Rev1 overexpression accelerates N-methyl-N-nitrosourea (MNU)-induced thymic lymphoma by increasing mutagenesis.

Rev1 has two important functions in the translesion synthesis pathway, including dCMP transferase activity, and acts as a scaffolding protein for other polymerases involved in translesion synthesis. However, the role of Rev1 in mutagenesis and tumorigenesis in vivo remains unclear. We previously generated Rev1-overexpressing (Rev1-Tg) mice and reported that they exhibited a significantly increased incidence of intestinal adenoma and thymic lymphoma (TL) after N-methyl-N-nitrosourea (MNU) treatment. In this study, we investigated mutagenesis of MNU-induced TL tumorigenesis in wild-type (WT) and Rev1-Tg mice using diverse approaches, including whole-exome sequencing (WES). In Rev1-Tg TLs, the mutation frequency was higher than that in WT TL in most cases. However, no difference in the number of nonsynonymous mutations in the Catalogue of Somatic Mutations in Cancer (COSMIC) genes was observed, and mutations involved in Notch1 and MAPK signaling were similarly detected in both TLs. Mutational signature analysis of WT and Rev1-Tg TLs revealed cosine similarity with COSMIC mutational SBS5 (aging-related) and SBS11 (alkylation-related). Interestingly, the total number of mutations, but not the genotypes of WT and Rev1-Tg, was positively correlated with the relative contribution of SBS5 in individual TLs, suggesting that genetic instability could be accelerated in Rev1-Tg TLs. Finally, we demonstrated that preleukemic cells could be detected earlier in Rev1-Tg mice than in WT mice, following MNU treatment. In conclusion, Rev1 overexpression accelerates mutagenesis and increases the incidence of MNU-induced TL by shortening the latency period, which may be associated with more frequent DNA damage-induced genetic instability.

Morphology dynamics in intestinal crypt during postnatal development affect age-dependent susceptibility to radiation-induced intestinal tumorigenesis in ApcMin/+ mice: possible mechanisms of radiation tumorigenesis.

Age at exposure is a major modifier of radiation-induced carcinogenesis. We used mouse models to elucidate the mechanism underlying age-related susceptibility to radiation-induced tumorigenesis. Radiation exposure in infants was effective at inducing tumors in B6/B6-Chr18MSM-F1 ApcMin/+ mice. Loss of heterozygosity analysis revealed that interstitial deletion may be considered a radiation signature in this model and tumor number containing a deletion correlated with the susceptibility to radiation-induced tumorigenesis as a function of age. Furthermore, in Lgr5-eGFP-ires-CreERT2; Apcflox/flox mice, deletions of both floxed Apc alleles in Lgr5-positive stem cells in infants resulted in the formation of more tumors than in adults. These results suggest that tumorigenicity of Apc-deficient stem cells varies with age and is higher in infant mice. Three-dimensional immunostaining analyses indicated that the crypt architecture in the intestine of infants was immature and different from that in adults concerning crypt size and the number of stem cells and Paneth cells per crypt. Interestingly, the frequency of crypt fission correlated with the susceptibility to radiation-induced tumorigenesis as a function of age. During crypt fission, the percentage of crypts with lysozyme-positive mature Paneth cells was lower in infants than that in adults, whereas no difference in the behavior of stem cells or Paneth cells was observed regardless of age. These data suggest that morphological dynamics in intestinal crypts affect age-dependent susceptibility to radiation-induced tumorigenesis; oncogenic mutations in infant stem cells resulting from radiation exposure may acquire an increased proliferative potential for tumor induction compared with that in adults.

Apolipoprotein E genotype-dependent accumulation of amyloid ß in APP-knock-in mouse model of Alzheimer's disease.

