[Show abstract][Hide abstract] ABSTRACT: Mps One Binder Kinase Activator (MOB)1A/1B are core components of the Hippo pathway that coactivate large tumor suppressor homolog (LATS) kinases. Mob1a/1b double deficiency in mouse liver (LMob1DKO) results in hyperplasia of oval cells and immature cholangiocytes accompanied by inflammatory cell infiltration and fibrosis. More than half of mutant mice die within 3 wk of birth. All survivors eventually develop liver cancers, particularly combined hepatocellular and cholangiocarcinomas (cHC-CCs) and intrahepatic cholangiocellular carcinomas (ICCs), and die by age 60 wk. Because this phenotype is the most severe among mutant mice lacking a Hippo signaling component, MOB1A/1B constitute the critical hub of Hippo signaling in mammalian liver. LMob1DKO liver cells show hyperproliferation, increased cell saturation density, hepatocyte dedifferentiation, enhanced epithelial-mesenchymal transition and cell migration, and elevated transforming growth factor beta(TGF-β)2/3 production. These changes are strongly dependent on Yes-Associated Protein-1 (Yap1) and partially dependent on PDZ-binding motif (Taz) and Tgfbr2, but independent of connective tissue growth factor (Ctgf). In human liver cancers, YAP1 activation is frequent in cHC-CCs and ICCs and correlates with SMAD family member 2 activation. Drug screening revealed that antiparasitic macrocyclic lactones inhibit YAP1 activation in vitro and in vivo. Targeting YAP1/TAZ with these drugs in combination with inhibition of the TGF-β pathway may be effective treatment for cHC-CCs and ICCs.
Full-text · Article · Dec 2015 · Proceedings of the National Academy of Sciences
[Show abstract][Hide abstract] ABSTRACT: Global DNA hypomethylation and DNA hypermethylation of promoter regions are frequently detected in human cancers. Although many studies have suggested a contribution to carcinogenesis, it is still unclear whether the aberrant DNA hypomethylation observed in tumors is a consequence or a cause of cancer. Here, we show that the enforced expression of Stella (also known as PGC7 and Dppa3) induced not only global DNA demethylation but also transformation of NIH3T3 cells. Furthermore, overexpression of Stella enhanced the metastatic ability of B16 melanoma cells, presumably through the induction of metastasis-related genes. These results provide new insights into the function of global DNA hypomethylation in carcinogenesis.
[Show abstract][Hide abstract] ABSTRACT: Genomic imprinting is a major monoallelic gene expression regulatory mechanism in mammals, and depends on gamete-specific DNA methylation of specialized cis-regulatory elements called imprinting control regions (ICRs). Allele-specific DNA methylation of the ICRs is faithfully maintained at the imprinted loci throughout development, even in early embryos where genomes undergo extensive epigenetic reprogramming, including DNA demethylation, to acquire totipotency. We previously found that an ectopically introduced H19 ICR fragment in transgenic mice acquired paternal allele-specific methylation in the somatic cells of offspring, while it was not methylated in sperm, suggesting its gametic and postfertilization modifications are separable events. We hypothesized that this latter activity might contribute to maintenance of the methylation imprint in early embryos. Here we demonstrate that methylation of the paternally inherited transgenic H19 ICR commences soon after fertilization in a maternal Dnmt3a- and Dnmt3L-dependent manner. When its germline methylation was partially obstructed by insertion of insulator sequences, the endogenous, paternal H19 ICR also exhibited postfertilization methylation. Finally, we refined the responsible sequences for this activity in transgenic mice, and found that deletion of the 5' segment of the endogenous paternal H19 ICR decreased its methylation after fertilization, attenuated Igf2 gene expression. These results demonstrate that this segment of the H19 ICR is essential for its de novo post-fertilization DNA methylation, and that this activity contributes to the maintenance of imprinted methylation at the endogenous H19 ICR during early embryogenesis.
