研究者詳細 - 青山　和正

論文 - 青山　和正

分割表示 45 件中 21 - 40 件目／全件表示 >>

Ezh2 loss propagates hypermethylation at T cell differentiation-regulating genes to promote leukemic transformation

Wang, C., Oshima, M., Sato, D., Matsui, H., Kubota, S., Aoyama, K., Nakajima-Takagi, Y., Koide, S., Matsubayashi, J., Mochizuki-Kashio, M., Nakano-Yokomizo, T., Bai, J., Nagao, T., Kanai, A., Iwama, A. and Sashida, G.

J Clin Invest 128 （ 9 ） 3872 - 3886 2018年08月

研究論文（学術雑誌）, 査読有り, ISSN 0021-9738

Early T cell precursor acute lymphoblastic leukemia (ETP-ALL) is a new pathological entity with poor outcomes in T cell ALL (T-ALL) that is characterized by a high incidence of loss-of-function mutations in polycomb repressive complex 2 (PRC2) genes. We generated a mouse model of ETP-ALL by deleting Ezh2, one of the PRC2 genes, in p53-null hematopoietic cells. The loss of Ezh2 in p53-null hematopoietic cells impeded the differentiation of ETPs and eventually induced ETP-ALL-like disease in mice, indicating that PRC2 functions as a bona fide tumor suppressor in ETPs. A large portion of PRC2 target genes acquired DNA hypermethylation of their promoters following reductions in H3K27me3 levels upon the loss of Ezh2, which included pivotal T cell differentiation-regulating genes. The reactivation of a set of regulators by a DNA-demethylating agent, but not the transduction of single regulator genes, effectively induced the differentiation of ETP-ALL cells. Thus, PRC2 protects key T cell developmental regulators from DNA hypermethylation in order to keep them primed for activation upon subsequent differentiation phases, while its insufficiency predisposes ETPs to leukemic transformation. These results revealed a previously unrecognized epigenetic switch in response to PRC2 dysfunction and provide the basis for specific rational epigenetic therapy for ETP-ALL with PRC2 insufficiency.

Access to Document (DOI)

Dual inhibition of EZH1/2 breaks the quiescence of leukemia stem cells in acute myeloid leukemia

S. Fujita, D. Honma, N. Adachi, K. Araki, E. Takamatsu, T. Katsumoto, K. Yamagata, K. Akashi, K. Aoyama, A. Iwama, I. Kitabayashi

Leukemia （Nature Publishing Group） 32 （ 4 ） 855 - 864 2018年04月

研究論文（学術雑誌）, 査読有り, ISSN 1476-5551

Acute myeloid leukemia (AML) is an aggressive and lethal blood cancer originating from rare populations of leukemia stem cells (LSCs). AML relapse after conventional chemotherapy is caused by a remaining population of drug-resistant LSCs. Selective targeting of the chemoresistant population is a promising strategy for preventing and treating AML relapse. Polycomb repressive complex 2 (PRC2) trimethylates histone H3 at lysine 27 to maintain the stemness of LSCs. Here, we show that quiescent LSCs expressed the highest levels of enhancer of zeste (EZH) 1 and EZH2, the PRC2 catalytic subunits, in the AML hierarchy, and that dual inactivation of EZH1/2 eradicated quiescent LSCs to cure AML. Genetic deletion of Ezh1/2 in a mouse AML model induced cell cycle progression of quiescent LSCs and differentiation to LSCs, eventually eradicating AML LSCs. Quiescent LSCs showed PRC2-mediated suppression of Cyclin D, and Cyclin D-overexpressing AML was more sensitive to chemotherapy. We have developed a novel EZH1/2 dual inhibitor with potent inhibitory activity against both EZH1/2. In AML mouse models and patient-derived xenograft models, the inhibitor reduced the number of LSCs, impaired leukemia progression, and prolonged survival. Taken together, these results show that dual inhibition of EZH1/2 is an effective strategy for eliminating AML LSCs.

Access to Document (DOI)

Internal deletion of BCOR reveals a tumor suppressor function for BCOR in T lymphocyte malignancies

Tanaka, T., Nakajima-Takagi, Y., Aoyama, K., Tara, S., Oshima, M., Saraya, A., Koide, S., Si, S., Manabe, I., Sanada, M., Nakayama, M., Masuko, M., Sone, H., Koseki, H. and Iwama, A.

J Exp Med 214 （ 10 ） 2901 - 2913 2017年10月

研究論文（学術雑誌）, 筆頭著者, 査読有り, ISSN 0022-1007

Recurrent inactivating mutations have been identified in various hematological malignancies in the X-linked BCOR gene encoding BCL6 corepressor (BCOR); however, its tumor suppressor function remains largely uncharacterized. We generated mice missing Bcor exon 4, expressing a variant BCOR lacking the BCL6-binding domain. Although the deletion of exon 4 in male mice (Bcor(DeltaE4/y) ) compromised the repopulating capacity of hematopoietic stem cells, Bcor(DeltaE4/y) thymocytes had augmented proliferative capacity in culture and showed a strong propensity to induce acute T-cell lymphoblastic leukemia (T-ALL), mostly in a Notch-dependent manner. Myc, one of the critical NOTCH1 targets in T-ALL, was highly up-regulated in Bcor(DeltaE4/y) T-ALL cells. Chromatin immunoprecipitation/DNA sequencing analysis revealed that BCOR was recruited to the Myc promoter and restrained its activation in thymocytes. BCOR also targeted other NOTCH1 targets and potentially antagonized their transcriptional activation. Bcl6-deficient thymocytes behaved in a manner similar to Bcor(DeltaE4/y) thymocytes. Our results provide the first evidence of a tumor suppressor role for BCOR in the pathogenesis of T lymphocyte malignancies.

