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Summary We here demonstrate that microsatellite (MS) alterations are elevated in both mouse and human induced pluripotent stem cells (iPSCs), but importantly we have now identified a type of human iPSC in which these alterations are considerably reduced. We aimed in our present analyses to profile the InDels in iPSC/ntESC genomes, especially in MS regions. To detect somatic de novo mutations in particular, we generated 13 independent reprogramed stem cell lines (11 iPSC and 2 ntESC lines) from an identical parent somatic cell fraction of a C57BL/6 mouse. By using this cell set with an identical genetic background, we could comprehensively detect clone-specific alterations and, importantly, experimentally validate them. The effectiveness of employing sister clones for detecting somatic de novo mutations was thereby demonstrated. We then successfully applied this approach to human iPSCs. Our results require further careful genomic analysis but make an important inroad into solving the issue of genome abnormalities in iPSCs., Graphical abstract, Highlights • InDels and microsatellite alterations are elevated in iPSCs • These alterations are reduced in human iPSCs derived from cord blood erythroblasts • Employing sister clones is an effective way to detect somatic de novo mutations, In this article, Abe and colleagues conclusively demonstrate that InDels and microsatellite alterations are elevated in reprogrammed pluripotent stem cells, both mouse and human, by employing sister clones and conducting large-scale validation experiments. Furthermore, they show that these alterations are considerably reduced in human iPSCs derived from cord blood erythroblasts.

A number of point mutations have been identified in reprogrammed pluripotent stem cells such as iPSCs and ntESCs. The molecular basis for these mutations has remained elusive however, which is a considerable impediment to their potential medical application. Here we report a specific stage at which iPSC generation is not reduced in response to ionizing radiation, i.e. radio-resistance. Quite intriguingly, a G1/S cell cycle checkpoint deficiency occurs in a transient fashion at the initial stage of the genome reprogramming process. These cancer-like phenomena, i.e. a cell cycle checkpoint deficiency resulting in the accumulation of point mutations, suggest a common developmental pathway between iPSC generation and tumorigenesis. This notion is supported by the identification of specific cancer mutational signatures in these cells. We describe efficient generation of human integration-free iPSCs using erythroblast cells, which have only a small number of point mutations and INDELs, none of which are in coding regions., Point mutations have been found in induced pluripotent stem cells (iPSCs) but when they arise is unclear. Here, the authors show that a G1/S cell cycle checkpoint deficiency transiently occurs early in genome reprogramming, suggesting a common developmental pathway between iPSC and tumorigenesis, and generate genetic burden-free human iPSCs.

Genome instability is a hallmark of cancer cells and dysregulation or defects in DNA repair pathways cause genome instability and are linked to inherited cancer predisposition syndromes. Ionizing radiation can cause immediate effects such as mutation or cell death, observed within hours or a few days after irradiation. Ionizing radiation also induces delayed effects many cell generations after irradiation. Delayed effects include hypermutation, hyper-homologous recombination, chromosome instability and reduced clonogenic survival (delayed death). Delayed hyperrecombination (DHR) is mechanistically distinct from delayed chromosomal instability and delayed death. Using a green fluorescent protein (GFP) direct repeat homologous recombination system, time-lapse microscopy and colony-based assays, we demonstrate that DHR increases several-fold in response to low-LET X rays and high-LET carbon-ion radiation. Time-lapse analyses of DHR revealed two classes of recombinants not detected in colony-based assays, including cells that recombined and then senesced or died. With both low- and high-LET radiation, DHR was evident during the first two weeks postirradiation, but resolved to background levels during the third week. The results indicate that the risk of radiation-induced genome destabilization via DHR is time limited, and suggest that there is little or no additional risk of radiation-induced genome instability mediated by DHR with high-LET radiation compared to low-LET radiation.

