Search

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.

Male penis is required to become erect during copulation. In the upper (dorsal) part of penis, the erectile tissue termed corpus cavernosum (CC) plays fundamental roles for erection by regulating the inner blood flow. When blood flows into the CC, the microvascular complex termed sinusoidal space is reported to expand during erection. A novel in vitro explant system to analyze the dynamic erectile responses during contraction/relaxation is established. The current data show regulatory contraction/relaxation processes induced by phenylephrine (PE) and nitric oxide (NO) donor mimicking dynamic erectile responses by in vitro CC explants. Two-photon excitation microscopy (TPEM) observation shows the synchronous movement of sinusoidal space and the entire CC. By taking advantages of the CC explant system, tadalafil (Cialis) was shown to increase sinusoidal relaxation. Histopathological changes have been generally reported associating with erection in several pathological conditions. Various stressed statuses have been suggested to occur in the erectile responses by previous studies. The current CC explant model enables to analyze such conditions through directly manipulating CC in the repeated contraction/relaxation processes. Expression of oxidative stress marker and contraction-related genes, Hypoxia-inducible factor 1-alpha (Hif1a), glutathione peroxidase 1 (Gpx1), Ras homolog family member A (RhoA), and Rho-associated protein kinase (Rock), was significantly increased in such repeated contraction/relaxation. Altogether, it is suggested that the system is valuable for analyzing structural changes and physiological responses to several regulators in the field of penile medicine.

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.

Di- or tri-methylated H3K9 (H3K9me2/3) is an epigenetic mark of heterochromatin. Heterochromatin protein 1 (HP1) specifically recognizes H3K9me2/3, contributing to transcriptional suppression and spread of H3K9me2/3. Here, we demonstrate another role of HP1 in heterochromatin organization: regulation of protein stability of H3K9 methyltransferases (H3K9 MTs) and demethylases (H3K9 DMs). We show that HP1 interaction-defective mutants of H3K9 MTs, Suv39h1 and Setdb1, undergo protein degradation. We further establish mouse embryonic stem cell lines lacking all three HP1 paralogs. In the HP1-deficient cells, Suv39h1, Suv39h2, Setdb1, and G9a/GLP complex decrease at the protein level, and the enzymes are released from chromatin. HP1 mutants that cannot recognize H3K9me2/3 or form dimers cannot stabilize these enzymes, indicating that the tethering of H3K9 MTs to chromatin is critical for their protein stability. We show that HP1 also stabilizes H3K9 DMs, Jmjd1a and Jmjd1b. Our study indicates that mammalian HP1 forms a heterochromatin hub that governs protein stability of H3K9 MTs and H3K9 DMs.

Two rather than one For several decades, it has been believed that the mammalian sex-determining gene Sry contains a single exon. Miyawaki et al. have now identified a cryptic second exon of mouse Sry . Loss- and gain-of-function analyses revealed that the two-exon SRY (SRY-T), not the canonical single exon–encoded SRY (SRY-S), is the bona fide testis-determining factor. Sry exon2 is composed of retrotransposon-derived sequences. The SRY-S carboxyl terminus contains a degradation sequence (degron), whereas the SRY-T carboxyl terminus encoded in the Sry exon2 is degron free, thereby conferring protein stability on SRY-T. Science , this issue p. 121

