CDYL reinforces male gonadal sex determination through epigenetically repressing Wnt4 transcription in mice.

In mammals, male and female gonads initially develop from bipotential progenitor cells, which can differentiate into either testicular or ovarian cells. The decision to adopt a testicular or ovarian fate relies on robust genetic forces, i.e., activation of the testis-determining gene Sry, as well as a delicate balance of expression levels for pro-testis and pro-ovary factors. Recently, epigenetic regulation has been found to be a key element in activation of Sry. Nevertheless, the mechanism by which epigenetic regulation controls the expression balance of pro-testis and pro-ovary factors remains unclear. Chromodomain Y-like protein (CDYL) is a reader protein for repressive histone H3 methylation marks. We found that a subpopulation of Cdyl-deficient mice exhibited XY sex reversal. Gene expression analysis revealed that the testis-promoting gene Sox9 was downregulated in XY Cdyl-deficient gonads during the sex determination period without affecting Sry expression. Instead, we found that the ovary-promoting gene Wnt4 was derepressed in XY Cdyl-deficient gonads prior to and during the sex-determination period. Wnt4 heterozygous deficiency restored SOX9 expression in Cdyl-deficient XY gonads, indicating that derepressed Wnt4 is a cause of the repression of Sox9. We found that CDYL directly bound to the Wnt4 promoter and maintained its H3K27me3 levels during the sex-determination period. These findings indicate that CDYL reinforces male gonadal sex determination by repressing the ovary-promoting pathway in mice.

Transcriptional Regulation of the Y-Linked Mammalian Testis-Determining Gene SRY.

Mammalian male sex differentiation is triggered during embryogenesis by the activation of the Y-linked testis-determining gene SRY. Since insufficient or delayed expression of SRY results in XY gonadal sex reversal, accurate regulation of SRY is critical for male development in XY animals. In humans, dysregulation of SRY may cause disorders of sex development. Mouse Sry is the most intensively studied mammalian model of sex determination. Sry expression is controlled in a spatially and temporally stringent manner. Several transcription factors play a key role in sex determination as trans-acting factors for Sry expression. In addition, recent studies have shown that several epigenetic modifications of Sry are involved in sex determination as cis-acting factors for Sry expression. Herein, we review the current understanding of transcription factor- and epigenetic modifier-mediated regulation of SRY/Sry expression.

The mouse Sry locus harbors a cryptic exon that is essential for male sex determination.

The mammalian sex-determining gene Sry induces male development. Since its discovery 30 years ago, Sry has been believed to be a single-exon gene. Here, we identified a cryptic second exon of mouse Sry and a corresponding two-exon type Sry (Sry-T) transcript. XY mice lacking Sry-T were sex-reversed, and ectopic expression of Sry-T in XX mice induced male development. Sry-T messenger RNA is expressed similarly to that of canonical single-exon type Sry (Sry-S), but SRY-T protein is expressed predominantly because of the absence of a degron in the C terminus of SRY-S. Sry exon2 appears to have evolved recently in mice through acquisition of a retrotransposon-derived coding sequence to replace the degron. Our findings suggest that in nature, SRY-T, not SRY-S, is the bona fide testis-determining factor.

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.

TET2 catalyzes active DNA demethylation of the Sry promoter and enhances its expression.

SRY is the master regulator of male sex determination in eutherian mammals. In mice, Sry expression is transcriptionally and epigenetically controlled in a developmental stage-specific manner. The Sry promoter undergoes demethylation in embryonic gonadal somatic cells at the sex-determining period. However, its molecular mechanism and in vivo significance remain unclear. Here, we report that the Sry promoter is actively demethylated during gonadal development, and TET2 plays a fundamental role in Sry demethylation. Tet2-deficient mice showed absence of 5-hydroxymethylcytosine in the Sry promoter. Furthermore, Tet2 deficiency diminished Sry expression, indicating that TET2-mediated DNA demethylation regulates Sry expression positively. We previously showed that the deficiency of the H3K9 demethylase Jmjd1a compromises Sry expression and induces male-to-female sex reversal. Tet2 deficiency enhanced the sex reversal phenotype of Jmjd1a-deficient mice. Thus, TET2-mediated active DNA demethylation and JMJD1A-mediated H3K9 demethylation contribute synergistically to sex determination.

Combined Loss of JMJD1A and JMJD1B Reveals Critical Roles for H3K9 Demethylation in the Maintenance of Embryonic Stem Cells and Early Embryogenesis.

Histone H3 lysine 9 (H3K9) methylation is unevenly distributed in mammalian chromosomes. However, the molecular mechanism controlling the uneven distribution and its biological significance remain to be elucidated. Here, we show that JMJD1A and JMJD1B preferentially target H3K9 demethylation of gene-dense regions of chromosomes, thereby establishing an H3K9 hypomethylation state in euchromatin. JMJD1A/JMJD1B-deficient embryos died soon after implantation accompanying epiblast cell death. Furthermore, combined loss of JMJD1A and JMJD1B caused perturbed expression of metabolic genes and rapid cell death in embryonic stem cells (ESCs). These results indicate that JMJD1A/JMJD1B-meditated H3K9 demethylation has critical roles for early embryogenesis and ESC maintenance. Finally, genetic rescue experiments clarified that H3K9 overmethylation by G9A was the cause of the cell death and perturbed gene expression of JMJD1A/JMJD1B-depleted ESCs. We summarized that JMJD1A and JMJD1B, in combination, ensure early embryogenesis and ESC viability by establishing the correct H3K9 methylated epigenome.

Rescuing the aberrant sex development of H3K9 demethylase Jmjd1a-deficient mice by modulating H3K9 methylation balance.

Histone H3 lysine 9 (H3K9) methylation is a hallmark of heterochromatin. H3K9 demethylation is crucial in mouse sex determination; The H3K9 demethylase Jmjd1a deficiency leads to increased H3K9 methylation at the Sry locus in embryonic gonads, thereby compromising Sry expression and causing male-to-female sex reversal. We hypothesized that the H3K9 methylation level at the Sry locus is finely tuned by the balance in activities between the H3K9 demethylase Jmjd1a and an unidentified H3K9 methyltransferase to ensure correct Sry expression. Here we identified the GLP/G9a H3K9 methyltransferase complex as the enzyme catalyzing H3K9 methylation at the Sry locus. Based on this finding, we tried to rescue the sex-reversal phenotype of Jmjd1a-deficient mice by modulating GLP/G9a complex activity. A heterozygous GLP mutation rescued the sex-reversal phenotype of Jmjd1a-deficient mice by restoring Sry expression. The administration of a chemical inhibitor of GLP/G9a enzyme into Jmjd1a-deficient embryos also successfully rescued sex reversal. Our study not only reveals the molecular mechanism underlying the tuning of Sry expression but also provides proof on the principle of therapeutic strategies based on the pharmacological modulation of epigenetic balance.

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.