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.

The G9a histone methyltransferase represses osteoclastogenesis and bone resorption by regulating NFATc1 function.

Epigenetic modifications affect cell differentiation via transcriptional regulation. G9a/EHMT2 is an important epigenetic modifier that catalyzes the methylation of histone 3 lysine 9 (H3K9) and interacts with various nuclear proteins. In this study, we investigated the role of G9a in osteoclast differentiation. When we deleted G9a by infection of Cre-expressing adenovirus into bone marrow macrophages (BMMs) from G9afl/fl (Ehmt2fl/fl) and induced osteoclastic differentiation by the addition of macrophage colony-stimulating factor (M-CSF) and receptor activator of NF-κB ligand (RANKL), the number of TRAP-positive multinucleated osteoclasts significantly increased compared with control. Furthermore, the mRNA expression of osteoclast markers, TRAP, and cathepsin K, and to a lesser extent, NFATc1, a critical transcription factor, increased in G9a KO cells. Infection of wild-type (WT) G9a-expressing adenovirus in G9a KO cells restored the number of TRAP-positive multinucleated cells. In G9a KO cells, increased nuclear accumulation of NFATc1 protein and decreased H3K9me2 accumulation were observed. Furthermore, ChIP experiments revealed that NFATc1 binding to its target, Ctsk promoter, was enhanced by G9a deletion. For in vivo experiments, we created G9a conditional knock-out (cKO) mice by crossing G9afl/fl mice with Rank Cre/+ (Tnfrsf11aCre/+) mice, in which G9a is deleted in osteoclast lineage cells. The trabecular bone volume was significantly reduced in female G9a cKO mice. The serum concentration of the C-terminal telopeptide of type I collagen (CTX), a bone-resorbing indicator, was higher in G9a cKO mice. In addition, osteoclasts differentiated from G9a cKO BMMs exhibited greater bone-resorbing activity. Our findings suggest that G9a plays a repressive role in osteoclastogenesis by modulating NFATc1 function.

HP1 maintains protein stability of H3K9 methyltransferases and demethylases.

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.

Dynamic erectile responses of a novel penile organ model utilizing TPEM†.

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.

DNA damage signaling triggers degradation of histone methyltransferases through APC/C(Cdh1) in senescent cells.

Both the DNA damage response (DDR) and epigenetic mechanisms play key roles in the implementation of senescent phenotypes, but very little is known about how these two mechanisms are integrated to establish senescence-associated gene expression. Here we show that, in senescent cells, the DDR induces proteasomal degradation of G9a and GLP, major histone H3K9 mono- and dimethyltransferases, through Cdc14B- and p21(Waf1/Cip1)-dependent activation of APC/C(Cdh1) ubiquitin ligase, thereby causing a global decrease in H3K9 dimethylation, an epigenetic mark for euchromatic gene silencing. Interestingly, induction of IL-6 and IL-8, major players of the senescence-associated secretory phenotype (SASP), correlated with a decline of H3K9 dimethylation around the respective gene promoters and knockdown of Cdh1 abolished IL-6/IL-8 expression in senescent cells, suggesting that the APC/C(Cdh1)-G9a/GLP axis plays crucial roles in aspects of senescent phenotype. These findings establish a role for APC/C(Cdh1) and reveal how the DDR integrates with epigenetic processes to induce senescence-associated gene expression.

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.

HIF-KDM3A-MMP12 regulatory circuit ensures trophoblast plasticity and placental adaptations to hypoxia.

The hemochorial placenta develops from the coordinated multilineage differentiation of trophoblast stem (TS) cells. An invasive trophoblast cell lineage remodels uterine spiral arteries, facilitating nutrient flow, failure of which is associated with pathological conditions such as preeclampsia, intrauterine growth restriction, and preterm birth. Hypoxia plays an instructive role in influencing trophoblast cell differentiation and regulating placental organization. Key downstream hypoxia-activated events were delineated using rat TS cells and tested in vivo, using trophoblast-specific lentiviral gene delivery and genome editing. DNA microarray analyses performed on rat TS cells exposed to ambient or low oxygen and pregnant rats exposed to ambient or hypoxic conditions showed up-regulation of genes characteristic of an invasive/vascular remodeling/inflammatory phenotype. Among the shared up-regulated genes was matrix metallopeptidase 12 (MMP12). To explore the functional importance of MMP12 in trophoblast cell-directed spiral artery remodeling, we generated an Mmp12 mutant rat model using transcription activator-like nucleases-mediated genome editing. Homozygous mutant placentation sites showed decreased hypoxia-dependent endovascular trophoblast invasion and impaired trophoblast-directed spiral artery remodeling. A link was established between hypoxia/HIF and MMP12; however, evidence did not support Mmp12 as a direct target of HIF action. Lysine demethylase 3A (KDM3A) was identified as mediator of hypoxia/HIF regulation of Mmp12 Knockdown of KDM3A in rat TS cells inhibited the expression of a subset of the hypoxia-hypoxia inducible factor (HIF)-dependent transcripts, including Mmp12, altered H3K9 methylation status, and decreased hypoxia-induced trophoblast cell invasion in vitro and in vivo. The hypoxia-HIF-KDM3A-MMP12 regulatory circuit is conserved and facilitates placental adaptations to environmental challenges.

