論文 - 青山　和正

Inhibition of TOPORS ubiquitin ligase augments the efficacy of DNA hypomethylating agents through DNMT1 stabilization

Kaito S, Aoyama K, Oshima M, Tsuchiya A, Miyota M, Yamashita M, Koide S, Nakajima-Takagi Y, Kozuka-Hata H, Oyama M, Yabushita T, Ueno M, Hirao A, Tohyama K, Yamaguchi K, Furukawa Y, Kosako H, Yoshimi A, Goyama S, Nannya Y, Ogawa S, Agger K, Helin K, Koseki H, Nishiyama A, Nakanishi M, and Iwama A,

Nature Communications （Springer Science and Business Media LLC）  15 （ 1 ）  2024年08月

研究論文（学術雑誌）, 共著, 査読有り

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Abstract

DNA hypomethylating agents (HMAs) are used for the treatment of myeloid malignancies, although their therapeutic effects have been unsatisfactory. Here we show that CRISPR-Cas9 screening reveals that knockout of topoisomerase 1-binding arginine/serine-rich protein (TOPORS), which encodes a ubiquitin/SUMO E3 ligase, augments the efficacy of HMAs on myeloid leukemic cells with little effect on normal hematopoiesis, suggesting that TOPORS is involved in resistance to HMAs. HMAs are incorporated into the DNA and trap DNA methyltransferase-1 (DNMT1) to form DNA-DNMT1 crosslinks, which undergo SUMOylation, followed by proteasomal degradation. Persistent crosslinking is cytotoxic. The TOPORS RING finger domain, which mediates ubiquitination, is responsible for HMA resistance. In TOPORS knockout cells, DNMT1 is stabilized by HMA treatment due to inefficient ubiquitination, resulting in the accumulation of unresolved SUMOylated DNMT1. This indicates that TOPORS ubiquitinates SUMOylated DNMT1, thereby promoting the resolution of DNA-DNMT1 crosslinks. Consistently, the ubiquitination inhibitor, TAK-243, and the SUMOylation inhibitor, TAK-981, show synergistic effects with HMAs through DNMT1 stabilization. Our study provides a novel HMA-based therapeutic strategy that interferes with the resolution of DNA-DNMT1 crosslinks.

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The pathogenetic significance of exhausted T cells in a mouse model of mature B cell neoplasms

Shibamiya, A., Miyamoto-Nagai, Y., Koide, S., Oshima, M., Rizq, O., Aoyama, K., Nakajima-Takagi, Y., Kato, R., Kayamori, K., Isshiki, Y., Oshima-Hasegawa, N., Muto, T., Tsukamoto, S., Takeda, Y., Koyama-Nasu, R., Chiba, T., Honda, H., Yokote, K., Iwama, A., Sakaida, E. and Mimura, N.

Cancer Immunol Immunother 72 （ 8 ） 2635 - 2648 2023年08月

研究論文（学術雑誌）, 査読有り,  ISSN  0340-7004

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Dysfunctional anti-tumor immunity has been implicated in the pathogenesis of mature B cell neoplasms, such as multiple myeloma and B cell lymphoma; however, the impact of exhausted T cells on disease development remains unclear. Therefore, the present study investigated the features and pathogenetic significance of exhausted T cells using a mouse model of de novo mature B cell neoplasms, which is likely to show immune escape similar to human patients. The results revealed a significant increase in PD-1(+) Tim-3(-) and PD-1(+) Tim-3(+) T cells in sick mice. Furthermore, PD-1(+) Tim-3(+) T cells exhibited direct cytotoxicity with a short lifespan, showing transcriptional similarities to terminally exhausted T cells. On the other hand, PD-1(+) Tim-3(-) T cells not only exhibited immunological responsiveness but also retained stem-like transcriptional features, suggesting that they play a role in the long-term maintenance of anti-tumor immunity. In PD-1(+) Tim-3(-) and PD-1(+) Tim-3(+) T cells, the transcription factors Tox and Nr4a2, which reportedly contribute to the progression of T cell exhaustion, were up-regulated in vivo. These transcription factors were down-regulated by IMiDs in our in vitro T cell exhaustion analyses. The prevention of excessive T cell exhaustion may maintain effective anti-tumor immunity to cure mature B cell neoplasms.

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Polycomb repressive complex 1.1 coordinates homeostatic and emergency myelopoiesis

Nakajima-Takagi, Y., Oshima, M., Takano, J., Koide, S., Itokawa, N., Uemura, S., Yamashita, M., Andoh, S., Aoyama, K., Isshiki, Y., Shinoda, D., Saraya, A., Arai, F., Yamaguchi, K., Furukawa, Y., Koseki, H., Ikawa, T. and Iwama, A.

Elife 12 2023年06月

研究論文（学術雑誌）, 査読有り,  ISSN  2050-084x

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Polycomb repressive complex (PRC) 1 regulates stem cell fate by mediating mono-ubiquitination of histone H2A at lysine 119. While canonical PRC1 is critical for hematopoietic stem and progenitor cell (HSPC) maintenance, the role of non-canonical PRC1 in hematopoiesis remains elusive. PRC1.1, a non-canonical PRC1, consists of PCGF1, RING1B, KDM2B, and BCOR. We recently showed that PRC1.1 insufficiency induced by the loss of PCGF1 or BCOR causes myeloid-biased hematopoiesis and promotes transformation of hematopoietic cells in mice. Here we show that PRC1.1 serves as an epigenetic switch that coordinates homeostatic and emergency hematopoiesis. PRC1.1 maintains balanced output of steady-state hematopoiesis by restricting C/EBPα-dependent precocious myeloid differentiation of HSPCs and the HOXA9- and β-catenin-driven self-renewing network in myeloid progenitors. Upon regeneration, PRC1.1 is transiently inhibited to facilitate formation of granulocyte-macrophage progenitor (GMP) clusters, thereby promoting emergency myelopoiesis. Moreover, constitutive inactivation of PRC1.1 results in unchecked expansion of GMPs and eventual transformation. Collectively, our results define PRC1.1 as a novel critical regulator of emergency myelopoiesis, dysregulation of which leads to myeloid transformation.