Access to Document (DOI)

Enhancement of TGF-β-induced Smad3 activity by c-Abl-mediated tyrosine phosphorylation of its coactivator SKI-interacting protein (SKIP)

Kuki, K., Yamaguchi, N., Iwasawa, S., Takakura, Y., Aoyama, K., Yuki, R., Nakayama, Y., Kuga, T., Hashimoto, Y., Tomonaga, T. and Yamaguchi, N.

Biochem Biophys Res Commun 490 （ 3 ） 1045 - 1051 2017年08月

研究論文（学術雑誌）, 査読有り, ISSN 0006-291x

c-Abl is a non-receptor-type tyrosine kinase that plays an important role in cell proliferation, migration, apoptosis, and fibrosis. Furthermore, although c-Abl is involved in transforming growth factor-β (TGF-β) signaling, its molecular functions in TGF-β signaling are not fully understood. Here, we found that c-Abl phosphorylates SKI-interacting protein (SKIP), a nuclear cofactor of the transcription factor Smad3. The c-Abl inhibitor imatinib suppressed TGF-β-induced expression of Smad3 targets as well as SKIP/Smad3 interaction. TGF-β-stimulation induced tyrosine phosphorylation of SKIP, and this phosphorylation was suppressed by imatinib. Tyr(292), Tyr(430), and Tyr(433) residues in SKIP were shown to be involved in c-Abl-mediated phosphorylation. Phosphomimetic glutamic acid substitution at Tyr(292) in SKIP enhanced, whereas its phospho-dead phenylalanine substitution attenuated TGF-β-induced SKIP/Smad3 interaction. Moreover, the phosphomimetic mutant of SKIP augmented transcriptional activity of Smad3. Taken together, these results suggest that c-Abl phosphorylates SKIP mainly at Tyr(292) and promotes SKIP/Smad3 interaction for the full activation of TGF-β/Smad3 signaling.

Access to Document (DOI)

Dual Inhibition of EZH2 and EZH1 Sensitizes PRC2-Dependent Tumors to Proteasome Inhibition

Rizq, O., Mimura, N., Oshima, M., Saraya, A., Koide, S., Kato, Y., Aoyama, K., Nakajima-Takagi, Y., Wang, C., Chiba, T., Ma, A., Jin, J., Iseki, T., Nakaseko, C. and Iwama, A.

Clin Cancer Res 23 （ 16 ） 4817 - 4830 2017年08月

研究論文（学術雑誌）, 査読有り, ISSN 1078-0432

Purpose: EZH2 and EZH1, the catalytic components of polycomb repressive complex 2 (PRC2), trigger trimethylation of H3K27 (H3K27me3) to repress the transcription of target genes and are implicated in the pathogenesis of various cancers including multiple myeloma and prostate cancer. Here, we investigated the preclinical effects of UNC1999, a dual inhibitor of EZH2 and EZH1, in combination with proteasome inhibitors on multiple myeloma and prostate cancer.Experimental Design:In vitro and in vivo efficacy of UNC1999 and the combination with proteasome inhibitors was evaluated in multiple myeloma cell lines, primary patient cells, and in a xenograft model. RNA-seq and ChIP-seq were performed to uncover the targets of UNC1999 in multiple myeloma. The efficacy of the combination therapy was validated in prostate cancer cell lines.Results: Proteasome inhibitors repressed EZH2 transcription via abrogation of the RB-E2F pathway, thereby sensitizing EZH2-dependent multiple myeloma cells to EZH1 inhibition by UNC1999. Correspondingly, combination of proteasome inhibitors with UNC1999, but not with an EZH2-specific inhibitor, induced synergistic antimyeloma activity in vitro Bortezomib combined with UNC1999 remarkably inhibited the growth of myeloma cells in vivo Comprehensive analyses revealed several direct targets of UNC1999 including the tumor suppressor gene NR4A1 Derepression of NR4A1 by UNC1999 resulted in suppression of MYC, which was enhanced by the combination with bortezomib, suggesting the cooperative blockade of PRC2 function. Notably, this combination also exhibited strong synergy in prostate cancer cells.Conclusions: Our results identify dual inhibition of EZH2 and EZH1 together with proteasome inhibition as a promising epigenetics-based therapy for PRC2-dependent cancers. Clin Cancer Res; 23(16); 4817-30. ©2017 AACR.

Access to Document (DOI)

Hmga2 collaborates with JAK2V617F in the development of myeloproliferative neoplasms

Ueda, K., Ikeda, K., Ikezoe, T., Harada-Shirado, K., Ogawa, K., Hashimoto, Y., Sano, T., Ohkawara, H., Kimura, S., Shichishima-Nakamura, A., Nakamura, Y., Shikama, Y., Mori, T., Mason, P. J., Bessler, M., Morishita, S., Komatsu, N., Shide, K., Shimoda, K., Koide, S., Aoyama, K., Oshima, M., Iwama, A. and Takeishi, Y.

