affect embryo implantation, growth and development, leading to the occurrence of RPL. In addition, the unique immune tolerogenic microenvironment formed at the maternal-fetal interface has an irreplaceable effect on the maintenance of pregnancy. In view of these, changes in the cellular components of the maternal-fetal immune microenvironment and the regulation of DNA methylation have attracted a lot of research interest. This review summarizes the research progress of DNA methylation involved in the occurrence of RPL and the regulation of the maternal-fetal immune microenvironment. The review provides insights into the personalized diagnosis and treatment of RPL.

Discovery of a first-in-class reversible DNMT1-selective inhibitor with improved tolerability and efficacy in acute myeloid leukemia

DNA methylation, a key epigenetic driver of transcriptional silencing, is universally dysregulated in cancer. Reversal of DNA methylation by hypomethylating agents, such as the cytidine analogs decitabine or azacytidine, has demonstrated clinical benefit in hematologic malignancies. These nucleoside analogs are incorporated into replicating DNA where they inhibit DNA cytosine methyltransferases DNMT1, DNMT3A and DNMT3B through irreversible covalent interactions. These agents induce notable toxicity to normal blood cells thus limiting their clinical doses. Herein we report the discovery of GSK3685032, a potent first-in-class DNMT1-selective inhibitor that was shown via crystallographic studies to compete with the active-site loop of DNMT1 for penetration into hemi-methylated DNA between two CpG base pairs. GSK3685032 induces robust loss of DNA methylation, transcriptional activation and cancer cell growth inhibition in vitro. Due to improved in vivo tolerability compared with decitabine, GSK3685032 yields superior tumor regression and survival mouse models of acute myeloid leukemia.

Variants in 46,XY DSD-Related Genes in Syndromic and Non-Syndromic Small for Gestational Age Children with Hypospadias

Hypospadias is a common congenital disorder of male genital formation. Children born small for gestational age (SGA) present a high frequency of hypospadias of undetermined etiology. No previous study investigated the molecular etiology of hypospadias in boys born SGA using massively parallel sequencing. Our objective is to report the genetic findings of a cohort of patients born SGA with medium or proximal hypospadias. We identified 46 individuals with this phenotype from a large cohort of 46,XY DSD patients, including 5 individuals with syndromic features. DNA samples from subjects were studied by either whole exome sequencing or target gene panel approach. Three of the syndromic patients have 5 main clinical features of Silver-Russell syndrome (SRS) and were first studied by MLPA. Among the syndromic patients, loss of DNA methylation at the imprinting control region H19/IGF2 was identified in 2 individuals with SRS clinical diagnosis. Two novel pathogenic variants in compound heterozygous state were identified in the CUL7 gene establishing the diagnosis of 3M syndrome in one patient, and a novel homozygous variant in TRIM37 was identified in another boy with Mulibrey nanism phenotype. Among the non-syndromic subjects, 7 rare heterozygous variants were identified in 6 DSD-related genes. However, none of the variants found can explain the phenotype by themselves. In conclusion, a genetic defect that clarifies the etiology of hypospadias was not found in most of the non-syndromic SGA children, supporting the hypothesis that multifactorial causes, new genes, and/or unidentified epigenetic defects may have an influence in this condition.

Multiomics Analysis of Neuroblastoma Cells Reveals a Diversity of Malignant Transformations

Neuroblastoma (NB) is a pediatric cancer of the developing sympathetic nervous system that exhibits significant variation in the stage of differentiation and cell composition of tumors. Global loss of DNA methylation and genomic 5-hydroxymethylcytosine (5hmC) is a hallmark of human cancers. Here, we used our recently developed single-base resolution approaches, hmTOP-seq and uTOP-seq, for construction of 5hmC maps and identification of large partially methylated domains (PMDs) in different NB cell subpopulations. The 5hmC profiles revealed distinct signatures characteristic to different cell lineages and stages of malignant transformation of NB cells in a conventional and oxygen-depleted environment, which often occurs in tumors. The analysis of the cell-type-specific PMD distribution highlighted differences in global genome organization among NB cells that were ascribed to the same lineage identity by transcriptomic networks. Collectively, we demonstrated a high informativeness of the integrative epigenomic and transcriptomic research and large-scale genome structure in investigating the mechanisms that regulate cell identities and developmental stages of NB cells. Such multiomics analysis, as compared with mutational studies, open new ways for identification of novel disease-associated features which bring prognostic and therapeutic value in treating this aggressive pediatric disease.