Summary Histone H3 lysine 9 (H3K9) methylation is dynamically regulated by methyltransferases and demethylases. In spermatogenesis, prospermatogonia differentiate into differentiating or undifferentiated spermatogonia after birth. However, the epigenetic regulation of prospermatogonia to spermatogonia transition is largely unknown. We found that perinatal prospermatogonia have extremely low levels of di-methylated H3K9 (H3K9me2) and that H3K9 demethylases, JMJD1A and JMJD1B, catalyze H3K9me2 demethylation in perinatal prospermatogonia. Depletion of JMJD1A and JMJD1B in the embryonic germline resulted in complete loss of male germ cells after puberty, indicating that H3K9me2 demethylation is essential for male germline maintenance. JMJD1A/JMJD1B-depleted germ cells were unable to differentiate into functional spermatogonia. JMJD1 isozymes contributed to activation of several spermatogonial stem cell maintenance genes through H3K9 demethylation during the prospermatogonia to spermatogonia transition, which we propose is key for spermatogonia development. In summary, JMJD1A/JMJD1B-mediated H3K9me2 demethylation promotes prospermatogonia to differentiate into functional spermatogonia by establishing proper gene expression profiles., Highlights • JMJD1A/JMJD1B catalyze H3K9 demethylation in prospermatogonia • JMJD1A/JMJD1B are required for prospermatogonia to spermatogonia transition • JMJD1A/JMJD1B activate genes required for spermatogonial stem cell maintenance, In this article, Kuroki et al. show that the H3K9 demethylases, JMJD1A and JMJD1B, are essential for differentiation and maintenance of male germ cells in mice. JMJD1A/JMJD1B-mediated H3K9 demethylation regulates prospermatogonia to spermatogonia transition by ensuring proper gene expression.

Background/Aim: The entire mechanisms by which epigenetic modifiers contribute to the development of pancreatic cancer remain unknown. Although the histone methyltransferase G9a is a promising target in human cancers, its role in pancreatic carcinogenesis has been under-studied. The aim of the study was to examine the role of G9a in pancreatic carcinogenesis by a gene-targeting mouse model. Materials and Methods: We established pancreas-specific G9a(flox/flox )mice and crossed them with Ptf1a(Cre/); Kras(G12D/+ )(KC) mice, which spontaneously develop pancreatic cancer. The phenotypes of the resulting KC mice with G9a deletion were examined. We analyzed transcriptomic data by microarray and genome-wide chromatin accessibility by transposase-accessible chromatin using sequencing. We established pancreatic organoids from KC mice. Results: G9a deficiency impaired the progression of pancreatic intraepithelial neoplasia (PanIN) and prolonged the survival of KC mice. The number of phosphorylated Erk-positive cells and Dclk1-positive cells, which are reported to be essential for the progression of PanIN, were decreased by G9a deletion. UNC0638, an inhibitor of G9a, suppressed the growth of organoids and increased global chromatin accessibility, especially around the regions including the protein phosphatase 2A genes. Conclusion: Thus, our study suggested the functional interaction of G9a, Dclk1 and Mapk pathway in the Kras-driven pancreatic carcinogenesis. The inhibition of G9a may suppress the initiation of oncogenic Kras-driven pancreatic carcinogenesis.

We report thermal conductivity (κ) above 300 K for perovskite ferroelectrics BaTiO3 (T c ≈ 402 K) and PbTiO3 (763 K), as well as antiferroelectrics PbZrO3 (503 K) and PbHfO3 (476 and 433 K). BaTiO3 and PbTiO3 show similar κ in the paraelectric phase. In contrast, smaller and glasslike κ is found above T c for PbZrO3 and PbHfO3, signifying the presence of large anharmonic distortions in the paraelectric phase. Low-temperature heat capacity on PbZrO3 shows a lack of glasslike thermal behavior in the antiferroelectric phase.

Reconstituting the ovarian follicle Recent advances have enabled the generation of oocytes from pluripotent stem cells in vitro. However, these cells require a somatic environment to develop fully as reproductive cells. Yoshino et al. applied what is known about differentiation processes in vivo to determine a culture condition to differentiate embryonic stem cells into gonadal somatic cell–like cells (see the Perspective by Yang and Ng). When the embryonic stem cell–generated ovarian gonadal tissue was combined with early primordial germ cells or in vitro–derived primordial germ cell–like cells, germ cells developed into viable oocytes within the reconstituted follicles that could be fertilized and result in viable offspring. This system enables an alternative method for mouse gamete production and advances our understanding of mammalian reproduction and development. Science , abe0237, this issue p. eabe0237 ; see also abj8347, p. 282

