The PRDM14-CtBP1/2-PRC2 complex regulates transcriptional repression during the transition from primed to naïve pluripotency.

The pluripotency-associated transcriptional network is regulated by a core circuitry of transcription factors. The PR domain-containing protein PRDM14 maintains pluripotency by activating and repressing transcription in a target gene-dependent manner. However, the mechanisms underlying dichotomic switching of PRDM14-mediated transcriptional control remain elusive. Here, we identified C-terminal binding protein 1 and 2 (CtBP1 and CtBP2; generically referred to as CtBP1/2) as components of the PRDM14-mediated repressive complex. CtBP1/2 binding to PRDM14 depends on CBFA2T2, a core component of the PRDM14 complex. The loss of Ctbp1/2 impaired the PRDM14-mediated transcriptional repression required for pluripotency maintenance and transition from primed to naïve pluripotency. Furthermore, CtBP1/2 interacted with the PRC2 complexes, and the loss of Ctbp1/2 impaired Polycomb repressive complex 2 (PRC2) and H3K27me3 enrichment at target genes after Prdm14 induction. These results provide evidence that the target gene-dependent transcriptional activity of PRDM14 is regulated by partner switching to ensure the transition from primed to naïve pluripotency.This article has an associated First Person interview with the first author of the paper.

Co-option of the PRDM14-CBFA2T complex from motor neurons to pluripotent cells during vertebrate evolution.

Gene regulatory networks underlying cellular pluripotency are controlled by a core circuitry of transcription factors in mammals, including POU5F1. However, the evolutionary origin and transformation of pluripotency-related transcriptional networks have not been elucidated in deuterostomes. PR domain-containing protein 14 (PRDM14) is specifically expressed in pluripotent cells and germ cells, and is required for establishing embryonic stem cells (ESCs) and primordial germ cells in mice. Here, we compared the functions and expression patterns of PRDM14 orthologues within deuterostomes. Amphioxus PRDM14 and zebrafish PRDM14, but not sea urchin PRDM14, compensated for mouse PRDM14 function in maintaining mouse ESC pluripotency. Interestingly, sea urchin PRDM14 together with sea urchin CBFA2T, an essential partner of PRDM14 in mouse ESCs, complemented the self-renewal defect in mouse Prdm14 KO ESCs. Contrary to the Prdm14 expression pattern in mouse embryos, Prdm14 was expressed in motor neurons of amphioxus embryos, as observed in zebrafish embryos. Thus, Prdm14 expression in motor neurons was conserved in non-tetrapod deuterostomes and the co-option of the PRDM14-CBFA2T complex from motor neurons into pluripotent cells may have maintained the transcriptional network for pluripotency during vertebrate evolution.This article has an associated 'The people behind the papers' interview.

Maintenance of cell fates and regulation of the histone variant H3.3 by TLK kinase in Caenorhabditis elegans.

Cell-fate maintenance is important to preserve the variety of cell types that are essential for the formation and function of tissues. We previously showed that the acetylated histone-binding protein BET-1 maintains cell fate by recruiting the histone variant H2A.z. Here, we report that Caenorhabditis elegans TLK-1 and the histone H3 chaperone CAF1 prevent the accumulation of histone variant H3.3. In addition, TLK-1 and CAF1 maintain cell fate by repressing ectopic expression of transcription factors that induce cell-fate specification. Genetic analyses suggested that TLK-1 and BET-1 act in parallel pathways. In tlk-1 mutants, the loss of SIN-3, which promotes histone acetylation, suppressed a defect in cell-fate maintenance in a manner dependent on MYST family histone acetyltransferase MYS-2 and BET-1. sin-3 mutation also suppressed abnormal H3.3 incorporation. Thus, we propose a hypothesis that the regulation and interaction of histone variants play crucial roles in cell-fate maintenance through the regulation of selector genes.

PRDM14 Is a Unique Epigenetic Regulator Stabilizing Transcriptional Networks for Pluripotency.

PR-domain containing protein 14 (PRDM14) is a site-specific DNA-binding protein and is required for establishment of pluripotency in embryonic stem cells (ESCs) and primordial germ cells (PGCs) in mice. DNA methylation status is regulated by the balance between de novo methylation and passive/active demethylation, and global DNA hypomethylation is closely associated with cellular pluripotency and totipotency. PRDM14 ensures hypomethylation in mouse ESCs and PGCs through two distinct layers, transcriptional repression of the DNA methyltransferases Dnmt3a/b/l and active demethylation by recruitment of TET proteins. However, the function of PRDM14 remains unclear in other species including humans. Hence, here we focus on the unique characteristics of mouse PRDM14 in the epigenetic regulation of pluripotent cells and primordial germ cells. In addition, we discuss the expression regulation and function of PRDM14 in other species compared with those in mice.