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UTX inactivation in germinal center B cells promotes the development of multiple myeloma with extramedullary disease.

Ola Rizq, Naoya Mimura, Motohiko Oshima, Shuji Momose, Naoya Takayama, Naoki Itokawa, Shuhei Koide, Asuka Shibamiya, Yurie Miyamoto-Nagai, Mohamed Rizk, Yaeko Nakajima-Takagi, Kazumasa Aoyama, Changshan Wang, Atsunori Saraya, Masanori Seimiya, Mariko Watanabe, Satoshi Yamasaki, Tatsuhiro Shibata, Kiyoshi Yamaguchi, Yoichi Furukawa, Tetsuhiro Chiba, Emiko Sakaida, Chiaki Nakaseko, Jun-Ichi Tamaru, Yu-Tzu Tai, Kenneth C Anderson, Hiroaki Honda, Atsushi Iwama

Leukemia 2023年05月

研究論文（学術雑誌）, 査読有り

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UTX/KDM6A, a histone H3K27 demethylase and a key component of the COMPASS complex, is frequently lost or mutated in cancer; however, its tumor suppressor function remains largely uncharacterized in multiple myeloma (MM). Here, we show that the conditional deletion of the X-linked Utx in germinal center (GC) derived cells collaborates with the activating BrafV600E mutation and promotes induction of lethal GC/post-GC B cell malignancies with MM-like plasma cell neoplasms being the most frequent. Mice that developed MM-like neoplasms showed expansion of clonal plasma cells in the bone marrow and extramedullary organs, serum M proteins, and anemia. Add-back of either wild-type UTX or a series of mutants revealed that cIDR domain, that forms phase-separated liquid condensates, is largely responsible for the catalytic activity-independent tumor suppressor function of UTX in MM cells. Utx loss in concert with BrafV600E only slightly induced MM-like profiles of transcriptome, chromatin accessibility, and H3K27 acetylation, however, it allowed plasma cells to gradually undergo full transformation through activation of transcriptional networks specific to MM that induce high levels of Myc expression. Our results reveal a tumor suppressor function of UTX in MM and implicate its insufficiency in the transcriptional reprogramming of plasma cells in the pathogenesis of MM.

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A culture platform to study quiescent hematopoietic stem cells following genome editing

Kohei Shiroshita, Hiroshi Kobayashi, Shintaro Watanuki, Daiki Karigane, Yuriko Sorimachi, Shinya Fujita, Shinpei Tamaki, Miho Haraguchi, Naoki Itokawa, Kazumasa Aoyama, Shuhei Koide, Yosuke Masamoto, Kenta Kobayashi, Ayako Nakamura-Ishizu, Mineo Kurokawa, Atsushi Iwama, Shinichiro Okamoto, Keisuke Kataoka, Keiyo Takubo

Cell Reports Methods 2022年12月

査読有り

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YAP1/TAZ activity maintains vascular integrity and organismal survival

Uemura, S., Yamashita, M., Aoyama, K., Yokomizo-Nakano, T., Oshima, M., Nishio, M., Masuko, M., Takizawa, J., Sone, H., Yamada, Y., Suzuki, A. and Iwama, A.

Biochem Biophys Res Commun 619   117 - 123 2022年09月

研究論文（学術雑誌）, 査読有り,  ISSN  0006-291x

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Radiation therapy is one of the major treatment modalities for patients with cancers. However, ionizing radiation (IR) damages not only cancer cells but also the surrounding vascular endothelial cells (ECs). Hippo pathway effector genes Yap1 and Taz are the two transcriptional coactivators that have crucial roles in tissue homeostasis and vascular integrity in various organs. However, their function in adult ECs at the steady state and after IR is poorly understood. Here, we report sex- and context-dependent roles of endothelial YAP1/TAZ in maintaining vascular integrity and organismal survival. EC-specific Yap1/Taz deletion compromised systemic vascular integrity, resulting in lethal circulation failure preferentially in male mice. Furthermore, EC-specific Yap1/Taz deletion induced acute lethality upon sublethal IR that was closely associated with exacerbated systemic vascular dysfunction and circulation failure. Consistent with these findings, RNA-seq analysis revealed downregulation of tight junction genes in Yap1/Taz-deleted ECs. Collectively, our findings highlight the importance of endothelial YAP1/TAZ for maintaining adult vascular function, which may provide clinical implications for preventing organ injury after radiation therapy.

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Epigenetic traits inscribed in chromatin accessibility in aged hematopoietic stem cells.

Naoki Itokawa, Motohiko Oshima, Shuhei Koide, Naoya Takayama, Wakako Kuribayashi, Yaeko Nakajima-Takagi, Kazumasa Aoyama, Satoshi Yamazaki, Kiyoshi Yamaguchi, Yoichi Furukawa, Koji Eto, Atsushi Iwama

Nature communications 13 （ 1 ） 2691 - 2691 2022年05月

研究論文（学術雑誌）, 査読有り

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Hematopoietic stem cells (HSCs) exhibit considerable cell-intrinsic changes with age. Here, we present an integrated analysis of transcriptome and chromatin accessibility of aged HSCs and downstream progenitors. Alterations in chromatin accessibility preferentially take place in HSCs with aging, which gradually resolve with differentiation. Differentially open accessible regions (open DARs) in aged HSCs are enriched for enhancers and show enrichment of binding motifs of the STAT, ATF, and CNC family transcription factors that are activated in response to external stresses. Genes linked to open DARs show significantly higher levels of basal expression and their expression reaches significantly higher peaks after cytokine stimulation in aged HSCs than in young HSCs, suggesting that open DARs contribute to augmented transcriptional responses under stress conditions. However, a short-term stress challenge that mimics infection is not sufficient to induce persistent chromatin accessibility changes in young HSCs. These results indicate that the ongoing and/or history of exposure to external stresses may be epigenetically inscribed in HSCs to augment their responses to external stimuli.