Blood Adv 1 （ 15 ） 1001 - 1015 2017年06月

研究論文（学術雑誌）, 査読有り, ISSN 2473-9529

High-mobility group AT-hook 2 (HMGA2) is crucial for the self-renewal of fetal hematopoietic stem cells (HSCs) but is downregulated in adult HSCs via repression by MIRlet-7 and the polycomb-recessive complex 2 (PRC2) including EZH2. The HMGA2 messenger RNA (mRNA) level is often elevated in patients with myelofibrosis that exhibits an advanced myeloproliferative neoplasm (MPN) subtype, and deletion of Ezh2 promotes the progression of severe myelofibrosis in JAK2(V617F) mice with upregulation of several oncogenes such as Hmga2. However, the direct role of HMGA2 in the pathogenesis of MPNs remains unknown. To clarify the impact of HMGA2 on MPNs carrying the driver mutation, we generated ΔHmga2/JAK2(V617F) mice overexpressing Hmga2 due to deletion of the 3' untranslated region. Compared with JAK2(V617F) mice, ΔHmga2/JAK2(V617F) mice exhibited more severe leukocytosis, anemia and splenomegaly, and shortened survival, whereas severity of myelofibrosis was comparable. ΔHmga2/JAK2(V617F) cells showed a greater repopulating ability that reproduced the severe MPN compared with JAK2(V617F) cells in serial bone marrow transplants, indicating that Hmga2 promotes MPN progression at the HSC level. Hmga2 also enhanced apoptosis of JAK2(V617F) erythroblasts that may worsen anemia. Relative to JAK2(V617F) hematopoietic stem and progenitor cells (HSPCs), over 30% of genes upregulated in ΔHmga2/JAK2(V617F) HSPCs overlapped with those derepressed by Ezh2 loss in JAK2(V617F)/Ezh2(Δ/Δ) HSPCs, suggesting that Hmga2 may facilitate upregulation of Ezh2 targets. Correspondingly, deletion of Hmga2 ameliorated anemia and splenomegaly in JAK2(V617F)/Ezh2(Δ/wild-type) mice, and MIRlet-7 suppression and PRC2 mutations correlated with the elevated HMGA2 mRNA levels in patients with MPNs, especially myelofibrosis. These findings suggest the crucial role of HMGA2 in MPN progression.

Access to Document (DOI)

Histone lysine methyltransferase G9a is a novel epigenetic target for the treatment of hepatocellular carcinoma

Yokoyama, M., Chiba, T., Zen, Y., Oshima, M., Kusakabe, Y., Noguchi, Y., Yuki, K., Koide, S., Tara, S., Saraya, A., Aoyama, K., Mimura, N., Miyagi, S., Inoue, M., Wakamatsu, T., Saito, T., Ogasawara, S., Suzuki, E., Ooka, Y., Tawada, A., Otsuka, M., Miyazaki, M., Yokosuka, O. and Iwama, A.

Oncotarget 8 （ 13 ） 21315 - 21326 2017年03月

研究論文（学術雑誌）, 査読有り, ISSN 1949-2553

Histone H3 lysine 9 dimethylation (H3K9me2) is mainly regulated by the histone lysine methyltransferase G9a and is associated with the repression of transcription. However, both the role of G9a and the significance of H3K9me2 in hepatocellular carcinoma (HCC) cells remain unclear. In this study, we conducted loss-of-function assay of G9a using short-hairpin RNA and pharmacological interference. Knockdown of G9a reduced H3K9me2 levels and impaired both HCC cell growth and sphere formation. However, transforming growth factor β1-induced epithelial mesenchymal transition (EMT) was not suppressed by G9a knockdown. Combined analyses of chromatin immunoprecipitation followed by sequencing and RNA-sequencing led to successful identification of 96 candidate epigenetic targets of G9a. Pharmacological inhibition of G9a by BIX-01294 resulted in both cell growth inhibition and induction of apoptosis in HCC cells. Intraperitoneal administration of BIX-01294 suppressed the growth of xenograft tumors generated by implantation of HCC cells in non-obese diabetic/severe combined immunodeficient mice. Immunohistochemical analyses revealed high levels of G9a and H3K9me2 in 36 (66.7%) and 35 (64.8%) primary HCC tissues, respectively. G9a expression levels were significantly positively correlated with H3K9me2 levels in tumor tissues. In contrast, in non-tumor tissues, G9a and H3K9me2 were only observed in biliary epithelial cells and periportal hepatocytes. In conclusion, G9a inhibition impairs anchorage-dependent and -independent cell growth, but not EMT in HCC cells. Our data indicate that pharmacological interference of G9a might be a novel epigenetic approach for the treatment of HCC.

Access to Document (DOI)

Protective role for lipid modifications of Src-family kinases against chromosome missegregation

Honda, T., Soeda, S., Tsuda, K., Yamaguchi, C., Aoyama, K., Morinaga, T., Yuki, R., Nakayama, Y., Yamaguchi, N. and Yamaguchi, N.