PRDM14 Drives OCT3/4 Recruitment via Active Demethylation in the Transition from Primed to Naive Pluripotency.

Primordial germ cells (PGCs) are specified from epiblast cells in mice. Genes associated with naive pluripotency are repressed in the transition from inner cell mass to epiblast cells, followed by upregulation after PGC specification. However, the molecular mechanisms underlying the reactivation of pluripotency genes are poorly characterized. Here, we exploited the in vitro differentiation of epiblast-like cells (EpiLCs) from embryonic stem cells (ESCs) to elucidate the molecular and epigenetic functions of PR domain-containing 14 (PRDM14). We found that Prdm14 overexpression in EpiLCs induced their conversion to ESC-like cells even in the absence of leukemia inhibitory factor in adherent culture. This was impaired by the loss of Kruppel-like factor 2 and ten-eleven translocation (TET) proteins. Furthermore, PRDM14 recruited OCT3/4 to the enhancer regions of naive pluripotency genes via TET-base excision repair-mediated demethylation. Our results provide evidence that PRDM14 establishes a transcriptional network for naive pluripotency via active DNA demethylation.

PRDM14 maintains pluripotency of embryonic stem cells through TET-mediated active DNA demethylation.

Pluripotency and self-renewal of mouse embryonic stem cells (ESCs) depend on a network of transcription factors maintained by exogenous leukaemia inhibitory factor (LIF). PR-domain containing transcriptional regulator 14 (PRDM14), is essential for maintenance of ESC self-renewal when the cells are cultured in serum plus LIF, but not in 2i medium plus LIF. Here, we show that pluripotency of ESCs is maintained by enforced expression of PRDM14 at a high level, as observed in ESCs in 2i plus LIF and developing primordial germ cells in the absence of LIF. Constitutive expression of PRDM14 represses de novo DNA methylation in pluripotency-associated genes, resulting in the maintenance of gene expression after withdrawal of LIF, while also repressing the upregulation of differentiation markers. Further, knockdown of Tet1/Tet2 and administration of base excision repair (BER) pathway inhibitors impairs the PRDM14-induced resistance of ESCs to differentiation. We conclude that, in the absence of LIF, PRDM14 governs the retention of pluripotency-associated genes through the regulation of TET functions in the BER-mediated active demethylation pathway, while acting to exert TET-independent transcriptional repressive activity of several differentiation markers.

PRDM14 promotes active DNA demethylation through the ten-eleven translocation (TET)-mediated base excision repair pathway in embryonic stem cells.

Ten-eleven translocation (TET) proteins oxidize 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). 5fC and 5caC can be excised and repaired by the base excision repair (BER) pathway, implicating 5mC oxidation in active DNA demethylation. Genome-wide DNA methylation is erased in the transition from metastable states to the ground state of embryonic stem cells (ESCs) and in migrating primordial germ cells (PGCs), although some resistant regions become demethylated only in gonadal PGCs. Understanding the mechanisms underlying global hypomethylation in naive ESCs and developing PGCs will be useful for realizing cellular pluripotency and totipotency. In this study, we found that PRDM14, the PR domain-containing transcriptional regulator, accelerates the TET-BER cycle, resulting in the promotion of active DNA demethylation in ESCs. Induction of Prdm14 expression transiently elevated 5hmC, followed by the reduction of 5mC at pluripotency-associated genes, germline-specific genes and imprinted loci, but not across the entire genome, which resembles the second wave of DNA demethylation observed in gonadal PGCs. PRDM14 physically interacts with TET1 and TET2 and enhances the recruitment of TET1 and TET2 at target loci. Knockdown of TET1 and TET2 impaired transcriptional regulation and DNA demethylation by PRDM14. The repression of the BER pathway by administration of pharmacological inhibitors of APE1 and PARP1 and the knockdown of thymine DNA glycosylase (TDG) also impaired DNA demethylation by PRDM14. Furthermore, DNA demethylation induced by PRDM14 takes place normally in the presence of aphidicolin, which is an inhibitor of G1/S progression. Together, our analysis provides mechanistic insight into DNA demethylation in naive pluripotent stem cells and developing PGCs.