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EZH1/2 inhibition augments the anti-tumor effects of sorafenib in hepatocellular carcinoma

Kusakabe, Y., Chiba, T., Oshima, M., Koide, S., Rizq, O., Aoyama, K., Ao, J., Kaneko, T., Kanzaki, H., Kanayama, K., Maeda, T., Saito, T., Nakagawa, R., Kobayashi, K., Kiyono, S., Nakamura, M., Ogasawara, S., Suzuki, E., Nakamoto, S., Yasui, S., Mikata, R., Muroyama, R., Kanda, T., Maruyama, H., Kato, J., Mimura, N., Ma, A., Jin, J., Zen, Y., Otsuka, M., Kaneda, A., Iwama, A. and Kato, N.

Sci Rep 11 （ 1 ） 21396 - 21396 2021年11月

研究論文（学術雑誌）, 査読有り,  ISSN  2045-2322

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Both EZH2 and its homolog EZH1 function as histone H3 Lysine 27 (H3K27) methyltransferases and repress the transcription of target genes. Dysregulation of H3K27 trimethylation (H3K27me3) plays an important role in the development and progression of cancers such as hepatocellular carcinoma (HCC). This study investigated the relationship between the expression of EZH1/2 and the level of H3K27me3 in HCC. Additionally, the role of EZH1/2 in cell growth, tumorigenicity, and resistance to sorafenib were also analyzed. Both the lentiviral knockdown and the pharmacological inhibition of EZH1/2 (UNC1999) diminished the level of H3K27me3 and suppressed cell growth in liver cancer cells, compared with EZH1 or EZH2 single knockdown. Although a significant association was observed between EZH2 expression and H3K27me3 levels in HCC samples, overexpression of EZH1 appeared to contribute to enhanced H3K27me3 levels in some EZH2(low)H3K27me3(high) cases. Akt suppression following sorafenib treatment resulted in an increase of the H3K27me3 levels through a decrease in EZH2 phosphorylation at serine 21. The combined use of sorafenib and UNC1999 exhibited synergistic antitumor effects in vitro and in vivo. Combination treatment canceled the sorafenib-induced enhancement in H3K27me3 levels, indicating that activation of EZH2 function is one of the mechanisms of sorafenib-resistance in HCC. In conclusion, sorafenib plus EZH1/2 inhibitors may comprise a novel therapeutic approach in HCC.

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Insufficiency of non-canonical PRC1 synergizes with JAK2V617F in the development of myelofibrosis.

Daisuke Shinoda, Yaeko Nakajima-Takagi, Motohiko Oshima, Shuhei Koide, Kazumasa Aoyama, Atsunori Saraya, Hironori Harada, Bahityar Rahmutulla, Atsushi Kaneda, Kiyoshi Yamaguchi, Yoichi Furukawa, Haruhiko Koseki, Kazuya Shimoda, Tomoaki Tanaka, Goro Sashida, Atsushi Iwama

Leukemia 36 （ 2 ） 452 - 463 2021年09月

研究論文（学術雑誌）, 査読有り

　概要を見る

Insufficiency of polycomb repressive complex 2 (PRC2), which trimethylates histone H3 at lysine 27, is frequently found in primary myelofibrosis and promotes the development of JAK2V617F-induced myelofibrosis in mice by enhancing the production of dysplastic megakaryocytes. Polycomb group ring finger protein 1 (Pcgf1) is a component of PRC1.1, a non-canonical PRC1 that monoubiquitylates H2A at lysine 119 (H2AK119ub1). We herein investigated the impact of PRC1.1 insufficiency on myelofibrosis. The deletion of Pcgf1 in JAK2V617F mice strongly promoted the development of lethal myelofibrosis accompanied by a block in erythroid differentiation. Transcriptome and chromatin immunoprecipitation sequence analyses showed the de-repression of PRC1.1 target genes in Pcgf1-deficient JAK2V617F hematopoietic progenitors and revealed Hoxa cluster genes as direct targets. The deletion of Pcgf1 in JAK2V617F hematopoietic stem and progenitor cells (HSPCs), as well as the overexpression of Hoxa9, restored the attenuated proliferation of JAK2V617F progenitors. The overexpression of Hoxa9 also enhanced JAK2V617F-mediated myelofibrosis. The expression of PRC2 target genes identified in PRC2-insufficient JAK2V617F HSPCs was not largely altered in Pcgf1-deleted JAK2V617F HSPCs. The present results revealed a tumor suppressor function for PRC1.1 in myelofibrosis and suggest that PRC1.1 insufficiency has a different impact from that of PRC2 insufficiency on the pathogenesis of myelofibrosis.

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PRC2 insufficiency causes p53-dependent dyserythropoiesis in myelodysplastic syndrome.

Aoyama K, Shinoda D, Suzuki E, Nakajima-Takagi Y, Oshima M, Koide S, Rizq O, Si S, Tara S, Sashida G, Iwama A

Leukemia 35 （ 4 ） 1156 - 1165 2021年04月

研究論文（学術雑誌）, 筆頭著者, 責任著者, 査読有り,  ISSN  0887-6924

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EZH1 and EZH2 are enzymatic components of polycomb repressive complex (PRC) 2, which catalyzes histone H3K27 tri-methylation (H3K27me3) to repress the transcription of PRC2 target genes. We previously reported that the hematopoietic cell-specific Ezh2 deletion (Ezh2(Δ/Δ)) induced a myelodysplastic syndrome (MDS)-like disease in mice. We herein demonstrated that severe PRC2 insufficiency induced by the deletion of one allele Ezh1 in Ezh2-deficient mice (Ezh1(+/-)Ezh2(Δ/Δ)) caused advanced dyserythropoiesis accompanied by a differentiation block and enhanced apoptosis in erythroblasts. p53, which is activated by impaired ribosome biogenesis in del(5q) MDS, was specifically activated in erythroblasts, but not in hematopoietic stem or progenitor cells in Ezh1(+/-)Ezh2(Δ/Δ) mice. Cdkn2a, a major PRC2 target encoding p19(Arf), which activates p53 by inhibiting MDM2 E3 ubiquitin ligase, was de-repressed in Ezh1(+/-)Ezh2(Δ/Δ) erythroblasts. The deletion of Cdkn2a as well as p53 rescued dyserythropoiesis in Ezh1(+/-)Ezh2(Δ/Δ) mice, indicating that PRC2 insufficiency caused p53-dependent dyserythropoiesis via the de-repression of Cdkn2a. Since PRC2 insufficiency is often involved in the pathogenesis of MDS, the present results suggest that p53-dependent dyserythropoiesis manifests in MDS in the setting of PRC2 insufficiency.