Apolipoprotein E4 (APOE4), the strongest risk factor for late-onset Alzheimer's disease (AD), has been revealed to cause greater accumulation of extracellular amyloid ß (Aß) aggregates than does APOE3 in traditional transgenic mouse models of AD. However, concerns that the overexpression paradigm might have affected the phenotype remain. Amyloid precursor protein (APP)-knock-in (KI) mice, incorporating APP mutations associated with AD development, offer an alternative approach for overproducing pathogenic Aß without needing overexpression of APP. Here, we present the results of comprehensive analyses of pathological and biochemical traits in the brains of APP-KI mice harboring APP-associated familial AD mutations (APPNL-G-F/NL-G-F mice) crossed with human APOE-KI mice. Immunohistochemical and biochemical analyses revealed the APOE genotype-dependent increase in Aß pathology and glial activation, which was evident within 8 months in the mouse model. These results suggested that this mouse model may be valuable for investigating APOE pathobiology within a reasonable experimental time frame. Thus, this model can be considered in investigating the interaction between APOE and Aß in vivo, which may not be addressed appropriately by using other transgenic mouse models.

UTX maintains the functional integrity of the murine hematopoietic system by globally regulating aging-associated genes.

Epigenetic regulation is essential for the maintenance of the hematopoietic system, and its deregulation is implicated in hematopoietic disorders. In this study, UTX, a demethylase for lysine 27 on histone H3 (H3K27) and a component of COMPASS-like and SWI/SNF complexes, played an essential role in the hematopoietic system by globally regulating aging-associated genes. Utx-deficient (UtxΔ/Δ) mice exhibited myeloid skewing with dysplasia, extramedullary hematopoiesis, impaired hematopoietic reconstituting ability, and increased susceptibility to leukemia, which are the hallmarks of hematopoietic aging. RNA-sequencing (RNA-seq) analysis revealed that Utx deficiency converted the gene expression profiles of young hematopoietic stem-progenitor cells (HSPCs) to those of aged HSPCs. Utx expression in hematopoietic stem cells declined with age, and UtxΔ/Δ HSPCs exhibited increased expression of an aging-associated marker, accumulation of reactive oxygen species, and impaired repair of DNA double-strand breaks. Pathway and chromatin immunoprecipitation analyses coupled with RNA-seq data indicated that UTX contributed to hematopoietic homeostasis mainly by maintaining the expression of genes downregulated with aging via demethylase-dependent and -independent epigenetic programming. Of note, comparison of pathway changes in UtxΔ/Δ HSPCs, aged muscle stem cells, aged fibroblasts, and aged induced neurons showed substantial overlap, strongly suggesting common aging mechanisms among different tissue stem cells.

miR-582-5p targets Skp1 and regulates NF-κB signaling-mediated inflammation.

A well-tuned inflammatory response is crucial for an effective immune process. Nuclear factor-kappa B (NF-κB) is a key mediator of inflammatory and innate immunity responses, and its dysregulation is closely associated with immune-related diseases. MicroRNAs (miRNAs) are important inflammation modulators. However, miRNA-regulated mechanisms that implicate NF-κB activity are not fully understood. This study aimed to identify a potential miRNA that could modulate the dysregulated NF-κB signaling during inflammation. We identified miR-582-5p that was significantly downregulated in inflamed murine adipose tissues and RAW264.7 cells. S-phase kinase-associated protein 1 (SKP1), a core component of an E3 ubiquitin ligase that regulates the NF-κB pathway, was proposed as a biological target of miR-582-5p by using TargetScan. The binding of miR-582-5p to a 3'-untranslated region site on Skp1 was confirmed using a dual-luciferase reporter assay; in addition, transfection with a miR-582-5p mimic suppressed SKP1 expression in RAW264.7 cells. Importantly, exogenous miR-582-5p attenuated the production of pro-inflammatory cytokines such as tumor necrosis factor-alpha, interleukin-1 beta, and interleukin-6 through suppressing the degradation of the NF-κB inhibitor alpha, followed by the nuclear translocation of NF-κB. Therefore, exogenously applied miR-582-5p can attenuate the NF-κB signaling pathway via targeting Skp1; this provides a prospective therapeutic strategy for treating inflammatory and immune diseases.