Epigenetic Therapies in Ovarian Cancer Alter Repetitive Element Expression in a TP53-Dependent Manner.

Epithelial ovarian carcinomas are particularly deadly due to intratumoral heterogeneity, resistance to standard-of-care therapies, and poor response to alternative treatments such as immunotherapy. Targeting the ovarian carcinoma epigenome with DNA methyltransferase inhibitors (DNMTi) or histone deacetylase inhibitors (HDACi) increases immune signaling and recruits CD8+ T cells and natural killer cells to fight ovarian carcinoma in murine models. This increased immune activity is caused by increased transcription of repetitive elements (RE) that form double-stranded RNA (dsRNA) and trigger an IFN response. To understand which REs are affected by epigenetic therapies in ovarian carcinoma, we assessed the effect of DNMTi and HDACi on ovarian carcinoma cell lines and patient samples. Subfamily-level (TEtranscripts) and individual locus-level (Telescope) analysis of REs showed that DNMTi treatment upregulated more REs than HDACi treatment. Upregulated REs were predominantly LTR and SINE subfamilies, and SINEs exhibited the greatest loss of DNA methylation upon DNMTi treatment. Cell lines with TP53 mutations exhibited significantly fewer upregulated REs with epigenetic therapy than wild-type TP53 cell lines. This observation was validated using isogenic cell lines; the TP53-mutant cell line had significantly higher baseline expression of REs but upregulated fewer upon epigenetic treatment. In addition, p53 activation increased expression of REs in wild-type but not mutant cell lines. These data give a comprehensive, genome-wide picture of RE chromatin and transcription-related changes in ovarian carcinoma after epigenetic treatment and implicate p53 in RE transcriptional regulation. SIGNIFICANCE: This study identifies the repetitive element targets of epigenetic therapies in ovarian carcinoma and indicates a role for p53 in this process.

Age patterns of intra-pair DNA methylation discordance in twins: Sex difference in epigenomic instability and implication on survival.

Aging is a biological process linked to specific patterns and changes in the epigenome. We hypothesize that age‐related variation in the DNA methylome could reflect cumulative environmental modulation to the epigenome which could impact epigenomic instability and survival differentially by sex. To test the hypothesis, we performed sex‐stratified epigenome‐wide association studies on age‐related intra‐pair DNA methylation discordance in 492 twins aged 56–80 years. We identified 3084 CpGs showing increased methylation variability with age (FDR < 0.05, 7 CpGs with p < 1e‐07) in male twins but no significant site found in female twins. The results were replicated in an independent cohort of 292 twins aged 30–74 years with 37% of the discovery CpGs successfully replicated in male twins. Functional annotation showed that genes linked to the identified CpGs were significantly enriched in signaling pathways, neurological functions, extracellular matrix assembly, and cancer. We further explored the implication of discovery CpGs on individual survival in an old cohort of 224 twins (220 deceased). In total, 264 CpGs displayed significant association with risk of death in male twins. In female twins, 175 of the male discovery CpGs also showed non‐random correlation with mortality. Intra‐pair comparison showed that majority of the discovery CpGs have higher methylation in the longer‐lived twins suggesting that loss of DNA methylation during aging contributes to increased risk of death which is more pronounced in male twins. In conclusion, age‐related epigenomic instability in the DNA methylome is more evident in males than in females and could impact individual survival and contribute to sex difference in human lifespan.

H3K9 methyltransferase EHMT2/G9a controls ERVK-driven noncanonical imprinted genes.

