Summary Primate germ cell development remains largely unexplored due to limitations in sample collection and the long duration of development. In mice, primordial germ cell-like cells (PGCLCs) derived from pluripotent stem cells (PSCs) can develop into functional gametes by in vitro culture or in vivo transplantation. Such PGCLC-mediated induction of mature gametes in primates is highly useful for understanding human germ cell development. Since marmosets generate functional sperm earlier than other species, recapitulating the whole male germ cell development process is technically more feasible. Here, we induced the differentiation of iPSCs into gonocyte-like cells via PGCLCs in marmosets. First, we developed an mRNA transfection-based method to efficiently generate PGCLC. Subsequently, to promote PGCLC differentiation, xenoreconstituted testes (xrtestes) were generated in the mouse kidney capsule. PGCLCs show progressive DNA demethylation and stepwise expression of developmental marker genes. This study provides an efficient platform for the study of marmoset germ cell development. Highlights An mRNA transfection-based PGCLC induction method is developed Marmoset PGCLCs are efficiently induced from iPSCs Marmoset PGCLCs differentiate into gonocyte-like cells in mouse kidneys Developmentally regulated expression and demethylation are recapitulated eTOC blurb Watanabe et al. efficiently induced marmoset primordial germ cell-like cells (PGCLCs) using an mRNA transfection-based approach. PGCLCs further develop into gonocyte-like cells in the xenoreconstituted testes constructed under mouse kidney capsules. This system faithfully reproduced in vivo developmental processes (e.g., stage-specific expression of developmentally regulated genes and DNA demethylation).
Primate germ cell development remains largely unexplored due to limitations in sample collection and the long duration of development. In mice, primordial germ cell-like cells (PGCLCs) derived from pluripotent stem cells (PSCs) can develop into functional gametes by in vitro culture or in vivo transplantation. Such PGCLC-mediated induction of mature gametes in primates is highly useful for understanding human germ cell development. Since marmosets generate functional sperm earlier than other species, recapitulating the whole male germ cell development process is technically more feasible. Here, we induced the differentiation of iPSCs into gonocyte-like cells via PGCLCs in marmosets. First, we developed an mRNA transfection-based method to efficiently generate PGCLCs. Subsequently, to promote PGCLC differentiation, xenoreconstituted testes (xrtestes) were generated in the mouse kidney capsule. PGCLCs show progressive DNA demethylation and stepwise expression of developmental marker genes. This study provides an efficient platform for the study of marmoset germ cell development.
DNA methyltransferases and Ten-Eleven Translocation (TET) proteins regulate the DNA methylation and demethylation cycles during mouse embryonic development. Although DNMT1 mainly plays a role in the maintenance of DNA methylation after DNA replication, it is also reported to possess de novo methyltransferase capacity. However, its physiological significance remains unclear. Here, we demonstrate that full-length DNMT1 (FL) and a mutant lacking the N-terminus necessary for its maintenance activity (602) confer the differentiation potential of mouse Dnmt1, Dnmt3a, and Dnmt3b (Dnmts-TKO) embryonic stem cells (ESCs). Both FL and 602 inhibit the spontaneous differentiation of Dnmts-TKO ESCs in the undifferentiated state. Dnmts-TKO ESCs showed loss of DNA methylation and de-repression of primitive endoderm-related genes, but these defects were partially restored in Dnmts-TKO + FL and Dnmts-TKO + 602 ESCs. Upon differentiation, Dnmts-TKO + FL ESCs show increased 5mC and 5hmC levels across chromosomes, including pericentromeric regions. In contrast, Dnmts-TKO + 602 ESCs didn't accumulate 5mC, and sister chromatids showed 5hmC asynchronously. Furthermore, in comparison with DNMT1_602, DNMT1_FL effectively promoted commitment to the epiblast-like cells and beyond, driving cell-autonomous mesendodermal and germline differentiation through embryoid body-based methods. With precise target selectivity achieved by its N-terminal region, DNMT1 may play a role in gene regulation leading to germline development.