GPR3 expression in retinal ganglion cells contributes to neuron survival and accelerates axonal regeneration after optic nerve crush in mice.

Glaucoma is an optic neuropathy and is currently one of the most common diseases that leads to irreversible blindness. The axonal degeneration that occurs before retinal ganglion neuronal loss is suggested to be involved in the pathogenesis of glaucoma. G protein-coupled receptor 3 (GPR3) belongs to the class A rhodopsin-type GPCR family and is highly expressed in various neurons. GPR3 is unique in its ability to constitutively activate the Gαs protein without a ligand, which elevates the basal intracellular cAMP level. Our earlier reports suggested that GPR3 enhances both neurite outgrowth and neuronal survival. However, the potential role of GPR3 in axonal regeneration after neuronal injury has not been elucidated. Herein, we investigated retinal GPR3 expression and its possible involvement in axonal regeneration after retinal injury in mice. GPR3 was relatively highly expressed in retinal ganglion cells (RGCs). Surprisingly, RGCs in GPR3 knockout mice were vulnerable to neural death during aging without affecting high intraocular pressure (IOP) and under ischemic conditions. Primary cultured neurons from the retina showed that GPR3 expression was correlated with neurite outgrowth and neuronal survival. Evaluation of the effect of GPR3 on axonal regeneration using GPR3 knockout mice revealed that GPR3 in RGCs participates in axonal regeneration after optic nerve crush (ONC) under zymosan stimulation. In addition, regenerating axons were further stimulated when GPR3 was upregulated in RGCs, and the effect was further augmented when combined with zymosan treatment. These results suggest that GPR3 expression in RGCs helps maintain neuronal survival and accelerates axonal regeneration after ONC in mice.

Disruption of piRNA machinery by deletion of ASZ1/GASZ results in the expression of aberrant chimeric transcripts in gonocytes.

In the male germline, the machinery to repress retrotransposons that threaten genomic integrity via the piRNA pathway is established in gonocytes. It has been reported that disruption of the piRNA pathway leads to activation of retrotransposons and arrests spermatogenesis before it enters the second meiosis; however, its effects on gonocytes have not been fully elucidated. In this study, we analyzed the effects of Asz1 deletion, which is a crucial component of the piRNA pathway, on the gonocyte transcriptome. In Asz1-null gonocytes, MIWI2, which is responsible for introducing DNA methylation to retrotransposons in a piRNA-dependent manner, disappeared from the nuclei of fetal gonocytes. Transcriptome analysis revealed that retrotransposons targeted by the piRNA pathway and non-annotated transcript variants were upregulated in gonocytes from neonatal Asz1-/- mice. These non-annotated transcript variants were chimeras generated by joining exons transcribed from retrotransposons and canonical genes. DNA methylation analysis showed that retrotransposons that induce the expression of aberrant chimeric transcripts are not fully methylated. This was consistent with the impaired nuclear localization of MIWI2 in Asz1-null gonocytes. Furthermore, heterogeneity of DNA methylation status in retrotransposons was observed in both gonocytes and their descendants. This suggests that the piRNA system in gonocytes can potentially prevent spermatogenic cell populations bearing aberrant chimeric transcripts from propagating later in spermatogenesis. In conclusion, Asz1 is required to repress retrotransposons and retrotransposon-driven aberrant chimeric transcripts in gonocytes through the piRNA pathway.

Optineurin defects cause TDP43-pathology with autophagic vacuolar formation.