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Bmi1 counteracts hematopoietic stem cell aging by repressing target genes and enforcing the stem cell gene signature

Nitta, E., Itokawa, N., Yabata, S., Koide, S., Hou, L. B., Oshima, M., Aoyama, K., Saraya, A. and Iwama, A.

Biochem Biophys Res Commun 521 （ 3 ） 612 - 619 2020年01月

研究論文（学術雑誌）, 査読有り,  ISSN  0006-291x

　概要を見る

Polycomb-group proteins are critical regulators of stem cells. We previously demonstrated that Bmi1, a component of polycomb repressive complex 1, defines the regenerative capacity of hematopoietic stem cells (HSCs). Here, we attempted to ameliorate the age-related decline in HSC function by modulating Bmi1 expression. The forced expression of Bmi1 did not attenuate myeloid-biased differentiation of aged HSCs. However, single cell transplantation assays revealed that the sustained expression of Bmi1 augmented the multi-lineage repopulating capacity of aged HSCs. Chromatin immunoprecipitation-sequencing of Bmi1 combined with an RNA sequence analysis showed that the majority of Bmi1 direct target genes are developmental regulator genes marked with a bivalent histone domain. The sustained expression of Bmi1 strictly maintained the transcriptional repression of their target genes and enforced expression of HSC signature genes in aged HSCs. Therefore, the manipulation of Bmi1 expression is a potential approach against impairments in HSC function with aging.

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KDM2B in polycomb repressive complex 1.1 functions as a tumor suppressor in the initiation of T-cell leukemogenesis.

Yusuke Isshiki, Yaeko Nakajima-Takagi, Motohiko Oshima, Kazumasa Aoyama, Mohamed Rizk, Shuhei Kurosawa, Atsunori Saraya, Takashi Kondo, Emiko Sakaida, Chiaki Nakaseko, Koutaro Yokote, Haruhiko Koseki, Atsushi Iwama

Blood advances 3 （ 17 ） 2537 - 2549 2019年09月

研究論文（学術雑誌）, 査読有り

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KDM2B together with RING1B, PCGF1, and BCOR or BCORL1 comprise polycomb repressive complex 1.1 (PRC1.1), a noncanonical PRC1 that catalyzes H2AK119ub1. It binds to nonmethylated CpG islands through its zinc finger-CxxC DNA binding domain and recruits the complex to target gene loci. Recent studies identified the loss of function mutations in the PRC1.1 gene, BCOR and BCORL1 in human T-cell acute lymphoblastic leukemia (T-ALL). We previously reported that Bcor insufficiency induces T-ALL in mice, supporting a tumor suppressor role for BCOR. However, the function of BCOR responsible for tumor suppression, either its corepressor function for BCL6 or that as a component of PRC1.1, remains unclear. We herein examined mice specifically lacking the zinc finger-CxxC domain of KDM2B in hematopoietic cells. Similar to Bcor-deficient mice, Kdm2b-deficient mice developed lethal T-ALL mostly in a NOTCH1-dependent manner. A chromatin immunoprecipitation sequence analysis of thymocytes revealed the binding of KDM2B at promoter regions, at which BCOR and EZH2 colocalized. KDM2B target genes markedly overlapped with those of NOTCH1 in human T-ALL cells, suggesting that noncanonical PRC1.1 antagonizes NOTCH1-mediated gene activation. KDM2B target genes were expressed at higher levels than the others and were marked with high levels of H2AK119ub1 and H3K4me3, but low levels of H3K27me3, suggesting that KDM2B target genes are transcriptionally active or primed for activation. These results indicate that PRC1.1 plays a key role in restricting excessive transcriptional activation by active NOTCH1, thereby acting as a tumor suppressor in the initiation of T-cell leukemogenesis.

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Bmi1 restricts the adipogenic differentiation of bone marrow stromal cells to maintain the integrity of the hematopoietic stem cell niche

Kato, Y., Hou, L. B., Miyagi, S., Nitta, E., Aoyama, K., Shinoda, D., Yamazaki, S., Kuribayashi, W., Isshiki, Y., Koide, S., Si, S., Saraya, A., Matsuzaki, Y., van Lohuizen, M. and Iwama, A.

Exp Hematol 76   24 - 37 2019年08月

研究論文（学術雑誌）, 査読有り,  ISSN  0301-472x

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The polycomb group protein Bmi1 maintains hematopoietic stem cell (HSC) functions. We previously reported that Bmi1-deficient mice exhibited progressive fatty changes in bone marrow (BM). A large portion of HSCs reside in the perivascular niche created partly by endothelial cells and leptin receptor(+) (LepR(+)) BM stromal cells. To clarify how Bmi1 regulates the HSC niche, we specifically deleted Bmi1 in LepR(+) cells in mice. The Bmi1 deletion promoted the adipogenic differentiation of LepR(+) stromal cells and caused progressive fatty changes in the BM of limb bones with age, resulting in reductions in the numbers of HSCs and progenitors in BM and enhanced extramedullary hematopoiesis. This adipogenic change was also evident during BM regeneration after irradiation. Several adipogenic regulator genes appeared to be regulated by Bmi1. Our results indicate that Bmi1 keeps the adipogenic differentiation program repressed in BM stromal cells to maintain the integrity of the HSC niche.

