Erasure Of DNA Methylation Research Articles

Erasure of DNA methylation in rat fetal germ cells is sex-specific and sensitive to maternal high-fat diet.

In mammals, DNA methylation (DNAme) erasure and reinstatement during embryo development and germline establishment are sensitive to the intrauterine environment. Maternal intake of a high-fat diet (HFD), associated with excessive gestational weight gain, has transgenerational effects on offspring health, which may be mediated by changes in DNAme in the germline. Here, we tested the impact of a maternal HFD on embryonic germline DNAme erasure using a rat strain that expresses green fluorescent protein specifically in germ cells. DNAme was analysed by methyl-seq capture in germ cells collected from male and female F1 gonads at gestational day 16. Our data show that although HFD induced global hypomethylation in both sexes, DNAme erasure in female germ cells was more advanced compared to male germ cells. The delay in DNAme erasure in males and the greater impact of HFD suggest that male germ cells are more vulnerable to alterations by exogenous factors.

Calling the question: what is mammalian transgenerational epigenetic inheritance?

ABSTRACT While transgenerational epigenetic inheritance has been extensively documented in plants, nematodes, and fruit flies, its existence in mammals remains controversial. Several factors have contributed to this debate, including the lack of a clear distinction between intergenerational and transgenerational epigenetic inheritance (TEI), the inconsistency of some studies, the potential confounding effects of in-utero vs. epigenetic factors, and, most importantly, the biological challenge of epigenetic reprogramming. Two waves of epigenetic reprogramming occur: in the primordial germ cells and the developing embryo after fertilization, characterized by global erasure of DNA methylation and remodelling of histone modifications. Consequently, TEI can only occur if specific genetic regions evade this reprogramming and persist through embryonic development. These challenges have revived the long-standing debate about the possibility of inheriting acquired traits, which has been strongly contested since the Lamarckian and Darwinian eras. As a result, coupled with the absence of universally accepted criteria for transgenerational epigenetic studies, a vast body of literature has emerged claiming evidence of TEI. Therefore, the goal of this study is to advocate for establishing fundamental criteria that must be met for a study to qualify as evidence of TEI. We identified five criteria based on the consensus of studies that critically evaluated TEI. To assess whether published original research papers adhere to these criteria, we examined 80 studies that either claimed or were cited as supporting TEI. The findings of this analysis underscore the widespread confusion in this field and highlight the urgent need for a unified scientific consensus on TEI requirements.

A role for DNA methylation in bumblebee morphogenesis hints at female-specific developmental erasure.

Epigenetic mechanisms, such as DNA methylation, are crucial factors in animal development. In some mammals, almost all DNA methylation is erased during embryo development and re-established in a sex- and cell-specific manner. This erasure and re-establishment is thought to primarily be a vertebrate-specific trait. Insects are particularly interesting in terms of development as many species often undergo remarkable morphological changes en route to maturity, that is, morphogenesis. However, little is known about the role of epigenetic mechanisms in this process across species. We have used whole-genome bisulfite sequencing to track genome-wide DNA methylation changes through the development of an economically and environmentally important pollinator species, the bumblebee Bombus terrestris (Hymenoptera:Apidae Linnaeus). We find overall levels of DNA methylation vary throughout development, and we find developmentally relevant differentially methylated genes throughout. Intriguingly, we have identified a depletion of DNA methylation in ovaries/eggs and an enrichment of highly methylated genes in sperm. We suggest this could represent a sex-specific DNA methylation erasure event. To our knowledge, this is the first suggestion of possible developmental DNA methylation erasure in an insect species. This study lays the required groundwork for functional experimental work to determine if there is a causal nature to the DNA methylation differences identified. Additionally, the application of single-cell methylation sequencing to this system will enable more accurate identification of if or when DNA methylation is erased during development.