Sci Rep 6 38751 - 38751 2016年12月

研究論文（学術雑誌）, 査読有り, ISSN 2045-2322

Src-family tyrosine kinases, which are expressed in various cell types, play critical roles in cell signalling at the cytoplasmic side of the plasma membrane through their lipid modifications. Src-family kinases are cotranslationally myristoylated and posttranslationally palmitoylated in the amino-terminal region. The Src-family member Lyn contains a myristoylation site at glycine-2 and a palmitoylation site at cysteine-3, whereas c-Src has a myristoylation site at glycine-2 but not any palmitoylation sites. However, little is known about the role for lipid modifications of Src-family kinases in cell division. Here, we show that non-lipid-modified Lyn and c-Src, Lyn(G2A/C3A) and c-Src(G2A), are delocalized from membranes to the cytoplasm and the nucleus, which gives rise to a significant increase in the rate of chromosome missegregation, such as chromosome lagging and anaphase chromosome bridging, in a tyrosine kinase activity-dependent manner. Treatment with the Src inhibitor PP2 shows that the kinase activity of non-lipid-modified, non-membrane-bound Src during M phase is critical for giving rise to chromosome missegregation. Given that only a fraction of Src-family kinases fails in lipid modifications during biosynthesis, these results suggest that Src's membrane anchorage through their lipid modifications from prophase to anaphase plays a protective role against induction of chromosome missegregation.

Access to Document (DOI)

Possible role of intragenic DNA hypermethylation in gene silencing of the tumor suppressor gene NR4A3 in acute myeloid leukemia.

Ryo Shimizu, Tomoya Muto, Kazumasa Aoyama, Kwangmin Choi, Masahiro Takeuchi, Shuhei Koide, Nagisa Hasegawa, Yusuke Isshiki, Emi Togasaki, Chika Kawajiri-Manako, Yuhei Nagao, Shokichi Tsukamoto, Shio Sakai, Yusuke Takeda, Naoya Mimura, Chikako Ohwada, Emiko Sakaida, Tohru Iseki, Daniel T Starczynowski, Atsushi Iwama, Koutaro Yokote, Chiaki Nakaseko

Leukemia research 50 85 - 94 2016年11月

研究論文（学術雑誌）, 査読有り

Expression of the tumor suppressor gene NR4A3 is silenced in the blasts of acute myeloid leukemia (AML), irrespective of the karyotype. Although the transcriptional reactivation of NR4A3 is considered to have a broad-spectrum anti-leukemic effect, the therapeutic modalities targeting this gene have been hindered by our minimal understanding of the transcriptional mechanisms regulating its expression, particularly in human AML. Here we show the role of intragenic DNA hypermethylation in reducing the expression of NR4A3 in AML. Bisulfite sequencing analysis revealed that CpG sites at the intragenic region encompassing exon 3 of NR4A3, but not the promoter region, are hypermethylated in AML cell lines and primary AML cells. A DNA methyltransferase inhibitor restored the expression of NR4A3 following a reduction in DNA methylation levels at intragenic CpG sites. The in silico data revealed an enrichment of H3K4me1 and H2A.Z at exon 3 of NR4A3 in human non-malignant cells but that was excluded specifically in leukemia cells with CpG hypermethylation. This suggests that exon 3 represents a functional regulatory element involved in the transcriptional regulation of NR4A3. Our findings improve the current understanding of the mechanism underlying NR4A3 silencing and facilitate the development of NR4A3-targeted therapy.

Access to Document (DOI)

Setdb1 maintains hematopoietic stem and progenitor cells by restricting the ectopic activation of nonhematopoietic genes

Koide, S., Oshima, M., Takubo, K., Yamazaki, S., Nitta, E., Saraya, A., Aoyama, K., Kato, Y., Miyagi, S., Nakajima-Takagi, Y., Chiba, T., Matsui, H., Arai, F., Suzuki, Y., Kimura, H., Nakauchi, H., Suda, T., Shinkai, Y. and Iwama, A.

Blood 128 （ 5 ） 638 - 49 2016年08月

研究論文（学術雑誌）, 査読有り, ISSN 0006-4971

Setdb1, also known as Eset, is a methyltransferase that catalyzes trimethylation of H3K9 (H3K9me3) and plays an essential role in the silencing of endogenous retroviral elements (ERVs) in the developing embryo and embryonic stem cells (ESCs). Its role in somatic stem cells, however, remains unclear because of the early death of Setdb1-deficient embryos. We demonstrate here that Setdb1 is the first H3K9 methyltransferase shown to be essential for the maintenance of hematopoietic stem and progenitor cells (HSPCs) in mice. The deletion of Setdb1 caused the rapid depletion of hematopoietic stem and progenitor cells (HSPCs), as well as leukemic stem cells. In contrast to ESCs, ERVs were largely repressed in Setdb1-deficient HSPCs. A list of nonhematopoietic genes was instead ectopically activated in HSPCs after reductions in H3K9me3 levels, including key gluconeogenic enzyme genes fructose-1,6-bisphosphatase 1 (Fbp1) and Fbp2 The ectopic activation of gluconeogenic enzymes antagonized glycolysis and impaired ATP production, resulting in a compromised repopulating capacity of HSPCs. Our results demonstrate that Setdb1 maintains HSPCs by restricting the ectopic activation of nonhematopoietic genes detrimental to their function and uncover that the gluconeogenic pathway is one of the critical targets of Setdb1 in HSPCs.