A replication-dependent passive mechanism modulates DNA demethylation in mouse primordial germ cells.

Germline cells reprogramme extensive epigenetic modifications to ensure the cellular totipotency of subsequent generations and to prevent the accumulation of epimutations. Notably, primordial germ cells (PGCs) erase genome-wide DNA methylation and H3K9 dimethylation marks in a stepwise manner during migration and gonadal periods. In this study, we profiled DNA and histone methylation on transposable elements during PGC development, and examined the role of DNA replication in DNA demethylation in gonadal PGCs. CpGs in short interspersed nuclear elements (SINEs) B1 and B2 were substantially demethylated in migrating PGCs, whereas CpGs in long interspersed nuclear elements (LINEs), such as LINE-1, were resistant to early demethylation. By contrast, CpGs in both LINE-1 and SINEs were rapidly demethylated in gonadal PGCs. Four major modifiers of DNA and histone methylation, Dnmt3a, Dnmt3b, Glp and Uhrf1, were actively repressed at distinct stages of PGC development. DNMT1 was localised at replication foci in nascent PGCs, whereas the efficiency of recruitment of DNMT1 into replication foci was severely impaired in gonadal PGCs. Hairpin bisulphite sequencing analysis showed that strand-specific hemi-methylated CpGs on LINE-1 were predominant in gonadal PGCs. Furthermore, DNA demethylation in SINEs and LINE-1 was impaired in Cbx3-deficient PGCs, indicating abnormalities in G1 to S phase progression. We propose that PGCs employ active and passive mechanisms for efficient and widespread erasure of genomic DNA methylation.

Effects of dppa3 on DNA methylation dynamics during primordial germ cell development in mice.

DNA methylation is a central epigenetic event that regulates cellular differentiation, reprogramming, and pathogenesis. Genomewide DNA demethylation occurs in preimplantation embryos and in embryonic germ cell precursors called primordial germ cells (PGCs). We previously showed that Dppa3, also known as Stella and PGC7, protects the maternal genome from tet methylcytosine dioxygenase 3 (Tet3)-mediated conversion of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) in zygotes. Here, we demonstrated that retrotransposon genes, such as long interspersed nuclear element-1 (Line-1) and intracisternal A particle (IAP), showed higher 5mC levels in Dppa3-null PGCs. In contrast, oxidative bisulfite sequence analysis revealed that the amounts of 5hmC in Line-1 and IAP were slightly reduced in the Dppa3-deficient PGCs. From our findings, we propose that Dppa3 is involved in the Tet-mediated active demethylation process during reprogramming of PGCs.

Locus- and domain-dependent control of DNA methylation at mouse B1 retrotransposons during male germ cell development.

In mammals, germ cells undergo striking dynamic changes in DNA methylation during their development. However, the dynamics and mode of methylation are poorly understood for short interspersed elements (SINEs) dispersed throughout the genome. We investigated the DNA methylation status of mouse B1 SINEs in male germ cells at different developmental stages. B1 elements showed a large locus-to-locus variation in methylation; loci close to RNA polymerase II promoters were hypomethylated, while most others were hypermethylated. Interestingly, a mutation that eliminates Piwi-interacting RNAs (piRNAs), which are involved in methylation of long interspersed elements (LINEs), did not affect the level of B1 methylation, implying a piRNA-independent mechanism. Methylation at B1 loci in SINE-poor genomic domains showed a higher dependency on the de novo DNA methyltransferase DNMT3A but not on DNMT3B, suggesting that DNMT3A plays a major role in methylation of these domains. We also found that many genes specifically expressed in the testis possess B1 elements in their promoters, suggesting the involvement of B1 methylation in transcriptional regulation. Taken altogether, our results not only reveal the dynamics and mode of SINE methylation but also suggest how the DNA methylation profile is created in the germline by a pair of DNA methyltransferases.

Specification of the germ cell lineage in mice: a process orchestrated by the PR-domain proteins, Blimp1 and Prdm14.