Aim: Paternal allele-specific expression of noncanonical imprinted genes in the extraembryonic lineages depends on an H3K27me3-based imprint in the oocyte, which is not a lasting mark. We hypothesized that EHMT2, the main euchromatic H3K9 dimethyltransferase, also has a role in controlling noncanonical imprinting. Methods: We carried out allele-specific total RNA-seq analysis in the ectoplacental cone of somite-matched 8.5days post coitumembryos using reciprocal mouse crosses. Results: We found that the maternal allele of noncanonical imprinted genes was derepressed from its ERVK promoter in the Ehmt2-/-ectoplacental cone. In Ehmt2-/- embryos, loss of DNA methylation accompanied biallelic derepression of the ERVK promoters. Canonical imprinting and imprinted X chromosome inactivation were generally undisturbed. Conclusion: EHMT2 is essential for repressing the maternal allele in noncanonical imprinting.

Kaiso Regulates DNA Methylation Homeostasis.

Gain and loss of DNA methylation in cells is a dynamic process that tends to achieve an equilibrium. Many factors are involved in maintaining the balance between DNA methylation and demethylation. Previously, it was shown that methyl-DNA protein Kaiso may attract NCoR, SMRT repressive complexes affecting histone modifications. On the other hand, the deficiency of Kaiso resulted in reduced methylation of ICR in H19/Igf2 locus and Oct4 promoter in mouse embryonic fibroblasts. However, nothing is known about how Kaiso influences DNA methylation at the genome level. Here we show that deficiency of Kaiso led to whole-genome hypermethylation, using Kaiso deficient human renal cancer cell line obtained via CRISPR/CAS9 genome editing. However, Kaiso serves to protect genic regions, enhancers, and regions with a low level of histone modifications from demethylation. We detected hypomethylation of binding sites for Oct4 and Nanog in Kaiso deficient cells. Kaiso immunoprecipitated with de novo DNA methyltransferases DNMT3a/3b, but not with maintenance methyltransferase DNMT1. Thus, Kaiso may attract methyltransferases to surrounding regions and modulate genome methylation in renal cancer cells apart from being methyl DNA binding protein.

Paternal MTHFR deficiency leads to hypomethylation of young retrotransposons and reproductive decline across two successive generations.

ABSTRACT5,10-Methylenetetrahydrofolate reductase (MTHFR) is a crucial enzyme in the folate metabolic pathway with a key role in generating methyl groups. As MTHFR deficiency impacts male fertility and sperm DNA methylation, there is the potential for epimutations to be passed to the next generation. Here, we assessed whether the impact of MTHFR deficiency on testis morphology and sperm DNA methylation is exacerbated across generations in mouse. Although MTHFR deficiency in F1 fathers has only minor effects on sperm counts and testis weights and histology, F2 generation sons show further deterioration in reproductive parameters. Extensive loss of DNA methylation is observed in both F1 and F2 sperm, with >80% of sites shared between generations, suggestive of regions consistently susceptible to MTHFR deficiency. These regions are generally methylated during late embryonic germ cell development and are enriched in young retrotransposons. As retrotransposons are resistant to reprogramming of DNA methylation in embryonic germ cells, their hypomethylated state in the sperm of F1 males could contribute to the worsening reproductive phenotype observed in F2 MTHFR-deficient males, compatible with the intergenerational passage of epimutations.