Summary A large number of point mutations have been identified in induced pluripotent stem cell (iPSC) genomes to date. Whether these mutations are associated with iPSC generation is an important and controversial issue. In this study, we approached this critical issue in different ways, including an assessment of iPSCs versus embryonic stem cells (ESCs), and an investigation of variant allele frequencies and the heterogeneity of point mutations within a single iPSC clone. Through these analyses, we obtained strong evidence that iPSC-generation-associated point mutations occur frequently in a transversion-predominant manner just after the onset of cell lineage conversion. The heterogeneity of the point mutation profiles within an iPSC clone was also revealed and reflects the history of the emergence of each mutation. Further, our results suggest a possible approach for establishing iPSCs with fewer point mutations., Graphical Abstract, Highlights • iPSCs versus ESCs—an obvious difference in the frequency and mode of point mutations • A large number of iPSC generation-associated, not preexisting, point mutations • Heterogeneity of point mutation profiles in an iPSC clone • No common point mutations among iPSC clones, Sugiura et al. have obtained strong evidence that iPSC generation-associated point mutations occur frequently in a transversion-predominant manner just after the onset of cell lineage conversion. The heterogeneity of the point mutation profiles within an iPSC clone was also revealed and reflects the history of the emergence of each mutation.

Xist RNA responsible for X inactivation is one of the most important epigenetic players for embryogenesis of female mammals. Of the several repeats conserved in Xist RNA, the A-repeat has been shown to be essential for its silencing function in differentiating ES cells. Here, we introduced a new Xist allele into the mouse, which produces mutated Xist RNA lacking the A-repeat (XistCAGΔ5'). XistCAGΔ5' RNA expressed in the embryo coated the X chromosome but failed to silence it. Although imprinted X inactivation was substantially compromised upon paternal transmission, allele-specific RNA-seq in the trophoblast revealed that XistCAGΔ5' RNA still retained some silencing ability. Furthermore, the failure of imprinted X inactivation had more significant impacts than expected on gene expression genome-wide. It is likely that dosage compensation is required for not only equalizing the X-linked gene expression between the sexes but also proper global gene regulation in differentiated female somatic cells.

Immune rejection may limit the therapeutic use of induced pluripotent stem cells (iPSCs); here, terminally differentiated mouse iPSCs are shown to generate negligible immune rejection in their host. Induced pluripotent stem cells (iPSCs) derived from a patient's own somatic cells could have great therapeutic potential. The hope is that iPSC-derived differentiated cells would avoid any immunogenic responses. In this study, Masumi Abe and colleagues assess the immunogenicity of skin and bone marrow tissues derived from a large set of isogenic mouse embryonic stem cell and iPSC lines. Their results are consistent with negligible immune rejection by the host. The advantages of using induced pluripotent stem cells (iPSCs) instead of embryonic stem (ES) cells in regenerative medicine centre around circumventing concerns about the ethics of using ES cells and the likelihood of immune rejection of ES-cell-derived tissues1,2. However, partial reprogramming and genetic instabilities in iPSCs3,4,5,6 could elicit immune responses in transplant recipients even when iPSC-derived differentiated cells are transplanted. iPSCs are first differentiated into specific types of cells in vitro for subsequent transplantation. Although model transplantation experiments have been conducted using various iPSC-derived differentiated tissues7,8,9,10 and immune rejections have not been observed, careful investigation of the immunogenicity of iPSC-derived tissue is becoming increasingly critical, especially as this has not been the focus of most studies done so far. A recent study reported immunogenicity of iPSC- but not ES-cell-derived teratomas11 and implicated several causative genes. Nevertheless, some controversy has arisen regarding these findings12. Here we examine the immunogenicity of differentiated skin and bone marrow tissues derived from mouse iPSCs. To ensure optimal comparison of iPSCs and ES cells, we established ten integration-free iPSC and seven ES-cell lines using an inbred mouse strain, C57BL/6. We observed no differences in the rate of success of transplantation when skin and bone marrow cells derived from iPSCs were compared with ES-cell-derived tissues. Moreover, we observed limited or no immune responses, including T-cell infiltration, for tissues derived from either iPSCs or ES cells, and no increase in the expression of the immunogenicity-causing Zg16 and Hormad1 genes in regressing skin and teratoma tissues. Our findings suggest limited immunogenicity of transplanted cells differentiated from iPSCs and ES cells.