We previously showed that optineurin (OPTN) mutations lead to the development of amyotrophic lateral sclerosis. The association between OPTN mutations and the pathogenesis of amyotrophic lateral sclerosis remains unclear. To investigate the mechanism underlying its pathogenesis, we generated Optn knockout mice. We evaluated histopathological observations of these mice and compared with those of OPTN- amyotrophic lateral sclerosis cases to investigate the mechanism underlying the pathogenesis of amyotrophic lateral sclerosis caused by OPTN mutations. The Optn (-/-) mice presented neuronal autophagic vacuoles immunopositive for charged multivesicular body protein 2b, one of the hallmarks of granulovacuolar degenerations, in the cytoplasm of spinal cord motor neurons at the age of 8 months and the OPTN- amyotrophic lateral sclerosis case with homozygous Q398X mutation. In addition, Optn (-/-) mice showed TAR-DNA binding protein 43/sequestosome1/p62 -positive cytoplasmic inclusions and the clearance of nuclear TAR-DNA binding protein 43. The axonal degeneration of the sciatic nerves was observed in Optn (-/-) mice. However, we could not observe significant differences in survival time, body weight, and motor functions, at 24 months. Our findings suggest that homozygous OPTN deletion or mutations might result in autophagic dysfunction and TAR-DNA binding protein 43 mislocalization, thereby leading to neurodegeneration of motor neurons. These findings indicate that the Optn (-/-) mice recapitulate both common and specific pathogenesis of amyotrophic lateral sclerosis associated with autophagic abnormalities. Optn (-/-) mice could serve as a mouse model for the development of therapeutic strategies.

Overexpression of miR-125b in Osteoblasts Improves Age-Related Changes in Bone Mass and Quality through Suppression of Osteoclast Formation.

We recently reported an unexpected role of osteoblast-derived matrix vesicles in the delivery of microRNAs to bone matrix. Of such microRNAs, we found that miR-125b inhibited osteoclast formation by targeting Prdm1 encoding a transcriptional repressor of anti-osteoclastogenesis factors. Transgenic (Tg) mice overexpressing miR-125b in osteoblasts by using human osteocalcin promoter grow normally but exhibit high trabecular bone mass. We have now further investigated the effects of osteoblast-mediated miR-125b overexpression on skeletal morphogenesis and remodeling during development, aging and in a situation of skeletal repair, i.e., fracture healing. There were no significant differences in the growth plate, primary spongiosa or lateral (periosteal) bone formation and mineral apposition rate between Tg and wild-type (WT) mice during early bone development. However, osteoclast number and medial (endosteal) bone resorption were less in Tg compared to WT mice, concomitant with increased trabecular bone mass. Tg mice were less susceptible to age-dependent changes in bone mass, phosphate/amide I ratio and mechanical strength. In a femoral fracture model, callus formation progressed similarly in Tg and WT mice, but callus resorption was delayed, reflecting the decreased osteoclast numbers associated with the Tg callus. These results indicate that the decreased osteoclastogenesis mediated by miR-125b overexpression in osteoblasts leads to increased bone mass and strength, while preserving bone formation and quality. They also suggest that, in spite of the fact that single miRNAs may target multiple genes, the miR-125b axis may be an attractive therapeutic target for bone loss in various age groups.

Oncogenic mutation in RAS-RAF axis leads to increased expression of GREB1, resulting in tumor proliferation in colorectal cancer.

BRAFV600E mutation accounts for up to 90% of all BRAF mutations in human colorectal cancer (CRC), and constitutively activates the MEK-MAPK pathway. It is recognized that neutralizing mAbs for epidermal growth factor receptor alone are not effective for CRC with BRAFV600E mutation. Therefore, there is increasing interest in identification of the possible therapeutic targets in downstream of BRAF mutation in CRCs. To address this, we studied genome engineered mouse models for colonic neoplasia that has BrafV600E mutation on the basis of Apc inactivation, induced in 2 distinct Cre mouse models, CDX2P-G22Cre and CDX2P-CreERT2 mice. We carried out oligonucleotide microarray analysis for colonic neoplasia generated in these mouse models, and compared gene expression profiles among Kras/Braf WT, Kras-mutated, and Braf-mutated mouse colon tumors to seek new molecular targets corresponding to the KRAS-BRAF-MAPK axis. We found that the expression of the growth regulation by estrogen in breast cancer protein 1 (Greb1) was the most upregulated gene in Braf-mutated mouse tumors compared to Kras/Braf WT counterparts. The silencing of GREB1 significantly reduced the proliferation and tumorigenesis of CRC cell lines, whereas the overexpression of GREB1 promoted cell proliferation. Although GREB1 was first identified as a hormone-responsive gene mediating estrogen-stimulated cell proliferation in endometriosis, breast, and ovarian cancers, these results suggest that RAS-RAF-MAPK signaling upregulates GREB1 expression in CRC, resulting in cellular proliferation. Thus, GREB1 is a possible therapeutic target for CRCs with BrafV600E mutation.