H3K9 methyltransferase G9a and the related molecule GLP.

The discovery of Suv39h1, the first SET domain-containing histone lysine methyltransferase (HKMT), was reported in 2000. Since then, research on histone methylation has progressed rapidly. Among the identified HKMTs in mammals, G9a and GLP are the primary enzymes for mono- and dimethylation at Lys 9 of histone H3 (H3K9me1 and H3K9me2), and exist predominantly as a G9a-GLP heteromeric complex that appears to be a functional H3K9 methyltransferase in vivo. Recently, many important studies have reported that G9a and GLP play critical roles in various biological processes. The physiological relevance of G9a/GLP-mediated epigenetic gene regulation is discussed.

Essential roles of G9a in cell proliferation and differentiation during tooth development.

Teeth develop through interactions between epithelial and mesenchymal tissues mediated by a signaling network comprised of growth factors and transcription factors. However, little is known about how epigenetic modifiers affect signaling pathways and thereby regulate tooth formation. We previously reported that the histone 3 lysine 9 (H3K9) methyltransferase (MTase) G9a is specifically enriched in the tooth mesenchyme during mouse development. In this study, we investigated the functions of G9a in tooth development using G9a conditional knockout (KO) mice. We used Sox9-Cre mice to delete G9a in the tooth mesenchyme because Sox9 is highly expressed in the mesenchyme derived from the cranial neural crest. Immunohistochemical analyses revealed that G9a expression was significantly decreased in the mesenchyme of Sox9-Cre;G9afl/fl (G9a cKO) mice compared with that in Sox9-Cre;G9a fl/+(control) mice. Protein levels of the G9a substrate H3K9me2 were also decreased in the tooth mesenchyme. G9a cKO mice showed smaller tooth germ after embryonic day (E) 16.5 and E17.5, but not at E15.5. The developing cusp tips, which were visible in control mice, were absent in G9a cKO mice at E17.5. At 3 weeks after birth, small first molars with smaller cusps and unseparated roots were formed. Organ culture of tooth germs derived from E15.5 cKO mouse embryos showed impaired tooth development, suggesting that tooth development per se is affected independently of skull development. BrdU labeling experiments revealed that the proliferation rates were decreased in the mesenchyme in G9a cKO mice at E17.5. In addition, the proliferation rates in the tooth inner enamel epithelium were also decreased. In situ hybridization revealed altered localization of genes associated with tooth development. In cKO mice, intensively localized expression of mRNAs encoding bone morphogenic protein (Bmp2 and Bmp4) was observed in the tooth mesenchyme at E17.5, similar to the expression patterns observed in control mice at E15.5. Localization of Shh and related signaling components, including Gli1, Ptch1, and Ptch2, in the tooth mesenchyme of cKO mice was generally similar to that at earlier stages in control mice. In addition, expression of Fgf3 and Fgf10 in the mesenchyme was decreased in G9a cKO mice at P0. Expression levels of Fgf9 and p21, both of which were expressed in the secondary enamel-knot, were also decreased. Thus, the expression of genes associated with tooth development was delayed in cKO mice. Our results suggest that H3K9MTase G9a regulates cell proliferation and timing of differentiation and that G9a expression in the tooth mesenchyme is required for proper tooth development.

PGC7 binds histone H3K9me2 to protect against conversion of 5mC to 5hmC in early embryos.