OTEH-10. Evolutionary trajectory of epigenomic of gliomas

Gliomas are the most common malignant brain tumor, have an aggressive behavior, and invariably relapse and progress. Despite the recent advancements, little is known about the role of the epigenome in glioma disease progression and recurrence. To investigate the molecular dynamics over time and in response to therapeutic pressures, the Glioma Longitudinal AnalySiS (GLASS) Consortium, a multinational collaboration, is investigating epigenome-wide molecular data from primary and recurrent matched pairs, including IDH mutant (IDHmut) and IDH wildtype (IDHwt) gliomas. We have compiled a total of 357 samples comprising 143 primary-recurrent pairs profiled by DNA methylation, of which 157 samples have genomic data (WXS/WGS) and 120 have transcriptomic data (RNAseq). IDHwt gliomas have a distinct epigenetic evolution compared to IDHmut after treatment. IDHwt gliomas are more epigenetically stable over time, while IDHmut gliomas display a loss of DNA methylation throughout disease progression. Next, we investigated the molecular drivers of longitudinal gliomas by integration of DNA methylation and gene expression data. We identified epigenetic activation of cell cycle pathways in recurrent IDHmut compared to initial tumors. Transcription factors musculin, ZNF367, and ZNF682 are enriched among recurrent IDHmut gliomas and potentially regulate IDHmut recurrence and/or progression. We next used a DNA methylation-based deconvolution approach to estimate the tumor microenvironment (TME) composition. We found that the TME among IDHmut subtypes (Codel, GCIMP-high, and GCIMP-low) presented less immune infiltration than IDHwt (Classic-like, Mesenchymal-like, and PA-like). Post-treatment, we found a decrease of CD4+T and an increase of CD8+T cells in IDHmut. In conclusion, IDHmut gliomas present a more unstable epigenome, while the epigenome of IDHwt gliomas seems relatively preserved after treatment. We identified potential master regulators of cell cycle deregulation of IDHmut recurrence. Finally, the TME differs across IDHmut and IDHwt gliomas and the cell composition changes over time.

Acute Deletion of Tet2 or Tet3 Transiently Dysregulate Murine Erythropoiesis

The majority of palindromic CpG dinucleotides in mammalian DNA are symmetrically methylated on cytosine (5mC). Erythroid differentiation is associated with replication-dependent genome-wide loss of ~30-50% of all 5mC (1). One potential mechanism of demethylation involves Tet dioxygneases, which oxidize 5 methylcytosine (5mC) to 5-hydroxy-methylcytosine (5hmC), thereby interfering with maintenance methylation by DNMT1 (2). Tet2 and Tet3 are the two major Tet proteins expressed in hematopoietic cells. Tet2 is one of the most commonly mutated genes in hematological malignancies. Germline deletion of Tet2 results in increased hematopoietic stem cell self-renewal, erythroid and myeloid hyperplasia and myeloid and lymphoid malignancies (3, 4). Germline deletion of Tet3 is embryonic lethal (5). Here we investigated the potential role of Tet2 and Tet3 in the global loss of DNA methylation during erythroid differentiation, using mouse genetic models.We bred Tet2fl/fl and Tet3fl/fl mice onto the Rosa26-CreERT2 background, enabling acute deletion in vivo of either Tet2, Tet3 or both genes in adult and fetal mice. Within 4 to 10 days of deletion, we noted a rapid decline in total bone marrow cellularity in all genotypes; bone-marrow erythroblasts decreased to 15% of control value in Tet2-deleted (Tet2del) mice (p<0.0001), and to 43% (p=0.05) and 20% (p<0.0001) of controls in the Tet3 del and Tet2/Tet3 double deleted mice (DKO), respectively. These findings were associated with decreased reticulocyte counts in peripheral blood, and a decline in hematocrit, in Tet2del (36% vs. 46% in controls, p<0.004) and the DKO (31%, p=0.03). Bone-marrow CFU-e progenitors, but not BFU-e progenitors, also decreased in number. These losses partially improved over the next 2 to 4 weeks in mice deleted for either Tet2 or Tet3, but not in the DKO mice, in which anemia persisted. Megakaryocyte CFU-Mk progenitors increase in the DKO but are unaffected in the single deletions. Lymphoid lineage cells (CD4+, CD8+ and CD19+) showed similar transient losses, but the numbers of granylocytic and monocytic progenitors in the bone marrow was not affected. Similarly, fetal liver cell number decreased in all genotypes within 4 days of deletion, compared with controls, in all genotypes, and most severely in the DKO.We used three approaches to determine the potential effect of Tet proteins on erythroid DNA demethylation: Mass spectrometry, to measure global levels of 5mC and 5hmC in fetal liver erythroblasts; 5hMe-DIP-seq (5hyrdoxymethyl-DNA immune precipitation- sequencing), to determine the genomic distribution of hydroxymethylation in erythroblasts; and pyrosequencing for DNA methylation/hydroxymethylation at specific genomic loci including repetitive LINE_1 elements. We found that 5hmC is highly enriched in previously identified erythroid enhancers. Tet2 accelerates, but is not essential, for DNA demethylation of specific erythroid enhancers. Neither Tet2 nor Tet3 are required for global DNA demethylation.Taken together, we conclude that Tet2 and Tet3 are required during erythropoiesis, where they play partially redundant roles, possibly contributing to the changing chromatin environment during activation of erythroid terminal differentiation. Their acute deletion leads to transient loss of erythroblast viability and to anemia, but this is followed by a process of adaptation and chronically compensated erythropoiesis. These results raise the possibility that the adaptation required for the rapid partial recovery in the erythroid and lymphoid lineages following acute Tet2 loss may contribute, over the long term, to the development of myeloid and lymphoid malignancies.1. J. R. Shearstone et al., Global DNA demethylation during mouse erythropoiesis in vivo. Science334, 799-802 (2011).2. M. Ko et al., Impaired hydroxylation of 5-methylcytosine in myeloid cancers with mutant TET2. Nature468, 839-843 (2010).3. M. Ko et al., TET proteins and 5-methylcytosine oxidation in hematological cancers. Immunol Rev263, 6-21 (2015).4. J. An et al., Acute loss of TET function results in aggressive myeloid cancer in mice. Nature communications6, 10071 (2015).5. T. P. Gu et al., The role of Tet3 DNA dioxygenase in epigenetic reprogramming by oocytes. Nature477, 606-610 (2011). DisclosuresNo relevant conflicts of interest to declare.

