Dynamic Reprogramming Of DNA Methylation Research Articles

Pre-implantation alcohol exposure induces lasting sex-specific DNA methylation programming errors in the developing forebrain

BackgroundPrenatal alcohol exposure is recognized for altering DNA methylation profiles of brain cells during development, and to be part of the molecular basis underpinning Fetal Alcohol Spectrum Disorder (FASD) etiology. However, we have negligible information on the effects of alcohol exposure during pre-implantation, the early embryonic window marked with dynamic DNA methylation reprogramming, and on how this may rewire the brain developmental program.ResultsUsing a pre-clinical in vivo mouse model, we show that a binge-like alcohol exposure during pre-implantation at the 8-cell stage leads to surge in morphological brain defects and adverse developmental outcomes during fetal life. Genome-wide DNA methylation analyses of fetal forebrains uncovered sex-specific alterations, including partial loss of DNA methylation maintenance at imprinting control regions, and abnormal de novo DNA methylation profiles in various biological pathways (e.g., neural/brain development).ConclusionThese findings support that alcohol-induced DNA methylation programming deviations during pre-implantation could contribute to the manifestation of neurodevelopmental phenotypes associated with FASD.

DNA methylation dynamics during epigenetic reprogramming of medaka embryo

ABSTRACTPost-fertilization epigenome reprogramming erases epigenetic marks transmitted through gametes and establishes new marks during mid-blastula stages. The mouse embryo undergoes dynamic DNA methylation reprogramming after fertilization, while in zebrafish, the paternal DNA methylation pattern is maintained throughout the early embryogenesis and the maternal genome is reprogrammed in a pattern similar to that of sperm during the mid-blastula transition. Here, we show DNA methylation dynamics in medaka embryos, the biomedical model fish, during epigenetic reprogramming of embryonic genome. The sperm genome was hypermethylated and the oocyte genome hypomethylated prior to fertilization. After fertilization, the methylation marks of sperm genome were erased within the first cell cycle and embryonic genome remained hypomethylated from the zygote until 16-cell stage. The DNA methylation level gradually increased from 16-cell stage through the gastrula. The 5-hydroxymethylation (5hmC) levels showed an opposite pattern to DNA methylation (5-mC). The mRNA levels for DNA methyltransferase (DNMT) 1 remained high in oocytes and maintained the same level through late blastula stage and was reduced thereafter. DNMT3BB.1 mRNA levels increased prior to remethylation. The mRNA levels for ten-eleven translocation methylcytosine dioxygenases (TET2 & TET3) were detected in sperm and embryos at cleavage stages, whereas TET1 and TET3 mRNAs decreased during gastrulation. The pattern of genome methylation in medaka was identical to mammalian genome methylation but not to zebrafish. The present study suggests that a medaka embryo resets its DNA methylation pattern by active demethylation and by a gradual remethylation similar to mammals.

DNA demethylation by ROS1a in rice vegetative cells promotes methylation in sperm

Epigenetic reprogramming is required for proper regulation of gene expression in eukaryotic organisms. In Arabidopsis, active DNA demethylation is crucial for seed viability, pollen function, and successful reproduction. The DEMETER (DME) DNA glycosylase initiates localized DNA demethylation in vegetative and central cells, so-called companion cells that are adjacent to sperm and egg gametes, respectively. In rice, the central cell genome displays local DNA hypomethylation, suggesting that active DNA demethylation also occurs in rice; however, the enzyme responsible for this process is unknown. One candidate is the rice REPRESSOR OF SILENCING1a (ROS1a) gene, which is related to DME and is essential for rice seed viability and pollen function. Here, we report genome-wide analyses of DNA methylation in wild-type and ros1a mutant sperm and vegetative cells. We find that the rice vegetative cell genome is locally hypomethylated compared with sperm by a process that requires ROS1a activity. We show that many ROS1a target sequences in the vegetative cell are hypomethylated in the rice central cell, suggesting that ROS1a also demethylates the central cell genome. Similar to Arabidopsis, we show that sperm non-CG methylation is indirectly promoted by DNA demethylation in the vegetative cell. These results reveal that DNA glycosylase-mediated DNA demethylation processes are conserved in Arabidopsis and rice, plant species that diverged 150 million years ago. Finally, although global non-CG methylation levels of sperm and egg differ, the maternal and paternal embryo genomes show similar non-CG methylation levels, suggesting that rice gamete genomes undergo dynamic DNA methylation reprogramming after cell fusion.