A Novel Approach to Epigenetic Profiling in Acute Myeloid Leukemia: Mapping Methylation and Hydroxymethylation in Genomic and Cell-Free DNA

Introduction: Acute Myeloid Leukemia (AML) is marked by uncontrolled growth of undifferentiated myeloid cells. DNA methylation patterns in AML have been thoroughly researched, however, studies on hydroxy methylation patterns, indicating active DNA methylation erasure, are still evolving. Our principal goal was to detect differential methylation and hydroxy methylation regions between AML and non-malignant samples. We proposed that simultaneous profiling of blood cells and circulating cell-free DNA would offer more insights into the disease mechanism. Therefore, we combined these data sources to provide a comprehensive epigenetic picture, assuming that this dual approach could reveal new AML biomarkers and enhance our understanding regarding AML pathobiology. Methods: We collected peripheral blood samples from 16 newly diagnosed AML patients (median age 64, age range 36-85) treated at Tel Aviv Sourasky Medical Center, Israel, and 33 age-matched healthy volunteers (NCT05735704). We utilized novel microarray technology to examine methylation and hydroxy methylation patterns in both genomic and cell-free DNA. Following differential methylation region extraction, we used DAVID functional annotation tool to interpret our results' biological significance. Results: This report presents the preliminary methylation data results. Methylation analysis unveiled 474 and 475 differentially methylated regions between AML and controls in genomic and cell-free DNA analysis, respectively, with only 18 common regions. The clear AML-control distinction is displayed in hierarchical clustering heatmaps (Figure 1a, 1b). Further DAVID tool analysis revealed enrichment in biological processes such as aging, cell cycle regulation, cell morphogenesis, and MAP kinase activity - processes closely linked with AML. Notably, genomic data analysis showed significant enrichment of the ‘transcriptional misregulation in cancer’ pathway, involving genes encoding vital transcription factors for cell development and differentiation. Figure 2 demonstrates the biological pathways for genomic and cell free data analysis. Conclusions: This research underscores the importance of combined epigenetic data from malignant tissue and its microenvironment, obtained by examining genomic and cell-free DNA. Both datasets exhibit striking differences between AML and control samples. We detected enrichment of AML-related biological pathways within differential regions. Our findings of enriched biological pathways align with existing literature, reinforce our analysis validity, and support our combined view of methylation patterns in genomic and cell-free DNA. By suggesting a combined view of these two types of DNA, we provide a more comprehensive perspective on AML's complex epigenetic landscape. It underscores the value of such integrative epigenetic profiling in shedding new light on the intricacies of AML development and progression, which may eventually guide the discovery of novel therapeutic targets.

Evolutionary conservation of embryonic DNA methylome remodelling in distantly related teleost species.

Methylation of cytosines in the CG context (mCG) is the most abundant DNA modification in vertebrates that plays crucial roles in cellular differentiation and identity. After fertilization, DNA methylation patterns inherited from parental gametes are remodelled into a state compatible with embryogenesis. In mammals, this is achieved through the global erasure and re-establishment of DNA methylation patterns. However, in non-mammalian vertebrates like zebrafish, no global erasure has been observed. To investigate the evolutionary conservation and divergence of DNA methylation remodelling in teleosts, we generated base resolution DNA methylome datasets of developing medaka and medaka-zebrafish hybrid embryos. In contrast to previous reports, we show that medaka display comparable DNA methylome dynamics to zebrafish with high gametic mCG levels (sperm: ∼90%; egg: ∼75%), and adoption of a paternal-like methylome during early embryogenesis, with no signs of prior DNA methylation erasure. We also demonstrate that non-canonical DNA methylation (mCH) reprogramming at TGCT tandem repeats is a conserved feature of teleost embryogenesis. Lastly, we find remarkable evolutionary conservation of DNA methylation remodelling patterns in medaka-zebrafish hybrids, indicative of compatible DNA methylation maintenance machinery in far-related teleost species. Overall, these results suggest strong evolutionary conservation of DNA methylation remodelling pathways in teleosts, which is distinct from the global DNA methylome erasure and reestablishment observed in mammals.

