Zinc finger transcription factor Zfp57 recognizes the methylated CpG within the TGCCGC element. We determined the structure of the DNA-binding domain of Zfp57, consisting of two adjacent zinc ...fingers, in complex with fully methylated DNA at 1.0 Å resolution. The first zinc finger contacts the 5' half (TGC), and the second recognizes the 3' half (CGC) of the recognition sequence. Zfp57 recognizes the two 5-methylcytosines (5mCs) asymmetrically: One involves hydrophobic interactions with Arg178, which also interacts with the neighboring 3' guanine and forms a 5mC-Arg-G interaction, while the other involves a layer of ordered water molecules. Two point mutations in patients with transient neonatal diabetes abolish DNA-binding activity. Zfp57 has reduced binding affinity for unmodified DNA and the oxidative products of 5mC.
DNA methylation is an epigenetic modification that plays a crucial role in normal mammalian development, retrotransposon silencing, and cellular reprogramming. Although methylation mainly occurs on ...the cytosine in a CG site, non-CG methylation is prevalent in pluripotent stem cells, brain, and oocytes. We previously identified non-CG methylation in several CG-rich regions in mouse germinal vesicle oocytes (GVOs), but the overall distribution of non-CG methylation and the enzymes responsible for this modification are unknown. Using amplification-free whole-genome bisulfite sequencing, which can be used with minute amounts of DNA, we constructed the base-resolution methylome maps of GVOs, non-growing oocytes (NGOs), and mutant GVOs lacking the DNA methyltransferase Dnmt1, Dnmt3a, Dnmt3b, or Dnmt3L. We found that nearly two-thirds of all methylcytosines occur in a non-CG context in GVOs. The distribution of non-CG methylation closely resembled that of CG methylation throughout the genome and showed clear enrichment in gene bodies. Compared to NGOs, GVOs were over four times more methylated at non-CG sites, indicating that non-CG methylation accumulates during oocyte growth. Lack of Dnmt3a or Dnmt3L resulted in a global reduction in both CG and non-CG methylation, showing that non-CG methylation depends on the Dnmt3a-Dnmt3L complex. Dnmt3b was dispensable. Of note, lack of Dnmt1 resulted in a slight decrease in CG methylation, suggesting that this maintenance enzyme plays a role in non-dividing oocytes. Dnmt1 may act on CG sites that remain hemimethylated in the de novo methylation process. Our results provide a basis for understanding the mechanisms and significance of non-CG methylation in mammalian oocytes.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The methylation of cytosine at CG sites in the mammalian genome is dynamically reprogrammed during gametogenesis and preimplantation development. It was previously shown that oocyte-derived DNMT1 (a ...maintenance methyltransferase) is essential for maintaining and propagating CG methylation at imprinting control regions in preimplantation embryos. In mammalian somatic cells, hemimethylated-CG-binding protein UHRF1 plays a critical role in maintaining CG methylation by recruiting DNMT1 to hemimethylated CG sites. However, the role of UHRF1 in oogenesis and preimplantation development is unknown. In the present study, we show that UHRF1 is mainly, but not exclusively, localized in the cytoplasm of oocytes and preimplantation embryos. However, smaller amounts of UHRF1 existed in the nucleus, consistent with the expected role in DNA methylation. We then generated oocyte-specific Uhrf1 knockout (KO) mice and found that, although oogenesis was itself unaffected, a large proportion of the embryos derived from the KO oocytes died before reaching the blastocyst stage (a maternal effect). Whole genome bisulfite sequencing revealed that blastocysts derived from KO oocytes have a greatly reduced level of CG methylation, suggesting that maternal UHRF1 is essential for maintaining CG methylation, particularly at the imprinting control regions, in preimplantation embryos. Surprisingly, UHRF1 was also found to contribute to de novo CG and non-CG methylation during oocyte growth: in Uhrf1 KO oocytes, transcriptionally-inactive regions gained less methylation, while actively transcribed regions, including the imprinting control regions, were unaffected or only slightly affected. We also found that de novo methylation was defective during the late stage of oocyte growth. To the best of our knowledge, this is the first study to demonstrate the role of UHRF1 in de novo DNA methylation in vivo. Our study reveals multiple functions of UHRF1 during the global epigenetic reprogramming of oocytes and early embryos.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The human gut is colonized with a wide variety of microorganisms, including species, such as those belonging to the bacterial genus Bifidobacterium, that have beneficial effects on human physiology ...and pathology. Among the most distinctive benefits of bifidobacteria are modulation of host defence responses and protection against infectious diseases. Nevertheless, the molecular mechanisms underlying these effects have barely been elucidated. To investigate these mechanisms, we used mice associated with certain bifidobacterial strains and a simplified model of lethal infection with enterohaemorrhagic Escherichia coli O157:H7, together with an integrated 'omics' approach. Here we show that genes encoding an ATP-binding-cassette-type carbohydrate transporter present in certain bifidobacteria contribute to protecting mice against death induced by E. coli O157:H7. We found that this effect can be attributed, at least in part, to increased production of acetate and that translocation of the E. coli O157:H7 Shiga toxin from the gut lumen to the blood was inhibited. We propose that acetate produced by protective bifidobacteria improves intestinal defence mediated by epithelial cells and thereby protects the host against lethal infection.