Dynamic DNA methylation reconfiguration during seed development and germination

BackgroundUnlike animals, plants can pause their life cycle as dormant seeds. In both plants and animals, DNA methylation is involved in the regulation of gene expression and genome integrity. In animals, reprogramming erases and re-establishes DNA methylation during development. However, knowledge of reprogramming or reconfiguration in plants has been limited to pollen and the central cell. To better understand epigenetic reconfiguration in the embryo, which forms the plant body, we compared time-series methylomes of dry and germinating seeds to publicly available seed development methylomes.ResultsTime-series whole genome bisulfite sequencing reveals extensive gain of CHH methylation during seed development and drastic loss of CHH methylation during germination. These dynamic changes in methylation mainly occur within transposable elements. Active DNA methylation during seed development depends on both RNA-directed DNA methylation and heterochromatin formation pathways, whereas global demethylation during germination occurs in a passive manner. However, an active DNA demethylation pathway is initiated during late seed development.ConclusionsThis study provides new insights into dynamic DNA methylation reprogramming events during seed development and germination and suggests possible mechanisms of regulation. The observed sequential methylation/demethylation cycle suggests an important role of DNA methylation in seed dormancy.

Dynamic reprogramming of DNA methylation in SETD2-deregulated renal cell carcinoma.

Clear cell renal cell carcinomas (ccRCCs) harbor frequent mutations in epigenetic modifiers including SETD2, the H3K36me3 writer. We profiled DNA methylation (5mC) across the genome in cell line-based models of SETD2 inactivation and SETD2 mutant primary tumors because 5mC has been linked to H3K36me3 and is therapeutically targetable. SETD2 depleted cell line models (long-term and acute) exhibited a DNA hypermethylation phenotype coinciding with ectopic gains in H3K36me3 centered across intergenic regions adjacent to low expressing genes, which became upregulated upon dysregulation of the epigenome. Poised enhancers of developmental genes were prominent hypermethylation targets. SETD2 mutant primary ccRCCs, papillary renal cell carcinomas, and lung adenocarcinomas all demonstrated a DNA hypermethylation phenotype that segregated tumors by SETD2 genotype and advanced grade. These findings collectively demonstrate that SETD2 mutations drive tumorigenesis by coordinated disruption of the epigenome and transcriptome,and they have important implications for future therapeutic strategies targeting chromatin regulator mutant tumors.

6 ALTERED CHROMATIN CONFORMATION IN BOVINE SPERMATOZOA PERTURBS DYNAMIC DNA METHYLATION IN THE MALE PRONUCLEUS AFTER FERTILIZATION IN VITRO

Soon after fertilization, mammalian zygotes need proper DNA methylation reprogramming, at which time the epigenetic marks that the oocyte and sperm have acquired during gametogenesis are erased to allow totipotent zygotic development. Aberrant epigenetic marks in the paternal genome are thought to be associated with altered chromatin condensation in spermatozoa of suboptimal quality. We have recently reported that heat stress on bulls during germ cell development, especially at the spermiogenesis stage, altered sperm chromatin condensation. The objective of this study was to investigate dynamic DNA methylation reprogramming in the male pronucleus after fertilization of oocytes with sperm known to have altered chromatin conformation. To evaluate dynamic DNA methylation reprogramming, zygotes collected at 3 different time points [i.e. 12, 18, and 24 h post-insemination (hpi)] were immunocytochemically investigated using an antibody against 5-methylcytosine (5mC). The total fluorescence intensity of the male pronuclei (n = 89, ≥25 in each group) was measured by ImageJ and data were analyzed by ANOVA. The DNA methylation pattern in male pronuclei when oocytes were fertilized with heat-stressed sperm did not change between time points (P > 0.05), whereas control zygotes clearly showed demethylation and de novo methylation at 18 and 24 hpi, respectively. The results of this study indicated that dynamic DNA methylation reprogramming patterns such as DNA demethylation followed by de novo methylation in the male pronucleus soon after fertilization were altered when oocytes were fertilized with heat-stressed sperm. In conclusion, altered sperm chromatin conformation due to heat stress perturbs dynamic DNA methylation reprogramming in the male pronucleus, which may hamper nuclear totipotency and embryo survival.