79-OR: Targeted Erasure of DNA Methylation by TET3 with C/EBPδ Rewires Adipogenic Execution for Energy Metabolism

DNA methylation is one of the key epigenetic modifications in the establishment of cell-type-specific characteristics. However, how DNA methylation is selectively regulated at adipocyte-specific loci during adipogenesis remains unclear. We have demonstrated that the transcription factor, C/EBPδ, and the DNA methylation eraser, TET3, cooperatively control adipocyte differentiation. We performed whole-genome bisulfite sequencing to explore the DNA methylation in adipocyte differentiation. Intriguingly, DNA methylation selectively decreases at adipocyte-specific loci carrying the C/EBP binding motif, which correlates with the activity of adipogenic promoters and enhancers. Mechanistically, we find that C/EBPδ recruits a DNA methylation eraser, TET3, to catalyze DNA demethylation and stimulate the expression of key adipogenic genes. Ectopic expression of TET3 potentiates in vitro and in vivo adipocyte differentiation and restores insulin sensitivity and decreased adipogenic potential, which is observed in aged mice and humans. Together, our study highlights how targeted reprogramming of DNA methylation through cooperative action of the transcription factor C/EBPδ, and the DNA methylation eraser TET3, controls adipocyte differentiation for whole body energy metabolism. Disclosure J.Park: None. J.Kim: None. Funding National Research Foundation of Korea (2018R1A5A1024340 to J.B.K. and J.P), (2020R1A3B2078617 to J.B.K)

Transgenerational sperm DMRs escape DNA methylation erasure during embryonic development andepigenetic inheritance.

Germline transmission of epigenetic information is a critical component of epigenetic inheritance. Previous studies have suggested that an erasure of DNA methylation is required to develop stem cells in the morula embryo. An exception involves imprinted genes that escape this DNA methylation erasure. Transgenerational differential DNA methylation regions (DMRs) have been speculated to be imprinted-like and escape this erasure. The current study was designed to assess if morula embryos escape the erasure of dichlorodiphenyltrichloroethane-induced transgenerational sperm DMR methylation. Observations demonstrate that the majority (98%) of transgenerational sperm DMR sites retain DNA methylation and are not erased, so appearing similar to imprinted-like sites. Interestingly, observations also demonstrate that the majority of low-density CpG genomic sites had a significant increase in DNA methylation in the morula embryo compared to sperm. This is in contrast to the previously observed DNA methylation erasure of higher-density CpG sites. The general erasure of DNA methylation during embryogenesis appears applicable to high-density DNA methylation sites (e.g. CpG islands) but neither to transgenerational DMR methylation sites nor to low-density CpG deserts, which constitute the vast majority of the genome's DNA methylation sites. The role of epigenetics during embryogenesis appears more dynamic than the simple erasure of DNA methylation.

Targeted erasure of DNA methylation by TET3 drives adipogenic reprogramming and differentiation.

DNA methylation is a crucial epigenetic modification in the establishment of cell-type-specific characteristics. However, how DNA methylation is selectively reprogrammed at adipocyte-specific loci during adipogenesis remains unclear. Here, we show that the transcription factor, C/EBPδ, and the DNA methylation eraser, TET3, cooperatively control adipocyte differentiation. We perform whole-genome bisulfite sequencing to explore the dynamics and regulatory mechanisms of DNA methylation in adipocyte differentiation. During adipogenesis, DNA methylation selectively decreases at adipocyte-specific loci carrying the C/EBP binding motif, which correlates with the activity of adipogenic promoters and enhancers. Mechanistically, we find that C/EBPδ recruits a DNA methylation eraser, TET3, to catalyse DNA demethylation at the C/EBP binding motif and stimulate the expression of key adipogenic genes. Ectopic expression of TET3 potentiates in vitro and in vivo adipocyte differentiation and recovers downregulated adipogenic potential, which is observed in aged mice and humans. Taken together, our study highlights how targeted reprogramming of DNA methylation through cooperative action of the transcription factor C/EBPδ, and the DNA methylation eraser TET3, controls adipocyte differentiation.

Genome-wide screening for genes involved in the epigenetic basis of fragile X syndrome.

