Maintenance Of Methylation Research Articles

Antagonistic interactions safeguard mitotic propagation of genetic and epigenetic information in zebrafish

The stability of cellular phenotypes in developing organisms depends on error-free transmission of epigenetic and genetic information during mitosis. Methylation of cytosine residues in genomic DNA is a key epigenetic mark that modulates gene expression and prevents genome instability. Here, we report on a genetic test of the relationship between DNA replication and methylation in the context of the developing vertebrate organism instead of cell lines. Our analysis is based on the identification of hypomorphic alleles of dnmt1, encoding the DNA maintenance methylase Dnmt1, and pole1, encoding the catalytic subunit of leading-strand DNA polymerase epsilon holoenzyme (Pole). Homozygous dnmt1 mutants exhibit genome-wide DNA hypomethylation, whereas the pole1 mutation is associated with increased DNA methylation levels. In dnmt1/pole1 double-mutant zebrafish larvae, DNA methylation levels are restored to near normal values, associated with partial rescue of mutant-associated transcriptional changes and phenotypes. Hence, a balancing antagonism between DNA replication and maintenance methylation buffers against replicative errors contributing to the robustness of vertebrate development.

Epigenetics comprehensive experimental course based on the integration of science and education to cultivate students' ability of cutting-edge innovation.

The integration of science and education is an effective way for universities to cultivate students in cutting-edge innovative interests. Epigenetics is the expansion of classical genetics, the corresponding experimental courses of which have not been integrated into the current teaching system. In this paper, by taking advantage of our laboratory's research on the DNA methylation maintenance gene, OsMET1-2 in rice, we have integrated our innovative findings in the education curriculum, and built a comprehensive teaching system on experimentation research, which greatly stimulates the curiosity of the students. Taking the OsMET1-2 mutants and its isogenic wild-type rice plants as experimental materials, this course has successfully demonstrated a causal link between genetic mutation and epigenetic variation, a topic widely interested by the students in learning genetics and epigenetics. Through the practice of this course, students have a deeper understanding of the important role of epigenetic modifications, their scientific research capabilities have been greatly improved, thereby strongly supporting the cultivation of top innovative talents among the students.

MBD4 loss results in global reactivation of promoters and retroelements with low methylated CpG density

BackgroundInherited defects in the base-excision repair gene MBD4 predispose individuals to adenomatous polyposis and colorectal cancer, which is characterized by an accumulation of C > T transitions resulting from spontaneous deamination of 5’-methylcytosine.MethodsHere, we have investigated the potential role of MBD4 in regulating DNA methylation levels using genome-wide transcriptome and methylome analyses. Additionally, we have elucidated its function through a series of in vitro experiments.ResultsHere we show that the protein MBD4 is required for DNA methylation maintenance and G/T mismatch repair. Transcriptome and methylome analyses reveal a genome-wide hypomethylation of promoters, gene bodies and repetitive elements in the absence of MBD4 in vivo. Methylation mark loss is accompanied by a broad transcriptional derepression phenotype affecting promoters and retroelements with low methylated CpG density. MBD4 in vivo forms a complex with the mismatch repair proteins (MMR), which exhibits high bi-functional glycosylase/AP-lyase endonuclease specific activity towards methylated DNA substrates containing a G/T mismatch. Experiments using recombinant proteins reveal that the association of MBD4 with the MMR protein MLH1 is required for this activity.ConclusionsOur data identify MBD4 as an enzyme specifically designed to repair deaminated 5-methylcytosines and underscores its critical role in safeguarding against methylation damage. Furthermore, it illustrates how MBD4 functions in normal and pathological conditions.

Exploring the Effects of Rearing Densities on Epigenetic Modifications in the Zebrafish Gonads.

