Genome-wide DNA Demethylation Research Articles

Reinforcement of repressive marks in the chicken primordial germ cell epigenetic signature: divergence from basal state resetting in mammals

BackgroundIn mammals, primordial germ cells (PGCs), the embryonic precursors of the germline, arise from embryonic or extra-embryonic cells upon induction by the surrounding tissues during gastrulation, according to mechanisms which are elucidated in mice but remain controversial in primates. They undergo genome-wide epigenetic reprogramming, consisting of extensive DNA demethylation and histone post-translational modification (PTM) changes, toward a basal, euchromatinized state. In contrast, chicken PGCs are specified by preformation before gastrulation based on maternally-inherited factors. They can be isolated from the bloodstream during their migration to the genital ridges. Our prior research highlighted differences in the global epigenetic profile of cultured chicken PGCs compared with chicken somatic cells and mammalian PGCs. This study investigates the acquisition and evolution of this profile during development.ResultsQuantitative analysis of global DNA methylation and histone PTMs, including their distribution, during key stages of chicken early development revealed divergent PGC epigenetic changes compared with mammals. Unlike mammalian PGCs, chicken PGCs do not undergo genome-wide DNA demethylation or exhibit a decrease in histone H3 lysine 9 dimethylation. However, chicken PGCs show 5‑hydroxymethylcytosine loss, macroH2A redistribution, and chromatin decompaction, mirroring mammalian processes. Chicken PGCs initiate their epigenetic signature during migration, progressively accumulating high global levels of H3K9me3, with preferential enrichment in inactive genome regions. Despite apparent global chromatin decompaction, abundant heterochromatin marks, including repressive histone PTMs, HP1 variants, and DNA methylation, persists in chicken PGCs, contrasting with mammalian PGCs.ConclusionsChicken PGCs’ epigenetic signature does not align with the basal chromatin state observed in mammals, suggesting a departure from extensive epigenetic reprogramming. Despite disparities in early PGC development, the persistence of several epigenetic features shared with mammals implies their involvement in chromatin-regulated germ cell properties, with the distinctive elevation of chicken-specific H3K9me3 potentially participating in these processes.

DPPA3 facilitates genome-wide DNA demethylation in mouse primordial germ cells

BackgroundGenome-wide DNA demethylation occurs in mammalian primordial germ cells (PGCs) as part of the epigenetic reprogramming important for gametogenesis and resetting the epigenetic information for totipotency. Dppa3 (also known as Stella or Pgc7) is highly expressed in mouse PGCs and oocytes and encodes a factor essential for female fertility. It prevents excessive DNA methylation in oocytes and ensures proper gene expression in preimplantation embryos: however, its role in PGCs is largely unexplored. In the present study, we investigated whether or not DPPA3 has an impact on CG methylation/demethylation in mouse PGCs.ResultsWe show that DPPA3 plays a role in genome-wide demethylation in PGCs even before sex differentiation. Dppa3 knockout female PGCs show aberrant hypermethylation, most predominantly at H3K9me3-marked retrotransposons, which persists up to the fully-grown oocyte stage. DPPA3 works downstream of PRDM14, a master regulator of epigenetic reprogramming in embryonic stem cells and PGCs, and independently of TET1, an enzyme that hydroxylates 5-methylcytosine.ConclusionsThe results suggest that DPPA3 facilitates DNA demethylation through a replication-coupled passive mechanism in PGCs. Our study identifies DPPA3 as a novel epigenetic reprogramming factor in mouse PGCs.

Epigenetic reshaping through damage: promoting cell fate transition by BrdU and IdU incorporation

BackgroundThymidine analogs have long been recognized for their ability to randomly incorporate into DNA. However, the precise mechanisms through which thymidine analogs facilitate cell fate transition remains unclear.ResultsHere, we discovered a strong correlation between the dosage dependence of thymidine analogs and their ability to overcome reprogramming barrier. The extraembryonic endoderm (XEN) state seems to be a cell's selective response to DNA damage repair (DDR), offering a shortcut to overcome reprogramming barriers. Meanwhile, we found that homologous recombination repair (HRR) pathway causes an overall epigenetic reshaping of cells and enabling them to overcome greater barriers. This response leads to the creation of a hypomethylated environment, which facilitates the transition of cell fate in various reprogramming systems. We term this mechanism as Epigenetic Reshaping through Damage (ERD).ConclusionOverall, our study finds that BrdU/IdU can activate the DNA damage repair pathway (HRR), leading to increased histone acetylation and genome-wide DNA demethylation, regulating somatic cell reprogramming. This offers valuable insights into mechanisms underlying cell fate transition.Graphical

DNA methylation restricts coordinated germline and neural fates in embryonic stem cell differentiation.