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DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
DNA methylation is globally reprogrammed after fertilization, and as a result, the parental genomes have similar DNA-methylation profiles after implantation except at the germline differentially ...methylated regions (gDMRs). We and others have previously shown that human blastocysts might contain thousands of transient maternally methylated gDMRs (transient mDMRs), whose maternal methylation is lost in embryonic tissues after implantation. In this study, we performed genome-wide allelic DNA methylation analyses of purified trophoblast cells from human placentas and, surprisingly, found that more than one-quarter of the transient-in-embryo mDMRs maintained their maternally biased DNA methylation. RNA-sequencing-based allelic expression analyses revealed that some of the placenta-specific mDMRs were associated with expression of imprinted genes (e.g., TIGAR, SLC4A7, PROSER2-AS1, and KLHDC10), and three imprinted gene clusters were identified. This approach also identified some X-linked gDMRs. Comparisons of the data with those from other mammals revealed that genomic imprinting in the placenta is highly variable. These findings highlight the incomplete erasure of germline DNA methylation in the human placenta; understanding this erasure is important for understanding normal placental development and the pathogenesis of developmental disorders with imprinting effects.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Because spermatogonial stem cells (SSCs) are immortal by serial transplantation, SSC aging in intact testes is considered to be caused by a deteriorated microenvironment. Here, we report a ...cell-intrinsic mode of SSC aging by glycolysis activation. Using cultured SSCs, we found that aged SSCs proliferated more actively than young SSCs and showed enhanced glycolytic activity. Moreover, they remained euploid and exhibited stable androgenetic imprinting patterns with robust SSC activity despite having shortened telomeres. Aged SSCs showed increased Wnt7b expression, which was associated with decreased Polycomb complex 2 activity. Our results suggest that aberrant Wnt7b expression activated c-jun N-terminal kinase (JNK), which down-regulated mitochondria numbers by suppressing Ppargc1a. Down-regulation of Ppargc1a probably decreased reactive oxygen species and enhanced glycolysis. Analyses of the Klotho-deficient aging mouse model and 2-y-old aged rats confirmed JNK hyperactivation and increased glycolysis. Therefore, not only microenvironment but also intrinsic activation of JNK-mediated glycolysis contributes to SSC aging.
