Publications by authors named "Atsunori Saraya"

37 Publications

The combination of the tubulin binding small molecule PTC596 and proteasome inhibitors suppresses the growth of myeloma cells.

Sci Rep 2021 Jan 22;11(1):2074. Epub 2021 Jan 22.

Department of Hematology, Chiba University Hospital, Chiba, Japan.

The novel small molecule PTC596 inhibits microtubule polymerization and its clinical development has been initiated for some solid cancers. We herein investigated the preclinical efficacy of PTC596 alone and in combination with proteasome inhibitors in the treatment of multiple myeloma (MM). PTC596 inhibited the proliferation of MM cell lines as well as primary MM samples in vitro, and this was confirmed with MM cell lines in vivo. PTC596 synergized with bortezomib or carfilzomib to inhibit the growth of MM cells in vitro. The combination treatment of PTC596 with bortezomib exerted synergistic effects in a xenograft model of human MM cell lines in immunodeficient mice and exhibited acceptable tolerability. Mechanistically, treatment with PTC596 induced cell cycle arrest at G2/M phase followed by apoptotic cell death, associated with the inhibition of microtubule polymerization. RNA sequence analysis also revealed that PTC596 and the combination with bortezomib affected the cell cycle and apoptosis in MM cells. Importantly, endoplasmic reticulum stress induced by bortezomib was enhanced by PTC596, providing an underlying mechanism of action of the combination therapy. Our results indicate that PTC596 alone and in combination with proteasome inhibition are potential novel therapeutic options to improve outcomes in patients with MM.
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http://dx.doi.org/10.1038/s41598-021-81577-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7822878PMC
January 2021

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

Biochem Biophys Res Commun 2020 01 1;521(3):612-619. Epub 2019 Nov 1.

Department of Cellular and Molecular Medicine, Graduate School of Medicine, Chiba University, Chiba, Japan; Division of Stem Cell and Molecular Medicine, Center for Stem Cell Biology and Regenerative Medicine, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan. Electronic address:

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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http://dx.doi.org/10.1016/j.bbrc.2019.10.153DOI Listing
January 2020

Akt inhibition synergizes with polycomb repressive complex 2 inhibition in the treatment of multiple myeloma.

Cancer Sci 2019 Dec 22;110(12):3695-3707. Epub 2019 Oct 22.

Department of Transfusion Medicine and Cell Therapy, Chiba University Hospital, Chiba, Japan.

Polycomb repressive complex 2 (PRC2) components, EZH2 and its homolog EZH1, and PI3K/Akt signaling pathway are focal points as therapeutic targets for multiple myeloma. However, the exact crosstalk between their downstream targets remains unclear. We herein elucidated some epigenetic interactions following Akt inhibition and demonstrated the efficacy of the combined inhibition of Akt and PRC2. We found that TAS-117, a potent and selective Akt inhibitor, downregulated EZH2 expression at the mRNA and protein levels via interference with the Rb-E2F pathway, while EZH1 was compensatively upregulated to maintain H3K27me3 modifications. Consistent with these results, the dual EZH2/EZH1 inhibitor, UNC1999, but not the selective EZH2 inhibitor, GSK126, synergistically enhanced TAS-117-induced cytotoxicity and provoked myeloma cell apoptosis. RNA-seq analysis revealed the activation of the FOXO signaling pathway after TAS-117 treatment. FOXO3/4 mRNA and their downstream targets were upregulated with the enhanced nuclear localization of FOXO3 protein after TAS-117 treatment. ChIP assays confirmed the direct binding of FOXO3 to EZH1 promoter, which was enhanced by TAS-117 treatment. Moreover, FOXO3 knockdown repressed EZH1 expression. Collectively, the present results reveal some molecular interactions between Akt signaling and epigenetic modulators, which emphasize the benefits of targeting PRC2 full activity and the Akt pathway as a therapeutic option for multiple myeloma.
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http://dx.doi.org/10.1111/cas.14207DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6890440PMC
December 2019

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

Blood Adv 2019 09;3(17):2537-2549

Department of Cellular and Molecular Medicine, Graduate School of Medicine, Chiba University, Chiba, Japan.