As embryonic stem cells (ESCs) transition from naive to primed pluripotency during early mammalian development, they acquire high DNA methylation levels. During this transition, the germline is specified and undergoes genome-wide DNA demethylation, while emergence of the three somatic germ layers is preceded by acquisition of somatic DNA methylation levels in the primed epiblast. DNA methylation is essential for embryogenesis, but the point at which it becomes critical during differentiation and whether all lineages equally depend on it is unclear. Here, using culture modeling of cellular transitions, we found that DNA methylation-free mouse ESCs with triple DNA methyltransferase knockout (TKO) progressed through the continuum of pluripotency states but demonstrated skewed differentiation abilities toward neural versus other somatic lineages. More saliently, TKO ESCs were fully competent for establishing primordial germ cell-like cells, even showing temporally extended and self-sustained capacity for the germline fate. By mapping chromatin states, we found that neural and germline lineages are linked by a similar enhancer dynamic upon exit from the naive state, defined by common sets of transcription factors, including methyl-sensitive ones, that fail to be decommissioned in the absence of DNA methylation. We propose that DNA methylation controls the temporality of a coordinated neural-germline axis of the preferred differentiation route during early development.

Perturbation of 3D nuclear architecture, epigenomic dysregulation and aging, and cannabinoid synaptopathy reconfigures conceptualization of cannabinoid pathophysiology: part 1-aging and epigenomics.

Much recent attention has been directed toward the spatial organization of the cell nucleus and the manner in which three-dimensional topologically associated domains and transcription factories are epigenetically coordinated to precisely bring enhancers into close proximity with promoters to control gene expression. Twenty lines of evidence robustly implicate cannabinoid exposure with accelerated organismal and cellular aging. Aging has recently been shown to be caused by increased DNA breaks. These breaks rearrange and maldistribute the epigenomic machinery to weaken and reverse cellular differentiation, cause genome-wide DNA demethylation, reduce gene transcription, and lead to the inhibition of developmental pathways, which contribute to the progressive loss of function and chronic immune stimulation that characterize cellular aging. Both cell lineage-defining superenhancers and the superanchors that control them are weakened. Cannabis exposure phenocopies the elements of this process and reproduces DNA and chromatin breakages, reduces the DNA, RNA protein and histone synthesis, interferes with the epigenomic machinery controlling both DNA and histone modifications, induces general DNA hypomethylation, and epigenomically disrupts both the critical boundary elements and the cohesin motors that create chromatin loops. This pattern of widespread interference with developmental programs and relative cellular dedifferentiation (which is pro-oncogenic) is reinforced by cannabinoid impairment of intermediate metabolism (which locks in the stem cell-like hyper-replicative state) and cannabinoid immune stimulation (which perpetuates and increases aging and senescence programs, DNA damage, DNA hypomethylation, genomic instability, and oncogenesis), which together account for the diverse pattern of teratologic and carcinogenic outcomes reported in recent large epidemiologic studies in Europe, the USA, and elsewhere. It also accounts for the prominent aging phenotype observed clinically in long-term cannabis use disorder and the 20 characteristics of aging that it manifests. Increasing daily cannabis use, increasing use in pregnancy, and exponential dose-response effects heighten the epidemiologic and clinical urgency of these findings. Together, these findings indicate that cannabinoid genotoxicity and epigenotoxicity are prominent features of cannabis dependence and strongly indicate coordinated multiomics investigations of cannabinoid genome-epigenome-transcriptome-metabolome, chromatin conformation, and 3D nuclear architecture. Considering the well-established exponential dose-response relationships, the diversity of cannabinoids, and the multigenerational nature of the implications, great caution is warranted in community cannabinoid penetration.