In germ cells, early embryos, and stem cells of animals, PIWI-interacting RNAs (piRNAs) have an important role in silencing retrotransposons, which are vicious genomic parasites, through transcriptional and post-transcriptional mechanisms. To examine whether the piRNA pathway can be used to silence genes of interest in germ cells, we have generated knock-in mice in which a foreign DNA fragment was inserted into a region generating pachytene piRNAs. The knock-in sequence was transcribed, and the resulting RNA was processed to yield piRNAs in postnatal testes. When reporter genes possessing a sequence complementary to portions of the knock-in sequence were introduced, they were greatly repressed after the time of pachytene piRNA generation. This repression mainly occurred at the post-transcriptional level, as degradation of the reporter RNAs was accelerated. Our results show that the piRNA pathway can be used as a tool for sequence-specific gene silencing in germ cells and support the idea that the piRNA generating regions serve as traps for retrotransposons, enabling the host cell to generate piRNAs against active retrotransposons.

X chromosome inactivation (X-inactivation) in female mammals is triggered by differential upregulation of the Xist gene on one of the two X chromosomes and subsequent coating of the X in cis with its non-coding transcripts. Although targeted mutation has clearly shown that Xist is essential for X-inactivation in cis, the molecular mechanism by which Xist RNA induces chromosome silencing is largely unknown. Here, we demonstrate that an Xist mutant generated previously in mouse by gene targeting, Xist IVS , is unique in that it partially retains the capacity to silence the X chromosome. Although Xist IVS is differentially upregulated and its mutated transcript coats the X chromosome in cis in embryonic and extra-embryonic tissues, X-inactivation thus initiated does not seem to be fully established. The state of such incomplete inactivation is probably unstable and the mutated X is apparently reactivated in a subset of extra-embryonic tissues and, perhaps, early epiblastic cells. Xist IVS , which can be referred to as a partial loss-of-function mutation, would provide an opportunity to dissect the molecular mechanism of Xist RNA-mediated chromosome silencing.

Genomic imprinting causes parental origin-specific monoallelic gene expression through differential DNA methylation established in the parental germ line. However, the mechanisms underlying how specific sequences are selectively methylated are not fully understood. We have found that the components of the PIWI-interacting RNA (piRNA) pathway are required for de novo methylation of the differentially methylated region (DMR) of the imprinted mouse Rasgrf1 locus, but not other paternally imprinted loci. A retrotransposon sequence within a noncoding RNA spanning the DMR was targeted by piRNAs generated from a different locus. A direct repeat in the DMR, which is required for the methylation and imprinting of Rasgrf1, served as a promoter for this RNA. We propose a model in which piRNAs and a target RNA direct the sequence-specific methylation of Rasgrf1.

MITOPLD is a member of the phospholipase D superfamily proteins conserved among diverse species. Zucchini (Zuc), the Drosophila homolog of MITOPLD, has been implicated in primary biogenesis of Piwi-interacting RNAs (piRNAs). By contrast, MITOPLD has been shown to hydrolyze cardiolipin in the outer membrane of mitochondria to generate phosphatidic acid, which is a signaling molecule. To assess whether the mammalian MITOPLD is involved in piRNA biogenesis, we generated Mitopld mutant mice. The mice display meiotic arrest during spermatogenesis, demethylation and derepression of retrotransposons, and defects in primary piRNA biogenesis. Furthermore, in mutant germ cells, mitochondria and the components of the nuage, a perinuclear structure involved in piRNA biogenesis/function, are mislocalized to regions around the centrosome, suggesting that MITOPLD may be involved in microtubule-dependent localization of mitochondria and these proteins. Our results indicate a conserved role for MITOPLD/Zuc in the piRNA pathway and link mitochondrial membrane metabolism/signaling to small RNA biogenesis.