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, and in human T-cell acute lymphoblastic leukemia (T-ALL). We previously reported that 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 -deficient mice, -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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http://dx.doi.org/10.1182/bloodadvances.2018028522DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6737409PMC
September 2019

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

Exp Hematol 2019 08 11;76:24-37. Epub 2019 Aug 11.

Department of Cellular and Molecular Medicine, Graduate School of Medicine, Chiba University, Chiba, Japan; Division of Stem Cell and Molecular Medicine, Institute of Medical Science, University of Tokyo, Tokyo, Japan. Electronic address:

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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http://dx.doi.org/10.1016/j.exphem.2019.07.006DOI Listing
August 2019

Genome-Wide Mapping of Bivalent Histone Modifications in Hepatic Stem/Progenitor Cells.

Stem Cells Int 2019 1;2019:9789240. Epub 2019 Apr 1.

Department of Gastroenterology, Graduate School of Medicine, Chiba University, Chiba, Japan.

The "bivalent domain," a distinctive histone modification signature, is characterized by repressive trimethylation of histone H3 at lysine 27 (H3K27me3) and active trimethylation of histone H3 at lysine 4 (H3K4me3) marks. Maintenance and dynamic resolution of these histone marks play important roles in regulating differentiation processes in various stem cell systems. However, little is known regarding their roles in hepatic stem/progenitor cells. In the present study, we conducted the chromatin immunoprecipitation (ChIP) assay followed by high-throughput DNA sequencing (ChIP-seq) analyses in purified delta-like 1 protein (Dlk) hepatic stem/progenitor cells and successfully identified 562 genes exhibiting bivalent domains within 2 kb of the transcription start site. Gene ontology analysis revealed that these genes were enriched in developmental functions and differentiation processes. Microarray analyses indicated that many of these genes exhibited derepression after differentiation toward hepatocyte and cholangiocyte lineages. Among these, 72 genes, including and , were significantly upregulated after differentiation toward hepatocyte or cholangiocyte lineages. Knockdown of in Dlk cells suppressed colony propagation and resulted in increased numbers of albumin/cytokeratin 7 progenitor cells in colonies. These findings implicate that derepression of expression is required to induce normal differentiation processes. In conclusion, combined ChIP-seq and microarray analyses successfully identified bivalent genes. Functional analyses of these genes will help elucidate the epigenetic machinery underlying the terminal differentiation of hepatic stem/progenitor cells.
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http://dx.doi.org/10.1155/2019/9789240DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6466853PMC
April 2019

The chromatin-binding protein Phf6 restricts the self-renewal of hematopoietic stem cells.

Blood 2019 06 27;133(23):2495-2506. Epub 2019 Mar 27.

Department of Cellular and Molecular Medicine, Graduate School of Medicine, Chiba University, Chiba, Japan.

Recurrent inactivating mutations have been identified in the X-linked plant homeodomain finger protein 6 () gene, encoding a chromatin-binding transcriptional regulator protein, in various hematological malignancies. However, the role of PHF6 in normal hematopoiesis and its tumor-suppressor function remain largely unknown. We herein generated mice carrying a floxed allele and inactivated in hematopoietic cells at various developmental stages. The deletion in embryos augmented the capacity of hematopoietic stem cells (HSCs) to proliferate in cultures and reconstitute hematopoiesis in recipient mice. The deletion in neonates and adults revealed that cycling HSCs readily acquired an advantage in competitive repopulation upon the deletion, whereas dormant HSCs only did so after serial transplantations. -deficient HSCs maintained an enhanced repopulating capacity during serial transplantations; however, they did not induce any hematological malignancies. Mechanistically, Phf6 directly and indirectly activated downstream effectors in tumor necrosis factor α (TNFα) signaling. The deletion repressed the expression of a set of genes associated with TNFα signaling, thereby conferring resistance against the TNFα-mediated growth inhibition on HSCs. Collectively, these results not only define Phf6 as a novel negative regulator of HSC self-renewal, implicating inactivating mutations in the pathogenesis of hematological malignancies, but also indicate that a deficiency alone is not sufficient to induce hematopoietic transformation.
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http://dx.doi.org/10.1182/blood.2019000468DOI Listing
June 2019

Genetic and transcriptional landscape of plasma cells in POEMS syndrome.