Imprinting fidelity in mouse iPSCs depends on sex of donor cell and medium formulation

Reprogramming of somatic cells into induced Pluripotent Stem Cells (iPSCs) is a major leap towards personalised approaches to disease modelling and cell-replacement therapies. However, we still lack the ability to fully control the epigenetic status of iPSCs, which is a major hurdle for their downstream applications. Epigenetic fidelity can be tracked by genomic imprinting, a phenomenon dependent on DNA methylation, which is frequently perturbed in iPSCs by yet unknown reasons. To try to understand the causes underlying these defects, we conducted a thorough imprinting analysis using IMPLICON, a high-throughput method measuring DNA methylation levels, in multiple female and male murine iPSC lines generated under different experimental conditions. Our results show that imprinting defects are remarkably common in iPSCs, but their nature depends on the sex of donor cells and their response to culture conditions. Imprints in female iPSCs resist the initial genome-wide DNA demethylation wave during reprogramming, but ultimately cells accumulate hypomethylation defects irrespective of culture medium formulations. In contrast, imprinting defects on male iPSCs depends on the experimental conditions and arise during reprogramming, being mitigated by the addition of vitamin C (VitC). Our findings are fundamental to further optimise reprogramming strategies and generate iPSCs with a stable epigenome.

Nucleome programming is required for the foundation of totipotency in mammalian germline development.

Germ cells are unique in engendering totipotency, yet the mechanisms underlying this capacity remain elusive. Here, we perform comprehensive and in-depth nucleome analysis of mouse germ-cell development in vitro, encompassing pluripotent precursors, primordial germ cells (PGCs) before and after epigenetic reprogramming, and spermatogonia/spermatogonial stem cells (SSCs). Although epigenetic reprogramming, including genome-wide DNA de-methylation, creates broadly open chromatin with abundant enhancer-like signatures, the augmented chromatin insulation safeguards transcriptional fidelity. These insulatory constraints are then erased en masse for spermatogonial development. Notably, despite distinguishing epigenetic programming, including global DNA re-methylation, the PGCs-to-spermatogonia/SSCs development entails further euchromatization. This accompanies substantial erasure of lamina-associated domains, generating spermatogonia/SSCs with a minimal peripheral attachment of chromatin except for pericentromeres-an architecture conserved in primates. Accordingly, faulty nucleome maturation, including persistent insulation and improper euchromatization, leads to impaired spermatogenic potential. Given that PGCs after epigenetic reprogramming serve as oogenic progenitors as well, our findings elucidate a principle for the nucleome programming that creates gametogenic progenitors in both sexes, defining a basis for nuclear totipotency.

Temporal changes in transcripts of miniature inverted-repeat transposable elements during rice endosperm development.

SUMMARYThe repression of transcription from transposable elements (TEs) by DNA methylation is necessary to maintain genome integrity and prevent harmful mutations. However, under certain circumstances, TEs may escape from the host defense system and reactivate their transcription. In Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa), DNA demethylases target the sequences derived from TEs in the central cell, the progenitor cell for the endosperm in the female gametophyte. Genome‐wide DNA demethylation is also observed in the endosperm after fertilization. In the present study, we used a custom microarray to survey the transcripts generated from TEs during rice endosperm development and at selected time points in the embryo as a control. The expression patterns of TE transcripts are dynamically up‐ and downregulated during endosperm development, especially those of miniature inverted‐repeat TEs (MITEs). Some TE transcripts were directionally controlled, whereas the other DNA transposons and retrotransposons were not. We also discovered the NUCLEAR FACTOR Y binding motif, CCAAT, in the region near the 5′ terminal inverted repeat of Youren, one of the transcribed MITEs in the endosperm. Our results uncover dynamic changes in TE activity during endosperm development in rice.