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Genetic and transcriptional landscape of plasma cells in POEMS syndrome

Nagao, Y., Mimura, N., Takeda, J., Yoshida, K., Shiozawa, Y., Oshima, M., Aoyama, K., Saraya, A., Koide, S., Rizq, O., Hasegawa, Y., Shiraishi, Y., Chiba, K., Tanaka, H., Nishijima, D., Isshiki, Y., Kayamori, K., Kawajiri-Manako, C., Oshima-Hasegawa, N., Tsukamoto, S., Mitsukawa, S., Takeda, Y., Ohwada, C., Takeuchi, M., Iseki, T., Misawa, S., Miyano, S., Ohara, O., Yokote, K., Sakaida, E., Kuwabara, S., Sanada, M., Iwama, A., Ogawa, S. and Nakaseko, C.

Leukemia 33 （ 7 ） 1723 - 1735 2019年07月

研究論文（学術雑誌）, 査読有り,  ISSN  0887-6924

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POEMS syndrome is a rare paraneoplastic disease associated with monoclonal plasma cells; however, the pathogenic importance of plasma cells remains unclear. We performed comprehensive genetic analyses of plasma cells in 20 patients with POEMS syndrome. Whole exome sequencing was performed in 11 cases and found a total of 308 somatic mutations in 285 genes. Targeted sequencing was performed in all 20 cases and identified 20 mutations in 7 recurrently mutated genes, namely KLHL6, LTB, EHD1, EML4, HEPHL1, HIPK1, and PCDH10. None of the driver gene mutations frequently found in multiple myeloma (MM) such as NRAS, KRAS, BRAF, and TP53 was detected. Copy number analysis showed chromosomal abnormalities shared with monoclonal gammopathy of undetermined significance (MGUS), suggesting a partial overlap in the early development of MGUS and POEMS syndrome. RNA sequencing revealed a transcription profile specific to POEMS syndrome when compared with normal plasma cells, MGUS and MM. Unexpectedly, disease-specific VEGFA expression was not increased in POEMS syndrome. Our study illustrates that the genetic and transcriptional profiles of plasma cells in POEMS syndrome are distinct from MM and MGUS, indicating unique function of clonal plasma cells in its pathogenesis.

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Role for tyrosine phosphorylation of SUV39H1 histone methyltransferase in enhanced trimethylation of histone H3K9 via neuregulin-1/ErbB4 nuclear signaling.

Haruna Nakajo, Kenichi Ishibashi, Kazumasa Aoyama, Sho Kubota, Hitomi Hasegawa, Noritaka Yamaguchi, Naoto Yamaguchi

Biochemical and biophysical research communications 511 （ 4 ） 765 - 771 2019年04月

研究論文（学術雑誌）, 査読有り

　概要を見る

Protein-tyrosine kinases transmit signals by phosphorylating their substrates in diverse cellular events. The receptor-type tyrosine kinase ErbB4, a member of the epidermal growth factor receptor subfamily, is activated and proteolytically cleaved upon ligand stimulation, and the cleaved ErbB4 intracellular domain (4ICD) is released into the cytoplasm and the nucleus. We previously showed that generation of nuclear 4ICD by neuregulin-1 (NRG-1) stimulation enhances the levels of trimethylation of histone H3 at lysine 9 (H3K9me3). However, it remains unclear how nuclear 4ICD enhances H3K9me3 levels. Here we show that the histone H3K9 methyltransferase SUV39H1 associates with NRG-1/ErbB4-mediated H3K9me3. Knockdown of SUV39H1 blocked NRG-1-mediated enhancement of the levels of H3K9me3. Nuclear 4ICD was found to phosphorylate SUV39H1 primarily at Tyr-297, -303, and -308 that are conserved among humans, mice, and flies. Furthermore, knockdown-rescue experiments showed that the unphosphorylatable SUV39H1 mutant (3 YF) was incapable of enhancing the levels of H3K9me3 upon NRG-1 stimulation. These results suggest that nuclear ErbB4 enhances H3K9me3 levels through tyrosine phosphorylation of SUV39H1 in NRG-1/ErbB4 signal-mediated chromatin remodeling.

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PARP1 inhibition alleviates injury in ARH3-deficient mice and human cells

Mashimo, M., Bu, X., Aoyama, K., Kato, J., Ishiwata-Endo, H., Stevens, L. A., Kasamatsu, A., Wolfe, L. A., Toro, C., Adams, D., Markello, T., Gahl, W. A. and Moss, J.

JCI Insight 4 （ 4 ）  2019年02月

研究論文（学術雑誌）, 筆頭著者, 査読有り,  ISSN  2379-3708

　概要を見る

Poly(ADP-ribosyl)ation refers to the covalent attachment of ADP-ribose to protein, generating branched, long chains of ADP-ribose moieties, known as poly(ADP-ribose) (PAR). Poly(ADP-ribose) polymerase 1 (PARP1) is the main polymerase and acceptor of PAR in response to DNA damage. Excessive intracellular PAR accumulation due to PARP1 activation leads cell death in a pathway known as parthanatos. PAR degradation is mainly controlled by poly(ADP-ribose) glycohydrolase (PARG) and ADP-ribose-acceptor hydrolase 3 (ARH3). Our previous results demonstrated that ARH3 confers protection against hydrogen peroxide (H2O2) exposure, by lowering cytosolic and nuclear PAR levels and preventing apoptosis-inducing factor (AIF) nuclear translocation. We identified a family with an ARH3 gene mutation that resulted in a truncated, inactive protein. The 8-year-old proband exhibited a progressive neurodegeneration phenotype. In addition, parthanatos was observed in neurons of the patient's deceased sibling, and an older sibling exhibited a mild behavioral phenotype. Consistent with the previous findings, the patient's fibroblasts and ARH3-deficient mice were more sensitive, respectively, to H2O2 stress and cerebral ischemia/reperfusion-induced PAR accumulation and cell death. Further, PARP1 inhibition alleviated cell death and injury resulting from oxidative stress and ischemia/reperfusion. PARP1 inhibitors may attenuate the progression of neurodegeneration in affected patients with ARH3 deficiency.