Riluzole Administration to Rats with Levodopa-Induced Dyskinesia Leads to Loss of DNA Methylation in Neuronal Genes.

Dyskinesias are characterized by abnormal repetitive involuntary movements due to dysfunctional neuronal activity. Although levodopa-induced dyskinesia, characterized by tic-like abnormal involuntary movements, has no clinical treatment for Parkinson’s disease patients, animal studies indicate that Riluzole, which interferes with glutamatergic neurotransmission, can improve the phenotype. The rat model of Levodopa-Induced Dyskinesia is a unilateral lesion with 6-hydroxydopamine in the medial forebrain bundle, followed by the repeated administration of levodopa. The molecular pathomechanism of Levodopa-Induced Dyskinesia is still not deciphered; however, the implication of epigenetic mechanisms was suggested. In this study, we investigated the striatum for DNA methylation alterations under chronic levodopa treatment with or without co-treatment with Riluzole. Our data show that the lesioned and contralateral striata have nearly identical DNA methylation profiles. Chronic levodopa and levodopa + Riluzole treatments led to DNA methylation loss, particularly outside of promoters, in gene bodies and CpG poor regions. We observed that several genes involved in the Levodopa-Induced Dyskinesia underwent methylation changes. Furthermore, the Riluzole co-treatment, which improved the phenotype, pinpointed specific methylation targets, with a more than 20% methylation difference relative to levodopa treatment alone. These findings indicate potential new druggable targets for Levodopa-Induced Dyskinesia.

Melatonin-induced DNA demethylation of metal transporters and antioxidant genes alleviates lead stress in radish plants

Melatonin (MT) is a tryptophan-derived natural product that plays a vital role in plant response to abiotic stresses, including heavy metals (HMs). However, it remains elusive how exogenous MT mediates lead (Pb) accumulation and detoxification at the methylation and transcriptional levels in radish. In this study, decreased Pb accumulation and increased antioxidant enzyme activity were detected under MT treatment in radish. Single-base resolution maps of DNA methylation under Pb stress (Pb200) and Pb plus MT treatment (Pb_50MT) were first generated. The genome-wide methylation level was increased under Pb stress, while an overall loss of DNA methylation was observed under MT treatment. The differentially methylated region (DMR)-associated genes between Pb_50MT and Pb200 were uniquely enriched in ion binding terms, including cation binding, iron ion binding, and transition metal ion binding. Hyper-DMRs between Pb200 and Control exhibited a decreasing trend of methylation under Pb_50MT treatment. A few critical upregulated antioxidant genes (e.g., RsAPX2, RsPOD52 and RsGST) exhibited decreased methylation levels under MT treatment, which enabled the radish plants to scavenge lead-induced reactive oxygen species (ROS) and decrease oxidative stress. Notably, several MT-induced HM transporter genes with low methylation (e.g., RsABCF5, RsYSL7 and RsHMT) and transcription factors (e.g., RsWRKY41 and RsMYB2) were involved in reducing Pb accumulation in radish roots. These findings could facilitate comprehensive elucidation of the molecular mechanism underlying MT-mediated Pb accumulation and detoxification in radish and other root vegetable crops.