Analysis of DNA methylation in potato tuber in response to light exposure during storage

Exposure to light induces tuber greening and the accumulation of the toxic alkaloid Solanine in potato (Solanum tuberosum L) during storage greatly reduce tuber value. While the mechanism of this greening process remains unclear, it is well understood that DNA methylation plays an important role in regulating gene expression in response to environmental conditions. In this study, methylation-sensitive amplified polymorphism was used to assess the effect of light exposure on DNA methylation during storage of potato tubers. Light-induced genome-wide DNA demethylation and the rate of DNA methylation decreased with long storage times. Following, the sequencing of 14 differentially amplified fragments and analysis using the Basic Local Alignment Search Tool, eight genomic sequences and six annotated fragment sequences were identified. The latter included ADP glucose pyrophosphorylase 1/2, chlorophyllide a oxygenase 1 (CAO1), receptor-like protein kinase HAIKU2, and repressor of GA4, all of which are involved in starch biosynthesis, chlorophyll synthesis, endosperm development, and gibberellic acid signaling, respectively. Demethylation was observed in the CpG island (−273 to −166 bp) of the CAO1 promoter in response to light, which further confirmed that the variations in genome methylation are dependent upon the light exposure and suggests a direct role for DNA methylation. Our results provide an epigenetic perspective for further exploring the mechanism of light-induced tuber greening.

Vitamin C Rescues in vitro Embryonic Development by Correcting Impaired Active DNA Demethylation.

During preimplantation development, a wave of genome-wide DNA demethylation occurs to acquire a hypomethylated genome of the blastocyst. As an essential epigenomic event, postfertilization DNA demethylation is critical to establish full developmental potential. Despite its importance, this process is prone to be disrupted due to environmental perturbations such as manipulation and culture of embryos during in vitro fertilization (IVF), and thus leading to epigenetic errors. However, since the first case of aberrant DNA demethylation reported in IVF embryos, its underlying mechanism remains unclear and the strategy for correcting this error remains unavailable in the past decade. Thus, understanding the mechanism responsible for DNA demethylation defects, may provide a potential approach for preventing or correcting IVF-associated complications. Herein, using mouse and bovine IVF embryos as the model, we reported that ten-eleven translocation (TET)-mediated active DNA demethylation, an important contributor to the postfertilization epigenome reprogramming, was impaired throughout preimplantation development. Focusing on modulation of TET dioxygenases, we found vitamin C and α-ketoglutarate, the well-established important co-factors for stimulating TET enzymatic activity, were synthesized in both embryos and the oviduct during preimplantation development. Accordingly, impaired active DNA demethylation can be corrected by incubation of IVF embryos with vitamin C, and thus improving their lineage differentiation and developmental potential. Together, our data not only provides a promising approach for preventing or correcting IVF-associated epigenetic errors, but also highlights the critical role of small molecules or metabolites from maternal paracrine in finetuning embryonic epigenomic reprogramming during early development.

Intrauterine exposure to 2,3′,4,4′,5-pentachlorobiphenyl alters spermatogenesis and testicular DNA methylation levels in F1 male mice

Polychlorinated biphenyls (PCBs) are synthetic biphenyl compounds with high toxicity. There are a total of 209 homologs, among which 2,3’,4,4’,5-pentachlorobiphenyl (PCB118) is one of the dioxin-like PCBs. PCB118 can accumulate in pregnant mice, leading to fetus directly exposure during development. The stage of migration of mouse primordial germ cells ranges from 8.5 to 13.5 days of pregnancy, which is the stage undergoing a genome-wide DNA demethylation process. In this study, the mice were exposed to 20 μg/kg/day and 100 μg/kg/day PCB118 from 8.5 to 13.5 days of pregnancy. During the embryo stage at 18.5 days (E18.5 days), the expression level of DNA methyltransferase 1 (Dnmt1) was reduced in the testes, and the DNA methylation level in mouse testes were also decreased. We found that the seminiferous tubules showed vacuolization and that the sperm deformity rate increased in the treated groups compared with the control group in 7-week-old mice. Because exposure to PCB118 during pregnancy causes damage to the reproductive system of male offspring mice, attention should be devoted to the toxicity transmission of persistent environmental pollutants such as PCBs.