[Show abstract][Hide abstract] ABSTRACT: Endogenous bornavirus-like nucleoprotein elements (EBLNs) are sequences within vertebrate genomes derived from reverse transcription and integration of ancient bornaviral nucleoprotein mRNA via the host retrotransposon machinery. While species with EBLNs appear relatively resistant to bornaviral disease, the nature of this association is unclear. We hypothesized that EBLNs could give rise to antiviral interfering RNA in the form of PIWI-interacting RNAs (piRNAs), a class of small RNA known to silence transposons but not exogenous viruses. We found that in both rodents and primates, which acquired their EBLNs independently some 25–40 million years ago, EBLNs are present within piRNA-generating regions of the genome far more often than expected by chance alone (ℙ = 8 × 10−3–6 × 10−8). Three of the seven human EBLNs fall within annotated piRNA clusters and two marmoset EBLNs give rise to bona fide piRNAs. In both rats and mice, at least two of the five EBLNs give rise to abundant piRNAs in the male gonad. While no EBLNs are syntenic between rodent and primate, some of the piRNA clusters containing EBLNs are; thus we deduce that EBLNs were integrated into existing piRNA clusters. All true piRNAs derived from EBLNs are antisense relative to the proposed ancient bornaviral nucleoprotein mRNA. These observations are consistent with a role for EBLN-derived piRNA-like RNAs in interfering with ancient bornaviral infection. They raise the hypothesis that retrotransposon-dependent virus-to-host gene flow could engender RNA-mediated, sequence-specific antiviral immune memory in metazoans analogous to the CRISPR/Cas system in prokaryotes.
[Show abstract][Hide abstract] ABSTRACT: Mouse parthenogenetic haploid embryonic stem cells (ESCs) are pluripotent cells generated from chemically activated oocytes. Haploid ESCs provide an opportunity to study the effect of genetic alterations because of their hemizygotic characteristics. However, their further application for the selection of unique phenotypes remains limited since ideal reporters to monitor biological processes such as cell differentiation are missing. Here, we report the application of CRISPR/Cas9-mediated knock-in of a reporter cassette, which does not disrupt endogenous target genes in mouse haploid ESCs. We first validated the system by inserting the P2A-Venus reporter cassette into the housekeeping gene locus. In addition to the conventional strategy using the Cas9 nuclease, we employed the Cas9 nickase and truncated sgRNAs to reduce off-target mutagenesis. These strategies induce targeted insertions with an efficiency that correlated with sgRNA guiding activity. We also engineered the neural marker gene Sox1 locus and verified the precise insertion of the P2A-Venus reporter cassette and its functionality by monitoring neural differentiation. Our data demonstrate the successful application of the CRISPR/Cas9-mediated knock-in system for establishing haploid knock-in ESC lines carrying gene specific reporters. Genetically modified haploid ESCs have potential for applications in forward genetic screening of developmental pathways.
Full-text · Article · Jun 2015 · Scientific Reports
[Show abstract][Hide abstract] ABSTRACT: Although mammalian cloning by somatic cell nuclear transfer (SCNT) has been established in various species, the low developmental efficiency has hampered its practical applications. Treatment of SCNT-derived embryos with histone deacetylase (HDAC) inhibitors can improve their development, but the underlying mechanism is still unclear. To address this question, we analysed gene expression profiles of SCNT-derived 2-cell mouse embryos treated with trichostatin A (TSA), a potent HDAC inhibitor that is best used for mouse cloning. Unexpectedly, TSA had no effect on the numbers of aberrantly expressed genes or the overall gene expression pattern in the embryos. However, in-depth investigation by gene ontology and functional analyses revealed that TSA treatment specifically improved the expression of a small subset of genes encoding transcription factors and their regulatory factors, suggesting their positive involvement in de novo RNA synthesis. Indeed, introduction of one of such transcription factors, Spi-C, into the embryos at least partially mimicked the TSA-induced improvement in embryonic development by activating gene networks associated with transcriptional regulation. Thus, the effects of TSA treatment on embryonic gene expression did not seem to be stochastic, but more specific than expected, targeting genes that direct development and trigger zygotic genome activation at the 2-cell stage.
