Toshie Shinagawa

Role of PML and PML-RARα in Mad-Mediated Transcriptional Repression

Abstract Fusion of the promyelocytic leukemia (PML) protein to the retinoic acid receptor-α (RARα) generates the transforming protein of acute promyelocytic leukemias. PML appears to be involved in multiple functions, including apoptosis and transcriptional activation by RAR, whereas PML-RARα blocks these functions of PML. However, the mechanisms of leukemogenesis by PML-RARα remain elusive. Here we show that PML interacts with multiple corepressors (c-Ski, N-CoR, and mSin3A) and histone deacetylase 1, and that this interaction is required for transcriptional repression mediated by the tumor suppressor Mad. PML-RARα has the two corepressor-interacting sites and inhibits Mad-mediated repression, suggesting that aberrant binding of PML-RARα to the corepressor complexes may lead to abrogation of the corepressor function. These mechanisms may contribute to events leading to leukemogenesis.

The sno gene, which encodes a component of the histone deacetylase complex, acts as a tumor suppressor in mice

The Ski and Sno oncoproteins are components of a macromolecular complex containing the co‐repressor N‐CoR/SMRT, mSin3 and histone deacetylase. This complex has been implicated in the transcriptional repression exerted by a number of repressors including nuclear hormone receptors and Mad. Further more, Ski and Sno negatively regulate transforming growth factor‐β (TGF‐β) signaling by recruiting this complex to Smads. Here we show that loss of one copy of sno increases susceptibility to tumorigenesis in mice. Mice lacking sno died at an early stage of embryogenesis, and sno was required for blastocyst formation. Heterozygous (sno+/−) mice developed spontaneous lymphomas at a low frequency and showed an increased level of tumor formation relative to wild‐type mice when challenged with a chemical carcinogen. sno+/− embryonic fibroblasts had an increased proliferative capacity and the introduction of activated Ki‐ras into these cells resulted in neoplastic transformation. The B cells, T cells and embryonic fibroblasts of sno+/− mice had a decreased sensitivity to apoptosis or cell cycle arrest. These findings demonstrate that sno acts as a tumor suppressor at least in some types of cells.

Histone Variants Enriched in Oocytes Enhance Reprogramming to Induced Pluripotent Stem Cells

Expression of Oct3/4, Sox2, Klf4, and c-Myc (OSKM) can reprogram somatic cells into induced pluripotent stem cells (iPSCs). Somatic cell nuclear transfer (SCNT) can also be used for reprogramming, suggesting that factors present in oocytes could potentially augment OSKM-mediated induction of pluripotency. Here, we report that two histone variants, TH2A and TH2B, which are highly expressed in oocytes and contribute to activation of the paternal genome after fertilization, enhance OSKM-dependent generation of iPSCs and can induce reprogramming with Klf4 and Oct3/4 alone. TH2A and TH2B are enriched on the X chromosome during the reprogramming process, and their expression in somatic cells increases the DNase I sensitivity of chromatin. In addition, Xist deficiency, which was reported to enhance SCNT reprogramming efficiency, stimulates iPSC generation using TH2A/TH2B in conjunction with OSKM, but not OSKM alone. Thus, TH2A/TH2B may enhance reprogramming by introducing processes that normally operate in zygotes and during SCNT.

Increased susceptibility to tumorigenesis of ski-deficient heterozygous mice

The c-ski proto-oncogene product (c-Ski) acts as a co-repressor and binds to other co-repressors N-CoR/SMRT and mSin3A which form a complex with histone deacetylase (HDAC). c-Ski mediates the transcriptional repression by a number of repressors, including nuclear hormone receptors and Mad. c-Ski also directly binds to, and recruits the HDAC complex to Smads, leading to inhibition of tumor growth factor-β (TGF-β) signaling. This is consistent with the function of ski as an oncogene. Here we show that loss of one copy of c-ski increases susceptibility to tumorigenesis in mice. When challenged with a chemical carcinogen, c-ski heterozygous mice showed an increased level of tumor formation relative to wild-type mice. In addition, c-ski-deficient mouse embryonic fibroblasts (MEFs) had increased proliferative capacity, whereas overexpression of c-Ski suppressed the proliferation. Furthermore, the introduction of activated Ki-ras into c-ski-deficient MEFs resulted in neoplastic transformation. These findings demonstrate that c-ski acts as a tumor suppressor in some types of cells. The level of cdc25A mRNA, which is down regulated by two tumor suppressor gene products, Rb and Mad, was upregulated in c-ski-deficient MEFs, whereas it decreased by overexpressing c-Ski in MEFs. This is consistent with the fact that c-Ski acts as a co-repressor of Mad and Rb. These results support the view that the decreased activities of Mad and Rb in ski-deficient cells at least partly contribute to enhanced proliferation and susceptibility to tumorigenesis. Human c-ski gene was mapped to a region close to the p73 tumor suppressor gene at the 1p36.3 locus, which is already known to contain multiple uncharacterized tumor suppressor genes.