Abstract Background DNA methylation is a significant epigenetic modification that is evolutionarily conserved in various species and often serves as a repressive mark for transcription. DNA methylation levels and patterns are regulated by a balance of opposing enzyme functions, DNA methyltransferases, DNMT1/3A/3B and methylcytosine dioxygenases, TET1/2/3. In mice, the TET enzyme converts DNA cytosine methylation (5mC) to 5-hydroxymethylcytosine (5hmC) at the beginning of fertilisation and gastrulation and initiates a global loss of 5mC, while the 5mC level is increased on the onset of cell differentiation during early embryonic development. Objective Global loss and gain of DNA methylation may be differently regulated in diverged species. Methods Chicken B-cell lymphoma DT40 cells were used as an avian model to compare differences in the overall regulation of DNA modification with mammals. Results We found that DNA methylation is maintained at high levels in DT40 cells through compact chromatin formation, which inhibits TET-mediated demethylation. Human and mouse chromosomes introduced into DT40 cells by cell fusion lost the majority of 5mC, except for human subtelomeric repeats. Conclusion Our attempt to elucidate the differences in the epigenetic regulatory mechanisms between birds and mammals explored the evidence that they share a common chromatin-based regulation of TET–DNA access, while chicken DNMT1 is involved in different target sequence recognition systems, suggesting that factors inducing DNMT–DNA association have already diverged.
Abstract Beckwith-Wiedemann syndrome (BWS) is caused by a gain of methylation (GOM) at the imprinting control region within the Igf2-H19 domain on the maternal allele ( H19 -ICR GOM). Mutations in the binding sites of several transcription factors are involved in H19 -ICR GOM and BWS. However, the responsible sequence(s) for H19 -ICR GOM with BWS-like overgrowth has not been identified in mice. Here, we report that a mutation in the SOX-OCT binding site (SOBS) causes partial H19 -ICR GOM, which does not extend beyond CTCF binding site 3 (CTS3). Moreover, simultaneously mutating both SOBS and CTS3 causes complete GOM of the entire H19 -ICR, leading to the misexpression of the imprinted genes, and frequent BWS-like overgrowth. In addition, CTS3 is critical for CTCF/cohesin-mediated chromatin conformation. These results indicate that SOBS and CTS3 are the sequences responsible for H19 -ICR GOM leading to BWS-like overgrowth and are essential for maintaining the unmethylated state of maternal H19 -ICR.
DNA methylation (DNAme; 5-methylcytosine, 5mC) plays an essential role in mammalian development, and the 5mC profile is regulated by a balance of opposing enzymatic activities: DNA methyltransferases (DNMTs) and Ten-eleven translocation dioxygenases (TETs). In mouse embryonic stem cells (ESCs), de novo DNAme by DNMT3 family enzymes, demethylation by the TET-mediated conversion of 5mC to 5-hydroxymethylation (5hmC), and maintenance of the remaining DNAme by DNMT1 are actively repeated throughout cell cycles, dynamically forming a constant 5mC profile. Nevertheless, the detailed mechanism and physiological significance of this active cyclic DNA modification in mouse ESCs remain unclear. Here by visualizing the localization of DNA modifications on metaphase chromosomes and comparing whole-genome methylation profiles before and after the mid-S phase in ESCs lacking Dnmt1 (1KO ESCs), we demonstrated that in 1KO ESCs, DNMT3-mediated remethylation was interrupted during and after DNA replication. This results in a marked asymmetry in the distribution of 5hmC between sister chromatids at mitosis, with one chromatid being almost no 5hmC. When introduced in 1KO ESCs, the catalytically inactive form of DNMT1 (DNMT1 CI ) induced an increase in DNAme in pericentric heterochromatin and the DNAme-independent repression of IAPEz, a retrotransposon family, in 1KO ESCs. However, DNMT1 CI could not restore the ability of DNMT3 to methylate unmodified dsDNA de novo in S phase in 1KO ESCs. Furthermore, during in vitro differentiation into epiblasts, 1KO ESCs expressing DNMT1 CI showed an even stronger tendency to differentiate into the primitive endoderm than 1KO ESCs and were readily reprogrammed into the primitive streak via an epiblast-like cell state, reconfirming the importance of DNMT1 enzymatic activity at the onset of epiblast differentiation. These results indicate a novel function of DNMT1, in which DNMT1 actively regulates the timing and genomic targets of de novo methylation by DNMT3 in an enzymatic activity-dependent and independent manner, respectively.