The modification of DNA by 5-methylcytosine (5mC) has essential roles in cell differentiation and development through epigenetic gene regulation. 5mC can be converted to another modified base, 5-hydroxymethylcytosine (5hmC), by the tet methylcytosine dioxygenase (Tet) family of enzymes. Notably, the balance between 5hmC and 5mC in the genome is linked with cell-differentiation processes such as pluripotency and lineage commitment. We have previously reported that the maternal factor PGC7 (also known as Dppa3, Stella) is required for the maintenance of DNA methylation in early embryogenesis, and protects 5mC from conversion to 5hmC in the maternal genome. Here we show that PGC7 protects 5mC from Tet3-mediated conversion to 5hmC by binding to maternal chromatin containing dimethylated histone H3 lysine 9 (H3K9me2) in mice. In addition, imprinted loci that are marked with H3K9me2 in mature sperm are protected by PGC7 binding in early embryogenesis. This type of regulatory mechanism could be involved in DNA modifications in somatic cells as well as in early embryos.

Pericentric H3K9me3 Formation by HP1 Interaction-defective Histone Methyltransferase Suv39h1.

Pericentric regions form epigenetically organized, silent heterochromatin structures that accumulate histone H3 lysine 9 tri-methylation (H3K9me3) and heterochromatin protein 1 (HP1), a methylated H3K9-binding protein. At pericentric regions, Suv39h is the major enzyme that generates H3K9me3. Suv39h also interacts directly with HP1. However, the importance of HP1 interaction for Suv39h-mediated H3K9me3 formation at the pericentromere is not well characterized. To address this question, we introduced HP1 binding-defective, N-terminally truncated mouse Suv39h1 (ΔN) into Suv39h-deficient cells. Pericentric H3K9me3-positive cells were not detected by endogenous-level expression of ΔN. Notably, ΔN could induce pericentric accumulation of H3K9me3 as wild type Suv39h1 did if it was overexpressed. These findings demonstrate that the N-terminal region of Suv39h1, presumably via HP1-Suv39h1 interaction, is required for Suv39h1-mediated pericentric H3K9me3 formation, but can be overridden if Suv39h1 is overproduced, indicating that Suv39h1-mediated heterochromatin formation is controlled by multiple modules, including HP1.

Transgenic expression of Map3k4 rescues T-associated sex reversal (Tas) in mice.

Disorders of sex development in the human population range in severity from mild genital defects to gonadal sex reversal. XY female development has been associated with heterozygous mutations in several genes, including SOX9, WT1 and MAP3K1. In contrast, XY sex reversal in mice usually requires complete absence of testis-determining gene products. One exception to this involves T-associated sex reversal (Tas), a phenomenon characterized by the formation of ovotestes or ovaries in XY mice hemizygous for the hairpin-tail (T(hp)) or T-Orleans (T(Orl)) deletions on proximal mouse chromosome 17. We recently reported that mice heterozygous for a null allele of Map3k4, which resides in the T(hp) deletion, exhibit XY ovotestis development and occasional gonadal sex reversal on the sensitized C57BL/6J-Y(AKR) (B6-Y(AKR)) genetic background, reminiscent of the Tas phenotype. However, these experiments did not exclude the possibility that loss of other loci in the T(hp) deletion, or other effects of the deletion itself, might contribute to Tas. Here, we show that disruption to Sry expression underlies XY gonadal defects in B6-Y(AKR) embryos harbouring the T(hp) deletion and that a functional Map3k4 bacterial artificial chromosome rescues these abnormalities by re-establishing a normal Sry expression profile. These data demonstrate that Map3k4 haploinsufficiency is the cause of T-associated sex reversal and that levels of this signalling molecule are a major determinant of the expression profile of Sry.

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.

Proviral silencing in embryonic stem cells requires the histone methyltransferase ESET.

Endogenous retroviruses (ERVs), retrovirus-like elements with long terminal repeats, are widely dispersed in the euchromatic compartment in mammalian cells, comprising approximately 10% of the mouse genome. These parasitic elements are responsible for >10% of spontaneous mutations. Whereas DNA methylation has an important role in proviral silencing in somatic and germ-lineage cells, an additional DNA-methylation-independent pathway also functions in embryonal carcinoma and embryonic stem (ES) cells to inhibit transcription of the exogenous gammaretrovirus murine leukaemia virus (MLV). Notably, a recent genome-wide study revealed that ERVs are also marked by histone H3 lysine 9 trimethylation (H3K9me3) and H4K20me3 in ES cells but not in mouse embryonic fibroblasts. However, the role that these marks have in proviral silencing remains unexplored. Here we show that the H3K9 methyltransferase ESET (also called SETDB1 or KMT1E) and the Krüppel-associated box (KRAB)-associated protein 1 (KAP1, also called TRIM28) are required for H3K9me3 and silencing of endogenous and introduced retroviruses specifically in mouse ES cells. Furthermore, whereas ESET enzymatic activity is crucial for HP1 binding and efficient proviral silencing, the H4K20 methyltransferases Suv420h1 and Suv420h2 are dispensable for silencing. Notably, in DNA methyltransferase triple knockout (Dnmt1(-/-)Dnmt3a(-/-)Dnmt3b(-/-)) mouse ES cells, ESET and KAP1 binding and ESET-mediated H3K9me3 are maintained and ERVs are minimally derepressed. We propose that a DNA-methylation-independent pathway involving KAP1 and ESET/ESET-mediated H3K9me3 is required for proviral silencing during the period early in embryogenesis when DNA methylation is dynamically reprogrammed.