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BFBNIB, NMLJ, NUK, PNG, SAZU, UL, UM, UPUK
DNA methylation at CG sites is important for gene regulation and embryonic development. In mouse oocytes, de novo CG methylation requires preceding transcription-coupled histone mark H3K36me3 and is ...mediated by a DNA methyltransferase DNMT3A. DNMT3A has a PWWP domain, which recognizes H3K36me2/3, and heterozygous mutations in this domain, including D329A substitution, cause aberrant CG hypermethylation of regions marked by H3K27me3 in somatic cells, leading to a dwarfism phenotype. We herein demonstrate that D329A homozygous mice show greater CG hypermethylation and severer dwarfism. In oocytes, D329A substitution did not affect CG methylation of H3K36me2/3-marked regions, including maternally methylated imprinting control regions; rather, it caused aberrant hypermethylation in regions lacking H3K36me2/3, including H3K27me3-marked regions. Thus, the role of the PWWP domain in CG methylation seems similar in somatic cells and oocytes; however, there were cell-type-specific differences in affected regions. The major satellite repeat was also hypermethylated in mutant oocytes. Contrary to the CA hypomethylation in somatic cells, the mutation caused hypermethylation at CH sites, including CA sites. Surprisingly, oocytes expressing only the mutated protein could support embryonic and postnatal development. Our study reveals that the DNMT3A PWWP domain is important for suppressing aberrant CG hypermethylation in both somatic cells and oocytes but that D329A mutation has little impact on the developmental potential of oocytes.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Among the various pathogenic Escherichia coli strains, enterohemorrhagic E. coli (EHEC) is the most devastating. Although serotype O157:H7 strains are the most prevalent, strains of different ...serotypes also possess similar pathogenic potential. Here, we present the results of a genomic comparison between EHECs of serotype O157, O26, O111, and O103, as well as 21 other, fully sequenced E. coli/Shigella strains. All EHECs have much larger genomes (5.5-5.9 Mb) than the other strains and contain surprisingly large numbers of prophages and integrative elements (IEs). The gene contents of the 4 EHECs do not follow the phylogenetic relationships of the strains, and they share virulence genes for Shiga toxins and many other factors. We found many lambdoid phages, IEs, and virulence plasmids that carry the same or similar virulence genes but have distinct evolutionary histories, indicating that independent acquisition of these mobile genetic elements has driven the evolution of each EHEC. Particularly interesting is the evolution of the type III secretion system (T3SS). We found that the T3SS of EHECs is composed of genes that were introduced by 3 different types of genetic elements: an IE referred to as the locus of enterocyte effacement, which encodes a central part of the T3SS; SpLE3-like IEs; and lambdoid phages carrying numerous T3SS effector genes and other T3SS-related genes. Our data demonstrate how E. coli strains of different phylogenies can independently evolve into EHECs, providing unique insights into the mechanisms underlying the parallel evolution of complex virulence systems in bacteria.
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BFBNIB, NMLJ, NUK, PNG, SAZU, UL, UM, UPUK
Epigenetic modifications established in mammalian gametes are largely reprogrammed during early development, however, are partly inherited by the embryo to support its development. In this study, we ...examine CpG island (CGI) sequences to predict whether a mouse blastocyst CGI inherits oocyte-derived DNA methylation from the maternal genome. Recurrent neural networks (RNNs), including that based on gated recurrent units (GRUs), have recently been employed for variable-length inputs in classification and regression analyses. One advantage of this strategy is the ability of RNNs to automatically learn latent features embedded in inputs by learning their model parameters. However, the available CGI dataset applied for the prediction of oocyte-derived DNA methylation inheritance are not large enough to train the neural networks. We propose a GRU-based model called CMIC (CGI Methylation Inheritance Classifier) to augment CGI sequence by converting it into variable-length k-mers, where the length k is randomly selected from the range formula omitted to formula omitted, N times, which were then used as neural network input. N was set to 1000 in the default setting. In addition, we proposed a new embedding vector generator for k-mers called splitDNA2vec. The randomness of this procedure was higher than the previous work, dna2vec. We found that CMIC can predict the inheritance of oocyte-derived DNA methylation at CGIs in the maternal genome of blastocysts with a high F-measure (0.93). We also show that the F-measure can be improved by increasing the parameter N, that is, the number of sequences of variable-length k-mers derived from a single CGI sequence. This implies the effectiveness of augmenting input data by converting a DNA sequence to N sequences of variable-length k-mers. This approach can be applied to different DNA sequence classification and regression analyses, particularly those involving a small amount of data.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The human gut harbors a large and diverse community of commensal bacteria. Among them, Bifidobacterium is known to exhibit various probiotic effects including protection of hosts from infectious ...diseases. We recently discovered that genes encoding an ATP-binding-cassette-type carbohydrate transporter present in certain bifidobacteria contribute to protecting gnotobiotic mice from death induced by enterohemorrhagic Escherichia coli O157:H7. We elucidated the molecular mechanism on lethal infection in mice associated with several bifidobacterial strains by a multi-omics approach combining genomics, transcriptomics and metabolomics. The combined data clearly show that acetate produced by protective bifidobacteria acts in vivo to promote defense functions of the host epithelial cells and thereby protects the host from lethal infection. As demonstrated here, our multi-omics approach provides a powerful strategy for evaluation of host-microbial interactions in the complex gut ecosystem.
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