Although the induction of genome integration-free induced pluripotent stem cells (iPSCs) has been reported, c-Myc was still required for the efficient generation of these cells. Herein, we report mouse strain-dependent differences in the c-Myc dependence for iPSC generation and the successful generation of genome integration-free iPSCs without c-Myc transduction using C57BL/6 mouse embryonic fibroblasts. We performed 49 independent experiments and obtained a total of 24 iPSC clones, including 18 genome integration-free iPSC clones. These iPSCs were indistinguishable from embryonic stem cells and from iPSCs generated using other methods. Furthermore, the generation of three-factor iPSCs free of virus vectors revealed the contribution of c-Myc to the genomic integration of external genes. C57BL/6 is an inbred mouse strain with substantial advantages for use in genetic and molecular biological studies due to its use in the whole mouse genome sequencing project. Thus, the present series of C57BL/6 iPSCs generated by various procedures will serve as a valuable resource for future genetic studies of iPSC generation.

The emergence of induced pluripotent stem cells (iPSCs) from an ancestral somatic cell is one of the most important processes underlying their generation, but the mechanism has yet to be identified. This is principally because these cells emerge at a low frequency, about 0.1% in the case of fibroblasts, and in a stochastic manner. In our current study, we succeeded in identifying ancestral fibroblasts and the subsequent processes leading to their conversion to iPSCs. The ancestral fibroblasts were found to divide several times in a morphologically symmetric manner, maintaining a fibroblastic shape, and then gradually transform into embryonic stem-like cells. Interestingly, this conversion occurred within 48 hours after gene introduction in most iPSC generations. This is the first report to directly observe a cell lineage conversion of somatic cells to stem cells and provides a critical new insight into the “black box” of iPSCs, that is, the first three days of their generation.

Expression of Xist, which triggers X inactivation, is negatively regulated in cis by an antisense gene, Tsix, transcribed along the entire Xist gene. We recently demonstrated that Tsixsilences Xist through modification of the chromatin structure in the Xist promoter region. This finding prompted us to investigate the role of antisense transcription across the Xist promoter in Tsix-mediated silencing. Here, we prematurely terminated Tsix transcription before the Xist promoter and addressed its effect on Xist silencing in mouse embryos. We found that although 93% of the region encoding Tsix was transcribed, truncation of Tsix abolished the antisense regulation of Xist. This resulted in a failure to establish the repressive chromatin configuration at the Xist promoter on the mutated X, including DNA methylation and repressive histone modifications, especially in extraembryonic tissues. These results suggest a crucial role for antisense transcription across the Xist promoter in Xist silencing.

Differential induction of the X-linked non-coding Xist gene is a key event in the process of X inactivation occurring in female mammalian embryos. Xist is negatively regulated in cis by its antisense gene Tsix through modification of the chromatin structure. The maternal Xist allele, which is normally silent in the extraembryonic lineages, is ectopically activated when Tsix is disrupted on the same chromosome, and subsequently the maternal X chromosome undergoes inactivation in the extraembryonic lineages even in males. However, it is still unknown whether the single Tsix-deficient X chromosome (XΔTsix) in males is also inactivated in the embryonic lineage. Here, we show that both male and female embryos carrying a maternally derived XΔTsix could survive if the extraembryonic tissues were complemented by wild-type tetraploid cells. In addition, Xist on the XΔTsix was properly silenced and methylated at CpG sites in adult male somatic cells. These results indicate that the embryonic lethality caused by the maternal XΔTsix is solely attributable to the defects in the extraembryonic lineages. XΔTsix does not seem to undergo inactivation in the embryonic lineage in males, suggesting the presence of a Tsix-independent silencing mechanism for Xist in the embryonic lineage.