The DME demethylase regulates sporophyte gene expression, cell proliferation, differentiation, and meristem resurrection

The flowering plant life cycle consists of alternating haploid (gametophyte) and diploid (sporophyte) generations, where the sporophytic generation begins with fertilization of haploid gametes. In Arabidopsis, genome-wide DNA demethylation is required for normal development, catalyzed by the DEMETER (DME) DNA demethylase in the gamete companion cells of male and female gametophytes. In the sporophyte, postembryonic growth and development are largely dependent on the activity of numerous stem cell niches, or meristems. Analyzing Arabidopsis plants homozygous for a loss-of-function dme-2 allele, we show that DME influences many aspects of sporophytic growth and development. dme-2 mutants exhibited delayed seed germination, variable root hair growth, aberrant cellular proliferation and differentiation followed by enhanced de novo shoot formation, dysregulation of root quiescence and stomatal precursor cells, and inflorescence meristem (IM) resurrection. We also show that sporophytic DME activity exerts a profound effect on the transcriptome of developing Arabidopsis plants, including discrete groups of regulatory genes that are misregulated in dme-2 mutant tissues, allowing us to potentially link phenotypes to changes in specific gene expression pathways. These results show that DME plays a key role in sporophytic development and suggest that DME-mediated active DNA demethylation may be involved in the maintenance of stem cell activities during the sporophytic life cycle in Arabidopsis.

TRIM28 maintains genome imprints and regulates development of porcine SCNT embryos.

Pre-implantation embryos undergo genome-wide DNA demethylation, however certain regions, like imprinted loci remain methylated. Further, the mechanisms ensuring demethylation resistance by TRIM28 in epigenetic reprogramming remain poorly understood. Here, TRIM28 was knocked down in oocytes, and its effects on porcine somatic cell nuclear transfer (SCNT) embryo development was examined. Our results showed that SCNT embryos constructed from TRIM28 knockdown oocytes had significantly lower cleavage (53.9 ± 3.4% vs 64.8 ± 2.7%) and blastocyst rates (12.1 ± 4.3% vs 19.8 ± 1.9%) than control-SCNT embryos. The DNA methylation levels at the promoter regions of the imprinting gene IGF2 and H19 were significantly decreased in the 4-cell stage, and the transcript abundance of other imprinting gene was substantially increased. We also identified an aberrant two-fold decrease in the expression of CXXC1and H3K4me3 methyltransferase (ASH2L and MLL2), and the signal intensity of H3K4me3 had a transient drop in SCNT 2-cell embryos. Our results indicated that maternal TRIM28 knockdown disrupted the genome imprints and caused epigenetic variability in H3K4me3 levels, which blocked the transcription activity of zygote genes and affected the normal developmental progression of porcine SCNT embryos.

Recent evolution of a TET-controlled and DPPA3/STELLA-driven pathway of passive DNA demethylation in mammals

Genome-wide DNA demethylation is a unique feature of mammalian development and naïve pluripotent stem cells. Here, we describe a recently evolved pathway in which global hypomethylation is achieved by the coupling of active and passive demethylation. TET activity is required, albeit indirectly, for global demethylation, which mostly occurs at sites devoid of TET binding. Instead, TET-mediated active demethylation is locus-specific and necessary for activating a subset of genes, including the naïve pluripotency and germline marker Dppa3 (Stella, Pgc7). DPPA3 in turn drives large-scale passive demethylation by directly binding and displacing UHRF1 from chromatin, thereby inhibiting maintenance DNA methylation. Although unique to mammals, we show that DPPA3 alone is capable of inducing global DNA demethylation in non-mammalian species (Xenopus and medaka) despite their evolutionary divergence from mammals more than 300 million years ago. Our findings suggest that the evolution of Dppa3 facilitated the emergence of global DNA demethylation in mammals.

Dux-Mediated Corrections of Aberrant H3K9ac during 2-Cell Genome Activation Optimize Efficiency of Somatic Cell Nuclear Transfer.

Differentiated somatic cells can be reprogrammed to totipotent embryos through somatic cell nuclear transfer (SCNT) with low efficiency. The histone deacetylase inhibitor trichostatin A (TSA) has been found to improve SCNT efficiency, but the underlying mechanism remains undetermined. Here, we examined genome-wide H3K9ac during SCNT embryo development and found that aberrant H3K9ac regions resulted in reduced 2-cell genome activation. TSA treatment largely corrects aberrant acetylation in SCNT embryos with an efficiency that is dictated by the native epigenetic environment. We further identified that the overexpression of Dux greatly improves SCNT efficiency by correcting the aberrant H3K9ac signal at its target sites, ensuring appropriate 2-cell genome activation. Intriguingly, the improvement in development mediated by TSA and Kdm4b is impeded by Dux knockout in SCNT embryos. Together, our study reveals that reprogramming of H3K9ac is important for optimal SCNT efficiency and identifies Dux as a crucial transcription factor in this process.