Epigenetics of sex determination in mammals.

Epigenetics is the study of changes in gene function that cannot be explained by changes in DNA sequence. A mammalian body contains more than two hundred types of cells. Since all of them are derived from a single fertilized egg, their genotypes are identical. However, the gene expression patterns are different between the cell types, indicating that each cell type has unique own "epigenotype". Epigenetic gene regulation mechanisms essentially contribute to various processes of mammalian development. The essence of epigenetic regulation is the structural change of chromatin to modulate gene activity in a spatiotemporal manner. DNA methylation and histone modifications are the major epigenetic mechanisms. Sex determination is the process for gender establishment. There are two types of sex-determining mechanisms in animals, environmental sex determination (ESD) and genotypic sex determination (GSD). Recent studies have provided some evidence that epigenetic mechanisms play indispensable roles in ESD and GSD. Some fishes undergo ESD, in which DNA methylation is essentially involved. GSD is employed in therian mammals, where Sry (sex-determining region on the Y chromosome) triggers testis differentiation from undifferentiated gonads. Sry expression is tightly regulated in a spatiotemporal manner. A recent study demonstrated that histone modification is involved in Sry regulation. In this review, we discuss the role of epigenetic mechanisms for sex determination in mammals and other vertebrates.

Development of a general-purpose method for cell purification using Cre/loxP-mediated recombination.

A mammalian body is composed of more than 200 different types of cells. The purification of a certain cell type from tissues/organs enables a wide variety of studies. One popular cell purification method is immunological isolation, using antibodies against specific cell surface antigens. However, this is not a general-purpose method, since suitable antigens have not been found in certain cell types, including embryonic gonadal somatic cells and Sertoli cells. To address this issue, we established a knock-in mouse line, named R26 KI, designed to express the human cell surface antigen hCD271 through Cre/loxP-mediated recombination. First, we used the R26 Kl mouse line to purify embryonic gonadal somatic cells. Gonadal somatic cells were purified from the R26 KI; Nr5a1-Cre-transgenic (tg) embryos almost equally as efficiently as from Nr5a1-hCD271-tg embryos. Second, we used the R26 KI mouse line to purify Sertoli cells successfully from R26 KI; Amh-Cre-tg testes. In summary, we propose that the R26 KI mouse line is a powerful tool for the purification of various cell types.

Hippocampal dysfunction in the Euchromatin histone methyltransferase 1 heterozygous knockout mouse model for Kleefstra syndrome.

Euchromatin histone methyltransferase 1 (EHMT1) is a highly conserved protein that catalyzes mono- and dimethylation of histone H3 lysine 9, thereby epigenetically regulating transcription. Kleefstra syndrome (KS), is caused by haploinsufficiency of the EHMT1 gene, and is an example of an emerging group of intellectual disability (ID) disorders caused by genes encoding epigenetic regulators of neuronal gene activity. Little is known about the mechanisms underlying this disorder, prompting us to study the Euchromatin histone methyltransferase 1 heterozygous knockout (Ehmt1(+/-)) mice as a model for KS. In agreement with the cognitive disturbances observed in patients with KS, we detected deficits in fear extinction learning and both novel and spatial object recognition in Ehmt1(+/-) mice. These learning and memory deficits were associated with a significant reduction in dendritic arborization and the number of mature spines in hippocampal CA1 pyramidal neurons of Ehmt1(+/-) mice. In-depth analysis of the electrophysiological properties of CA3-CA1 synapses revealed no differences in basal synaptic transmission or theta-burst induced long-term potentiation (LTP). However, paired-pulse facilitation (PPF) was significantly increased in Ehmt1(+/-) neurons, pointing to a potential deficiency in presynaptic neurotransmitter release. Accordingly, a reduction in the frequency of miniature excitatory post-synaptic currents (mEPSCs) was observed in Ehmt1(+/-) neurons. These data demonstrate that Ehmt1 haploinsufficiency in mice leads to learning deficits and synaptic dysfunction, providing a possible mechanism for the ID phenotype in patients with KS.