SummaryFragile X syndrome (FXS), the most prevalent heritable form of intellectual disability, is caused by the transcriptional silencing of the FMR1 gene. The epigenetic factors responsible for FMR1 inactivation are largely unknown. Here, we initially demonstrated the feasibility of FMR1 reactivation by targeting a single epigenetic factor, DNMT1. Next, we established a model system for FMR1 silencing using a construct containing the FXS-related mutation upstream to a reporter gene. This construct was methylated in vitro and introduced into a genome-wide loss-of-function (LOF) library established in haploid human pluripotent stem cells (PSCs), allowing the identification of genes whose functional loss reversed the methylation-induced silencing of the FMR1 reporter. Selected candidate genes were further analyzed in haploid- and FXS-patient-derived PSCs, highlighting the epigenetic and metabolic pathways involved in FMR1 regulation. Our work sheds light on the mechanisms responsible for CGG-expansion-mediated FMR1 inactivation and offers novel targets for therapeutic FMR1 reactivation.

Paternal diet induces transgenerational epigenetic inheritance of DNA methylation signatures and phenotypes in sheep model.

Transgenerational epigenetic inheritance (TEI) requires transmission of environmentally induced epigenetic changes and associated phenotypes to subsequent generations without continued exposure to the environmental factor that originated the change. TEI is well-established in plants and Caenorhabditis elegans; however, occurrence in mammals is debated and poorly understood. Here, we examined whether paternal diet from weaning to puberty-induced changes in sperm DNA methylation that were transmitted to subsequent generations. Over 100 methylated cytosines, environmentally altered in the F0 generation, were inherited by the F1 and F2 generations. Furthermore, the F0 paternal diet was associated with growth and male fertility phenotypes in subsequent generations. Differentially methylated cytosines were correlated with gene expression. Our results demonstrate that some sperm methylation sites may escape DNA methylation erasure and are transmitted to subsequent generations despite the 2 waves of epigenetic programming: in primordial germ cells and in embryos after fertilization. These results advance our understanding of the complex relationships between nature and nurture.

DNA demethylases remodel DNA methylation in rice gametes and zygote and are required for reproduction

Fertilization constitutes a critical step in the plant life cycle during which the gamete genomes undergo chromatin dynamics in preparation for embryogenesis. In mammals, parental chromatin is extensively reprogrammed through the global erasure of DNA methylation. However, in flowering plants it remains unclear whether and how DNA methylation is remodeled in gametes and after fertilization in the zygote. In this study, we characterize DNA methylation patterns and investigate the function of DNA glycosylases in rice eggs, sperm, and unicellular zygotes and during embryogenesis. We found that DNA methylation is locally reconfigured after fertilization and is intensified during embryogenesis. Genetic, epigenomic, and transcriptomic analysis revealed that three rice DNA glycosylases, DNG702, DNG701, and DNG704, demethylate DNA at distinct genomic regions in the gametes and the zygote, and are required for zygotic gene expression and development. Collectively, these results indicate that active DNA demethylation takes place in the gametes and the zygote to locally remodel DNA methylation, which is critical for egg and zygote gene expression and reproduction in rice.

Transient exposure to miR-203 enhances the differentiation capacity of established pluripotent stem cells.

Full differentiation potential along with self-renewal capacity is a major property of pluripotent stem cells (PSCs). However, the differentiation capacity frequently decreases during expansion of PSCs in vitro. We show here that transient exposure to a single microRNA, expressed at early stages during normal development, improves the differentiation capacity of already-established murine and human PSCs. Short exposure to miR-203 in PSCs (miPSCs) induces a transient expression of 2C markers that later results in expanded differentiation potency to multiple lineages, as well as improved efficiency in tetraploid complementation and human-mouse interspecies chimerism assays. Mechanistically, these effects are at least partially mediated by direct repression of de novo DNA methyltransferases Dnmt3a and Dnmt3b, leading to transient and reversible erasure of DNA methylation. These data support the use of transient exposure to miR-203 as a versatile method to reset the epigenetic memory in PSCs, and improve their effectiveness in regenerative medicine.

Dppa2 and Dppa4 counteract de novo methylation to establish a permissive epigenome for development.