Optineurin regulates osteoblastogenesis through STAT1.

A sophisticated and delicate balance between bone resorption by osteoclasts and bone formation by osteoblasts regulates bone metabolism. Optineurin (OPTN) is a gene involved in primary open-angle glaucoma and amyotrophic lateral sclerosis. Although its function has been widely studied in ophthalmology and neurology, recent reports have shown its possible involvement in bone metabolism through negative regulation of osteoclast differentiation. However, little is known about the role of OPTN in osteoblast function. Here, we demonstrated that OPTN controls not only osteoclast but also osteoblast differentiation. Different parameters involved in osteoblastogenesis and osteoclastogenesis were assessed in Optn-/- mice. The results showed that osteoblasts from Optn-/- mice had impaired alkaline phosphatase activity, defective mineralized nodules, and inability to support osteoclast differentiation. Moreover, OPTN could bind to signal transducer and activator of transcription 1 (STAT1) and regulate runt-related transcription factor 2 (RUNX2) nuclear localization by modulating STAT1 levels in osteoblasts. These data suggest that OPTN is involved in bone metabolism not only by regulating osteoclast function but also by regulating osteoblast function by mediating RUNX2 nuclear translocation via STAT1.

The Role of Cysteine String Protein α Phosphorylation at Serine 10 and 34 by Protein Kinase Cγ for Presynaptic Maintenance.

Protein kinase Cγ (PKCγ) knock-out (KO) animals exhibit symptoms of Parkinson's disease (PD), including dopaminergic neuronal loss in the substantia nigra. However, the PKCγ substrates responsible for the survival of dopaminergic neurons in vivo have not yet been elucidated. Previously, we found 10 potent substrates in the striatum of PKCγ-KO mice. Here, we focused on cysteine string protein α (CSPα), a protein from the heat shock protein (HSP) 40 cochaperone families localized on synaptic vesicles. We found that in cultured cells, PKCγ phosphorylates CSPα at serine (Ser) 10 and Ser34. Additionally, apoptosis was found to have been enhanced by the overexpression of a phosphorylation-null mutant of CSPα, CSPα(S10A/S34A). Compared with wild-type (WT) CSPα, the CSPα(S10A/S34A) mutant had a weaker interaction with HSP70. However, in sharp contrast, a phosphomimetic CSPα(S10D/S34D) mutant, compared with WT CSPα, had a stronger interaction with HSP70. In addition, total levels of synaptosomal-associated protein (SNAP) 25, a main downstream target of the HSC70/HSP70 chaperone complex, were found to have decreased by the CSPα(S10A/S34A) mutant through increased ubiquitination of SNAP25 in PC12 cells. In the striatum of 2-year-old male PKCγ-KO mice, decreased phosphorylation levels of CSPα and decreased SNAP25 protein levels were observed. These findings indicate the phosphorylation of CSPα by PKCγ may protect the presynaptic terminal from neurodegeneration. The PKCγ-CSPα-HSC70/HSP70-SNAP25 axis, because of its role in protecting the presynaptic terminal, may provide a new therapeutic target for the treatment of PD.SIGNIFICANCE STATEMENT Cysteine string protein α (CSPα) is a protein belonging to the heat shock protein (HSP) 40 cochaperone families localized on synaptic vesicles, which maintain the presynaptic terminal. However, the function of CSPα phosphorylation by protein kinase C (PKC) for neuronal cell survival remains unclear. The experiments presented here demonstrate that PKCγ phosphorylates CSPα at serine (Ser) 10 and Ser34. CSPα phosphorylation at Ser10 and Ser34 by PKCγ protects the presynaptic terminal by promoting HSP70 chaperone activity. This report suggests that CSPα phosphorylation, because of its role in modulating HSP70 chaperone activity, may be a target for the treatment of neurodegeneration.