Full-text · Article · May 2015 · Scientific Reports
[Show abstract][Hide abstract] ABSTRACT: Primordial germ cells (PGCs) can give rise to pluripotent stem cells known as embryonic germ cells (EGCs) when cultured with basic fibroblast growth factor (bFGF), stem cell factor (SCF), and leukemia inhibitory factor (LIF). Somatic cells can give rise to induced pluripotent stem cells (iPSCs) by introduction of the reprogramming transcription factors Oct4, Sox2, and Klf4. The effects of Sox2 and Klf4 on somatic cell reprogramming can be reproduced using the small molecule compounds, transforming growth factor-β receptor (TGFβR) inhibitor and Kempaullone, respectively. Here we examined the effects of TGFβR inhibitor and Kempaullone on EGC derivation from PGCs. Treatment of PGCs with TGFβR inhibitor and/or Kempaullone generated pluripotent stem cells under standard embryonic stem cell (ESC) culture conditions without bFGF and SCF, which we termed induced EGCs (iEGCs). The derivation efficiency of iEGCs was dependent on the differentiation stage and sex. DNA methylation levels of imprinted genes in iEGCs were reduced, with the exception of the H19 gene. The promoters of genes involved in germline development were generally hypomethylated in PGCs, but three germline genes showed comparable DNA methylation levels among iEGs, ESCs, and iPSCs. These results show that PGCs can be reprogrammed into pluripotent state using small molecule compounds, and that DNA methylation of these germline genes is not maintained in iEGCs. Stem Cells 2014
[Show abstract][Hide abstract] ABSTRACT: The development of a mouse begins after fertilization, the fusion of the ovulated oocyte with a sperm. During the development of the preimplantation embryo, the parental genomes undergo extensive epigenetic reprogramming, including genome-wide DNA demethylation. Shortly after fertilization, DNA methylation of the paternal genome is rapidly removed by a replication-independent process. In contrast, DNA methylation of the maternal genome is reduced gradually, in a replication-dependent manner. Thus, DNA methylation status is quite different between the paternal and maternal genomes in the zygote stage; this difference is called “epigenetic asymmetry.” Recent studies have revealed that the maternal factor Stella (also known as PGC7 and Dppa3, “developmental pluripotency-associated 3”) is required for both the maintenance of DNA methylation status of some imprinted genes and retrotransposons and for the establishment of epigenetic asymmetry in zygotes.
[Show abstract][Hide abstract] ABSTRACT: Primordial germ cells (PGCs) are undifferentiated germ cells in embryos, the fate of which is to become gametes; however, mouse PGCs can easily be reprogrammed into pluripotent embryonic germ cells (EGCs) in culture in the presence of particular extracellular factors, such as combinations of Steel factor (KITL), LIF and bFGF (FGF2). Early PGCs form EGCs more readily than do later PGCs, and PGCs lose the ability to form EGCs by embryonic day (E) 15.5. Here, we examined the effects of activation of the serine/threonine kinase AKT in PGCs during EGC formation; notably, AKT activation, in combination with LIF and bFGF, enhanced EGC formation and caused ∼60% of E10.5 PGCs to become EGCs. The results indicate that the majority of PGCs at E10.5 could acquire pluripotency with an activated AKT signaling pathway. Importantly, AKT activation did not fully substitute for bFGF and LIF, and AKT activation without both LIF and bFGF did not result in EGC formation. These findings indicate that AKT signal enhances and/or collaborates with signaling pathways of bFGF and of LIF in PGCs for the acquisition of pluripotency.