Epigenetic reprogramming in the porcine germ line

BackgroundEpigenetic reprogramming is critical for genome regulation during germ line development. Genome-wide demethylation in mouse primordial germ cells (PGC) is a unique reprogramming event essential for erasing epigenetic memory and preventing the transmission of epimutations to the next generation. In addition to DNA demethylation, PGC are subject to a major reprogramming of histone marks, and many of these changes are concurrent with a cell cycle arrest in the G2 phase. There is limited information on how well conserved these events are in mammals. Here we report on the dynamic reprogramming of DNA methylation at CpGs of imprinted loci and DNA repeats, and the global changes in H3K27me3 and H3K9me2 in the developing germ line of the domestic pig.ResultsOur results show loss of DNA methylation in PGC colonizing the genital ridges. Analysis of IGF2-H19 regulatory region showed a gradual demethylation between E22-E42. In contrast, DMR2 of IGF2R was already demethylated in male PGC by E22. In females, IGF2R demethylation was delayed until E29-31, and was de novo methylated by E42. DNA repeats were gradually demethylated from E25 to E29-31, and became de novo methylated by E42. Analysis of histone marks showed strong H3K27me3 staining in migratory PGC between E15 and E21. In contrast, H3K9me2 signal was low in PGC by E15 and completely erased by E21. Cell cycle analysis of gonadal PGC (E22-31) showed a typical pattern of cycling cells, however, migrating PGC (E17) showed an increased proportion of cells in G2.ConclusionsOur study demonstrates that epigenetic reprogramming occurs in pig migratory and gonadal PGC, and establishes the window of time for the occurrence of these events. Reprogramming of histone H3K9me2 and H3K27me3 detected between E15-E21 precedes the dynamic DNA demethylation at imprinted loci and DNA repeats between E22-E42. Our findings demonstrate that major epigenetic reprogramming in the pig germ line follows the overall dynamics shown in mice, suggesting that epigenetic reprogramming of germ cells is conserved in mammals. A better understanding of the sequential reprogramming of PGC in the pig will facilitate the derivation of embryonic germ cells in this species.

Dynamic DNA methylation reprogramming: Active demethylation and immediate remethylation in the male pronucleus of bovine zygotes

DNA methylation reprogramming (DMR) is believed to be a key process by which mammalian zygotes gain nuclear totipotency through erasing epigenetic modifications acquired during gametogenesis. Nonetheless, DMR patterns do not seem to be conserved among mammals. To identify uniform rules underlying mammalian DMRs, we explored DMRs of diverse mammalian zygotes. Of the zygotes studied, of particular interest was the bovine zygote; the paternal DNA methylation first decreased and was then rapidly restored almost to the maternal methylation level even before the two-cell stage. The 5-azadeoxycytidine treatment led to complete demethylation of the male pronucleus. The unusually dramatic changes in DNA methylation levels indicate that the bovine male pronucleus undergoes active demethylation, which is followed by de novo methylation. Our results show that, in bovine, the compound processes of active DNA demethylation and de novo DNA methylation, along with de novo H3-K9 trimethylation also, take place altogether within this very narrow window of pronucleus development.

Dynamic reprogramming of DNA methylation at an epigenetically sensitive allele in mice.

There is increasing evidence in both plants and animals that epigenetic marks are not always cleared between generations. Incomplete erasure at genes associated with a measurable phenotype results in unusual patterns of inheritance from one generation to the next, termed transgenerational epigenetic inheritance. The Agouti viable yellow (Avy) allele is the best-studied example of this phenomenon in mice. The Avy allele is the result of a retrotransposon insertion upstream of the Agouti gene. Expression at this locus is controlled by the long terminal repeat (LTR) of the retrotransposon, and expression results in a yellow coat and correlates with hypomethylation of the LTR. Isogenic mice display variable expressivity, resulting in mice with a range of coat colours, from yellow through to agouti. Agouti mice have a methylated LTR. The locus displays epigenetic inheritance following maternal but not paternal transmission; yellow mothers produce more yellow offspring than agouti mothers. We have analysed the DNA methylation in mature gametes, zygotes, and blastocysts and found that the paternally and maternally inherited alleles are treated differently. The paternally inherited allele is demethylated rapidly, and the maternal allele is demethylated more slowly, in a manner similar to that of nonimprinted single-copy genes. Interestingly, following maternal transmission of the allele, there is no DNA methylation in the blastocyst, suggesting that DNA methylation is not the inherited mark. We have independent support for this conclusion from studies that do not involve direct analysis of DNA methylation. Haplo-insufficiency for Mel18, a polycomb group protein, introduces epigenetic inheritance at a paternally derived Avy allele, and the pedigrees reveal that this occurs after zygotic genome activation and, therefore, despite the rapid demethylation of the locus.