Overexpression of microRNAs from the Gtl2-Rian locus contributes to postnatal death in mice.

The Dlk1-Dio3 imprinted domain functions in embryonic development but the roles of noncoding RNAs expressed from this domain remain unclear. We addressed this question by generating transgenic (TG) mice harbouring a BAC carrying IG-DMR (intergenic-differentially methylated region), Gtl2-DMR, Gtl2, Rtl1/Rtl1as, and part of Rian. High postnatal lethality (>85%) of the BAC-TG pups was observed in the maternally transmitted individuals (MAT-TG), but not following paternal transmission (PAT-TG). The DNA methylation status of IG-DMR and Gtl2-DMR in the BAC-allele was paternally imprinted similar to the genomic allele. The mRNA-Seq and miRNA-Seq analysis revealed marked expression changes in the MAT-TG, with 1,500 upregulated and 2,131 downregulated genes. The long noncoding RNAs and 12 miRNAs containing the BAC locus were markedly enhanced in the MAT-TG. We identified the 24 target genes of the overexpressed miRNAs and confirmed the downregulation in the MAT-TG. Notably, overexpression of mir770, mir493, and mir665 from Gtl2 in the MAT-TG embryos led to decreased expression of the 3 target genes, Col5a1, Pcgf2, and Clip2. Our results suggest that decreased expression of the 3 target genes concomitant with overexpression of the miRNAs within Gtl2 may be involved in the postnatal death in the MAT-TG. Because this imprinted domain is well conserved between mice and humans, the results of genetic and molecular analysis in mice hold important implications for related human disorders such as Temple syndrome.

Transgenic mice specifically expressing amphiregulin in white adipose tissue showed less adipose tissue mass.

To determine adipocytokines that play a regulatory role during obesity development, we explored the genes that encode growth factors and investigated the physiological functions for adipose tissue development. Here, we isolated amphiregulin (Areg) gene whose expression was significantly up-regulated in obese adipose tissues. Areg mRNA level was positively correlated with macrophage marker gene expression in adipose tissues in vivo. Unexpectedly, Areg transgenic mice showed less adipose tissue mass with increased mRNA expression levels of Tnf-α and peroxisome proliferator-activated receptor γ coactivator 1α (Pgc-1α) and delayed white adipose tissue development during the convalescent stage in a dextran sodium sulfate-induced colitis model. This study showed that Areg mRNA expression was significantly up-regulated in obese adipose tissues and over-expression of Areg in white adipose tissue caused less adipose tissue mass.

XY oocytes of sex-reversed females with a Sry mutation deviate from the normal developmental process beyond the mitotic stage†.

The fertility of sex-reversed XY female mice is severely impaired by a massive loss of oocytes and failure of meiotic progression. This phenomenon remains an outstanding mystery. We sought to determine the molecular etiology of XY oocyte dysfunction by generating sex-reversed females that bear genetic ablation of Sry, a vital sex determination gene, on an inbred C57BL/6 background. These mutant mice, termed XYsry- mutants, showed severe attrition of germ cells during fetal development, resulting in the depletion of ovarian germ cells prior to sexual maturation. Comprehensive transcriptome analyses of primordial germ cells (PGCs) and postnatal oocytes demonstrated that XYsry- females had deviated significantly from normal developmental processes during the stages of mitotic proliferation. The impaired proliferation of XYsry- PGCs was associated with aberrant ß-catenin signaling and the excessive expression of transposable elements. Upon entry to the meiotic stage, XYsry- oocytes demonstrated extensive defects, including the impairment of crossover formation, the failure of primordial follicle maintenance, and no capacity for embryo development. Together, these results suggest potential molecular causes for germ cell disruption in sex-reversed female mice, thereby providing insights into disorders of sex differentiation in humans, such as "Swyer syndrome," in which patients with an XY karyotype present as typical females and are infertile.

