New insights into the role of Jmjd3 and Utx in axial skeletal formation in mice.

Jmjd3 and Utx are demethylases specific for lysine 27 of histone H3. Previous reports indicate that Jmjd3 is essential for differentiation of various cell types, such as macrophages and epidermal cells in mice, whereas Utx is involved in cancer and developmental diseases in humans and mice, as well as Hox regulation in zebrafish and nematodes. Here, we report that Jmjd3, but not Utx, is involved in axial skeletal formation in mice. A Jmjd3 mutant embryo (Jmjd3Δ18/Δ18), but not a catalytically inactive Utx truncation mutant (Utx-/y), showed anterior homeotic transformation. Quantitative RT-PCR and microarray analyses showed reduced Hox expression in both Jmjd3Δ18/Δ18 embryos and tailbuds, whereas levels of Hox activators, such as Wnt signaling factors and retinoic acid synthases, did not decrease, which suggests that Jmjd3 plays a regulatory role in Hox expression during axial patterning. Chromatin immunoprecipitation analyses of embryo tailbud tissue showed trimethylated lysine 27 on histone H3 to be at higher levels at the Hox loci in Jmjd3Δ18/Δ18 mutants compared with wild-type tailbuds. In contrast, trimethylated lysine 4 on histone H3 levels were found to be equivalent in wild-type and Jmjd3Δ18/Δ18 tailbuds. Demethylase-inactive Jmjd3 mutant embryos showed the same phenotype as Jmjd3Δ18/Δ18 mice. These results suggest that the demethylase activity of Jmjd3, but not that of Utx, affects mouse axial patterning in concert with alterations in Hox gene expression.-Naruse, C., Shibata, S., Tamura, M., Kawaguchi, T., Abe, K., Sugihara, K., Kato, T., Nishiuchi, T., Wakana, S., Ikawa, M., Asano, M. New insights into the role of Jmjd3 and Utx in axial skeletal formation in mice.

Synergy of Eed and Tsix in the repression of Xist gene and X-chromosome inactivation.

X-chromosome inactivation (XCI) depends on the noncoding Xist gene. Xist transcription is negatively regulated by its antisense partner Tsix, whose disruption results in nonrandom XCI in females. However, males can maintain Xist in a repressed state without Tsix, indicating participation of additional factor(s) in the protection of the single male X from inactivation. Here, we provide evidence that the histone methyltransferase Eed is also involved in the process. Male embryonic stem cells with Eed-null and Tsix mutations (X(Delta)Y Eed-/-) showed Xist hyperactivation upon differentiation, whereas cells with either mutation alone did not. Impaired X-linked gene expression was observed in the X(Delta)Y Eed-/- ES cells at the onset of differentiation. The Xist promoter in the X(Delta)Y Eed-/- cells showed elevated histone H3-dimethyl lysine 4 modifications and lowered CpG methylation, which are characteristics of open chromatin. Hence, we identified Eed as an additional major player in the regulation of Xist expression. The synergy of Polycomb group proteins and antisense Tsix transcription in Xist gene regulation explains why males can repress Xist without Tsix.

Alteration of histone tail modifications in the Xist locus in wild-type and Tsix-mutant male embryonic stem cells during differentiation.

The non-coding RNA Xist is indispensable for X chromosome inactivation. Transcriptional control of Xist gene depends on its antisense partner gene Tsix which prevents Xist up-regulation in cis. Previous studies proposed Tsix acts by regulating chromatin structure. Although histone modifications in the Xist locus during differentiation have been described in female embryonic stem (ES) cells, they remain unclear in males. Here we addressed histone modifications in the Xist locus in wild-type and Tsix-mutant male ES cells during differentiation. Their active and repressive modifications were attenuated upon differentiation, while the histone modification profile in males resembled that of females in an undifferentiated condition. These results provide implications in understanding the regulation of Xist gene, as well as other developmentally regulated genes, through chromatin structure.

Tsix transcription- versus RNA-based mechanisms in Xist repression and epigenetic choice.