Rearing density directly impacts fish welfare, which, in turn, affects productivity in aquaculture. Previous studies have indicated that high-density rearing during sexual development in fish can induce stress, resulting in a tendency towards male-biased sex ratios in the populations. In recent years, research has defined the relevance of the interactions between the environment and epigenetics playing a key role in the final phenotype. However, the underlying epigenetic mechanisms of individuals exposed to confinement remain elucidated. By using zebrafish (Danio rerio), the DNA methylation promotor region and the gene expression patterns of six genes, namely dnmt1, cyp19a1a, dmrt1, cyp11c1, hsd17b1, and hsd11b2, involved in the DNA maintenance methylation, reproduction, and stress were assessed. Zebrafish larvae were subjected to two high-density conditions (9 and 66 fish/L) during two periods of overlapping sex differentiation of this species (7 to 18 and 18 to 45 days post-fertilization, dpf). Results showed a significant masculinization in the populations of fish subjected to high densities from 18 to 45 dpf. In adulthood, the dnmt1 gene was differentially hypomethylated in ovaries and its expression was significantly downregulated in the testes of fish exposed to high-density. Further, the cyp19a1a gene showed downregulation of gene expression in the ovaries of fish subjected to elevated density, as previously observed in other studies. We proposed dnmt1 as a potential testicular epimarker and the expression of ovarian cyp19a1a as a potential biomarker for predicting stress originated from high densities during the early stages of development. These findings highlight the importance of rearing densities by long-lasting effects in adulthood conveying cautions for stocking protocols in fish hatcheries.

SAT274 Multilocus Imprinting Disorder Due To Genome-wide Paternal Uniparental Disomy Associated With Adrenocortical Tumors

Abstract Disclosure: E.M. Pinto: None. E. Lalli: None. G. Wu: None. R. Ribeiro: None. G. Zambetti: None. Pediatric adrenocortical tumor (ACT) is an uncommon malignancy frequently associated with Li-Fraumeni syndrome, a familial cancer predisposition disorder caused by germline mutations in the tumor suppressor TP53. ACT is also associated with other genetic constitutional disorders, such as Beckwith-Wiedemann syndrome (BWS) which results from the deregulation of a gene cluster on chromosome 11p15. We report here the identification of three pediatric patients with ACT and additional tumors, two of which received a clinical diagnosis of BWS. Family histories of these two probands include multiple unsuccessful pregnancies. All patients have wild-type TP53 sequence and no evidence for germline chromosome 11p15 alterations. Molecular analyses by whole genome sequencing revealed genome-wide copy neutral loss of heterozygosity (cnLOH) in all tumors. Microsatellite STR and MS-MLPA confirmed genome-wide cnLOH with the selective loss of all maternal chromosomes and duplication of paternal chromosomes in each tumor. Analysis of skin fibroblasts showed mosaic paternal uniparental disomy with partial LOH for all chromosomes, while no LOH was detected in blood DNA. The most prominent clinical feature of genome-wide cnLOH is an unbalanced monoallelic and parent-of-origin specific expression of imprinted genes known as multilocus imprinting disorder (MID). MID has been associated with maternal-effect variants that alter the maintenance of methylation at germline-derived differentially methylated regions (DMRs) in early embryogenesis. In addition, causative trans-acting factors in imprinting disorders, including the subcortical maternal complex (SCMC), have the potential to affect multiple DMRs across the genome, resulting in a multilocus imprinting disorder. Our study identifies three pediatric patients who developed ACT and other tumors that have undergone genome wide cnLOH that specifically selected against all maternal chromosomes. None of three patients presented with germline alterations in TP53 or 11p15, typical of pediatric ACT. WGS data revealed a germline mutation (p.M573T) in the histone methyltransferase NSD1 in the mother of patient #2. NSD1 mutations are reportedly associated with recurrent miscarriages, tissue overgrowth and increased cancer risk. Alterations in other epigenetic regulators, such as SETD1B (p.R226C), a component of a histone methyltransferase complex and TNRC18 (p.P454L), that enables chromatin binding activity were observed in both mother and child in each family. Further analysis of maternal-effect variants associated with abnormal pregnancies and MID will be important for understanding the mechanisms underlying genome-wide cnLOH. Our results support the molecular testing of multiple loci in atypical clinical presentations of patients with pediatric ACT. Presentation: Saturday, June 17, 2023

PLK1 maintains DNA methylation and cell viability by regulating phosphorylation-dependent UHRF1 protein stability