[Show abstract][Hide abstract] ABSTRACT: Primordial germ cells (PGCs) are embryonic germ cell precursors. Specification of PGCs occurs under the influence of mesodermal induction signaling during in vivo gastrulation. Although bone morphogenetic proteins and Wnt signaling play pivotal roles in both mesodermal and PGC specification, the signal regulating PGC specification remains unknown. Coculture of mouse embryonic stem cells (ESCs) with OP9 feeder cells induces mesodermal differentiation in vitro. Using this mesodermal differentiation system, we demonstrated that PGC-like cells were efficiently induced from mouse ESCs by ERK signaling inhibition. Inhibition of ERK signaling by a MEK inhibitor upregulated germ cell marker genes but downregulated mesodermal genes. In addition, the PGC-like cells showed downregulation of DNA methylation and formed pluripotent stem cell colonies upon treatment with retinoic acid. These results show that inhibition of ERK signaling suppresses mesodermal differentiation but activates germline differentiation program in this mesodermal differentiation system. Our findings provide a new insight into the signaling networks regulating PGC specification. Stem Cells 2014
[Show abstract][Hide abstract] ABSTRACT: DNA methylation is a key epigenetic regulator in mammals, and the dynamic balance between methylation and demethylation impacts various processes, from development to disease. DNA methylation is erased during replication when DNA methyltransferase 1 (DNMT1) fails to methylate the daughter strand, in a process known as passive DNA demethylation. We found that the enforced expression of Stella (also known as PGC7, Dppa3), a maternal factor required for the maintenance of DNA methylation in early embryos, induced global DNA demethylation in NIH3T3 cells. This demethylation was caused by the binding of Stella to Np95 (also known as Uhrf1, ICBP90) and the subsequent inhibition of DNMT1 recruitment. Considering that impaired DNA methylation profiles are associated with various developmental or disease phenomena, Stella may be a powerful tool with which to study the biological effects of global DNA hypomethylation.
Full-text · Article · Sep 2014 · Biochemical and Biophysical Research Communications
[Show abstract][Hide abstract] ABSTRACT: HSP90, found in all kingdoms of life, is a major chaperone protein regulating many client proteins. We demonstrated that HSP90α,
one of two paralogs duplicated in vertebrates, plays an important role in the biogenesis of fetal PIWI-interacting RNAs (piRNA),
which act against the transposon activities, in mouse male germ cells. The knockout mutation of Hsp90α resulted in a large reduction in the expression of primary and secondary piRNAs and mislocalization of MIWI2, a PIWI homolog.
Whereas the mutation in Fkbp6 encoding a co-chaperone reduced piRNAs of 28–32 nucleotides in length, the Hsp90α mutation reduced piRNAs of 24–32 nucleotides, suggesting the presence of both FKBP6-dependent and -independent actions of
HSP90α. Although DNA methylation and mRNA levels of L1 retrotransposon were largely unchanged in the Hsp90α mutant testes, the L1-encoded protein was increased, suggesting the presence of post-transcriptional regulation. This study
revealed the specialized function of the HSP90α isofom in the piRNA biogenesis and repression of retrotransposons during the
development of male germ cells in mammals.
Full-text · Article · Sep 2014 · Nucleic Acids Research
[Show abstract][Hide abstract] ABSTRACT: DNA methylation of retrotransposons and imprinted genes is accurately regulated in spermatogenesis. In particular, CpG methylation of long interspersed elements-1 (LINE1 or L1) and intracisternal A-particle (IAP) retrotransposons during spermatogenesis has been well characterized. CpG methylation of the regulatory regions of retrotransposons is acquired during embryonic testis development; however, reductions of DNA methylation in LINE1 and/or IAP and/or Rasgrf1, which is an imprinted gene, are observed in deficient mice of piRNA biogenesis concerning. Here, we describe two methods, bisulfite sequencing and Southern blotting using a methylation-sensitive restriction enzyme, for analysis of DNA methylation of LINE1, IAP, and imprinted genes in mouse testes.
Full-text · Article · Jan 2014 · Methods in molecular biology (Clifton, N.J.)