Access to Document (DOI)

The loss of Ezh2 drives the pathogenesis of myelofibrosis and sensitizes tumor-initiating cells to bromodomain inhibition.

Goro Sashida, Changshan Wang, Takahisa Tomioka, Motohiko Oshima, Kazumasa Aoyama, Akinori Kanai, Makiko Mochizuki-Kashio, Hironori Harada, Kazuya Shimoda, Atsushi Iwama

The Journal of experimental medicine 213 （ 8 ） 1459 - 77 2016年07月

研究論文（学術雑誌）, 査読有り

EZH2 is a component of polycomb repressive complex 2 (PRC2) and functions as an H3K27 methyltransferase. Loss-of-function mutations in EZH2 are associated with poorer outcomes in patients with myeloproliferative neoplasms (MPNs), particularly those with primary myelofibrosis (MF [PMF]). To determine how EZH2 insufficiency is involved in the pathogenesis of PMF, we generated mice compound for an Ezh2 conditional deletion and activating mutation in JAK2 (JAK2V617F) present in patients with PMF. The deletion of Ezh2 in JAK2(V617F) mice markedly promoted the development of MF, indicating a tumor suppressor function for EZH2 in PMF. The loss of Ezh2 in JAK2(V617F) hematopoietic cells caused significant reductions in H3K27 trimethylation (H3K27me3) levels, resulting in an epigenetic switch to H3K27 acetylation (H3K27ac). These epigenetic switches were closely associated with the activation of PRC2 target genes including Hmga2, an oncogene implicated in the pathogenesis of PMF. The treatment of JAK2(V617F)/Ezh2-null mice with a bromodomain inhibitor significantly attenuated H3K27ac levels at the promoter regions of PRC2 targets and down-regulated their expression, leading to the abrogation of MF-initiating cells. Therefore, an EZH2 insufficiency not only cooperated with active JAK2 to induce MF, but also conferred an oncogenic addiction to the H3K27ac modification in MF-initiating cells that was capable of being restored by bromodomain inhibition.

Access to Document (DOI)

Loss of Pcgf5 Affects Global H2A Monoubiquitination but Not the Function of Hematopoietic Stem and Progenitor Cells

Si, S., Nakajima-Takagi, Y., Aoyama, K., Oshima, M., Saraya, A., Sugishita, H., Nakayama, M., Ishikura, T., Koseki, H. and Iwama, A.

PLoS One 11 （ 5 ） e0154561 2016年

研究論文（学術雑誌）, 査読有り, ISSN 1932-6203

Polycomb-group RING finger proteins (Pcgf1-Pcgf6) are components of Polycomb repressive complex 1 (PRC1)-related complexes that catalyze monoubiquitination of histone H2A at lysine 119 (H2AK119ub1), an epigenetic mark associated with repression of genes. Pcgf5 has been characterized as a component of PRC1.5, one of the non-canonical PRC1, consisting of Ring1a/b, Rybp/Yaf2 and Auts2. However, the biological functions of Pcgf5 have not yet been identified. Here we analyzed the impact of the deletion of Pcgf5 specifically in hematopoietic stem and progenitor cells (HSPCs). Pcgf5 is expressed preferentially in hematopoietic stem cells (HSCs) and multipotent progenitors (MPPs) compared with committed myeloid progenitors and differentiated cells. We transplanted bone marrow (BM) cells from Rosa::Cre-ERT control and Cre-ERT;Pcgf5fl/fl mice into lethally irradiated recipient mice. At 4 weeks post-transplantation, we deleted Pcgf5 by injecting tamoxifen, however, no obvious changes in hematopoiesis were detected including the number of HSPCs during a long-term observation period following the deletion. Competitive BM repopulating assays revealed normal repopulating capacity of Pcgf5-deficient HSCs. Nevertheless, Pcgf5-deficient HSPCs showed a significant reduction in H2AK119ub1 levels compared with the control. ChIP-sequence analysis confirmed the reduction in H2AK119ub1 levels, but revealed no significant association of changes in H2AK119ub1 levels with gene expression levels. Our findings demonstrate that Pcgf5-containing PRC1 functions as a histone modifier in vivo, but its role in HSPCs is limited and can be compensated by other PRC1-related complexes in HSPCs.

Access to Document (DOI)

c-Abl-mediated tyrosine phosphorylation of JunB is required for Adriamycin-induced expression of p21

Yamaguchi, N., Yuki, R., Kubota, S., Aoyama, K., Kuga, T., Hashimoto, Y., Tomonaga, T. and Yamaguchi, N.