Early mammalian development entails genome-wide epigenome remodeling, including DNA methylation erasure and reacquisition, which facilitates developmental competence. To uncover the mechanisms that orchestrate DNA methylation dynamics, we coupled a single-cell ratiometric DNA methylation reporter with unbiased CRISPR screening in murine embryonic stem cells (ESCs). We identify key genes and regulatory pathways that drive global DNA hypomethylation, and characterize roles for Cop1 and Dusp6. We also identify Dppa2 and Dppa4 as essential safeguards of focal epigenetic states. In their absence, developmental genes and evolutionarily young LINE1 elements, which are specifically bound by DPPA2, lose H3K4me3 and gain ectopic de novo DNA methylation in pluripotent cells. Consequently, lineage-associated genes and LINE1 acquire a repressive epigenetic memory, which renders them incompetent for activation during future lineage specification. Dppa2/4 thereby sculpt the pluripotent epigenome by facilitating H3K4me3 and bivalency to counteract de novo methylation, a function co-opted by evolutionarily young LINE1 to evade epigenetic decommissioning.

Targeted reprogramming of H3K27me3 resets epigenetic memory in plant paternal chromatin.

Epigenetic marks are reprogrammed in the gametes to reset genomic potential in the next generation. In mammals, paternal chromatin is extensively reprogrammed through the global erasure of DNA methylation and the exchange of histones with protamines1,2. Precisely how the paternal epigenome is reprogrammed in flowering plants has remained unclear since DNA is not demethylated and histones are retained in sperm3,4. Here, we describe a multi-layered mechanism by which H3K27me3 is globally lost from histone-based sperm chromatin in Arabidopsis. This mechanism involves the silencing of H3K27me3 writers, activity of H3K27me3 erasers and deposition of a sperm-specific histone, H3.10 (ref. 5), which we show is immune to lysine 27 methylation. The loss of H3K27me3 facilitates the transcription of genes essential for spermatogenesis and pre-configures sperm with a chromatin state that forecasts gene expression in the next generation. Thus, plants have evolved a specific mechanism to simultaneously differentiate male gametes and reprogram the paternal epigenome.

An Extended Culture System that Supports Human Primordial Germ Cell-like Cell Survival and Initiation of DNA Methylation Erasure.

SummaryThe development of an in vitro system in which human primordial germ cell-like cells (hPGCLCs) are generated from human pluripotent stem cells (hPSCs) has been invaluable to further our understanding of human primordial germ cell (hPGC) specification. However, the means to evaluate the next fundamental steps in germ cell development have not been well established. In this study we describe a two dimensional extended culture system that promotes proliferation of specified hPGCLCs, without reversion to a pluripotent state. We demonstrate that hPGCLCs in extended culture undergo partial epigenetic reprogramming, mirroring events described in hPGCs in vivo, including a genome-wide reduction in DNA methylation and maintenance of depleted H3K9me2. This extended culture system provides a new approach for expanding the number of hPGCLCs for downstream technologies, including transplantation, molecular screening, or possibly the differentiation of hPGCLCs into gametes by in vitro gametogenesis.

DNA methylation dynamics at transposable elements in mammals

Transposable elements dominate the mammalian genome, but their contribution to genetic and epigenetic regulation has been largely overlooked. This was in part due to technical limitations, which made the study of repetitive sequences at single copy resolution difficult. The advancement of next-generation sequencing assays in the last decade has greatly enhanced our understanding of transposable element function. In some instances, specific transposable elements are thought to have been co-opted into regulatory roles during both mouse and human development, while in disease such regulatory potential can contribute to malignancy. DNA methylation is arguably the best characterised regulator of transposable element activity. DNA methylation is associated with transposable element repression, and acts to limit their genotoxic potential. In specific developmental contexts, erasure of DNA methylation is associated with a burst of transposable element expression. Developmental regulation of DNA methylation enables transposon activation, ensuring their survival and propagation throughout the host genome, and also allows the host access to regulatory sequences encoded within the elements. Here I discuss DNA methylation at transposable elements, describing its function and dynamic regulation throughout murine and human development.

The diverse roles of DNA methylation in mammalian development and disease.

DNA methylation is of paramount importance for mammalian embryonic development. DNA methylation has numerous functions: it is implicated in the repression of transposons and genes, but is also associated with actively transcribed gene bodies and, in some cases, with gene activation per se. In recent years, sensitive technologies have been developed that allow the interrogation of DNA methylation patterns from a small number of cells. The use of these technologies has greatly improved our knowledge of DNA methylation dynamics and heterogeneity in embryos and in specific tissues. Combined with genetic analyses, it is increasingly apparent that regulation of DNA methylation erasure and (re-)establishment varies considerably between different developmental stages. In this Review, we discuss the mechanisms and functions of DNA methylation and demethylation in both mice and humans at CpG-rich promoters, gene bodies and transposable elements. We highlight the dynamic erasure and re-establishment of DNA methylation in embryonic, germline and somatic cell development. Finally, we provide insights into DNA methylation gained from studying genetic diseases.