Transgenerational effect of mutants in the RNA-directed DNA methylation pathway on the triploid block in Arabidopsis

BackgroundHybridization of plants that differ in number of chromosome sets (ploidy) frequently causes endosperm failure and seed arrest, a phenomenon referred to as triploid block. In Arabidopsis, loss of function of NRPD1, encoding the largest subunit of the plant-specific RNA polymerase IV (Pol IV), can suppress the triploid block. Pol IV generates short RNAs required to guide de novo methylation in the RNA-directed DNA methylation (RdDM) pathway. Recent work suggests that suppression of the triploid block by mutants in RdDM components differs, depending on whether the diploid pollen is derived from tetraploid plants or from the omission in second division 1 (osd1) mutant. This study aims to understand this difference.ResultsIn this study, we find that the ability of mutants in the RdDM pathway to suppress the triploid block depends on their degree of inbreeding. While first homozygous generation mutants in RdDM components NRPD1, RDR2, NRPE1, and DRM2 have weak or no ability to rescue the triploid block, they are able to suppress the triploid block with successive generations of inbreeding. Inbreeding of nrpd1 was connected with a transgenerational loss of non-CG DNA methylation on sites jointly regulated by CHROMOMETHYLASES 2 and 3.ConclusionsOur data reveal that loss of RdDM function differs in its effect in early and late generations, which has important implications when interpreting the effect of RdDM mutants.

P148 Differential methylation of the TNF gene promoter, potential for the development of a diagnostic biomarker for RA

Abstract Background/Aims Diagnosing rheumatoid arthritis (RA) early is difficult despite the use of anti-citrullinated protein antibodies in the new EULAR-2010 classification criteria, particularly for antibody-negative patients. Alterations in epigenetic patterns have been associated with RA CD4+T-cells at the TNF gene locus (1). The second aim of my PhD project was to select a CpG site to develop a quantitative methylation sensitive PCR (qMSP) assay as a diagnostic biomarker test for RA classification. Methods Patients attending an early arthritis clinic were used (with full ethical approval). An 450K Illumina methylation genome-wide dataset analysing DNA from CD4+T-cells identified CpG on TNF promoter region as a differentially methylated CpG between controls and RA patients (1). A qMSP assay was designed using a TaqMan approach (primers and probe), using a reference gene (GAPHD) for normalisation. The assay was validated in DNA extracted from PBMC. The performance of the assay for RA classification was accessed using binary logistic regression. Results We noted differences in levels of methylation in CD4+T-cells in RA, notably affecting the TNF gene promoter in about 15% of cells. We validated this using bisulphite sequencing showing about 30% of cells with differential methylation in early RA compared to controls. This allowed us to select cg11484872, cg21370522, and cg01569083 as candidates. We optimised a qMSP assay using fully methylated/un-methylated DNA. PBMC samples were retrieved from our tissue bank for 65 early RA, and 64 non-RA patients (including 11 reactive arthritis, 37 undifferentiated arthritis, and 16 psoriatic arthritis). The methylation levels (%) detected by the TNF-qMSP assay were significantly lower in RA (Δ-DM =-10.28%, p = 7x10-7). The loss of TNF promoter gene methylation was associated with RA classification (p &amp;lt; 0.0001, unadjusted OR = 0.900 (95% CI: 0.85-0.94)) with a good classification performance (AUROC = 0.76 (95% CI: 0.67, 0.84)). An adjusted regression model using the demographic and clinical parameters associated with RA classification in this group of patients (age, smoking, counts for tender and swollen joints) showed that adding TNF to the clinical model improved the model’s performance (increasing AUROC from 0.86 , to 0.91). Comparing classification accuracy, adding the TNF-qMSP resulted in an increased correct classification of 5.7 % compared to the clinical model. A similar result was obtained in the antibody negative patients, with TNF-qMSP assay improving the model’s classification from AUROC 0.91 to 0.96 and 3.3% more patients correctly classified. Conclusion The loss of DNA methylation on the TNF gene promoter identified in early RA patients provided a valuable target for a biomarker assay development. The results will need to be confirmed in a larger cohort but the TNF-qMSP assay offers good opportunities for RA versus non-RA classification particular in antibody negative disease. Disclosure R. Pitaksalee: Grants/research support; ROYAL THAI GOVERNMENT SCHOLARSHIP. P. Emery: None. R. Hodgett: None. F. Ponchel: None.