Mutations in the Recql4 gene are very likely responsible for a subset of Rothmund-Thomson syndrome (RTS) cases, but until now there has been no animal model to confirm this. Knockout mice in which the Recql4 gene is disrupted at exons 5-8 exhibit embryonic lethality at embryonic day 3.5-6.5. We generated a helicase activity-inhibited mouse by deleting exon 13 of Recql4, which is one of the coding exons of the consensus RecQ-helicase domain. This domain is the primary site of mutations that have been identified in RTS patients. The exon 13-deleted Recql4-deficient mice are viable, but exhibit severe growth retardation and abnormalities in several tissues, and embryonic fibroblasts show a defect in cell proliferation. Abnormalities in the Recql4-deficient mice are similar to those in RTS patients, suggesting that defects in the Recql4 gene may indeed be responsible for RTS. We speculate that the loss of Recql4 helicase activity results in the prematurely aged appearance observed in some RecQ helicase diseases.

The dosage difference of X-linked genes between the sexes in mammals is compensated for by genetic inactivation of one of the X chromosomes in XX females. A noncoding RNA transcribed from the Xist gene at the onset of X chromosome inactivation coats the X chromosome in cis and induces chromosome-wide heterochromatinization. Here, we report a new Xist allele (Xist(CAG)) driven by a CAG promoter, which is known to be constitutively active in many types of cells. The paternal transmission of Xist(CAG) resulted in the preferential inactivation of the targeted paternal X (Xp) not only in the extra-embryonic but also the embryonic lineage, whereas maternal transmission ended with embryonic lethality at the early postimplantation stage with a phenotype that resembled mutant embryos carrying a maternal deficiency in Tsix, an antisense negative regulator of Xist, in both sexes. Interestingly, we found that the upregulation of Xist(CAG) in preimplantation embryos temporally differed depending on its parental origin: its expression started at the 4- to 8-cell stages when paternally inherited, and Xist(CAG) was upregulated at the blastocyst stage when maternally inherited. This might indicate that the Xist locus on Xp is permissive to transcription, but the Xist locus on the maternal X (Xm) is not. We extrapolated from these findings that the maternal Xist allele might manifest a chromatin structure inaccessible by transcription factors relative to the paternal allele. This might underlie the mechanism for the maternal repression of Xist at the early cleavage stage when Tsix expression has not yet occurred on Xm.

Ataxia-telangiectasia (AT) is an autosomal recessive disease. The relevant gene has been cloned and designated ATM. We studied the expression of both ATM mRNA and the ATM protein in unirradiated and X-irradiated EBV (Epstein-Barr virus)-transformed lymphoblastoid cell lines (LCLs) derived from donors who were normal (ATM + / + ), AT heterozygotes (ATM + / - ), or AT homozygotes (ATM - / - ), respectively. In ATM + / + LCLs, the levels of ATM mRNA were found to have increased by approximately 1.5-fold within 1 h of exposure to 10 Gy of X-rays, while the ATM protein levels had increased by 1.5- to 2.0-fold within 2 to 3 h of irradiation. The wild-type mRNA and protein levels both returned to their basal values fairly quickly after this time. The results obtained with the ATM + / - LCLs were quite different, however: neither the mRNA nor protein levels were found to have increased as a consequence of X-irradiation in any ATM + / - LCL. Twelve of the mutations in the ATM - / - LCLs we used were truncating mutations, and we suspected that the corresponding truncated ATM proteins would be too labile to be detected by western blot analysis. However, five of the ATM - / - LCLs produced mutant ATM proteins that were identical in molecular weight to the wild-type ATM protein. When cells from three of these five clones were exposed to X-rays, transcription of the mutant ATM genes appeared to reduce somewhat, as were the levels of protein being produced. These results suggest that the normal ATM gene responds to ionizing radiation by up-regulating its activity, whereas none of the mutant ATM genes we studied were able to respond in this way.