Abstract Beckwith-Wiedemann syndrome (BWS) is caused by a gain of methylation (GOM) at the imprinting control region within the Igf2-H19 domain on the maternal allele ( H19 -ICR GOM). Mutations in the binding sites of several transcription factors are involved in H19 -ICR GOM and BWS. However, the responsible sequence(s) for H19 -ICR GOM with BWS-like overgrowth has not been identified in mice. Here, we report that a mutation in the SOX-OCT binding site (SOBS) causes partial H19 -ICR GOM, which does not extend beyond CTCF binding site 3 (CTS3). Moreover, simultaneously mutating both SOBS and CTS3 causes complete GOM of the entire H19 -ICR, leading to the misexpression of the imprinted genes, and frequent BWS-like overgrowth. In addition, CTS3 is critical for CTCF/cohesin-mediated chromatin conformation. These results indicate that SOBS and CTS3 are the sequences in which mutations cause H19 -ICR GOM leading to BWS-like overgrowth and are essential for maintaining the unmethylated state of maternal H19 -ICR.
Abstract DNA methyltransferases and TET enzymes dynamically regulate the DNA methylation-demethylation cycles during mouse embryonic development. DNMT1 maintains cell-specific DNA methylation after DNA replication. However, little is known about the physiological significance of the de novo methylation enzymatic potential of DNMT1, which has attracted much attention. Here, we show that the full-length DNMT1 and a mutant form without the N-terminal necessary for its maintenance activity potentially inhibit the differentiation of mouse pluripotent embryonic stem cells lacking Dnmt1, 3a and 3b into primitive endoderm cells. However, the mutant cells could differentiate into epiblast-like cells and beyond, leading to cell-autonomous mesendoderm differentiation. Strikingly, we found that in response to Nodal signalling and the increased concurrent access of DNMT1 and TET enzymes resulting from chromatin relaxation activated primordial germ cell-specific genes. With the precise target selectivity by its N-terminal region, DNMT1 may be involved in gene regulation leading to germline development.
Abstract Cytochrome P450 enzymes (CYP) function in drug metabolism in the liver. To evaluate numerous drug candidates, a high‐content screening (HCS) system with hepatocyte‐like cells (HLCs) that can replace adult human hepatocytes is required. Human hepatocellular carcinoma HepaRG is the only cell line capable of providing HLCs with high CYP3A4 expression comparable to that in adult hepatocytes after cell differentiation. The aim of this study was to design an ideal multiwell culture system for HLCs using transgenic HepaRG cells expressing the EGFP coding an enhanced green fluorescent protein under CYP3A4 transcriptional regulation. HLCs were matured on five different types of 96‐well black plates. Culturing HLCs on glass‐bottom Optical CVG plates significantly promoted cell maturation and increased metabolic activity by twofold under two‐dimensional (2D) culture conditions, and these features were enhanced by 2% collagen coating. Three plates for three‐dimensional (3D) cell cultures with a gas‐exchangeable fabric or dimethylpolysiloxane membrane bottom formed multiple round colonies, whereas they were ineffective for CYP3A4 expression. Under optimized conditions presented here, HLCs lost responsiveness to nuclear receptor‐mediated transcriptional induction of CYP3A4 , suggesting that CYP3A4 transcription has already been fully upregulated. Therefore, HepaRG‐derived HLCs will provide an alternative to human hepatocytes with high levels of CYP3A4 enzyme activity even under 2D culture conditions. This will improve a variety of drug screening methods.
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