Reduced Levels of ATF-2 Predispose Mice to Mammary Tumors

ABSTRACT Transcription factor ATF-2 is a nuclear target of stress-activated protein kinases, such as p38, which are activated by various extracellular stresses, including UV light. Here, we show that ATF-2 plays a critical role in hypoxia- and high-cell-density-induced apoptosis and the development of mammary tumors. Compared to wild-type cells, Atf-2−/− mouse embryonic fibroblasts (MEFs) were more resistant to hypoxia- and anisomycin-induced apoptosis but remained equally susceptible to other stresses, including UV. Atf-2−/− and Atf-2+/− MEFs could not express a group of genes, such as Gadd45α, whose overexpression can induce apoptosis, in response to hypoxia. Atf-2−/− MEFs also had a higher saturation density than wild-type cells and expressed lower levels of Maspin, the breast cancer tumor suppressor, which is also known to enhance cellular sensitivity to apoptotic stimuli. Atf-2−/− MEFs underwent a lower degree of apoptosis at high cell density than wild-type cells. Atf-2+/− mice were highly prone to mammary tumors that expressed reduced levels of Gadd45α and Maspin. The ATF-2 mRNA levels in human breast cancers were lower than those in normal breast tissue. Thus, ATF-2 acts as a tumor susceptibility gene of mammary tumors, at least partly, by activating a group of target genes, including Maspin and Gadd45α.

ATF-2 controls transcription of Maspin and GADD45 alpha genes independently from p53 to suppress mammary tumors.

The activating transcription factor, ATF-2, is a target of p38 and JNK that are involved in stress-induced apoptosis. Heterozygous Atf-2 mutant (Atf-2+/−) mice are highly prone to mammary tumors. The apoptosis-regulated gene GADD45α and the breast cancer suppressor gene Maspin, both of which are known to be p53 target genes, are downregulated in the mammary tumors arisen in Atf-2+/− mice. Here, we have analysed how ATF-2 controls the transcription of GADD45α and Maspin. ATF-2 and p53 independently activate the GADD45α transcription. ATF-2 does not directly bind to the GADD45α promoter; instead, it is recruited via Oct-1 and NF-I. ATF-2 simultaneously binds to Oct-1, NF-I and breast cancer suppressor BRCA1 to activate transcription. With regard to Maspin, ATF-2 and p53 directly bind to different sites in the Maspin promoter to independently activate its transcription. Consistent with the observation that ATF-2 and p53 independently activate the transcription of Maspin and GADD45α is that the loss of one copy of p53 shortened the period required for mammary tumor development in Atf-2+/− mice. These studies suggest the functional link between the ATF-2 and the two tumor suppressors BRCA1 and p53.

A B-Myb complex containing clathrin and filamin is required for mitotic spindle function.

B‐Myb is one member of the vertebrate Myb family of transcription factors and is ubiquitously expressed. B‐Myb activates transcription of a group of genes required for the G2/M cell cycle transition by forming the dREAM/Myb–MuvB‐like complex, which was originally identified in Drosophila. Mutants of zebrafish B‐myb and Drosophila myb exhibit defects in cell cycle progression and genome instability. Although the genome instability caused by a loss of B‐Myb has been speculated to be due to abnormal cell cycle progression, the precise mechanism remains unknown. Here, we have purified a B‐Myb complex containing clathrin and filamin (Myb–Clafi complex). This complex is required for normal localization of clathrin at the mitotic spindle, which was previously reported to stabilize kinetochore fibres. The Myb–Clafi complex is not tightly associated with the mitotic spindles, suggesting that this complex ferries clathrin to the mitotic spindles. Thus, identification of the Myb–Clafi complex reveals a previously unrecognized function of B‐Myb that may contribute to its role in chromosome stability, possibly, tumour suppression.