Long-term expansion with germline potential of human primordial germ cell-like cells invitro.

Human germ cells perpetuate human genetic and epigenetic information. However, the underlying mechanism remains elusive, due to a lack of appropriate experimental systems. Here, we show that human primordial germ cell-like cells (hPGCLCs) derived from human-induced pluripotent stem cells (hiPSCs) can be propagated to at least ~106 -fold over a period of 4months under a defined condition invitro. During expansion, hPGCLCs maintain an early hPGC-like transcriptome and preserve their genome-wide DNA methylation profiles, most likely due to retention of maintenance DNA methyltransferase activity. These characteristics contrast starkly with those of mouse PGCLCs, which, under an analogous condition, show a limited propagation (up to ~50-fold) and persist only around 1week, yet undergo cell-autonomous genome-wide DNA demethylation. Importantly, upon aggregation culture with mouse embryonic ovarian somatic cells in xenogeneic-reconstituted ovaries, expanded hPGCLCs initiate genome-wide DNA demethylation and differentiate into oogonia/gonocyte-like cells, demonstrating their germline potential. By creating a paradigm for hPGCLC expansion, our study uncovers critical divergences in expansion potential and the mechanism for epigenetic reprogramming between the human and mouse germ cell lineage.

46 Presence of porcine TET3L isoform in oocytes: Potential involvement in the DNA demethylation process

Fertilized oocytes undergo genome-wide DNA demethylation with the exception of imprinted genes and certain repetitive elements. Ten-eleven translocation 3 (TET3) protein has been known to be responsible for the DNA demethylation process by catalysing oxidation of 5-methylcytosine. Recent studies in the mouse indicated that multiple Tet3 isoforms exist in oocytes, implying differential actions of the isoforms. Previously, we reported the sequence of TET3 in porcine oocytes (GenBank: KC137685). Here, we investigated the presence of TET3 isoforms in porcine oocytes and cumulus cells, and followed changes in the TET3 expression during oocyte maturation to further understand the mechanism of TET3 action in the DNA demethylation process. To identify porcine TET3 isoforms, 5′ RACE (rapid amplification of cDNA ends) was conducted using mRNAs isolated from oocytes and cumulus cells at both germinal vesicle (GV) and MII stages. Gene-specific primers for 5′RACE were designed to recognise conserved regions of TET3 that are present in all isoforms, based on EST databases and the cloned sequence in our previous study. The PCR-amplified 5′RACE products were cloned into a vector and subsequently sequenced. The 5′RACE revealed 3 different TET3 isoforms from GV and MII cumulus cells; no amplification was detected using oocytes, potentially due to a low amount of mRNA. Among the isoforms, the longest variant (TET3L) contained sequences for the CXXC domain, known to carry DNA binding properties. Then, RT-PCR was used to detect the presence of the isoforms in porcine oocytes. Interestingly, the expression of TET3 isoform containing the CXXC domain (TET3L) was only verified from the RT-PCR, suggesting that the isoform may be the predominant isoform in porcine oocytes. To characterise transcript abundance of porcine TET3L, RNAs were isolated from different cells and/or tissues including cumulus cells, oocytes, brain, spleen, and lung. The RT-qPCR was performed using the RNAs and ΔΔCT method was used to analyse the data; GAPDH was used as an internal control. Three biological replicates were used for analysis of RT-qPCR data and P-values of less than 0.05 from one-way analysis of variance were considered significant. The expression level of TET3L was much higher in MII oocytes compared with that in somatic tissues; MII oocytes expressed the TET3 isoform over 350-fold higher than MII cumulus cells and 18-fold higher than lung cells. Interestingly, the expression level of TET3L increased over 3-fold during oocyte maturation (i.e. from GV to MII stage oocytes), indicating that TET3L may have a significant role in DNA demethylation after fertilisation. In conclusion, the TET3 isoform containing CXXC domain (TET3L) is predominantly expressed in matured porcine oocytes, suggesting an important role of the TET3 CXXC domain in DNA demethylation in zygotes.