Biochem J 471 （ 1 ） 67 - 77 2015年10月

研究論文（学術雑誌）, 査読有り, ISSN 0264-6021

The non-receptor-type tyrosine kinase c-Abl functions as a cytoplasmic signal transducer upon activation of cell-surface receptors. c-Abl is also involved in DDR (DNA-damage response), which is initiated in the nucleus, whereas its molecular functions in DDR are not fully understood. In the present study, we found that c-Abl phosphorylates JunB, a member of the AP-1 (activator protein 1) transcription factor family. Because JunB was suggested to be involved in DDR, we analysed the role of c-Abl-mediated phosphorylation of JunB in DDR. We first analysed phosphorylation sites of JunB and found that c-Abl majorly phosphorylates JunB at Tyr(173), Tyr(182) and Tyr(188). Because c-Abl promotes expression of the cyclin-dependent kinase inhibitor p21 upon stimulation with the DNA-damaging agent Adriamycin (doxorubicin), we analysed the involvement of JunB in Adriamycin-induced p21 expression. We found that JunB suppresses p21 induction through inhibition of its promoter activity. The phosphomimetic JunB, which was generated by glutamic acid substitutions at the phosphorylation sites, failed to repress p21 induction. Recruitment of JunB to the p21 promoter was promoted by Adriamycin stimulation and was further enhanced by co-treatment with the c-Abl inhibitor imatinib. The phosphomimetic glutamic acid substitutions in JunB or Adriamycin treatment impaired the JunB-c-Fos transcription factor complex formation. Taken together, these results suggest that, although JunB represses p21 promoter activity, c-Abl phosphorylates JunB and conversely inhibits its suppressive role on p21 promoter activity upon Adriamycin stimulation. Therefore JunB is likely to be a key target of c-Abl in expression of p21 in Adriamycin-induced DDR.

Access to Document (DOI)

Ezh2 loss in hematopoietic stem cells predisposes mice to develop heterogeneous malignancies in an Ezh1-dependent manner

Mochizuki-Kashio, M., Aoyama, K., Sashida, G., Oshima, M., Tomioka, T., Muto, T., Wang, C. and Iwama, A.

Blood 126 （ 10 ） 1172 - 83 2015年09月

研究論文（学術雑誌）, 査読有り, ISSN 0006-4971

Recent genome sequencing revealed inactivating mutations in EZH2, which encodes an enzymatic component of polycomb-repressive complex 2 (PRC2), in patients with myelodysplastic syndrome (MDS), myeloproliferative neoplasms (MPNs), and MDS/MPN overlap disorders. We herein demonstrated that the hematopoietic-specific deletion of Ezh2 in mice induced heterogeneous hematopoietic malignancies. Myelodysplasia was detected in mice following the deletion of Ezh2, and resulted in the development of MDS and MDS/MPN. Thrombocytosis was induced by Ezh2 loss and sustained in some mice with myelodysplasia. Although less frequent, Ezh2 loss also induced T-cell acute lymphoblastic leukemia and the clonal expansion of B-1a B cells. Gene expression profiling showed that PRC2 target genes were derepressed upon the deletion of Ezh2 in hematopoietic stem and progenitor cells, but were largely repressed during the development of MDS and MDS/MPN. Chromatin immunoprecipitation-sequence analysis of trimethylation of histone H3 at lysine 27 (H3K27me3) revealed a compensatory function of Ezh1, another enzymatic component of PRC2, in this process. The deletion of Ezh1 alone did not cause dysplasia or any hematologic malignancies in mice, but abolished the repopulating capacity of hematopoietic stem cells when combined with Ezh2 loss. These results clearly demonstrated an essential role of Ezh1 in the pathogenesis of hematopoietic malignancies induced by Ezh2 insufficiency, and highlighted the differential functions of Ezh1 and Ezh2 in hematopoiesis.

Access to Document (DOI)

Gene expression profiling of loss of TET2 and/or JAK2V617F mutant hematopoietic stem cells from mouse models of myeloproliferative neoplasms

Kameda, T., Shide, K., Yamaji, T., Kamiunten, A., Sekine, M., Hidaka, T., Kubuki, Y., Sashida, G., Aoyama, K., Yoshimitsu, M., Abe, H., Miike, T., Iwakiri, H., Tahara, Y., Yamamoto, S., Hasuike, S., Nagata, K., Iwama, A., Kitanaka, A. and Shimoda, K.

Genom Data 4 102 - 8 2015年06月

研究論文（学術雑誌）, 査読有り, ISSN 2213-5960

Myeloproliferative neoplasms (MPNs) are clinically characterized by the chronic overproduction of differentiated peripheral blood cells and the gradual expansion of malignant intramedullary/extramedullary hematopoiesis. In MPNs mutations in JAK2 MPL or CALR are detected mutually exclusive in more than 90% of cases [1,2]. Mutations in them lead to the abnormal activation of JAK/STAT signaling and the autonomous growth of differentiated cells therefore they are considered as "driver" gene mutations. In addition to the above driver gene mutations mutations in epigenetic regulators such as TET2 DNMT3A ASXL1 EZH2 or IDH1/2 are detected in about 5%-30% of cases respectively [3]. Mutations in TET2 DNMT3A EZH2 or IDH1/2 commonly confer the increased self-renewal capacity on normal hematopoietic stem cells (HSCs) but they do not lead to the autonomous growth of differentiated cells and only exhibit subtle clinical phenotypes [4,6-8,5]. It was unclear how mutations in such epigenetic regulators influenced abnormal HSCs with driver gene mutations how they influenced the disease phenotype or whether a single driver gene mutation was sufficient for the initiation of human MPNs. Therefore we focused on JAK2V617F and loss of TET2-the former as a representative of driver gene mutations and the latter as a representative of mutations in epigenetic regulators-and examined the influence of single or double mutations on HSCs (Lineage(-)Sca-1(+)c-Kit(+) cells (LSKs)) by functional analyses and microarray whole-genome expression analyses [9]. Gene expression profiling showed that the HSC fingerprint genes [10] was statistically equally enriched in TET2-knockdown-LSKs but negatively enriched in JAK2V617F-LSKs compared to that in wild-type-LSKs. Double-mutant-LSKs showed the same tendency as JAK2V617F-LSKs in terms of their HSC fingerprint genes but the expression of individual genes differed between the two groups. Among 245 HSC fingerprint genes 100 were more highly expressed in double-mutant-LSKs than in JAK2V617F-LSKs. These altered gene expressions might partly explain the mechanisms of initiation and progression of MPNs which was observed in the functional analyses [9]. Here we describe gene expression profiles deposited at the Gene Expression Omnibus (GEO) under the accession number GSE62302 including experimental methods and quality control analyses.