Recent inquiries have revealed a surprisingly large number (>2500) of naturally occurring antisense transcripts, but their function remains largely undiscovered. A better understanding of antisense mechanisms is clearly needed because of their potentially diverse roles in gene regulation and disease. A well-documented case occurs in X inactivation, the mechanism by which X-linked gene expression is equalized between XX females and XY males. The antisense gene Tsix determines X chromosome choice and represses the noncoding silencer, Xist. In principle, Tsix action may involve RNA, the act of transcription, or local chromatin. Here, we create novel Tsix alleles to distinguish transcription- versus RNA-based mechanisms. When Tsix transcription is terminated before Xist (TsixTRAP), Tsix cannot block Xist upregulation, suggesting the importance of overlapping antisense transcription. To separate the act of transcription from RNA, we knocked in Tsix cDNA in the reverse orientation (Tsix(cDNA)) to restore RNA levels in cis without concurrent transcription across Xist. However, Tsix(cDNA) cannot complement TsixTRAP. Surprisingly, both mutations disrupt choice, indicating that this epigenetic step requires transcription. We conclude that the processed antisense RNA does not act alone and that Tsix function specifically requires antiparallel transcription through Xist. A mechanism of transcription-based feedback regulation is proposed.

Histone H3 Lysine 36 Trimethylation Is Established over the Xist Promoter by Antisense Tsix Transcription and Contributes to Repressing Xist Expression.

One of the two X chromosomes in female mammals is inactivated by the noncoding Xist RNA. In mice, X chromosome inactivation (XCI) is regulated by the antisense RNA Tsix, which represses Xist on the active X chromosome. In the absence of Tsix, PRC2-mediated histone H3 lysine 27 trimethylation (H3K27me3) is established over the Xist promoter. Simultaneous disruption of Tsix and PRC2 leads to derepression of Xist and in turn silencing of the single X chromosome in male embryonic stem cells. Here, we identified histone H3 lysine 36 trimethylation (H3K36me3) as a modification that is recruited by Tsix cotranscriptionally and extends over the Xist promoter. Reduction of H3K36me3 by expression of a mutated histone H3.3 with a substitution of methionine for lysine at position 36 causes a significant derepression of Xist. Moreover, depletion of the H3K36 methylase Setd2 leads to upregulation of Xist, suggesting H3K36me3 as a modification that contributes to the mechanism of Tsix function in regulating XCI. Furthermore, we found that reduction of H3K36me3 does not facilitate an increase in H3K27me3 over the Xist promoter, indicating that additional mechanisms exist by which Tsix blocks PRC2 recruitment to the Xist promoter.

Genomic structure of the mouse 2',5'-oligoadenylate synthetase gene family.

2',5'-Oligoadenylate synthetase (2-5OAS) is one of the interferon (IFN)-induced proteins and mediates the antiviral action of IFN. In human, three classes of 2-5OAS genes (OAS1, OAS2, and OAS3) and one OAS-like gene (OASL) are reported. In mice, however, OAS genes corresponding to human OAS2 and OAS3 have not been identified. In this report, we identified six novel OAS family genes in mice by screening mouse genomic library and expressed sequence tag (EST) database. These genes include three homologs of the human OAS1 and each homologous gene of the human OAS2, OAS3, and OASL, respectively. Each gene displays 52%-65% amino acid identity to the corresponding human homologs. Nine 2-5OAS genes, except for two OASL genes, locate within the 210-kb genomic region and form a cluster. Each novel 2-5OAS gene showed a characteristic expression pattern among different tissues, and all of them were induced by polyinosinic-polycytidylic acid. Biochemical analyses using recombinant proteins produced in Escherichia coli showed that all the novel mouse 2-5OAS molecules have double-stranded RNA (dsRNA) binding ability, but they do not have 2-5OAS activity except for the OAS2 and OAS3 mouse homologs. These results show that there are at least 11 OAS genes, which are classified into four groups, in the mouse.

Acute hepatic failure in IFN-gamma-deficient BALB/c mice after murine coronavirus infection.