H3K9MTase G9a is essential for the differentiation and growth of tenocytes in vitro.

Cell differentiation is controlled by specific transcription factors. The functions and expression levels of these transcription factors are regulated by epigenetic modifications, such as histone modifications and cytosine methylation of the genome. In tendon tissue, tendon-specific transcription factors have been shown to play functional roles in the regulation of tenocyte differentiation. However, the effects of epigenetic modifications on gene expression and differentiation in tenocytes are unclear. In this study, we investigated the epigenetic regulation of tenocyte differentiation, focusing on the enzymes mediating histone 3 lysine 9 (H3K9) methylation. In primary mouse tenocytes, six H3K9 methyltransferase (H3K9MTase) genes, i.e., G9a, G9a-like protein (GLP), PR domain zinc finger protein 2 (PRDM2), SUV39H1, SUV39H2, and SETDB1/ESET were all expressed, with increased mRNA levels observed during tenocyte differentiation. In mouse embryos, G9a and Prdm2 mRNAs were expressed in tenocyte precursor cells, which were overlapped with or were adjacent to cells expressing a tenocyte-specific marker, tenomodulin. Using tenocytes isolated from G9a-flox/flox mice, we deleted G9a by infecting the cells with Cre-expressing adenoviruses. Proliferation of G9a-null tenocytes was significantly decreased compared with that of control cells infected with GFP-expressing adenoviruses. Moreover, the expression levels of tendon transcription factors gene, i.e., Scleraxis (Scx), Mohawk (Mkx), Egr1, Six1, and Six2 were all suppressed in G9a-null tenocytes. The tendon-related genes Col1a1, tenomodulin, and periostin were also downregulated. Consistent with this, Western blot analysis showed that tenomodulin protein expression was significantly suppressed by G9a deletion. These results suggested that expression of the H3K9MTase G9a was essential for the differentiation and growth of tenocytes and that H3K9MTases may play important roles in tendinogenesis.

Pericentric heterochromatin generated by HP1 protein interaction-defective histone methyltransferase Suv39h1.

Pericentric regions form epigenetically organized silent heterochromatin structures that accumulate histone H3 lysine 9 trimethylation (H3K9me3) and HP1. At pericentric regions, Suv39h is the major enzyme that generates H3K9me3. Suv39h also interacts directly with HP1, a methylated H3K9-binding protein. However, it is not well characterized how HP1 interaction is important for Suv39h accumulation and Suv39h-mediated H3K9me3 formation at the pericentromere. To address this, we introduced the HP1 binding-defective N-terminally truncated mouse Suv39h1 (ΔN) into Suv39h-deficient embryonic stem cells. Interestingly, pericentric accumulation of ΔN and ΔN-mediated H3K9me3 was observed to recover, but HP1 accumulation was only marginally restored. ΔN also rescued DNA methyltransferase Dnmt3a and -3b accumulation and DNA methylation of the pericentromere. In contrast, other pericentric heterochromatin features, such as ATRX protein association and H4K20me3, were not recovered. Finally, derepressed major satellite repeats were partially silenced by ΔN expression. These findings clearly showed that the Suv39h-HP1 binding is dispensable for pericentric H3K9me3 and DNA methylation, but this interaction and HP1 recruitment/accumulation seem to be crucial for complete formation of heterochromatin.

HP1γ links histone methylation marks to meiotic synapsis in mice.

During meiosis, specific histone modifications at pericentric heterochromatin (PCH), especially histone H3 tri- and dimethylation at lysine 9 (H3K9me3 and H3K9me2, respectively), are required for proper chromosome interactions. However, the molecular mechanism by which H3K9 methylation mediates the synapsis is not yet understood. We have generated a Cbx3-deficient mouse line and performed comparative analysis on Suv39h1/h2-, G9a- and Cbx3-deficient spermatocytes. This study revealed that H3K9me2 at PCH depended on Suv39h1/h2-mediated H3K9me3 and its recognition by the Cbx3 gene product HP1γ. We further found that centromere clustering and synapsis were commonly affected in G9a- and Cbx3-deficient spermatocytes. These genetic observations suggest that HP1γ/G9a-dependent PCH-mediated centromere clustering is an axis for proper chromosome interactions during meiotic prophase. We propose that the role of the HP1γ/G9a axis is to retain centromeric regions of unpaired homologous chromosomes in close alignment and facilitate progression of their pairing in early meiotic prophase. This study also reveals considerable plasticity in the interplay between different histone modifications and suggests that such stepwise and dynamic epigenetic modifications may play a pivotal role in meiosis.