Germ cell specification in mice, which generates primordial germ cells (PGCs), the common source of the oocytes and spermatozoa, from the epiblast, integrates three key events: repression of the somatic program, re-acquisition of potential pluripotency, and genome-wide epigenetic reprogramming. A PR-domain containing protein, Blimp1 (also known as Prdm1), has been identified as a critical factor for PGC specification. Using a highly representative single-cell microarray technology, we identified a complex but highly ordered genome-wide transcription dynamics associated with PGC specification. This analysis not only demonstrated a dominant role of Blimp1 for the repression of the genes normally downregulated in PGCs relative to their somatic neighbors, but also revealed the presence of gene expression programs initiating independently from Blimp1. Among such programs, we identified Prdm14, another PR-domain containing protein, as a key regulator for the re-acquisition of potential pluripotency and genome-wide epigenetic reprogramming. The launch of the germ cell lineage in mice, therefore, is orchestrated by two independently acquired, PR domain-containing transcriptional regulators, Blimp1 and Prdm14.

Critical function of Prdm14 for the establishment of the germ cell lineage in mice.

Specification of germ cell fate is fundamental in development and heredity. Recent evidence indicates that in mice, specification of primordial germ cells (PGCs), the common source of both oocytes and spermatozoa, occurs through the integration of three key events: repression of the somatic program, reacquisition of potential pluripotency and ensuing genome-wide epigenetic reprogramming. Here we provide genetic evidence that Prdm14, a PR domain-containing transcriptional regulator with exclusive expression in the germ cell lineage and pluripotent cell lines, is critical in two of these events, the reacquisition of potential pluripotency and successful epigenetic reprogramming. In Prdm14 mutants, the failure of these two events manifests even in the presence of Prdm1 (also known as Blimp1), a key transcriptional regulator for PGC specification. Our combined evidence demonstrates that Prdm14 defines a previously unknown genetic pathway, initiating independently from Prdm1, for ensuring the launching of the mammalian germ cell lineage.

Cellular dynamics associated with the genome-wide epigenetic reprogramming in migrating primordial germ cells in mice.

We previously reported that primordial germ cells (PGCs) in mice erase genome-wide DNA methylation and histone H3 lysine9 dimethylation (H3K9me2), and instead acquire high levels of tri-methylation of H3K27 (H3K27me3) during their migration, a process that might be crucial for the re-establishment of potential totipotency in the germline. We here explored a cellular dynamics associated with this epigenetic reprogramming. We found that PGCs undergo erasure of H3K9me2 and upregulation of H3K27me3 in a progressive, cell-by-cell manner, presumably depending on their developmental maturation. Before or concomitant with the onset of H3K9 demethylation, PGCs entered the G2 arrest of the cell cycle, which apparently persisted until they acquired high H3K27me3 levels. Interestingly, PGCs exhibited repression of RNA polymerase II-dependent transcription, which began after the onset of H3K9me2 reduction in the G2 phase and tapered off after the acquisition of high-level H3K27me3. The epigenetic reprogramming and transcriptional quiescence were independent from the function of Nanos3. We found that before H3K9 demethylation, PGCs exclusively repress an essential histone methyltransferase, GLP, without specifically upregulating histone demethylases. We suggest the possibility that active repression of an essential enzyme and subsequent unique cellular dynamics ensures successful implementation of genome-wide epigenetic reprogramming in migrating PGCs.

Gene expression dynamics during germline specification in mice identified by quantitative single-cell gene expression profiling.

Germ cell fate in mice is induced in proximal epiblast cells at Embryonic Day (E) 6.5 by signaling molecules. Prdm1(also known as Blimp1)-positive lineage-restricted precursors of primordial germ cells (PGCs) initiate the formation of a cluster that differentiates into Dppa3 (also known as stella)-positive PGCs from around E7.0 onwards in the extra-embryonic mesoderm. Around E7.5, these PGCs begin migrating towards the definitive endoderm, with concomitant extensive epigenetic reprogramming. To gain a more precise insight into the mechanism of PGC specification and its subsequent development, we exploited quantitative, single-cell, gene expression profiling to explore gene expression dynamics during the 36 h of PGC differentiation from E6.75 to E8.25, in comparison with the corresponding profiles of somatic neighbors. This analysis revealed that the transitions from Prdm1-positive PGC precursors to Dppa3-positive PGCs and to more advanced migrating PGCs involve a highly dynamic, stage-dependent transcriptional orchestration that begins with the regaining of the pluripotency-associated gene network, followed by stepwise activation of PGC-specific genes, differential repression of the somatic mesodermal program, as well as potential modulations of signal transduction capacities and unique control of epigenetic regulators. The information presented here regarding the cascade of events involved in PGC development should serve as a basis for detailed functional analyses of the gene products associated with this process, as well as for appropriate reconstitution of PGCs and their descendant cells in culture.