Access to Document (DOI)

Overexpression of zinc-finger protein 777 (ZNF777) inhibits proliferation at low cell density through down-regulation of FAM129A

Yuki, R., Aoyama, K., Kubota, S., Yamaguchi, N., Kubota, S., Hasegawa, H., Morii, M., Huang, X., Liu, K., Williams, R., Fukuda, M. N. and Yamaguchi, N.

J Cell Biochem 116 （ 6 ） 954 - 68 2015年06月

研究論文（学術雑誌）, 査読有り, ISSN 0730-2312

Krüppel-associated box-containing zinc finger proteins (KRAB-ZFPs) regulate a wide range of cellular processes. KRAB-ZFPs have a KRAB domain, which binds to transcriptional corepressors, and a zinc finger domain, which binds to DNA to activate or repress gene transcription. Here, we characterize ZNF777, a member of KRAB-ZFPs. We show that ZNF777 localizes to the nucleus and inducible overexpression of ZNF777 inhibits cell proliferation in a manner dependent on its zinc finger domain but independent of its KRAB domain. Intriguingly, ZNF777 overexpression drastically inhibits cell proliferation at low cell density but slightly inhibits cell proliferation at high cell density. Furthermore, ZNF777 overexpression decreases the mRNA level of FAM129A irrespective of cell density. Importantly, the protein level of FAM129A strongly decreases at low cell density, but at high cell density the protein level of FAM129A does not decrease to that observed at low cell density. ZNF777-mediated inhibition of cell proliferation is attenuated by overexpression of FAM129A at low cell density. Furthermore, ZNF777-mediated down-regulation of FAM129A induces moderate levels of the cyclin-dependent kinase inhibitor p21. These results suggest that ZNF777 overexpression inhibits cell proliferation at low cell density and that p21 induction by ZNF777-mediated down-regulation of FAM129A plays a role in inhibition of cell proliferation.

Access to Document (DOI)

Role for Tyrosine Phosphorylation of A-kinase Anchoring Protein 8 (AKAP8) in Its Dissociation from Chromatin and the Nuclear Matrix.

Sho Kubota, Mariko Morii, Ryuzaburo Yuki, Noritaka Yamaguchi, Hiromi Yamaguchi, Kazumasa Aoyama, Takahisa Kuga, Takeshi Tomonaga, Naoto Yamaguchi

The Journal of biological chemistry 290 （ 17 ） 10891 - 904 2015年04月

研究論文（学術雑誌）, 査読有り

Protein-tyrosine phosphorylation regulates a wide variety of cellular processes at the plasma membrane. Recently, we showed that nuclear tyrosine kinases induce global nuclear structure changes, which we called chromatin structural changes. However, the mechanisms are not fully understood. In this study we identify protein kinase A anchoring protein 8 (AKAP8/AKAP95), which associates with chromatin and the nuclear matrix, as a nuclear tyrosine-phosphorylated protein. Tyrosine phosphorylation of AKAP8 is induced by several tyrosine kinases, such as Src, Fyn, and c-Abl but not Syk. Nucleus-targeted Lyn and c-Src strongly dissociate AKAP8 from chromatin and the nuclear matrix in a kinase activity-dependent manner. The levels of tyrosine phosphorylation of AKAP8 are decreased by substitution of multiple tyrosine residues on AKAP8 into phenylalanine. Importantly, the phenylalanine mutations of AKAP8 inhibit its dissociation from nuclear structures, suggesting that the association/dissociation of AKAP8 with/from nuclear structures is regulated by its tyrosine phosphorylation. Furthermore, the phenylalanine mutations of AKAP8 suppress the levels of nuclear tyrosine kinase-induced chromatin structural changes. In contrast, AKAP8 knockdown increases the levels of chromatin structural changes. Intriguingly, stimulation with hydrogen peroxide induces chromatin structural changes accompanied by the dissociation of AKAP8 from nuclear structures. These results suggest that AKAP8 is involved in the regulation of chromatin structural changes through nuclear tyrosine phosphorylation.

Access to Document (DOI)

c-Abl induces stabilization of histone deacetylase 1 (HDAC1) in a kinase activity-dependent manner

Aoyama, K., Yamaguchi, N., Yuki, R., Morii, M., Kubota, S., Hirata, K., Abe, K., Honda, T., Kuga, T., Hashimoto, Y., Tomonaga, T. and Yamaguchi, N.