Leukemia 2019 07 11;33(7):1723-1735. Epub 2019 Jan 11.

Department of Hematology, Chiba University Hospital, Chiba, Japan.

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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http://dx.doi.org/10.1038/s41375-018-0348-xDOI Listing
July 2019

insufficiency promotes initiation and progression of myelodysplastic syndrome.

Blood 2018 12 18;132(23):2470-2483. Epub 2018 Sep 18.

Department of Cellular and Molecular Medicine, Graduate School of Medicine, Chiba University, Chiba, Japan.

, 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 exon 4 ( ) in the hematopoietic compartment developed NOTCH-dependent acute T-cell lymphoblastic leukemia (T-ALL). Here, we analyzed mice lacking exons 9 and 10 ( ), which express a carboxyl-terminal truncated BCOR that fails to interact with core effector components of polycomb repressive complex 1.1. mice developed lethal T-ALL in a similar manner to mice, whereas hematopoietic cells showed a growth advantage in the myeloid compartment that was further enhanced by the concurrent deletion of mice developed lethal MDS with progressive anemia and leukocytopenia, inefficient hematopoiesis, and the morphological dysplasia of blood cells. 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 family and cluster genes in 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 insufficiency on the initiation and progression of MDS.
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http://dx.doi.org/10.1182/blood-2018-01-827964DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6450057PMC
December 2018

Hematopoietic insults damage bone marrow niche by activating p53 in vascular endothelial cells.

Exp Hematol 2018 07 27;63:41-51.e1. Epub 2018 Apr 27.

Department of Cellular and Molecular Medicine, Graduate School of Medicine, Chiba University, Chiba 260-8670, Japan; Division of Stem Cell and Molecular Medicine, Center for Stem Cell Biology and Regenerative Medicine, Institute of Medical Science, The University of Tokyo, Tokyo 108-8639, Japan. Electronic address:

Hematopoietic stem cells (HSCs) are exposed to various insults such as genotoxic stress, inflammation, and infection, which have a direct effect. These insults deplete, cause a functional decline in, and promote HSC aging and transformation. However, the impact of hematopoietic insults on niche cells remains largely unknown. We have reported previously that p53 is activated in blood vessels by various stresses, including hypoxia, inflammation, and aging, and contributes to tissue dysfunction and metabolic abnormalities. We hypothesized that hematopoietic insults also affect the bone marrow (BM) vascular niche. Here, we demonstrate that p53 becomes activated in BM endothelial cells upon hematopoietic stresses such as irradiation and chemotherapeutic treatments. The conditional activation of p53 in VE-cadherin vascular niche cells by deleting Mdm2 induces the expression of p53 target genes specifically in vascular endothelial cells, resulting in the dilation and collapse of vascular endothelial cells and reductions in perivascular mesenchymal stromal cell numbers. Consequently, hematopoietic stem cells (HSCs) fail to maintain dormancy, mobilize to the periphery, and are depleted significantly. Our results indicate that various hematopoietic insults affect HSCs, not only directly, but also indirectly by altering vascular integrity, which is critical for perivascular niche formation and maintenance of HSCs.
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http://dx.doi.org/10.1016/j.exphem.2018.04.006DOI Listing
July 2018

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

J Exp Med 2017 Oct 21;214(10):2901-2913. Epub 2017 Aug 21.