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Forkhead box protein A1 confers resistance to transforming growth factor-β-induced apoptosis in breast cancer cells through inhibition of Smad3 nuclear translocation

Hirata, K., Takakura, Y., Shibazaki, M., Morii, M., Honda, T., Oshima, M., Aoyama, K., Iwama, A., Nakayama, Y., Takano, H., Yamaguchi, N. and Yamaguchi, N.

J Cell Biochem 120 （ 2 ） 2259 - 2270 2019年02月

研究論文（学術雑誌）, 査読有り,  ISSN  0730-2312

　概要を見る

Transforming growth factor-β (TGF-β) induces apoptosis of normal epithelial cells, such as mammary epithelium. Although breast cancer progression associates with acquisition of resistance to TGF-β-induced apoptosis, the molecular mechanisms underlying this resistance are largely unknown. Here, we show that forkhead box protein A1 (FOXA1), which is known as a pioneer transcription factor, suppresses TGF-β-induced apoptosis of estrogen receptor-positive breast cancer cells. FOXA1 is found to inhibit nuclear translocation of Smad3, a key transcription factor downstream of TGF-β signaling, through suppression of the binding of Smad3 to the nuclear import receptor importin7. Furthermore, RNA sequencing analyses show that knockdown of FOXA1 upregulates Smad3-mediated proapoptotic gene expression. These results demonstrate that FOXA1 as a potent survival factor that suppresses TGF-β-induced apoptosis by inhibiting Smad3 signaling in estrogen receptor-positive breast cancer cells. Thus, we provide evidence for the first time that FOXA1 localizing to the cytoplasm negatively regulates Smad3-induced apoptosis in TGF-β-mediated signal transduction.

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Desuppression of TGF-β signaling via nuclear c-Abl-mediated phosphorylation of TIF1γ/TRIM33 at Tyr-524, -610, and -1048.

Yuki R, Tatewaki T, Yamaguchi N, Aoyama K, Honda T, Kubota S, Morii M, Manabe I, Kuga T, Tomonaga T, Yamaguchi N

Oncogene 38 （ 5 ） 637 - 655 2019年01月

研究論文（学術雑誌）, 査読有り,  ISSN  0950-9232

　概要を見る

Protein-tyrosine kinases regulate a broad range of intracellular processes occurring primarily just beneath the plasma membrane. With the greatest care to prevent dephosphorylation, we have shown that nuclear tyrosine phosphorylation regulates global chromatin structural states. However, the roles for tyrosine phosphorylation in the nucleus are poorly understood. Here we identify transcriptional intermediary factor 1-γ (TIF1γ/TRIM33/Ectodermin), which suppresses transforming growth factor-β (TGF-β) signaling through the association with Smad2/3 transcription factor, as a new nuclear substrate of c-Abl tyrosine kinase. Replacement of the three tyrosine residues Tyr-524, -610, and -1048 with phenylalanine (3YF) inhibits c-Abl-mediated phosphorylation of TIF1γ and enhances TIF1γ's association with Smad3. Importantly, knockdown-rescue experiments show that 3YF strengthens TIF1γ's ability to suppress TGF-β signaling. Intriguingly, activation of c-Abl by epidermal growth factor (EGF) induces desuppression of TGF-β signaling via enhancing the tyrosine phosphorylation level of TIF1γ. TGF-β together with EGF synergistically provokes desuppressive responses of epithelial-to-mesenchymal transition through tyrosine phosphorylation of TIF1γ. These results suggest that nuclear c-Abl-mediated tyrosine phosphorylation of TIF1γ has a desuppressive role in TGF-β-Smad2/3 signaling.

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Bcor insufficiency promotes initiation and progression of myelodysplastic syndrome.

Tara S, Isshiki Y, Nakajima-Takagi Y, Oshima M, Aoyama K, Tanaka T, Shinoda D, Koide S, Saraya A, Miyagi S, Manabe I, Matsui H, Koseki H, Bardwell VJ, Iwama A

Blood 132 （ 23 ） 2470 - 2483 2018年12月

研究論文（学術雑誌）, 査読有り,  ISSN  0006-4971

　概要を見る

BCOR, encoding BCL-6 corepressor (BCOR), is X-linked and targeted by somatic mutations in various hematological malignancies including myelodysplastic syndrome (MDS). We previously reported that mice lacking Bcor exon 4 (Bcor (ΔE4/y) ) in the hematopoietic compartment developed NOTCH-dependent acute T-cell lymphoblastic leukemia (T-ALL). Here, we analyzed mice lacking Bcor exons 9 and 10 (Bcor (ΔE9-10/y) ), which express a carboxyl-terminal truncated BCOR that fails to interact with core effector components of polycomb repressive complex 1.1. Bcor (ΔE9-10/y) mice developed lethal T-ALL in a similar manner to Bcor (ΔE4/y) mice, whereas Bcor (ΔE9-10/y) hematopoietic cells showed a growth advantage in the myeloid compartment that was further enhanced by the concurrent deletion of Tet2 Tet2 (Δ/Δ) Bcor (ΔE9-10/y) mice developed lethal MDS with progressive anemia and leukocytopenia, inefficient hematopoiesis, and the morphological dysplasia of blood cells. Tet2 (Δ/Δ) Bcor (ΔE9-10/y) MDS cells reproduced MDS or evolved into lethal MDS/myeloproliferative neoplasms in secondary recipients. Transcriptional profiling revealed the derepression of myeloid regulator genes of the Cebp family and Hoxa cluster genes in Bcor (ΔE9-10/y) progenitor cells and the activation of p53 target genes specifically in MDS erythroblasts where massive apoptosis occurred. Our results reveal a tumor suppressor function of BCOR in myeloid malignancies and highlight the impact of Bcor insufficiency on the initiation and progression of MDS.