Among caspase family members, Caspase-8 is unique, with associated critical activities to induce and suppress death receptor-mediated apoptosis and necroptosis, respectively. Caspase-8 inhibits necroptosis by suppressing the function of receptor-interacting protein kinase 1 (RIPK1 or RIP1) and RIPK3 to activate mixed lineage kinase domain-like (MLKL). Disruption ofCaspase-8expression causes embryonic lethality in mice, which is rescued by depletion of eitherRipk3orMlkl, indicating that the embryonic lethality is caused by activation of necroptosis. Here, we show that knockdown ofCaspase-8expression in embryoid bodies derived from ES cells markedly enhances retinoic acid (RA)-induced cell differentiation and necroptosis, both of which are dependent onRipk1andRipk3; however, the enhancement of RA-induced cell differentiation is independent ofMlkland necrosome formation. RA treatment obviously enhanced the expression of RA-specific target genes having the retinoic acid response element (RARE) in their promoter regions to induce cell differentiation, and induced marked expression of RIPK1, RIPK3, and MLKL to stimulate necroptosis.Caspase-8knockdown induced RIPK1 and RIPK3 to translocate into the nucleus and to form a complex with RA receptor (RAR), and RAR interacting with RIPK1 and RIPK3 showed much stronger binding activity toRAREthan RAR without RIPK1 or RIPK3. InCaspase-8-deficient as well asCaspase-8- andMlkl-deficient mouse embryos, the expression of RA-specific target genes was obviously enhanced. Thus, Caspase-8, RIPK1, and RIPK3 regulate RA-induced cell differentiation and necroptosis both in vitro and in vivo.

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.

While the significance of acquired genetic abnormalities in the initiation of hepatocellular carcinoma (HCC) has been established, the role of epigenetic modification remains unknown. Here we identified the pivotal role of histone methyltransferase G9a in the DNA damage-triggered initiation of HCC. Using liver-specific G9a-deficient (G9aΔHep) mice, we revealed that loss of G9a significantly attenuated liver tumor initiation caused by diethylnitrosamine (DEN). In addition, pharmacological inhibition of G9a attenuated the DEN-induced initiation of HCC. After treatment with DEN, while the induction of γH2AX and p53 were comparable in the G9aΔHep and wild-type livers, more apoptotic hepatocytes were detected in the G9aΔHep liver. Transcriptome analysis identified Bcl-G, a pro-apoptotic Bcl-2 family member, to be markedly upregulated in the G9aΔHep liver. In human cultured hepatoma cells, a G9a inhibitor, UNC0638, upregulated BCL-G expression and enhanced the apoptotic response after treatment with hydrogen peroxide or irradiation, suggesting an essential role of the G9a-Bcl-G axis in DNA damage response in hepatocytes. The proposed mechanism was that DNA damage stimuli recruited G9a to the p53-responsive element of the Bcl-G gene, resulting in the impaired enrichment of p53 to the region and the attenuation of Bcl-G expression. G9a deletion allowed the recruitment of p53 and upregulated Bcl-G expression. These results demonstrate that G9a allows DNA-damaged hepatocytes to escape p53-induced apoptosis by silencing Bcl-G, which may contribute to the tumor initiation. Therefore, G9a inhibition can be a novel preventive strategy for HCC.

SummaryDuring spermatogenesis, meiosis is accompanied by robust alteration in gene expression and chromatin status. However, it remained elusive how meiotic transcriptional program is established to ensure completion of meiotic prophase. Here, we identified a novel protein complex consisting of germ-cell-specific zinc-finger protein ZFP541 and its interactor KCTD19 as the key transcriptional regulator for meiotic prophase exit. Our genetic study showed that ZFP541 and KCTD19 are co-expressed from pachytene onward and play an essential role in the completion of meiotic prophase program in the testis. Furthermore, our ChIP-seq and transcriptome analyses revealed that ZFP541 binds to and suppresses a broad range of genes whose function is associated with biological processes of transcriptional regulation and covalent chromatin modification. The present study demonstrated that germ-cell specific ZFP541-KCTD19 containing complex promotes meiotic prophase exit in males, and triggers reconstruction of the transcription network and chromatin organization leading to post-meiotic development.