Divergent DNA methylation contributes to duplicated gene evolution and chilling response in tea plants.

The tea plant (Camellia sinensis) is a thermophilic cash crop and contains a highly duplicated and repeat-rich genome. It is still unclear how DNA methylation regulates the evolution of duplicated genes and chilling stress in tea plants. We therefore generated a single-base-resolution DNA methylation map of tea plants under chilling stress. We found that, compared with other plants, the tea plant genome is highly methylated in all three sequence contexts, including CG, CHG and CHH (where H=A, T, or C), which is further proven to be correlated with its repeat content and genome size. We show that DNA methylation in the gene body negatively regulates the gene expression of tea plants, whereas non-CG methylation in the flanking region enables a positive regulation of gene expression. We demonstrate that transposable element-mediated methylation dynamics significantly drives the expression divergence of duplicated genes in tea plants. The DNA methylation and expression divergence of duplicated genes in the tea plant increases with evolutionary age and selective pressure. Moreover, we detect thousands of differentially methylated genes, some of which are functionally associated with chilling stress. We also experimentally reveal that DNA methyltransferase genes of tea plants are significantly downregulated, whereas demethylase genes are upregulated at the initial stage of chilling stress, which is in line with the significant loss of DNA methylation of three well-known cold-responsive genes at their promoter and gene body regions. Overall, our findings underscore the importance of DNA methylation regulation and offer new insights into duplicated gene evolution and chilling tolerance in tea plants.

Молекулярные механизмы развития рака легкого

Рак легкого является многоэтапным процессом с участием генетических факторов и изменений в эпигенетическом регулировании клеточного цикла. Амплификация генов, сверхэкспрессия, точечные мутациии или перестройки ДНК могут привести к постоянной позитивной регуляции митогенных сигналов роста, вследствие чего усиливается онкогенная сигнализация в клетках. В данном обзоре рассматриваются нарушения в 3 сигнальных путях (RAS-RAF-MEK-ERK, PI3K-Akt-mTOR и Keap1-Nrf2) при раке легкого. Причинами неконтролируемого роста и пролиферации клеток являются мутации EGFR, KRAS и аномальное слияние EML4-ALK, вследствие чего активируются 2 основных пути внутриклеточной сигнализации RAS-RAF-MEK-ERK и PI3K-Akt-mTOR. Канцерогенез при раке легкого может быть вызван генетическими аберрациями в любых компонентах пути PI3K-Akt-mTOR, его отрицательных регуляторах, взаимосвязанных путях и передаче сигналов через рецепторы RTK. Нарушения сигнального пути Keap1-Nrf2, обнаруженные при раке легкого, вызваны соматическими мутациями, потерей гетерозиготности или метилированием ДНК в промоторной области Keap1 и мутациями Nrf2, что приводит к увеличению накопления Nrf2. Опухоли с высоким уровнем Nrf2 связаны с плохим прогнозом из-за радио- и химиорезистентности и агрессивной пролиферации раковых клеток. Гиперактивность Nrf2 помогает автономным злокачественным клеткам выдерживать высокие уровни эндогенных активных форм кислорода (АФК) и избегать апоптоза. В этой связи важным подходом для профилактики рака является усиление активности Nrf2, тогда как для лечения рака желательно ингибирование Nrf2. Lung cancer is a multistep process involving genetic factors and changes in the epigenetic regulation of cell cycle. Gene amplification, overexpression, point mutations or DNA rearrangements may lead to continuous positive regulation of mitogenic growth signals resulting in enhancement of oncogenic cell signaling. This review focuses on disorders of three signaling pathways (RAS-RAF-MEK-ERK, PI3K-Akt-mTOR, and Keap1-Nrf2) in lung cancer. The causes for the uncontrolled cell growth and proliferation are EGFR and KRAS mutations and the abnormal EML4-ALK merging, which activate two major pathways of intracellular signaling, RAS-RAF-MEK-ERK and PI3K-Akt-mTOR. Carcinogenesis in lung cancer may result from genetic aberrations in any component of the PI3K-Akt-mTOR pathway, its negative regulators, interconnected pathways, and the signal transduction through RTK receptors. The disorders of the Keap1-Nrf2 signaling pathway found in lung cancer are caused by somatic mutations, loss of heterozygosity or DNA methylation in the Keap1 promoter region and Nrf2 mutations, which lead to an increase in the accumulation of Nrf2. Tumors with high Nrf2 levels are associated with a poor prognosis due to their radio- and chemoresistance and aggressive proliferation of cancer cells. The Nrf2 hyperactivity helps autonomous malignant cells withstand high levels of endogenous reactive oxygen species (ROS) and evade apoptosis. In this regard, enhancing the Nrf2 activity is an important approach to prevention of cancer while Nrf2 inhibition is desirable for its treatment.