Methylome Dynamics of Bovine Gametes and in vivo Early Embryos.

DNA methylation undergoes drastic fluctuation during early mammalian embryogenesis. The dynamics of global DNA methylation in bovine embryos, however, have mostly been studied by immunostaining. We adopted the whole genome bisulfite sequencing (WGBS) method to characterize stage-specific genome-wide DNA methylation in bovine sperm, immature oocytes, oocytes matured in vivo and in vitro, as well as in vivo developed single embryos at the 2-, 4-, 8-, and 16-cell stages. We found that the major wave of genome-wide DNA demethylation was complete by the 8-cell stage when de novo methylation became prominent. Sperm and oocytes were differentially methylated in numerous regions (DMRs), which were primarily intergenic, suggesting that these non-coding regions may play important roles in gamete specification. DMRs were also identified between in vivo and in vitro matured oocytes, suggesting environmental effects on epigenetic modifications. In addition, virtually no (less than 1.5%) DNA methylation was found in mitochondrial DNA. Finally, by using RNA-seq data generated from embryos at the same developmental stages, we revealed a weak inverse correlation between gene expression and promoter methylation. This comprehensive analysis provides insight into the critical features of the bovine embryo methylome, and serves as an important reference for embryos produced in vitro, such as by in vitro fertilization and cloning. Lastly, these data can also provide a model for the epigenetic dynamics in human early embryos.

UHRF1 depletion and HDAC inhibition reactivate epigenetically silenced genes in colorectal cancer cells

BackgroundUbiquitin-like protein containing PHD and RING finger domains 1 (UHRF1) is a major regulator of epigenetic mechanisms and is overexpressed in various human malignancies. In this study, we examined the involvement of UHRF1 in aberrant DNA methylation and gene silencing in colorectal cancer (CRC).ResultsCRC cell lines were transiently transfected with siRNAs targeting UHRF1, after which DNA methylation was analyzed using dot blots, bisulfite pyrosequencing, and Infinium HumanMethylation450 BeadChip assays. Gene expression was analyzed using RT-PCR and gene expression microarrays. Depletion of UHRF1 rapidly induced genome-wide DNA demethylation in CRC cells. Infinium BeadChip assays and bisulfite pyrosequencing revealed significant demethylation across entire genomic regions, including CpG islands, gene bodies, intergenic regions, and repetitive elements. Despite the substantial demethylation, however, UHRF1 depletion only minimally reversed CpG island hypermethylation-associated gene silencing. By contrast, the combination of UHRF1 depletion and histone deacetylase (HDAC) inhibition reactivated the silenced genes and strongly suppressed CRC cell proliferation. The combination of UHRF1 depletion and HDAC inhibition also induced marked changes in the gene expression profiles such that cell cycle-related genes were strikingly downregulated.ConclusionsOur results suggest that (i) maintenance of DNA methylation in CRC cells is highly dependent on UHRF1; (ii) UHRF1 depletion rapidly induces DNA demethylation, though it is insufficient to fully reactivate the silenced genes; and (iii) dual targeting of UHRF1 and HDAC may be an effective new therapeutic strategy.

Effects of a single transient transfection of Ten-eleven translocation 1 catalytic domain on hepatocellular carcinoma.

Tumor suppressor genes (TSGs), including Ten-eleven translocation 1 (TET1), are hypermethylated in hepatocellular carcinoma (HCC). TET1 catalytic domain (TET1-CD) induces genome-wide DNA demethylation to activate TSGs, but so far, anticancer effects of TET1-CD are unclear. Here we showed that after HCC cells were transiently transfected with TET1-CD, the methylation levels of TSGs, namely APC, p16, RASSF1A, SOCS1 and TET1, were distinctly reduced, and their mRNA levels were significantly increased and HCC cells proliferation, migration and invasion were suppressed, but the methylation and mRNA levels of oncogenes, namely C-myc, Bmi1, EMS1, Kpna2 and c-fos, were not significantly change. Strikingly, HCC subcutaneous xenografts in nude mice remained to be significantly repressed even 54 days after transient transfection of TET1-CD. So, transient transfection of TET1-CD may be a great advance in HCC treatment due to its activation of multiple TSGs and persistent anticancer effects.