PLK1 is a key serine/threonine kinase as well as a master mitotic regulator, but it has never been reported that PLK1 regulates DNA methylation. In the present study, we for the first time found that PLK1 inhibition disrupted global DNA methylation and elevated the expression level of tumor suppressor genes. Mechanistically, we found that PLK1 interacts UHRF1 protein to induce its phosphorylation at serine 265. Phosphorylation is required for the maintenance of UHRF1 protein stability by recruiting a deubiquitinase USP7. Conversely, PLK1 inhibition decreases UHRF1 protein interaction with USP7 and activates the ubiquitin-proteasome pathway, thereby accelerating UHRF1 protein degradation. UHRF1 degradation decreases the recruitment of DNMT1 to chromatin, and decreases the level of genome-wide DNA methylation, thereby elevating the expression of tumor suppressor genes and decreasing cell viability. We here presented the first report on the novel role of PLK1 in DNA methylation maintenance through UHRF1-DNMT1 pathway, and revealed a novel anticancer mechanism of PLK1 inhibitors.

Coevolution of the CDCA7-HELLS ICF-related nucleosome remodeling complex and DNA methyltransferases

5-Methylcytosine (5mC) and DNA methyltransferases (DNMTs) are broadly conserved in eukaryotes but are also frequently lost during evolution. The mammalian SNF2 family ATPase HELLS and its plant ortholog DDM1 are critical for maintaining 5mC. Mutations in HELLS, its activator CDCA7, and the de novo DNA methyltransferase DNMT3B, cause immunodeficiency-centromeric instability-facial anomalies (ICF) syndrome, a genetic disorder associated with the loss of DNA methylation. We here examine the coevolution of CDCA7, HELLS and DNMTs. While DNMT3, the maintenance DNA methyltransferase DNMT1, HELLS, and CDCA7 are all highly conserved in vertebrates and green plants, they are frequently co-lost in other evolutionary clades. The presence-absence patterns of these genes are not random; almost all CDCA7 harboring eukaryote species also have HELLS and DNMT1 (or another maintenance methyltransferase, DNMT5). Coevolution of presence-absence patterns (CoPAP) analysis in Ecdysozoa further indicates coevolutionary linkages among CDCA7, HELLS, DNMT1 and its activator UHRF1. We hypothesize that CDCA7 becomes dispensable in species that lost HELLS or DNA methylation, and/or the loss of CDCA7 triggers the replacement of DNA methylation by other chromatin regulation mechanisms. Our study suggests that a unique specialized role of CDCA7 in HELLS-dependent DNA methylation maintenance is broadly inherited from the last eukaryotic common ancestor.

Coevolution of the CDCA7-HELLS ICF-related nucleosome remodeling complex and DNA methyltransferases.

5-Methylcytosine (5mC) and DNA methyltransferases (DNMTs) are broadly conserved in eukaryotes but are also frequently lost during evolution. The mammalian SNF2 family ATPase HELLS and its plant ortholog DDM1 are critical for maintaining 5mC. Mutations in HELLS, its activator CDCA7, and the de novo DNA methyltransferase DNMT3B, cause immunodeficiency-centromeric instability-facial anomalies (ICF) syndrome, a genetic disorder associated with the loss of DNA methylation. We here examine the coevolution of CDCA7, HELLS and DNMTs. While DNMT3, the maintenance DNA methyltransferase DNMT1, HELLS, and CDCA7 are all highly conserved in vertebrates and green plants, they are frequently co-lost in other evolutionary clades. The presence-absence patterns of these genes are not random; almost all CDCA7 harboring eukaryote species also have HELLS and DNMT1 (or another maintenance methyltransferase, DNMT5). Coevolution of presence-absence patterns (CoPAP) analysis in Ecdysozoa further indicates coevolutionary linkages among CDCA7, HELLS, DNMT1 and its activator UHRF1. We hypothesize that CDCA7 becomes dispensable in species that lost HELLS or DNA methylation, and/or the loss of CDCA7 triggers the replacement of DNA methylation by other chromatin regulation mechanisms. Our study suggests that a unique specialized role of CDCA7 in HELLS-dependent DNA methylation maintenance is broadly inherited from the last eukaryotic common ancestor.

Refined read-out: The hUHRF1 Tandem-Tudor domain prefers binding to histone H3 tails containing K4me1 in the context of H3K9me2/3.