Uhrf1 and dnmt1 Loss Induces an Immune Response in Zebrafish Livers Due to Viral Mimicry by Transposable Elements.

Activation of transposable elements (TEs) can cause cellular damage. Cytoplasmic nucleic acid sensing pathways evolved to detect pathogens, but can also serve to cull cells with inappropriate TE activation as TEs can be viral mimetics. Epigenetic silencing of TEs is mediated in part by DNA methylation, but it is not clear if TE activation or the immune system contribute to the cellular damage caused by loss of DNA methylation. Here, we provide mechanistic insight into the observation of an activated interferon response in the liver of zebrafish larvae with deletion in critical components of the DNA methylation machinery, uhrf1 and dnmt1. We focus on dissecting the relationship between DNA methylation, TE activation and induction of an immune response through cytoplasmic DNA and double stranded RNA sensing pathways and identify tnfa as a mediator of cell death in the liver of these mutants. Integrated RNAseq and methylome analysis identified LTR transposons as the most upregulated in these mutants and also the most methylated in control larvae, indicating a direct role of DNA methylation in suppressing this TE subclass. RNAseq analysis from these same samples revealed expression signatures of a type-I interferon response and of tnfa activation, mimicking the pattern of gene expression in virally infected cells. CRISPR/Cas9 mediated depletion of the cellular antiviral sensors sting and mavs reduced expression of interferon response genes and tnfa depletion dramatically reduced cell death in uhrf1 mutant livers. This suggests that the antiviral response induced by DNA hypomethylation and TE activation in the liver is mediated by the signaling pathways activated by both cytoplasmic double stranded RNA and DNA and that tnfa mediates cell death as a potential mechanism to eliminate these damaged cells.

Sex-specific effects of in vitro fertilization on adult metabolic outcomes and hepatic transcriptome and proteome in mouse.

Although in vitro fertilization (IVF) is associated with adverse perinatal outcomes, there is increasing concern about the long-term and sex-specific health implications. Augmenting our IVF mouse model to longitudinally investigate metabolic outcomes in offspring from optimal neonatal litter sizes, we found sex-specific metabolic outcomes in IVF offspring. IVF-conceived females had higher body weight and cholesterol levels compared to naturally conceived females, whereas IVF-conceived males had higher levels of triglycerides and insulin, and increased body fat composition. Through adult liver transcriptomics and proteomics, we identified sexually dimorphic dysregulation of the sterol regulatory element-binding protein (SREBP) pathways that are associated with the sex-specific phenotypes. We also found that global loss of DNA methylation in placenta was linked to higher cholesterol levels in IVF-conceived females. Our findings indicate that IVF procedures have long-lasting sex-specific effects on metabolic health of offspring and lay the foundation to utilize the placenta as a predictor of long-term outcomes.