We previously showed that an intraperitoneal infection with mouse hepatitis virus (MHV) persists in interferon-gamma (IFN-gamma)-deficient C57BL/6 (B6-GKO) mice and results in subacute fatal peritonitis, which bears a resemblance to feline infectious peritonitis. To examine the role of other host factors in MHV infection in mice, IFN-gamma-deficient mice with a BALB/c background (BALB-GKO) were infected intraperitoneally with MHV and compared with B6-GKO mice. In contrast to B6-GKO mice, BALB-GKO mice died within 1 week due to acute hepatic failure. The viral titer of the liver in BALB-GKO mice was significantly higher than that in B6-GKO mice. All hepatocytes in BALB-GKO mice were necrotic at 5 days post-infection, which was clearly distinct from large but limited lesion in the liver from infected B6-GKO mice. The serum alanine aminotransferase activity of infected BALB-GKO mice were higher than that of B6-GKO mice and was paralleled with the severity of the pathological changes and viral titers in infected mice. Administration of exogenous IFN-gamma to BALB-GKO partially inhibited the acute death. These results indicate that BALB-GKO and B6-GKO mice clearly show different diseases following MHV infection, although wild type counterparts of both mice apparently showed the same clinical course after MHV infection.

Transcript versus transcription?

Numerous sense-antisense gene pairs have been discovered in various organisms. Antisense genes play important roles in establishing parentally imprinted gene expression patterns in mammals. Typically, protein-coding sense genes are reciprocally regulated by their non-coding antisense partners. One example for antisense regulation is the Xist (X-inactive specific transcript) and Tsix gene pair, which is pivotal in X-inactivation. Xist works as a functional RNA molecule that recruits repressive chromatin factors towards one of the female Xs for inactivation. Antisense Tsix transcription negatively regulates Xist and protects one X-chromosome in cis from inactivation by Xist. Albeit, the precise molecular mechanism is still obscure it has been shown that Tsix transcription regulates the chromatin structure by altering histone tail modifications and DNA methylation at the Xist promoter. In addition, Xist and Tsix RNA form an RNA duplexes in vivo and are processed to small RNAs, which have a potential regulatory function. Here we review the latest findings and based on ample experimental data consider models for antisense-mediated gene regulation in X-inactivation.

Characterization and quantitation of differential Tsix transcripts: implications for Tsix function.

In dosage compensation of female mammals, the accumulation of Xist RNA initiates silencing of one X-chromosome. Xist action is repressed by the antisense gene, Tsix, whose full-length RNA product is complementary to Xist RNA in mice. While previous work showed that Tsix transcription blocks the accumulation of Xist RNA, it is still unclear whether this repression requires the antisense RNA product or whether the antisense transcriptional movement is sufficient. A better understanding of potential mechanisms requires elucidation of Tsix RNA structure and determination of Tsix RNA copy number relative to that of Xist RNA. Previous work indicated that at least some of murine Tsix is spliced and that human TSIX truncates within the 3' end of XIST. Here, further characterization and quantitation of murine Tsix RNA reveal three new findings: first, in undifferentiated embryonic stem cells, Tsix RNA is present at 10-100-fold molar excess over Xist RNA. Second, only 30-60% of Tsix RNA is spliced at known exon-intron junctions. The nearly equal abundance of spliced and unspliced species leaves open possible roles for both isoforms. Finally, Tsix is spliced heterogeneously at the 5' end and most detectable splice variants exhibit only a 1.9 kb region of complementarity between sense and antisense RNAs. Implications for Tsix's possible mechanisms of action are discussed.

Inhibition of B16 melanoma experimental metastasis by interferon-gamma through direct inhibition of cell proliferation and activation of antitumour host mechanisms.

Interferon-gamma (IFN-gamma) has pleiotropic activities other than its antivirus action, including cell growth inhibition, natural killer (NK) cell and cytotoxic T lymphocyte (CTL) activation, and angiogenesis inhibitory activity, and these activities are supposed to be involved in its antitumour activity. However, it has not been completely elucidated which activity is mainly involved in the tumour suppression in vivo. In this study, we analysed inhibitory mechanisms of endogenous IFN-gamma against B16 melanoma experimental metastasis. After intravenous injection of tumour cells, tumour deposits in the lungs and liver were increased and life span was shorter in IFN-gamma(-/-) mice, indicating important roles for IFN-gamma in antitumour mechanisms. Interestingly, tumour deposits were not increased in IFN-gamma receptor (R)(-/-) mice. Furthermore, only low levels of cell-mediated immunity against the tumour and activation of NK cells were observed, indicating that antimetastatic effects of IFN-gamma is not mediated by host cells. The survival period of B16 melanoma-bearing IFN-gamma R(-/-) mice was, however, shorter than wild-type mice. These observations suggest that IFN-gamma prevents B16 melanoma experimental metastasis by directly inhibiting the cell growth, although antitumour host functions may also be involved in a later phase.