Department of Cellular and Molecular Medicine, Graduate School of Medicine, Chiba University, Chiba, Japan

Recurrent inactivating mutations have been identified in various hematological malignancies in the X-linked gene encoding BCL6 corepressor (BCOR); however, its tumor suppressor function remains largely uncharacterized. We generated mice missing exon 4, expressing a variant BCOR lacking the BCL6-binding domain. Although the deletion of exon 4 in male mice ( ) compromised the repopulating capacity of hematopoietic stem cells, 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. , one of the critical NOTCH1 targets in T-ALL, was highly up-regulated in T-ALL cells. Chromatin immunoprecipitation/DNA sequencing analysis revealed that BCOR was recruited to the promoter and restrained its activation in thymocytes. BCOR also targeted other NOTCH1 targets and potentially antagonized their transcriptional activation. -deficient thymocytes behaved in a manner similar to 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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http://dx.doi.org/10.1084/jem.20170167DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5626398PMC
October 2017

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

Clin Cancer Res 2017 Aug 10;23(16):4817-4830. Epub 2017 May 10.

Department of Cellular and Molecular Medicine, Chiba University Graduate School of Medicine, Chiba, Japan.

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. and 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. Proteasome inhibitors repressed 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 Bortezomib combined with UNC1999 remarkably inhibited the growth of myeloma cells Comprehensive analyses revealed several direct targets of UNC1999 including the tumor suppressor gene Derepression of by UNC1999 resulted in suppression of , 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. Our results identify dual inhibition of EZH2 and EZH1 together with proteasome inhibition as a promising epigenetics-based therapy for PRC2-dependent cancers. .
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http://dx.doi.org/10.1158/1078-0432.CCR-16-2735DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5562278PMC
August 2017

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

Oncotarget 2017 Mar;8(13):21315-21326

Department of Cellular and Molecular Medicine, Graduate School of Medicine, Chiba University, Chiba, Japan.

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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http://dx.doi.org/10.18632/oncotarget.15528DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5400586PMC
March 2017

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

Blood 2016 08 14;128(5):638-49. Epub 2016 Jun 14.

Department of Cellular and Molecular Medicine, Graduate School of Medicine, Chiba University, Chiba, Japan;

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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http://dx.doi.org/10.1182/blood-2016-01-694810DOI Listing
August 2016

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

PLoS One 2016 2;11(5):e0154561. Epub 2016 May 2.

Department of Cellular and Molecular Medicine, Graduate School of Medicine, Chiba University, Chiba, Japan.

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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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0154561PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4852895PMC
July 2017

Ezh2 regulates the Lin28/let-7 pathway to restrict activation of fetal gene signature in adult hematopoietic stem cells.

Exp Hematol 2016 Apr 7;44(4):282-96.e3. Epub 2016 Jan 7.

Department of Cellular and Molecular Medicine, Graduate School of Medicine, Chiba University, Chiba, Japan; JST, CREST, Tokyo, Japan. Electronic address:

Fetal liver hematopoietic stem cells (HSCs) seed bone marrow (BM) and undergo reprograming into adult-type HSCs that are largely quiescent and restricted in their self-renewal activity. Here we report that in the absence of the polycomb-group gene Ezh2, a cohort of fetal-specific genes, including let-7 target genes, were activated in BM hematopoietic stem/progenitor cells (HSPCs), leading to acquisition of fetal phenotypes by BM HSPCs, such as enhanced self-renewal activity and production of fetal-type lymphocytes. The Lin28b/let-7 pathway determines developmentally timed changes in HSPC programs. Of note, many of the fetal-specific let-7 target genes, including Lin28, appear to be transcriptionally repressed by Ezh2-mediated H3K27me3 in BM HSPCs, and Ezh2 loss results in their ectopic expression, particularly in hematologic malignancies that develop in the absence of Ezh2. These findings suggest that Ezh2 cooperates with let-7 microRNAs in silencing the fetal gene signature in BM HSPCs and restricts their transformation.
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http://dx.doi.org/10.1016/j.exphem.2015.12.009DOI Listing
April 2016

Histone acetylation mediated by Brd1 is crucial for Cd8 gene activation during early thymocyte development.

Nat Commun 2014 Dec 18;5:5872. Epub 2014 Dec 18.

1] Department of Cellular and Molecular Medicine, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8670, Japan [2] JST, CREST, 7 Gobancho, Chiyoda-ku, Tokyo 102-0076, Japan.