UHRF1 is an essential chromatin protein required for DNA methylation maintenance, mammalian development, and gene regulation. We investigated the Tandem-Tudor domain (TTD) of human UHRF1 that is known to bind H3K9me2/3 histones and is a major driver of UHRF1 localization in cells. We verified binding to H3K9me2/3 but unexpectedly discovered stronger binding to H3 peptides and mononucleosomes containing K9me2/3 with additional K4me1. We investigated the combined binding of TTD to H3K4me1-K9me2/3 versus H3K9me2/3 alone, engineered mutants with specific and differential changes of binding, and discovered a novel read-out mechanism for H3K4me1 in an H3K9me2/3 context that is based on the interaction of R207 with the H3K4me1 methyl group and on counting the H-bond capacity of H3K4. Individual TTD mutants showed up to a 10,000-fold preference for the double-modified peptides, suggesting that after a conformational change, WT TTD could exhibit similar effects. The frequent appearance of H3K4me1-K9me2 regions in human chromatin demonstrated in our TTD chromatin pull-down and ChIP-western blot data suggests that it has specific biological roles. Chromatin pull-down of TTD from HepG2 cells and full-length murine UHRF1 ChIP-seq data correlate with H3K4me1 profiles indicating that the H3K4me1-K9me2/3 interaction of TTD influences chromatin binding of full-length UHRF1. We demonstrate the H3K4me1-K9me2/3 specific binding of UHRF1-TTD to enhancers and promoters of cell-type-specific genes at the flanks of cell-type-specific transcription factor binding sites, and provided evidence supporting an H3K4me1-K9me2/3 dependent and TTD mediated downregulation of these genes by UHRF1. All these findings illustrate the important physiological function of UHRF1-TTD binding to H3K4me1-K9me2/3 double marks in a cellular context.

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.

Downregulation of DNA methylation enhances differentiation of THP-1 cells and induces M1 polarization of differentiated macrophages

DNA methylation is an epigenetic modification that regulates gene expression and plays an essential role in hematopoiesis. UHRF1 and DNMT1 are both crucial for regulating genome-wide maintenance of DNA methylation. Specifically, it is well known that hypermethylation is crucial characteristic of acute myeloid leukemia (AML). However, the mechanism underlying how DNA methylation regulates the differentiation of AML cells, including THP-1 is not fully elucidated. In this study, we report that UHRF1 or DNMT1 depletion enhances the phorbol-12-myristate-13-acetate (PMA)-induced differentiation of THP-1 cells. Transcriptome analysis and genome-wide methylation array results showed that depleting UHRF1 or DNMT1 induced changes that made THP-1 cells highly sensitive to PMA. Furthermore, knockdown of UHRF1 or DNMT1 impeded solid tumor formation in xenograft mouse model. These findings suggest that UHRF1 and DNMT1 play a pivotal role in regulating differentiation and proliferation of THP-1 cells and targeting these proteins may improve the efficiency of differentiation therapy in AML patients.

Methylation is maintained specifically at imprinting control regions but not other DMRs associated with imprinted genes in mice bearing a mutation in the Dnmt1 intrinsically disordered domain.

Differential methylation of imprinting control regions in mammals is essential for distinguishing the parental alleles from each other and regulating their expression accordingly. To ensure parent of origin-specific expression of imprinted genes and thereby normal developmental progression, the differentially methylated states that are inherited at fertilization must be stably maintained by DNA methyltransferase 1 throughout subsequent somatic cell division. Further epigenetic modifications, such as the acquisition of secondary regions of differential methylation, are dependent on the methylation status of imprinting control regions and are important for achieving the monoallelic expression of imprinted genes, but little is known about how imprinting control regions direct the acquisition and maintenance of methylation at these secondary sites. Recent analysis has identified mutations that reduce DNA methyltransferase 1 fidelity at some genomic sequences but not at others, suggesting that it may function differently at different loci. We examined the impact of the mutant DNA methyltransferase 1 P allele on methylation at imprinting control regions as well as at secondary differentially methylated regions and non-imprinted sequences. We found that while the P allele results in a major reduction in DNA methylation levels across the mouse genome, methylation is specifically maintained at imprinting control regions but not at their corresponding secondary DMRs. This result suggests that DNA methyltransferase 1 may work differently at imprinting control regions or that there is an alternate mechanism for maintaining methylation at these critical regulatory regions and that maintenance of methylation at secondary DMRs is not solely dependent on the methylation status of the ICR.