Transcriptional regulator Bhlhe40 works as a cofactor of T-bet in the regulation of IFN-γ production in iNKT cells.

Invariant natural killer T (iNKT) cells are a subset of innate-like T cells that act as important mediators of immune responses. In particular, iNKT cells have the ability to immediately produce large amounts of IFN-γ upon activation and thus initiate immune responses in various pathological conditions. However, molecular mechanisms that control IFN-γ production in iNKT cells are not fully understood. Here, we report that basic helix-loop-helix transcription factor family, member e40 (Bhlhe40), is an important regulator for IFN-γ production in iNKT cells. Bhlhe40 is highly expressed in stage 3 thymic iNKT cells and iNKT1 subsets, and the level of Bhlhe40 mRNA expression is correlated with Ifng mRNA expression in the resting state. Although Bhlhe40-deficient mice show normal iNKT cell development, Bhlhe40-deficient iNKT cells show significant impairment of IFN-γ production and antitumor effects. Bhlhe40 alone shows no significant effects on Ifng promoter activities but contributes to enhance T-box transcription factor Tbx21 (T-bet)-mediated Ifng promoter activation. Chromatin immunoprecipitation analysis revealed that Bhlhe40 accumulates in the T-box region of the Ifng locus and contributes to histone H3-lysine 9 acetylation of the Ifng locus, which is impaired without T-bet conditions. These results indicate that Bhlhe40 works as a cofactor of T-bet for enhancing IFN-γ production in iNKT cells.

Dynamics of genomic 5-hydroxymethylcytosine during mouse oocyte growth.

Recent studies of the demethylation process in murine zygotes have shown that 5-methylcytosine (5mC) is first converted into 5-hydroxymethylcytosine (5hmC) or further-oxidized cytosines in the paternal genome by the maternal ten-eleven translocation 3 (TET3) enzyme. This process is crucial for normal embryogenesis, and our aim was to elucidate the effect of Tet3 on the maternal genome during female germ-line development. Immunofluorescence analysis showed that 5hmC was clearly present in fully grown oocytes but not in nongrowing and early growth-stage oocytes. The 5hmC in the maternal genome was clearly detectable in DNA methyltransferase 3-like enzyme (Dnmt3L)-null oocytes and their fertilized zygotes, although Dnmt3L is essential for DNA methylation in oocytes. An analysis using an enzyme digestion-based method showed that 5hmC was present in LTR retrotransposons from the late growth period of oocytes. Quantitative RT-PCR analysis showed that Tet3 expression was enhanced during oocyte growth and exhibited an approximately 40-fold increase between nongrowing and fully grown oocytes. Our results show that 5hmC is generated since the oocyte growth stage, accompanied by up-regulation of Tet3; 5hmC is located mainly in LTR retrotransposons, indicating that 5hmC generated in growth-stage oocytes is responsible for genomewide demethylation after fertilization.

Generation of transgenic non-human primates with germline transmission.

The common marmoset (Callithrix jacchus) is increasingly attractive for use as a non-human primate animal model in biomedical research. It has a relatively high reproduction rate for a primate, making it potentially suitable for transgenic modification. Although several attempts have been made to produce non-human transgenic primates, transgene expression in the somatic tissues of live infants has not been demonstrated by objective analyses such as polymerase chain reaction with reverse transcription or western blots. Here we show that the injection of a self-inactivating lentiviral vector in sucrose solution into marmoset embryos results in transgenic common marmosets that expressed the transgene in several organs. Notably, we achieved germline transmission of the transgene, and the transgenic offspring developed normally. The successful creation of transgenic marmosets provides a new animal model for human disease that has the great advantage of a close genetic relationship with humans. This model will be valuable to many fields of biomedical research.

Contribution of intragenic DNA methylation in mouse gametic DNA methylomes to establish oocyte-specific heritable marks.