During T-cell development, Cd8 expression is controlled via dynamic regulation of its cis-regulatory enhancer elements. Insufficiency of enhancer activity causes variegated Cd8 expression in CD4(+)CD8(+) double-positive (DP) thymocytes. Brd1 is a subunit of the Hbo1 histone acetyltransferase (HAT) complex responsible for acetylation of histone H3 at lysine 14 (H3K14). Here we show that deletion of Brd1 in haematopoietic progenitors causes variegated expression of Cd8, resulting in the appearance of CD4(+)CD8(-)TCRβ(-/low) thymocytes indistinguishable from DP thymocytes in their properties. Biochemical analysis confirms that Brd1 forms a HAT complex with Hbo1 in thymocytes. ChIP analysis demonstrates that Brd1 localizes at the known enhancers in the Cd8 genes and is responsible for acetylation at H3K14. These findings indicate that the Brd1-mediated HAT activity is crucial for efficient activation of Cd8 expression via acetylation at H3K14, which serves as an epigenetic mark that promotes the recruitment of transcription machinery to the Cd8 enhancers.
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http://dx.doi.org/10.1038/ncomms6872DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4490789PMC
December 2014

Ezh2 loss promotes development of myelodysplastic syndrome but attenuates its predisposition to leukaemic transformation.

Nat Commun 2014 Jun 23;5:4177. Epub 2014 Jun 23.

1] Department of Cellular and Molecular Medicine, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8670, Japan [2] JST, CREST, 7 Gobancho, Chiyoda-ku, Tokyo 102-0076, Japan.

Loss-of-function mutations of EZH2, a catalytic component of polycomb repressive complex 2 (PRC2), are observed in ~\n10% of patients with myelodysplastic syndrome (MDS), but are rare in acute myeloid leukaemia (AML). Recent studies have shown that EZH2 mutations are often associated with RUNX1 mutations in MDS patients, although its pathological function remains to be addressed. Here we establish an MDS mouse model by transducing a RUNX1S291fs mutant into hematopoietic stem cells and subsequently deleting Ezh2. Ezh2 loss significantly promotes RUNX1S291fs-induced MDS. Despite their compromised proliferative capacity of RUNX1S291fs/Ezh2-null MDS cells, MDS bone marrow impairs normal hematopoietic cells via selectively activating inflammatory cytokine responses, thereby allowing propagation of MDS clones. In contrast, loss of Ezh2 prevents the transformation of AML via PRC1-mediated repression of Hoxa9. These findings provide a comprehensive picture of how Ezh2 loss collaborates with RUNX1 mutants in the pathogenesis of MDS in both cell autonomous and non-autonomous manners.
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http://dx.doi.org/10.1038/ncomms5177DOI Listing
June 2014

Depletion of Sf3b1 impairs proliferative capacity of hematopoietic stem cells but is not sufficient to induce myelodysplasia.

Blood 2014 May 15;123(21):3336-43. Epub 2014 Apr 15.

Department of Cellular and Molecular Medicine, Graduate School of Medicine, Chiba University, Chiba, Japan; Japan Science and Technology Corporation, Core Research for Evolutional Science and Technology, Gobancho, Chiyoda-ku, Tokyo, Japan;

Numerous studies have recently reported mutations involving multiple components of the messenger RNA (mRNA) splicing machinery in patients with myelodysplastic syndrome (MDS). SF3B1 is mutated in 70% to 85% of refractory anemia with ringed sideroblasts (RARS) patients and is highly associated with the presence of RARS, although the pathological role of SF3B1 mutations in MDS-RARS has not been elucidated yet. Here, we analyzed the function of pre-mRNA splicing factor Sf3b1 in hematopoiesis. Sf3b1(+/-) mice maintained almost normal hematopoiesis and did not develop hematological malignancies during a long observation period. However, Sf3b1(+/-) cells had a significantly impaired capacity to reconstitute hematopoiesis in a competitive setting and exhibited some enhancement of apoptosis, but they did not show any obvious defects in differentiation. Additional depletion of Sf3b1 with shRNA in Sf3b1(+/-) hematopoietic stem cells (HSCs) severely compromised their proliferative capacity both in vitro and in vivo. Finally, we unexpectedly found no changes in the frequencies of sideroblasts in either Sf3b1(+/-) erythroblasts or cultured Sf3b1(+/-) erythroblasts expressing shRNA against Sf3b1. Our findings indicate that the level of Sf3b1 expression is critical for the proliferative capacity of HSCs, but the haploinsufficiency for Sf3b1 is not sufficient to induce a RARS-like phenotype.
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http://dx.doi.org/10.1182/blood-2013-12-544544DOI Listing
May 2014

The TIF1β-HP1 system maintains transcriptional integrity of hematopoietic stem cells.