The Notch1/Hes1 signaling pathway affects autophagy by adjusting DNA methyltransferases expression in a valproic acid-induced autism spectrum disorder model

As a pervasive neurodevelopmental disease, autism spectrum disorder (ASD) is caused by both hereditary and environmental elements. Research has demonstrated the functions of the Notch pathway and DNA methylation in the etiology of ASD. DNA methyltransferases DNMT3 and DNMT1 are responsible for methylation establishment and maintenance, respectively. In this study, we aimed to explore the association of DNA methyltransferases with the Notch pathway in ASD. Our results showed Notch1 and Hes1 were upregulated, while DNMT3A and DNMT3B were downregulated at the protein level in the prefrontal cortex (PFC), hippocampus (HC) and cerebellum (CB) of VPA-induced ASD rats compared with Control (Con) group. However, the protein levels of DNMT3A and DNMT3B were augmented after treatment with 3,5-difluorophenacetyl-L-alanyl-S-phenylglycine-2-butyl ester (DAPT), suggesting that abnormal Notch pathway activation may affect the expression of DNMT3A and DNMT3B. Besides, our previous findings revealed that the Notch pathway may participate in development of ASD by influencing autophagy. Therefore, we hypothesized the Notch pathway adjusts autophagy and contributes to ASD by affecting DNA methyltransferases. Our current results showed that after receiving the DNA methyltransferase inhibitor 5-Aza-2′-deoxycytidine (5-Aza-2'dc), the VPA + DAPT+5-Aza-2'dc (V + D + Aza) group exhibited reduced social interaction ability and increased stereotyped behaviors, and decreased expression of DNMT3A, DNMT3B and autophagy-related proteins, but did not show changes in Notch1 and Hes1 protein levels. Our results indicated that the Notch1/Hes1 pathway may adjust DNMT3A and DNMT3B expression and subsequently affect autophagy in the occurrence of ASD, providing new insight into the pathogenesis of ASD.

Heritable induced resistance in Arabidopsis thaliana: Tips and tools to improve effect size and reproducibility.

Over a decade ago, three independent studies reported that pathogen- and herbivore-exposed Arabidopsis thaliana produces primed progeny with increased resistance. Since then, heritable induced resistance (h-IR) has been reported across numerous plant-biotic interactions, revealing a regulatory function of DNA (de)methylation dynamics. However, the identity of the epi-alleles controlling h-IR and the mechanisms by which they prime defense genes remain unknown, while the evolutionary significance of the response requires confirmation. Progress has been hampered by the relatively high variability, low effect size, and sometimes poor reproducibility of h-IR, as is exemplified by a recent study that failed to reproduce h-IR in A.thaliana by Pseudomonas syringae pv. tomato (Pst). This study aimed to improve h-IR effect size and reproducibility in the A.thaliana-Pst interaction. We show that recurrent Pst inoculations of seedlings result in stronger h-IR than repeated inoculations of older plants and that disease-related growth repression in the parents is a reliable marker for h-IR effect size in F1 progeny. Furthermore, RT-qPCR-based expression profiling of genes controlling DNA methylation maintenance revealed that the elicitation of strong h-IR upon seedling inoculations is marked by reduced expression of the chromatin remodeler DECREASE IN DNA METHYLATION 1 (DDM1) gene, which is maintained in the apical meristem and transmitted to F1 progeny. Two additional genes, MET1 and CHROMOMETHYLASE3 (CMT3), displayed similar transcriptional repression in progeny from seedling-inoculated plants. Thus, reduced expression of DDM1, MET1, and CMT3 can serve as a marker of robust h-IR in F1 progeny. Our report offers valuable information and markers to improve the effect size and reproducibility of h-IR in the A.thaliana-Pst model interaction.

Parallel shift of DNA methylation and gene expression toward the mean in mouse spleen with aging.