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Ezh1 Targets Bivalent Genes to Maintain Self-Renewing Stem Cells in Ezh2-Insufficient Myelodysplastic Syndrome

Aoyama, K., Oshima, M., Koide, S., Suzuki, E., Mochizuki-Kashio, M., Kato, Y., Tara, S., Shinoda, D., Hiura, N., Nakajima-Takagi, Y., Sashida, G. and Iwama, A.

iScience 9   161 - 174 2018年10月

研究論文（学術雑誌）, 筆頭著者, 責任著者, 査読有り,  ISSN  2589-0042

　概要を見る

Polycomb repressive complex (PRC) 2 represses transcription through histone H3K27 trimethylation (H3K27me3). We previously reported that the hematopoietic-cell-specific deletion of Ezh2, encoding a PRC2 enzyme, induced myelodysplastic syndrome (MDS) in mice, whereas the concurrent Ezh1 deletion depleted hematopoietic stem and progenitor cells (HSPCs). We herein demonstrated that mice with only one Ezh1 allele (Ezh1(+/-)Ezh2(Delta/Delta)) maintained HSPCs. A chromatin immunopreciptation sequence analysis revealed that residual PRC2 preferentially targeted genes with high levels of H3K27me3 and H2AK119 monoubiquitination (H2AK119ub1) in HSPCs (designated as Ezh1 core target genes), which were mostly developmental regulators, and maintained H3K27me3 levels in Ezh1(+/-)Ezh2(Delta/Delta) HSPCs. Even upon the complete depletion of Ezh1 and Ezh2, H2AK119ub1 levels were largely retained, and only a minimal number of Ezh1 core targets were de-repressed. These results indicate that genes marked with high levels of H3K27me3 and H2AK119ub1 are the core targets of polycomb complexes in HSPCs as well as MDS stem cells.

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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

　概要を見る

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.

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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

　概要を見る

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.

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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

　概要を見る

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.

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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

　概要を見る

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.

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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

　概要を見る

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.

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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

　概要を見る

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.

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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

　概要を見る

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.

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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

　概要を見る

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.

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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

　概要を見る

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.

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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

　概要を見る

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.

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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

　概要を見る

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.

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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

　概要を見る

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.

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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

　概要を見る

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.

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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

　概要を見る

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.

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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

　概要を見る

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.

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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

　概要を見る

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.

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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

　概要を見る

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.

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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

　概要を見る

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.

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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

　概要を見る

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.

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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

　概要を見る

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.

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Formation of long and winding nuclear F-actin bundles by nuclear c-Abl tyrosine kinase

Aoyama, K., Yuki, R., Horiike, Y., Kubota, S., Yamaguchi, N., Morii, M., Ishibashi, K., Nakayama, Y., Kuga, T., Hashimoto, Y., Tomonaga, T. and Yamaguchi, N.

Exp Cell Res 319 （ 20 ） 3251 - 68 2013年12月

研究論文（学術雑誌）, 筆頭著者, 査読有り,  ISSN  0014-4827

　概要を見る

The non-receptor-type tyrosine kinase c-Abl is involved in actin dynamics in the cytoplasm. Having three nuclear localization signals (NLSs) and one nuclear export signal, c-Abl shuttles between the nucleus and the cytoplasm. Although monomeric actin and filamentous actin (F-actin) are present in the nucleus, little is known about the relationship between c-Abl and nuclear actin dynamics. Here, we show that nuclear-localized c-Abl induces nuclear F-actin formation. Adriamycin-induced DNA damage together with leptomycin B treatment accumulates c-Abl into the nucleus and increases the levels of nuclear F-actin. Treatment of c-Abl-knockdown cells with Adriamycin and leptomycin B barely increases the nuclear F-actin levels. Expression of nuclear-targeted c-Abl (NLS-c-Abl) increases the levels of nuclear F-actin even without Adriamycin, and the increased levels of nuclear F-actin are not inhibited by inactivation of Abl kinase activity. Intriguingly, expression of NLS-c-Abl induces the formation of long and winding bundles of F-actin within the nucleus in a c-Abl kinase activity-dependent manner. Furthermore, NLS-c-AblDeltaC, which lacks the actin-binding domain but has the full tyrosine kinase activity, is incapable of forming nuclear F-actin and in particular long and winding nuclear F-actin bundles. These results suggest that nuclear c-Abl plays critical roles in actin dynamics within the nucleus.

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Phosphorylation of KRAB-associated protein 1 (KAP1) at Tyr-449, Tyr-458, and Tyr-517 by nuclear tyrosine kinases inhibits the association of KAP1 and heterochromatin protein 1α (HP1α) with heterochromatin

Kubota, S., Fukumoto, Y., Aoyama, K., Ishibashi, K., Yuki, R., Morinaga, T., Honda, T., Yamaguchi, N., Kuga, T., Tomonaga, T. and Yamaguchi, N.

J Biol Chem 288 （ 24 ） 17871 - 83 2013年06月

研究論文（学術雑誌）, 査読有り,  ISSN  0021-9258

　概要を見る

Protein tyrosine phosphorylation regulates a wide range of cellular processes at the plasma membrane. Recently, we showed that nuclear tyrosine phosphorylation by Src family kinases (SFKs) induces chromatin structural changes. In this study, we identify KRAB-associated protein 1 (KAP1/TIF1β/TRIM28), a component of heterochromatin, as a nuclear tyrosine-phosphorylated protein. Tyrosine phosphorylation of KAP1 is induced by several tyrosine kinases, such as Src, Lyn, Abl, and Brk. Among SFKs, Src strongly induces tyrosine phosphorylation of KAP1. Nucleus-targeted Lyn potentiates tyrosine phosphorylation of KAP1 compared with intact Lyn, but neither intact Fyn nor nucleus-targeted Fyn phosphorylates KAP1. Substitution of the three tyrosine residues Tyr-449/Tyr-458/Tyr-517, located close to the HP1 binding-motif, into phenylalanine ablates tyrosine phosphorylation of KAP1. Immunostaining and chromatin fractionation show that Src and Lyn decrease the association of KAP1 with heterochromatin in a kinase activity-dependent manner. KAP1 knockdown impairs the association of HP1α with heterochromatin, because HP1α associates with KAP1 in heterochromatin. Intriguingly, tyrosine phosphorylation of KAP1 decreases the association of HP1α with heterochromatin, which is inhibited by replacement of endogenous KAP1 with its phenylalanine mutant (KAP1-Y449F/Y458F/Y517F, KAP1-3YF). In DNA damage, KAP1-3YF repressed transcription of p21. These results suggest that nucleus-localized tyrosine kinases, including SFKs, phosphorylate KAP1 at Tyr-449/Tyr-458/Tyr-517 and inhibit the association of KAP1 and HP1α with heterochromatin.