Stem Cell Reports 2014 Feb 23;2(2):145-52. Epub 2014 Jan 23.

Department of Cellular and Molecular Medicine, Graduate School of Medicine, Chiba University, Chiba 260-8670, Japan ; JST, CREST, Sanbancho, Chiyoda-ku, Tokyo 102-0075, Japan.

TIF1β is a transcriptional corepressor that recruits repressive chromatin modifiers to target genes. Its biological function and physiological targets in somatic stem cells remain largely unknown. Here, we show that TIF1β is essential for the maintenance of hematopoietic stem cells (HSCs). Deletion of Tif1b in mice induced active cycling and apoptosis of HSCs and promoted egression of HSCs from the bone marrow, leading to rapid depletion of HSCs. Strikingly, Tif1b-deficient HSCs showed a strong trend of ectopic expression of nonhematopoietic genes. Levels of heterochromatin protein 1 (HP1α, β and γ) proteins, which form a complex with TIF1β, were significantly reduced in the absence of TIF1β and depletion of HP1 recapitulated a part of the phenotypes of Tif1b-deficient HSCs. These results demonstrate that the TIF1β-HP1 system functions as a critical repressive machinery that targets genes not normally activated in the hematopoietic compartment, thereby maintaining the transcriptional signature specific to HSCs.
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http://dx.doi.org/10.1016/j.stemcr.2013.12.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3923190PMC
February 2014

Disulfiram eradicates tumor-initiating hepatocellular carcinoma cells in ROS-p38 MAPK pathway-dependent and -independent manners.

PLoS One 2014 13;9(1):e84807. Epub 2014 Jan 13.

Department of Gastroenterology and Nephrology, Graduate School of Medicine, Chiba University, Chiba, Japan.

Tumor-initiating cells (TICs) play a central role in tumor development, metastasis, and recurrence. In the present study, we investigated the effect of disulfiram (DSF), an inhibitor of aldehyde dehydrogenase, toward tumor-initiating hepatocellular carcinoma (HCC) cells. DSF treatment suppressed the anchorage-independent sphere formation of both HCC cells. Flow cytometric analyses showed that DSF but not 5-fluorouracil (5-FU) drastically reduces the number of tumor-initiating HCC cells. The sphere formation assays of epithelial cell adhesion molecule (EpCAM)(+) HCC cells co-treated with p38-specific inhibitor revealed that DSF suppresses self-renewal capability mainly through the activation of reactive oxygen species (ROS)-p38 MAPK pathway. Microarray experiments also revealed the enrichment of the gene set involved in p38 MAPK signaling in EpCAM(+) cells treated with DSF but not 5-FU. In addition, DSF appeared to downregulate Glypican 3 (GPC3) in a manner independent of ROS-p38 MAPK pathway. GPC3 was co-expressed with EpCAM in HCC cell lines and primary HCC cells and GPC3-knockdown reduced the number of EpCAM(+) cells by compromising their self-renewal capability and inducing the apoptosis. These results indicate that DSF impaired the tumorigenicity of tumor-initiating HCC cells through activation of ROS-p38 pathway and in part through the downregulation of GPC3. DSF might be a promising therapeutic agent for the eradication of tumor-initiating HCC cells.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0084807PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3890271PMC
September 2014

Concurrent loss of Ezh2 and Tet2 cooperates in the pathogenesis of myelodysplastic disorders.

J Exp Med 2013 Nov 11;210(12):2627-39. Epub 2013 Nov 11.