c-Myc transduction has been considered previously to be nonessential for induced pluripotent stem cell (iPSC) generation. In this study, we investigated the effects of c-Myc transduction on the generation of iPSCs from an inbred mouse strain using a genome integration-free vector to exclude the effects of the genetic background and the genomic integration of exogenous genes. Our findings reveal a clear difference between iPSCs generated using the four defined factors including c-Myc (4F-iPSCs) and those produced without c-Myc (3F-iPSCs). Molecular and cellular analyses did not reveal any differences between 3F-iPSCs and 4F-iPSCs, as reported previously. However, a chimeric mice formation test indicated clear differences, whereby few highly chimeric mice and no germline transmission was observed using 3F-iPSCs. Similar differences were also observed in the mouse line that has been widely used in iPSC studies. Furthermore, the defect in 3F-iPSCs was considerably improved by trichostatin A, a histone deacetyl transferase inhibitor, indicating that c-Myc plays a crucial role in iPSC generation through the control of histone acetylation. Indeed, low levels of histone acetylation were observed in 3F-iPSCs. Our results shed new light on iPSC generation mechanisms and strongly recommend c-Myc transduction for preparing high-quality iPSCs.

X-inactivation in female mammals is triggered by the association of non-coding Xist RNA in cis with the X chromosome. Although it has been suggested that the A-repeat located in the proximal part of the Xist RNA is required for chromosomal silencing in ES cells, its role in mouse has not yet been addressed. Here, we deleted the A-repeat in mouse and studied its effects on X-inactivation during embryogenesis. The deletion,when paternally transmitted, caused a failure of imprinted X-inactivation in the extraembryonic tissues, demonstrating the essential role of the A-repeat in X-inactivation in the mouse embryo. Unexpectedly, the failure of X-inactivation was caused by a lack of Xist RNA rather than by a defect in the silencing function of the mutated RNA, which we expected to be expressed from the mutated X. Interestingly, the normally silent paternal copy of Tsix, which is an antisense negative regulator of Xist,was ectopically activated in the preimplantation embryo. Furthermore, CpG sites in the promoter region of paternal Xist, which are essentially unmethylated in the extraembryonic tissues of the wild-type female embryo,acquire a significant level of methylation on the mutated paternal X. These findings demonstrate that the DNA sequence deleted on the mutated X, most probably the A-repeat, is essential as a genomic element for the appropriate transcriptional regulation of the Xist/Tsix loci and subsequent X-inactivation in the mouse embryo.

Dosage differences of X-linked genes between male and female mammals are compensated for by a mechanism known as X-inactivation, and the noncoding Xist gene plays a crucial role in this process. The expression of Xist is regulated in cis by its noncoding antisense gene, Tsix, whose transcripts (though a fraction of them stay unspliced),are processed like common proteincoding RNAs. It has been suggested that certain classes of sense-antisense pairs of RNA are causally involved in not only gene regulation but also higher order chromatin structure in various organisms. In fact, recent studies demonstrated that Tsix modulates Xist expression through modification of the chromatin structure. It is still unknown, however, whether the RNA product is important for the function of Tsix or whether the antisense transcription is sufficient. To obtain insight into this issue, we eliminated the splicing products of Tsix in the mouse and explored the effects of this elimination on Tsix-mediated Xist silencing. To our surprise, the Xist locus was stably repressed on the X carrying the splicing-defective Tsix allele. Moreover, the repressive chromatin configuration was properly established at the Xist locus. These unexpected results indicate that the splicing products are dispensable for Tsix-mediated Xist silencing.

Deletion mutations were efficiently recovered in mouse liver after total-body irradiation with X rays by using a transgenic mouse "gpt-delta" system that harbored a lambda EG10 shuttle vector with the red and gam genes for Spi- (sensitive to P2 lysogen interference) selection. We incorporated this system into homozygous Atm-knockout mice as a model of the radiosensitive hereditary disease ataxia telangiectasia (AT). Lambda phages recovered from the livers of X-irradiated mice with the Atm+/+ genotype showed a dose-dependent increase in the Spi- mutant frequency up to sixfold at 50 Gy over the unirradiated control of 2.8x10(-6). The livers from Atm-/- mice yielded a virtually identical dose-response curve for X rays with a background fraction of 2.4x10(-6). Structural analyses revealed no significant difference in the proportion of -1 frameshifts and larger deletions between Atm+/+ and Atm-/- mice, although larger deletions prevailed in X-ray-induced Spi- mutants irrespective of Atm status. While a possible defect in DNA repair after irradiation has been strongly indicated in the literature for nondividing cultured cells in vitro from AT patients, the Atm disruption does not significantly affect radiation mutagenesis in the stationary mouse liver in vivo.