Zebrafish preserve global germline DNA methylation while sex-linked rDNA is amplified and demethylated during feminisation

The germline is the only cellular lineage capable of transferring genetic information from one generation to the next. Intergenerational transmission of epigenetic memory through the germline, in the form of DNA methylation, has been proposed; however, in mammals this is largely prevented by extensive epigenetic erasure during germline definition. Here we report that, unlike mammals, the continuously-defined ‘preformed’ germline of zebrafish does not undergo genome-wide erasure of DNA methylation during development. Our analysis also uncovers oocyte-specific germline amplification and demethylation of an 11.5-kb repeat region encoding 45S ribosomal RNA (fem-rDNA). The peak of fem-rDNA amplification coincides with the initial expansion of stage IB oocytes, the poly-nucleolar cell type responsible for zebrafish feminisation. Given that fem-rDNA overlaps with the only zebrafish locus identified thus far as sex-linked, we hypothesise fem-rDNA expansion could be intrinsic to sex determination in this species.

Endo-siRNAs repress expression of SINE1B during in vitro maturation of porcine oocyte

Approximately 40% of mammalian genome is made of transposable elements (TEs), and during specific biological processes, such as gametogenesis, they may be activated by global demethylation, so strict silencing mechanism is indispensable for genomic stability. Here, we performed small RNA-seq on Dicer1 knockdown (KD) oocytes in pig, and observed short interspersed nuclear elements 1B (SINE1B) derived endogenous small interfering RNAs (endo-siRNAs), termed SINE1B-siRNAs, were significantly decreased and their biogenesis was dependent on Dicer1 and transcript of SINE1B. Furthermore, by injection of mimics and inhibitors of the SINE1B-siRNAs into germinal vesicle-stage (GV-stage) oocytes, we found the maturation rate was significantly decreased by SINE1B-siRNAs, indicating the SINE1B-siRNAs are indispensible for in vitro maturation (IVM) of porcine oocyte. To figure out the mechanism, we checked the expression pattern and DNA methylation status of SINE1B during IVM of porcine oocytes, and demonstrated the SINE1B-siRNAs could repress SINE1B expression induced by hypomethylation at a post-transcriptional level. Our results suggest that during gametogenesis when the erasure of DNA methylation occurs, endo-siRNAs act as a chronic response to limit retrotransposon activation.

Superovulation alters global DNA methylation in early mouse embryo development

ABSTRACTAssisted reproductive technologies are known to alter the developmental environment of gametes and early embryos during the most dynamic period of establishing the epigenome. This may result in the introduction of errors during active DNA methylation reprogramming. Controlled ovarian hyperstimulation, or superovulation, is a ubiquitously used intervention which has been demonstrated to alter the methylation of certain imprinted genes. The objective of this study was to investigate whether ovarian hyperstimulation results in genome-wide DNA methylation changes in mouse early embryos. Ovarian hyperstimulation was induced by treating mice with either low doses (5 IU) or high doses (10 IU) of PMSG and hCG. Natural mating (NM) control mice received no treatment. Zygotes and 8-cell embryos were collected from each group and DNA methylomes were generated by whole-genome bisulfite sequencing. In the NM group, mean CpG methylation levels slightly decreased from zygote to 8-cell stage, whereas a large decrease in mean CpG methylation level was observed in both superovulated groups. A separate analysis of the mean CpG methylation levels within each developmental stage confirmed that significant genome-wide erasure of CpG methylation from the zygote to 8-cell stage only occurred in the superovulation groups. Our results suggest that superovulation alters the genome-wide DNA methylation erasure process in mouse early pre-implantation embryos. It is not clear whether these changes are transient or persistent. Further studies are ongoing to investigate the impact of ovarian hyperstimulation on DNA methylation re-establishment in later stages of embryo development.