Modulation of M2-type pyruvate kinase activity by the cytoplasmic PML tumor suppressor protein.

The promyelocytic leukemia (PML) tumor suppressor protein accumulates in PML nuclear bodies (PML‐NBs), and can induce growth arrest, cellular senescence and apoptosis. PML has also been localized in the cytoplasm, although its function in this localization remains elusive. A general property of primary cancers is their high glycolytic rate which results from increased glucose consumption. However, the mechanism by which cancer cells up‐regulate glycolysis is not well understood. Here, we have shown that cytoplasmic PML (cPML) directly interacts with M2‐type pyruvate kinase (PKM2), a key regulator of carbon fate. PKM2 determines the proportion of carbons derived from glucose that are used for glycolytic energy production. Over‐expression of PML‐2KA mutant in the cytoplasm, which was generated by mutagenesis of the nuclear localization signals of PML, in MCF‐7 breast cancer cells suppressed PKM2 activity and the accumulation of lactate. PKM2 exists in either an active tetrameric form which has high affinity for its substrate phosphoenolpyruvate (PEP) or a less active dimeric form which has low affinity for its substrate. Over‐expression of PML‐2KA suppressed the activity of the tetrameric form of PKM2, but not the dimeric form. Our findings suggest that cPML plays a role in tumor metabolism through its interaction with PKM2.

Ubiquitination-deubiquitination by the TRIM27-USP7 complex regulates tumor necrosis factor alpha-induced apoptosis.

ABSTRACT Tumor necrosis factor alpha (TNF-α) plays a role in apoptosis and proliferation in multiple types of cells, and defects in TNF-α-induced apoptosis are associated with various autoimmune diseases. Here, we show that TRIM27, a tripartite motif (TRIM) protein containing RING finger, B-box, and coiled-coil domains, positively regulates TNF-α-induced apoptosis. Trim27-deficient mice are resistant to TNF-α–d-galactosamine-induced hepatocyte apoptosis. Trim27-deficient mouse embryonic fibroblasts (MEFs) are also resistant to TNF-α–cycloheximide-induced apoptosis. TRIM27 forms a complex with and ubiquitinates the ubiquitin-specific protease USP7, which deubiquitinates receptor-interacting protein 1 (RIP1), resulting in the positive regulation of TNF-α-induced apoptosis. Our findings indicate that the ubiquitination-deubiquitination cascade mediated by the TRIM27-USP7 complex plays an important role in TNF-α-induced apoptosis.

Sin1 binds to both ATF-2 and p38 and enhances ATF-2- dependent transcription in an SAPK signaling pathway

Yeast Sin1 binds to the Sty1 kinase, a member of the stress‐activated kinases (SAPKs), and is required for stress‐induced phosphorylation and activation of the transcription factor Atf1, a homolog of the vertebrate‐activating transcription factor‐2 (ATF‐2). Here we report that mammalian Sin1 plays an important role in the SAPK signaling pathway by binding to both ATF‐2 and p38. In response to stress, ATF‐2, a member of the ATF/cAMP response element‐binding protein family, is phosphorylated by p38/Jun NH2‐terminal protein kinase and activates the transcription of apoptosis‐related genes. In contrast, in response to serum stimulation, ATF‐2 is phosphorylated via the Ras effector pathway and leads to the induction of growth‐related genes. We found that Sin1 binds directly to both ATF‐2 and p38. Sin1 over‐expression enhanced osmotic stress‐induced phosphorylation of ATF‐2 and ATF‐2‐mediated transcription, whereas knockdown of Sin1 expression by siRNA suppressed these responses. Moreover, a reduction in Sin1 expression suppressed osmotic stress‐induced apoptosis and the expression of Gadd45β, one of the ATF‐2 target genes that is correlated with apoptosis. Decreased Sin1 expression, however, did not affect the serum stimulation‐induced phosphorylation of ATF‐2. Sin1 may contribute to ATF‐2 signaling specificity by acting as a nuclear scaffold.