During spermatogenesis, meiosis is accompanied by a robust alteration in gene expression and chromatin status. However, it remains elusive how the meiotic transcriptional program is established to ensure completion of meiotic prophase. Here, we identify a protein complex that consists of germ-cell-specific zinc-finger protein ZFP541 and its interactor KCTD19 as the key transcriptional regulators in mouse meiotic prophase progression. Our genetic study shows that ZFP541 and KCTD19 are co-expressed from pachytene onward and play an essential role in the completion of the meiotic prophase program in the testis. Furthermore, our ChIP-seq and transcriptome analyses identify that ZFP541 binds to and suppresses a broad range of genes whose function is associated with biological processes of transcriptional regulation and covalent chromatin modification. The present study demonstrates that a germ-cell specific complex that contains ZFP541 and KCTD19 promotes the progression of meiotic prophase towards completion in male mice, and triggers the reconstruction of the transcriptional network and chromatin organization leading to post-meiotic development., The authors add to our knowledge of the transcriptional regulation of the meiotic program in mice spermatocytes, showing ZFP541 regulates meiotic prophase and transition to the division phase by being the target for upstream factors MEIOSIN/STRA8.

We report on the development of a capacitance measuring system which allows measurements of capacitance in pulsed magnetic fields up to 61~T. By using this system, magnetic-field responses of various physical quantities such magnetostriction, magnetic-field-induced change in complex dielectric constant, and magneto-caloric effect can be investigated in pulsed-magnetic-field conditions. Here, we examine the validity of our system for investigations of these magnetic-field-induced phenomena in pulse magnets. For the magnetostriction measurement, magnetostriction of a specimen can be measured through a change in the capacitance between two aligned electrodes glued on the specimen and a dilatometer. We demonstrate a precise detection of valley polarization in semimetallic bismuth through a magnetostriction signal with a resolution better than 10$^{-6}$ of the relative length change. For the magnetic-field-induced change in complex dielectric constant, we successfully observed clear dielectric anomalies accompanied by magnetic/magnetoelectric phase transitions in multiferroic Pb(TiO)Cu$_4$(PO$_4$)$_4$. For the measurement of magneto-caloric effect, a magnetic-field-induced change in sample temperature was verified for Gd$_3$Ga$_5$O$_{12}$ with a capacitance thermometer made of a non-magnetic ferroelectric compound KTa$_{1-x}$Nb$_x$O$_3$ ($x$ = 0.02) whose capacitance is nearly field-independent. These results show that our capacitance measuring system is a promising tool to study various magnetic-field-induced phenomena which have been difficult to detect in pulsed magnetic fields., 10 pages, 5 figures, submitted to Review of Scientific Instruments

The mammalian sex-determining gene

Regulation of gene expression without changing the DNA sequence is governed by epigenetic mechanisms. Epigenetic regulation is important for changing chromatin structure in response to environmental cues as well as maintaining chromatin structure after cell division. The epigenetic machinery can reversibly change chromatin function, allowing a wide variety of biological processes in multicellular organisms to be controlled. Epigenetic regulation ensures spatial and temporal accuracy of the expression of developmentally regulated genes. So far, few studies have focused on the relationship between epigenetic regulation and mammalian sex development, despite this being an interesting area of research. Sex development consists of three sequential stages: the undifferentiated stage, gonadal differentiation into testes or ovaries, and differentiation of internal and external genitalia. Some genetic studies have revealed that epigenetic regulation is required for proper gonadal differentiation in mice. Particularly, the epigenetic machinery plays an integral part in sex determination, which is the first step of gonadal differentiation. Mammalian sex determination is triggered by activation of the mammalian sex-determining gene, Sry, in a spatially and temporally accurate manner. Several studies have demonstrated that expression of Sry is controlled not only by specific transcription factors but also by the epigenetic machinery. Here, we focus on the epigenetic regulation of Sry expression.