Camptothecin and its derivatives are specific inhibitors of eukaryotic topoisomerase I (top1) and are active in cancer patients against a variety of refractory solid tumors and leukemia. Purpose: The present study further investigated the relationship between multidrug resistance (MDR) mediated by P-glycoproteinMDR and potential resistance to camptothecin derivatives using two experimental systems: (1) MDR KB-V1 cells selected for vinblastine resistance, and (2) NIH3T3 cells transfected with a plasmid expressing wildtype P-glycoproteinMDR multidrug transporter (NIH-MDR-G185). Results: We found that both KBV-1 and NIH-MDR-G185 cells were resistant to topotecan, and that topotecan-induced cleavable complexes were reduced in KB-V1 cells, consistent with a role of P-glycoproteinMDR in cellular resistance to topotecan. By contrast, no significant resistance to camptothecin, 9-aminocamptothecin, 10, 11-methylenedioxycamptothecin, or SN-38 (the active metabolite of CPT-11) was observed in NIH-MDR-G185 cells, while KB-V1 cells were cross-resistant to these compounds but produced cleavable complexes similar to those produced by parental KB-3-1 cells. Conclusions: These results suggest that topotecan is the only camptothecin tested with significant susceptibility to MDR in cell culture, and that multidrug resistant cells such as KBV1 probably exhibit additional resistance mechanisms to camptothecins besides P-glycoproteinMDR overexpression.

SummaryX inactivation is controlled by Xist and its antisense gene, Tsix, neither of which encodes a protein. Xist is essential for X inactivation to occur in cis, and its differential expression is a key event in the initiation of X inactivation. Xist and Tsix are imprinted in the extraembryonic tissues of mouse embryos so that they are expressed from the paternal and maternal X, respectively, resulting in the preferential inactivation of the paternal X. Targeted disruption of Tsix causes ectopic expression of Xist, suggesting that Tsix negatively regulates Xist in cis. However, the molecular mechanism of this antisense regulation remains unknown. Here, we demonstrate that Tsix transcriptionally silences Xist in both embryonic and extraembryonic tissues of mouse embryos. Moreover, we show that disruption of Tsix impairs establishment of repressive epigenetic modifications and chromatin structure at the Xist locus. We propose that Tsix silences Xist through modification of the chromatin structure.

CENP-U (CENP-50) is a component of the CENP-O complex, which includes CENP-O, CENP-P, CENP-Q, CENP-R, and CENP-U and is constitutively localized at kinetochores throughout the cell cycle in vertebrates. Although CENP-U deficiency results in some mitotic defects in chicken DT40 cells, CENP-U-deficient chicken DT40 cells are viable. To examine the functional roles of CENP-U in an organism-dependent context, we generated CENP-U-deficient mice. The CENP-U-deficient mice died during early embryogenesis (approximately E7.5). Thus, conditional CENP-U-deficient mouse ES cells were generated to analyze CENP-U-deficient phenotypes at the cell level. When CENP-U was disrupted in the mouse ES cells, all CENP-O complex proteins disappeared from kinetochores. In contrast, other kinetochore proteins were recruited in CENP-U-deficient mouse ES cells as CENP-U-deficient DT40 cells. However, the CENP-U-deficient ES cells died after exhibiting abnormal mitotic behavior. Although CENP-U was essential for cell viability during mouse early embryogenesis, CENP-U-deficient mouse embryonic fibroblast cells were viable, similar to the DT40 cells. Thus, although both DT40 and ES cells with CENP-U deficiency have similar mitotic defects, cellular responses to mitotic defects vary among different cell types. Electronic supplementary material The online version of this article (doi:10.1007/s10577-014-9404-1) contains supplementary material, which is available to authorized users.