DNA methylation patterns in human tripronucleate zygotes

In mammals, the dynamic reprogramming of DNA methylation begins during gametogenesis and continues through embryogenesis. Recently, immunofluorescence staining with an antibody against 5-methylcytosine (anti-5-MeC) has revealed active demethylation of the male pronucleus in zygotes beginning at 4-6 h after fertilization. In this study, we characterized the DNA methylation patterns in mouse zygotes and in human tripronucleate (3 PN) zygotes discarded after conventional fertilization or following ICSI. Pronuclei were subjected to fluorescence in-situ hybridization to identify the X and/or Y chromosomes and then stained with anti-5-MeC. In diandric 3 PN zygotes from conventional IVF, we consistently observed one strongly and two weakly stained pronuclei. In contrast, the majority of 3 PN ICSI zygotes, mainly digynic zygotes, displayed two strongly and one weakly stained pronuclei. Two zygotes from ICSI failed to show any staining difference among the three pronuclei. Our results indicate that the active demethylation of male pronuclei occurs in both mouse and human zygotes. It is possible that the abnormal methylation patterns resulting from a dysfunctional cytoplasm may occur in a small number of oocytes and may affect embryonic viability.

DNA methylation patterns in mouse and human zygotes

Objective: Epigenetic modification during embryogenesis is characterized by selective gene expression achieved through methylation of 5-methylcytosine. In mammals dynamic reprogramming of DNA methylation occurs during gametogenesis and embryogenesis. Changes in environment, e.g., in vivo vs in vitro culture, can alter the process of epigenetic modification. Staining of mouse zygotes with an antibody against 5-methylcytosine (Anti-MeC) has revealed that by 4–6 h post-fertilization the chromatids in the male pronucleus (PN) have been actively demethylated. This study examines whether a similar pattern of DNA methylation takes place in human zygotes. Design: Normally fertilized (2PN) mouse and abnormally fertilized human (3PN) zygotes were examined with Anti-MeC immunofluorescence staining. Sex chromosome status, assessed by fluorescence in situ hybridization (FISH), was used to identify the parental origin of each pronucleus. Materials and Methods: Mouse zygotes were obtained from in vitro fertilization of oocytes harvested from oviducts 16 h post-hCG. Abnormally fertilized zygotes (3PN) were obtained from patients undergoing conventional IVF or ICSI with patient consent (IBRA Protocol H 6092). Each zygote was fixed with methanol:acetic acid (3:1). DNA methylation patterns were detected in mouse zygotes by immunofluorescence staining with an Anti-MeC antibody (Barton et al 2001, Human Molecular Genetics, 26, 2983–2987). Human pronuclei were subjected to FISH to identify the X and Y chromosomes prior to staining with the anti-MeC antibody. Results: Staining patterns in mouse revealed active demethylation between sperm head decondensation and male PN formation. In contrast to the decline in paternal chromatid staining intensity, maternal chromatid staining remained strong throughout PN formation. All 19 conventionally inseminated human zygotes displayed one strongly and two weakly stained PNs. All 21 PNs with a Y chromosome stained poorly as did 17 of 34 PNs with an X chromosome, most likely those of paternal origin. In contrast, 8 zygotes from ICSI were digynic, displaying two strongly and one weakly staining PNs. Interestingly, an ICSI zygote displayed one strongly and two weakly fluorescing PNs, but it was diandric with 2 Y chromosomes. However, 2 ICSI zygotes presented 3 brightly stained PNs with no differences in intensity; these zygotes were X/X/Y and XX/X/O. Conclusion: Selective, active demethylation of the male PN occurs in both mouse and human zygotes. However, abnormal methylation patterns can occur in some zygotes and may indicate a dysfunctional ooplasm. A full investigation of the methylation patterns in donated normal embryos will provide more insights into the epigenetic modifications that occur during in vitro embryogenesis.

Dynamic Reprogramming of DNA Methylation in the Early Mouse Embryo

Dynamic epigenetic modification of the genome occurs during early development of the mouse. Active demethylation of the paternal genome occurs in the zygote, followed by passive demethylation during cleavage stages, and de novo methylation, which is thought to happen after implantation. We have investigated these processes by using indirect immunofluoresence with an antibody to 5-methyl cytosine. In contrast to previous work, we show that demethylation of the male pronucleus is completed within 4 h of fertilisation. This activity is intricately linked with and not separable from pronucleus formation. In conditions permissive for polyspermy, up to five male pronuclei underwent demethylation in the same oocyte. Paternal demethylation in fertilised oocytes deficient for MBD2, the only candidate demethylase, occurred normally. Passive loss of methylation occurred in a stepwise fashion up to the morulae stage without any evidence of spatial compartmentalisation. De novo methylation was observed specifically in the inner cell mass (ICM) but not in the trophectoderm of the blastocyst and hence may have an important role in early lineage specification. This is the first complete and detailed analysis of the epigenetic reprogramming cycle during preimplantation development. The three phases of methylation reprogramming may have roles in imprinting, the control of gene expression, and the establishment of nuclear totipotency.