Cell Biol Int 39 （ 4 ） 446 - 56 2015年04月

研究論文（学術雑誌）, 筆頭著者, 査読有り, ISSN 1065-6995

c-Abl is a non-receptor-type tyrosine kinase that regulates various cellular events, including cell proliferation, differentiation, and apoptosis, through phosphorylation of cytoplasmic and nuclear targets. Although we showed that c-Abl induces histone deacetylation, the molecular mechanisms of this phenomenon are largely unknown. Here, we analyzed the effect of c-Abl on the expression of histone deacetylase 1 (HDAC1), because c-Abl was shown to be involved in maintenance of nuclear protein levels of HDAC1. Co-transfection of HDAC1 with c-Abl increased the levels of HDAC1 protein in a kinase activity-dependent manner without affecting its mRNA levels. Treatment with the proteasome inhibitor MG132 increased protein levels of HDAC1 in cells transfected with HDAC1 but not in cells co-transfected with HDAC1 and c-Abl. Among class I HDACs, knockdown of endogenous c-Abl preferentially suppressed endogenous protein levels of HDAC1, suggesting that c-Abl stabilizes HDAC1 protein by inhibiting its proteasomal degradation. Subcellular fractionation showed that the stabilization of HDAC1 by c-Abl occurred in the nucleus. Despite the fact that HDAC1 was phosphorylated by co-expression with c-Abl, stabilization of HDAC1 by c-Abl was not affected by mutations in its sites phosphorylated by c-Abl. Co-expression with HDAC1 and nuclear-targeted c-Abl did not affect HDAC1 stabilization. Therefore, these results suggest that c-Abl induces HDAC1 stabilization possibly through phosphorylation of a cytoplasmic target that is involved in proteasomal degradation of HDAC1.

Access to Document (DOI)

Loss of TET2 has dual roles in murine myeloproliferative neoplasms: disease sustainer and disease accelerator

Kameda, T., Shide, K., Yamaji, T., Kamiunten, A., Sekine, M., Taniguchi, Y., Hidaka, T., Kubuki, Y., Shimoda, H., Marutsuka, K., Sashida, G., Aoyama, K., Yoshimitsu, M., Harada, T., Abe, H., Miike, T., Iwakiri, H., Tahara, Y., Sueta, M., Yamamoto, S., Hasuike, S., Nagata, K., Iwama, A., Kitanaka, A. and Shimoda, K.

Blood 125 （ 2 ） 304 - 15 2015年01月

研究論文（学術雑誌）, 査読有り, ISSN 0006-4971

Acquired mutations of JAK2 and TET2 are frequent in myeloproliferative neoplasms (MPNs). We examined the individual and cooperative effects of these mutations on MPN development. Recipients of JAK2V617F cells developed primary myelofibrosis-like features; the addition of loss of TET2 worsened this JAK2V617F-induced disease, causing prolonged leukocytosis, splenomegaly, extramedullary hematopoiesis, and modestly shorter survival. Double-mutant (JAK2V617F plus loss of TET2) myeloid cells were more likely to be in a proliferative state than JAK2V617F single-mutant myeloid cells. In a serial competitive transplantation assay, JAK2V617F cells resulted in decreased chimerism in the second recipients, which did not develop MPNs. In marked contrast, cooperation between JAK2V617F and loss of TET2 developed and maintained MPNs in the second recipients by compensating for impaired hematopoietic stem cell (HSC) functioning. In-vitro sequential colony formation assays also supported the observation that JAK2V617F did not maintain HSC functioning over the long-term, but concurrent loss of TET2 mutation restored it. Transcriptional profiling revealed that loss of TET2 affected the expression of many HSC signature genes. We conclude that loss of TET2 has two different roles in MPNs: disease accelerator and disease initiator and sustainer in combination with JAK2V617F.

Access to Document (DOI)

Activation of the prereplication complex is blocked by mimosine through reactive oxygen species-activated ataxia telangiectasia mutated (ATM) protein without DNA damage

Kubota, S., Fukumoto, Y., Ishibashi, K., Soeda, S., Kubota, S., Yuki, R., Nakayama, Y., Aoyama, K., Yamaguchi, N. and Yamaguchi, N.

J Biol Chem 289 （ 9 ） 5730 - 46 2014年02月

研究論文（学術雑誌）, 査読有り, ISSN 0021-9258

Mimosine is an effective cell synchronization reagent used for arresting cells in late G1 phase. However, the mechanism underlying mimosine-induced G1 cell cycle arrest remains unclear. Using highly synchronous cell populations, we show here that mimosine blocks S phase entry through ATM activation. HeLa S3 cells are exposed to thymidine for 15 h, released for 9 h by washing out the thymidine, and subsequently treated with 1 mM mimosine for a further 15 h (thymidine → mimosine). In contrast to thymidine-induced S phase arrest, mimosine treatment synchronizes >90% of cells at the G1-S phase boundary by inhibiting the transition of the prereplication complex to the preinitiation complex. Mimosine treatment activates ataxia telangiectasia mutated (ATM)/ataxia telangiectasia and Rad3-related (ATR)-mediated checkpoint signaling without inducing DNA damage. Inhibition of ATM activity is found to induce mimosine-arrested cells to enter S phase. In addition, ATM activation by mimosine treatment is mediated by reactive oxygen species (ROS). These results suggest that, upon mimosine treatment, ATM blocks S phase entry in response to ROS, which prevents replication fork stalling-induced DNA damage.

Access to Document (DOI)

　前のページ - 次のページ　

青山 和正

論文 - 青山 和正

青山　和正

論文 - 青山　和正