Age-associated DNA-methylation drift (AMD) manifests itself in two ways in mammals: global decrease (hypomethylation) and local increase of DNA methylation (hypermethylation). To comprehend the principle behind this bidirectional AMD, we studied methylation states of spatially clustered CpG dinucleotides in mouse splenic DNA using reduced-representation-bisulfite-sequencing (RRBS). The mean methylation levels of whole CpGs declined with age. Promoter-resident CpGs, generally weakly methylated (<5%) in young mice, became hypermethylated in old mice, whereas CpGs in gene-body and intergenic regions, initially moderately (~33%) and extensively (>80%) methylated, respectively, were hypomethylated in the old. Chromosome-wise analysis of methylation revealed that inter-individual heterogeneities increase with age. The density of nearby CpGs was used to classify individual CpGs, which found hypermethylation in CpG-rich regions and hypomethylation in CpG-poor regions. When genomic regions were grouped by methylation level, high-methylation regions tended to become hypomethylated whereas low-methylation regions tended to become hypermethylated, regardless of genomic structure/function. Data analysis revealed that while methylation level and CpG density were interdependent, methylation level was a better predictor of the AMD pattern representing a shift toward the mean. Further analysis of gene-expression data showed a decrease in the expression of highly-expressed genes and an increase in the expression of lowly-expressed genes with age. This shift towards the mean in gene-expression changes was correlated with that of methylation changes, indicating a potential link between the two age-associated changes. Our findings suggest that age-associated hyper- and hypomethylation events are stochastic and attributed to malfunctioning intrinsic mechanisms for methylation maintenance in low- and high-methylation regions, respectively.

Inhibition of DNMT1 methyltransferase activity via glucose-regulated O-GlcNAcylation alters the epigenome.

The DNA methyltransferase activity of DNMT1 is vital for genomic maintenance of DNA methylation. We report here that DNMT1 function is regulated by O-GlcNAcylation, a protein modification that is sensitive to glucose levels, and that elevated O-GlcNAcylation of DNMT1 from high glucose environment leads to alterations to the epigenome. Using mass spectrometry and complementary alanine mutation experiments, we identified S878 as the major residue that is O-GlcNAcylated on human DNMT1. Functional studies in human and mouse cells further revealed that O-GlcNAcylation of DNMT1-S878 results in an inhibition of methyltransferase activity, resulting in a general loss of DNA methylation that preferentially occurs at partially methylated domains (PMDs). This loss of methylation corresponds with an increase in DNA damage and apoptosis. These results establish O-GlcNAcylation of DNMT1 as a mechanism through which the epigenome is regulated by glucose metabolism and implicates a role for glycosylation of DNMT1 in metabolic diseases characterized by hyperglycemia.

Estrodiol-17β and aromatase inhibitor treatment induced alternations of genome-wide DNA methylation pattern in Takifugu rubripes gonads

Estradiol-17β (E2) and aromatase inhibitor (AI) exposure can change the phenotypic sex of fish gonads. To investigated whether alterations in DNA methylation is involved in this process, the level of genome-wide DNA methylation in Takifugu rubripes gonads was quantitatively analyzed during the E2-induced feminization and AI-induced masculinization processes in this study. The methylation levels of the total cytosine (C) in control-XX(C-XX), control-XY (C-XY), E2-treated-XY (E-XY) and AI-treated-XX (AI-XX) were 9.11%, 9.19%, 8.63% and 9.23%, respectively. In the C-XX vs C-XY comparison, 4,196 differentially methylated regions (DMRs) overlapped with the gene body of 2,497 genes and 608 DMRs overlapped with the promoter of 575 genes. In the E-XY vs C-XY comparison, 6,539 DMRs overlapped with the gene body of 3,416 genes and 856 DMRs overlapped with the promoter of 776 genes. In the AI-XX vs C-XX comparison, 2,843 DMRs overlapped with the gene body of 1,831 genes and 461 DMRs overlapped with the promoter of 421 genes. Gonadal genomic methylation mainly occurred at CG sites and the genes that overlapped with DMRs on CG context were most enriched in the signaling pathways related to gonad differentiation, such as the Wnt, TGF-β, MAPK, CAM and GnRH pathways. The DNA methylation levels of steroid synthesis genes and estrogen receptor genes promoter or gene body were negative correlated with their expression. After bisulfite sequencing verification, the DNA methylation level of the amhr2 promoter in XY was increased after E2 treatment, which consistent with the data from the genome-wide DNA methylation sequencing. In C-XY group, the expression of amhr2 was significantly higher than that in E-XY (p < 0.05). Additionally, dnmt1, which is responsible for methylation maintenance, expressed at similar level in four groups (p > 0.05). dnmt3, tet2, and setd1b, which were responsible for methylation modification, expressed at significantly higher levels in E-XY compared to the C-XY (p < 0.05). Dnmt3 and tet2 were expressed at significantly higher levels in AI-XX than that in C-XX (p < 0.05). These results indicated that E2 and AI treatment lead to the aberrant genome-wide DNA methylation level and expression level of dnmt3, tet2, and setd1b in T. rubripes gonad.