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Nuclear ErbB4 signaling through H3K9me3 is antagonized by EGFR-activated c-Src

Ishibashi, K., Fukumoto, Y., Hasegawa, H., Abe, K., Kubota, S., Aoyama, K., Kubota, S., Nakayama, Y. and Yamaguchi, N.

J Cell Sci 126 （ Pt 2 ） 625 - 37 2013年01月

研究論文（学術雑誌）, 査読有り,  ISSN  0021-9533

　概要を見る

The ErbB family of receptor tyrosine kinases comprises four members: epidermal growth factor receptor (EGFR)/ErbB1, HER2/ErbB2, ErbB3 and ErbB4, and plays roles in signal transduction at the plasma membrane upon ligand stimulation. Stimulation with neuregulin-1 (NRG-1) cleaves ErbB4 and releases the ErbB4 intracellular domain (4ICD) that translocates into the nucleus to control gene expression. However, little is known about the regulation of 4ICD nuclear signaling through tyrosine phosphorylation. We show here that 4ICD nuclear signaling is antagonized by EGF-induced c-Src activation through EGFR. Generation of 4ICD by NRG-1 leads to increased levels of trimethylated histone H3 on lysine 9 (H3K9me3) in a manner dependent on the nuclear accumulation of 4ICD and its tyrosine kinase activity. Once EGF activates c-Src downstream of EGFR concomitantly with NRG-1-induced ErbB4 activation, c-Src associates with phospho-Tyr950 and phospho-Tyr1056 on 4ICD, thereby decreasing nuclear accumulation of 4ICD and inhibiting an increase of H3K9me3 levels. Moreover, 4ICD-induced transcriptional repression of the human telomerase reverse transcriptase (hTERT) is inhibited by EGF-EGFR-Src signaling. Thus, our findings reveal c-Src-mediated inhibitory regulation of ErbB4 nuclear signaling upon EGFR activation.

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Nuclear c-Abl-mediated tyrosine phosphorylation induces chromatin structural changes through histone modifications that include H4K16 hypoacetylation

Aoyama, K., Fukumoto, Y., Ishibashi, K., Kubota, S., Morinaga, T., Horiike, Y., Yuki, R., Takahashi, A., Nakayama, Y. and Yamaguchi, N.

Exp Cell Res 317 （ 20 ） 2874 - 903 2011年12月

研究論文（学術雑誌）, 筆頭著者, 査読有り,  ISSN  0014-4827

　概要を見る

c-Abl tyrosine kinase, which is ubiquitously expressed, has three nuclear localization signals and one nuclear export signal and can shuttle between the nucleus and the cytoplasm. c-Abl plays important roles in cell proliferation, adhesion, migration, and apoptosis. Recently, we developed a pixel imaging method for quantitating the level of chromatin structural changes and showed that nuclear Src-family tyrosine kinases are involved in chromatin structural changes upon growth factor stimulation. Using this method, we show here that nuclear c-Abl induces chromatin structural changes in a manner dependent on the tyrosine kinase activity. Expression of nuclear-targeted c-Abl drastically increases the levels of chromatin structural changes, compared with that of c-Abl. Intriguingly, nuclear-targeted c-Abl induces heterochromatic profiles of histone methylation and acetylation, including hypoacetylation of histone H4 acetylated on lysine 16 (H4K16Ac). The level of heterochromatic histone modifications correlates with that of chromatin structural changes. Adriamycin-induced DNA damage stimulates translocation of c-Abl into the nucleus and induces chromatin structural changes together with H4K16 hypoacetylation. Treatment with trichostatin A, a histone deacetylase inhibitor, blocks chromatin structural changes but not nuclear tyrosine phosphorylation by c-Abl. These results suggest that nuclear c-Abl plays an important role in chromatin dynamics through nuclear tyrosine phosphorylation-induced heterochromatic histone modifications.

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Cost-effective gene transfection by DNA compaction at pH 4.0 using acidified, long shelf-life polyethylenimine

Fukumoto, Y., Obata, Y., Ishibashi, K., Tamura, N., Kikuchi, I., Aoyama, K., Hattori, Y., Tsuda, K., Nakayama, Y. and Yamaguchi, N.

Cytotechnology 62 （ 1 ） 73 - 82 2010年01月

研究論文（学術雑誌）, 査読有り,  ISSN  0920-9069

　概要を見る

Introduction of genetic material into cells is an essential prerequisite for current research in molecular cell biology. Although transfection with commercially available reagents results in excellent gene expression, their high costs are obstacles to experimentation with a large number or large scales of transfection. The cationic polymer linear-polyethylenimine (MW 25,000) (PEI), one of the most cost-effective vehicles, facilitates DNA compaction by polyplex formation, which leads to efficient delivery of DNA into cells by endocytosis. However, the use of PEI is still limited because of substantial cytotoxicity and intolerable deterioration in transfection efficiency by its low stability. Here, we show that acidification of PEI is important for its transfection activity. Dissolving PEI powder in 0.2N HCl confers a long shelf-life for PEI storage at 4 and -80 degrees C, and the polyplex formation of plasmid DNA with PEI is optimized in lactate-buffered saline at pH 4.0. Furthermore, changing the culture medium at 8-12 h posttransfection can minimize the cytotoxicity of PEI without sacrificing the high transfection efficiency comparable to that of commercial reagents. The cost per test using acidified PEI is drastically reduced to approximately 1:10,000, compared with commercial reagents. Thus, we conclude that acidification of PEI satisfactorily accomplishes cost-effective, high-efficiency transfection.

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