Genome-wide dynamic changes in DNA methylation are indispensable for germline development and genomic imprinting in mammals. Here, we report single-base resolution DNA methylome and transcriptome maps of mouse germ cells, generated using whole-genome shotgun bisulfite sequencing and cDNA sequencing (mRNA-seq). Oocyte genomes showed a significant positive correlation between mRNA transcript levels and methylation of the transcribed region. Sperm genomes had nearly complete coverage of methylation, except in the CpG-rich regions, and showed a significant negative correlation between gene expression and promoter methylation. Thus, these methylome maps revealed that oocytes and sperms are widely different in the extent and distribution of DNA methylation. Furthermore, a comparison of oocyte and sperm methylomes identified more than 1,600 CpG islands differentially methylated in oocytes and sperm (germline differentially methylated regions, gDMRs), in addition to the known imprinting control regions (ICRs). About half of these differentially methylated DNA sequences appear to be at least partially resistant to the global DNA demethylation that occurs during preimplantation development. In the absence of Dnmt3L, neither methylation of most oocyte-methylated gDMRs nor intragenic methylation was observed. There was also genome-wide hypomethylation, and partial methylation at particular retrotransposons, while maintaining global gene expression, in oocytes. Along with the identification of the many Dnmt3L-dependent gDMRs at intragenic regions, the present results suggest that oocyte methylation can be divided into 2 types: Dnmt3L-dependent methylation, which is required for maternal methylation imprinting, and Dnmt3L-independent methylation, which might be essential for endogenous retroviral DNA silencing. The present data provide entirely new perspectives on the evaluation of epigenetic markers in germline cells.

Developmental expression of GPR3 in rodent cerebellar granule neurons is associated with cell survival and protects neurons from various apoptotic stimuli.

G-protein coupled receptor 3 (GPR3), GPR6, and GPR12 belong to a family of constitutively active Gs-coupled receptors that activate 3'-5'-cyclic adenosine monophosphate (cAMP) and are highly expressed in the brain. Among these receptors, the endogenous expression of GPR3 in cerebellar granule neurons (CGNs) is increased following development. GPR3 is important for neurite outgrowth and neural maturation; however, the physiological functions of GPR3 remain to be fully elucidated. Here, we investigated the survival and antiapoptotic functions of GPR3 under normal and apoptosis-inducing culture conditions. Under normal culture conditions, CGNs from GPR3-knockout mice demonstrated lower survival than did CGNs from wild-type or GPR3-heterozygous mice. Cerebellar sections from GPR3-/- mice at P7, P14, and P21 revealed more caspase-3-positive neurons in the internal granular layer than in cerebellar sections from wild-type mice. Conversely, in a potassium-deprivation model of apoptosis, increased expression of these three receptors promoted neuronal survival. The antiapoptotic effect of GPR3 was also observed under hypoxic (1% O2/5% CO2) and reactive oxygen species (ROS)-induced apoptotic conditions. We further investigated the signaling pathways involved in the GPR3-mediated antiapoptotic effect. The addition of the PKA inhibitor KT5720, the MAP kinase inhibitor U0126, and the PI3 kinase inhibitor LY294002 abrogated the GPR3-mediated antiapoptotic effect in a potassium-deprivation model of apoptosis, whereas the PKC inhibitor Gö6976 did not affect the antiapoptotic function of GPR3. Furthermore, downregulation of endogenous GPR3 expression in CGNs resulted in a marked reduction in the basal levels of ERK and Akt phosphorylation under normal culture conditions. Finally, we used a transient middle cerebral artery occlusion (tMCAO) model in wild-type and GPR3-knockout mice to determine whether GPR3 expression modulates neuronal survival after brain ischemia. After tMCAO, GPR3-knockout mice exhibited a significantly larger infarct area than did wild-type mice. Collectively, these in vitro and in vivo results suggest that the developmental expression of constitutively active Gs-coupled GPR3 activates the ERK and Akt signaling pathways at the basal level, thereby protecting neurons from apoptosis that is induced by various stimuli.