Five nucleotides found in RCTG motifs are essential for post-fertilization methylation imprinting of the H19 ICR in YAC transgenic mice.

Genomic imprinting at the mouse Igf2/H19 locus is controlled by the H19 ICR, within which paternal allele-specific DNA methylation originating in sperm is maintained throughout development in offspring. We previously found that a 2.9 kb transgenic H19 ICR fragment in mice can be methylated de novo after fertilization only when paternally inherited, despite its unmethylated state in sperm. When the 118 bp sequence responsible for this methylation in transgenic mice was deleted from the endogenous H19 ICR, the methylation level of its paternal allele was significantly reduced after fertilization, suggesting the activity involving this 118 bp sequence is required for methylation maintenance at the endogenous locus. Here, we determined protein binding to the 118 bp sequence using an in vitro binding assay and inferred the binding motif to be RCTG by using a series of mutant competitors. Furthermore, we generated H19 ICR transgenic mice with a 5-bp substitution mutation that disrupts the RCTG motifs within the 118 bp sequence, and observed loss of methylation from the paternally inherited transgene. These results indicate that imprinted methylation of the H19 ICR established de novo during the post-fertilization period involves binding of specific factors to distinct sequence motifs within the 118 bp sequence.

UHRF1 is essential for proper cytoplasmic architecture and function of mouse oocytes and derived embryos.

Ubiquitin-like with PHD and RING finger domains 1 (UHRF1) is a protein essential for the maintenance of DNA methylation in somatic cells. However, UHRF1 is predominantly localized in the cytoplasm of mouse oocytes and preimplantation embryos, where it may play a role unrelated to the nuclear function. We herein report that oocyte-specific Uhrf1 KO results in impaired chromosome segregation, abnormal cleavage division, and preimplantation lethality of derived embryos. Our nuclear transfer experiment showed that the phenotype is attributable to cytoplasmic rather than nuclear defects of the zygotes. A proteomic analysis of KO oocytes revealed the down-regulation of proteins associated with microtubules including tubulins, which occurred independently of transcriptomic changes. Intriguingly, cytoplasmic lattices were disorganized, and mitochondria, endoplasmic reticulum, and components of the subcortical maternal complex were mislocalized. Thus, maternal UHRF1 regulates the proper cytoplasmic architecture and function of oocytes and preimplantation embryos, likely through a mechanism unrelated to DNA methylation.

Impacts of DNA methylases and demethylases on the methylation and expression of Arabidopsis ethylene signal pathway genes.

Arabidopsis ethylene (ET) signal pathway plays important roles in various aspects. Cytosine DNA methylation is significant in controlling gene expression in plants. Here, we analyzed the bisulfite sequencing and mRNA sequencing data from Arabidopsis (de)methylase mutants met1, cmt3, drm1/2, ddm1, ros1-4, and rdd to investigate how DNA (de)methylases influence the DNA methylation and expression of Arabidopsis ET pathway genes. At least 32 genes are found to involved in Arabidopsis ET pathway by text mining. Among them, 14 genes are unmethylated or methylated with very low levels. ACS6 and ACS9 are conspicuously methylated within their upstream regions. The other 16 genes are predominantly methylated at the CG sites within gene body regions in wild-type plants, and mutation of MET1 resulted in almost entire elimination of the CG methylations. In addition, CG methylations within some genes are jointly maintained by MET1 and other (de)methylases. Analyses of mRNA-seq data indicated that some ET pathway genes were differentially expressed between wild-type and diverse mutants. PDF1.2, the marker gene of ET signal pathway, was found being regulated indirectly by the methylases. Eighty-two transposable elements (TEs) were identified to be associated to 15 ET pathway genes. ACS11 is found located in a heterochromatin region that contains 57 TEs, indicating its specific expression and regulation. Together, our results suggest that DNA (de)methylases are implicated in the regulation of CG methylation within gene body regions and transcriptional activity of some ET pathway genes and that maintenance of normal CG methylation is essential for ET pathway in Arabidopsis.