Extensive and orderly reprogramming of genome-wide chromatin modifications associated with specification and early development of germ cells in mice.

Induction of mouse germ cells occurs from the proximal epiblast at around embryonic day (E) 7.0. These germ cells then migrate to, and enter the gonads at about E10.5 after which they undergo epigenetic reprogramming including erasure of parental imprints. However, the epigenetic properties acquired by nascent germ cells and the potential remodeling of these epigenetic marks in the subsequent migratory period have been largely unexplored. Here we have used immunohistochemistry to examine several genome-wide epigenetic modifications occurring in germ cells from their specification to their colonization of the genital ridges. We show that at around E8.0, germ cells concomitantly and significantly reduce H3-K9 dimethylation and DNA methylation, two major repressive modifications for gene expression. These events are preceded by the transient loss of all the DNA methyltransferases from their nuclei. By contrast, germ cells substantially increase the levels of H3-K27 trimethylation, another repressive modification with more plasticity, at E8.5-9.0 and maintain this state until at least E12.5. H3-K4 methylation and H3-K9 acetylation, modifications associated with transcriptionally permissive/active chromatin, are similar in germ and surrounding somatic cells but germ cells transiently increase these marks sharply upon their entry into the genital ridge. H3-K9 trimethylation, a hallmark of centromeric heterochromatin, is kept relatively constant during the periods examined. We suggest that this orderly and extensive epigenetic reprogramming in premigratory and migratory germ cells might be necessary for their reacquisition of underlying totipotency, for subsequent specific epigenetic remodeling, including the resetting of parental imprints, and for the production of gametes with an appropriate epigenotype for supporting normal development.

Myeloid Elf-1-like factor, an ETS transcription factor, up-regulates lysozyme transcription in epithelial cells through interaction with promyelocytic leukemia protein.

Myeloid elf-1-like factor (MEF) or Elf4, which is a member of the ETS transcription factor family, up-regulates the basal expression of lysozyme gene in epithelial cells and is constitutively localized in the nucleus. The mammalian cell nucleus is organized into distinct nuclear domains or compartments that are essential for diverse physiological processes. Promyelocytic leukemia (PML) nuclear body or nuclear domain 10 is one of the nuclear domains and is involved in tumor suppression and regulation of transcription. Here, we investigate the role of PML nuclear body in MEF transactivation. We show that PML, but not Sp100, induced the accumulation of MEF in PML nuclear bodies and that MEF and PML physically interacted. This interaction stimulated MEF transcriptional activity, resulting in the up-regulation of endogenous lysozyme expression. Amino acids 348-517 of MEF were required for the accumulation of MEF in PML nuclear bodies and up-regulation of lysozyme transcription, which is enhanced by PML. Moreover, the C-terminal region of MEF spanning amino acids 477-517 was the putative region required for interaction between MEF and PML as determined with the use of the mammalian two-hybrid system. In addition, heat-shock treatment induced the accumulation of MEF in endogenous PML nuclear bodies and enhanced MEF transactivation of lysozyme gene. Thus, the recruitment of MEF to PML nuclear bodies may partly regulate lysozyme transcription in epithelial cells.

ETS2 is involved in protein kinase C-activated expression of granulocyte-macrophage colony-stimulating factor in human non-small lung carcinoma cell line, A549.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a cytokine expressed in the non-small lung carcinoma cells (NSCLC). However, transcriptional regulation of GM-CSF is not well characterized in NSCLC. In this study we found that two cis-acting ETS family consensus sites are important for transcriptional regulation of GM-CSF in A549 human lung carcinoma cells. These two sites are located separately at around -40 and -100 bp from the transcription start site. Results of transient transfection assays with A549 cells indicated that ETS2 had a strong positive effect on GM-CSF promoter activity. Furthermore, this activity was enhanced by protein kinase C activator, phorbol 12-myristate 13-acetate (PMA), in an ETS consensus-dependent manner, while PMA could also enhance the expression level of ETS2. The protein kinase C inhibitors decreased GM-CSF promoter activity induced by the protein kinase C activator PMA. We also found that antisense ETS2 mRNA decreased PMA-induced GM-CSF promoter activity, supporting the possibility that ETS2 is involved in protein kinase C-induced GM-CSF transcriptional function. Endogenous expression of GM-CSF mRNA was increased by ETS2 transfection and the increased expression was further enhanced by PMA. These data indicate that GM-CSF is up-regulated by ETS2, a target of protein kinase C.