Department of Cellular and Molecular Medicine and 2 Department of Clinical Cell Biology and Medicine, Graduate School of Medicine, Chiba University, Chiba 260-8670, Japan.

Polycomb group (PcG) proteins are essential regulators of hematopoietic stem cells. Recent extensive mutation analyses of the myeloid malignancies have revealed that inactivating somatic mutations in PcG genes such as EZH2 and ASXL1 occur frequently in patients with myelodysplastic disorders including myelodysplastic syndromes (MDSs) and MDS/myeloproliferative neoplasm (MPN) overlap disorders (MDS/MPN). In our patient cohort, EZH2 mutations were also found and often coincided with tet methylcytosine dioxygenase 2 (TET2) mutations. Consistent with these findings, deletion of Ezh2 alone was enough to induce MDS/MPN-like diseases in mice. Furthermore, concurrent depletion of Ezh2 and Tet2 established more advanced myelodysplasia and markedly accelerated the development of myelodysplastic disorders including both MDS and MDS/MPN. Comprehensive genome-wide analyses in hematopoietic progenitor cells revealed that upon deletion of Ezh2, key developmental regulator genes were kept transcriptionally repressed, suggesting compensation by Ezh1, whereas a cohort of oncogenic direct and indirect polycomb targets became derepressed. Our findings provide the first evidence of the tumor suppressor function of EZH2 in myeloid malignancies and highlight the cooperative effect of concurrent gene mutations in the pathogenesis of myelodysplastic disorders.
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http://dx.doi.org/10.1084/jem.20131144DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3832936PMC
November 2013

Bmi1 confers resistance to oxidative stress on hematopoietic stem cells.

PLoS One 2012 11;7(5):e36209. Epub 2012 May 11.

Department of Cellular and Molecular Medicine, Graduate School of Medicine, Chiba University, Chiba, Japan.

Background: The polycomb-group (PcG) proteins function as general regulators of stem cells. We previously reported that retrovirus-mediated overexpression of Bmi1, a gene encoding a core component of polycomb repressive complex (PRC) 1, maintained self-renewing hematopoietic stem cells (HSCs) during long-term culture. However, the effects of overexpression of Bmi1 on HSCs in vivo remained to be precisely addressed.

Methodology/principal Findings: In this study, we generated a mouse line where Bmi1 can be conditionally overexpressed under the control of the endogenous Rosa26 promoter in a hematopoietic cell-specific fashion (Tie2-Cre;R26Stop(FL)Bmi1). Although overexpression of Bmi1 did not significantly affect steady state hematopoiesis, it promoted expansion of functional HSCs during ex vivo culture and efficiently protected HSCs against loss of self-renewal capacity during serial transplantation. Overexpression of Bmi1 had no effect on DNA damage response triggered by ionizing radiation. In contrast, Tie2-Cre;R26Stop(FL)Bmi1 HSCs under oxidative stress maintained a multipotent state and generally tolerated oxidative stress better than the control. Unexpectedly, overexpression of Bmi1 had no impact on the level of intracellular reactive oxygen species (ROS).

Conclusions/significance: Our findings demonstrate that overexpression of Bmi1 confers resistance to stresses, particularly oxidative stress, onto HSCs. This thereby enhances their regenerative capacity and suggests that Bmi1 is located downstream of ROS signaling and negatively regulated by it.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0036209PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3350495PMC
October 2012

Dependency on the polycomb gene Ezh2 distinguishes fetal from adult hematopoietic stem cells.

Blood 2011 Dec 31;118(25):6553-61. Epub 2011 Oct 31.

Department of Cellular and Molecular Medicine, Graduate School of Medicine, Chiba University, Chiba, Japan.

Polycomb-group (PcG) proteins are essential regulators of hematopoietic stem cells (HSCs). In contrast to Bmi1, a component of Polycomb repressive complex 1 (PRC1), the role of PRC2 and its components in hematopoiesis remains elusive. Here we show that Ezh2, a core component of PRC2, is essential for fetal, but not adult, HSCs. Ezh2-deficient embryos died of anemia because of insufficient expansion of HSCs/progenitor cells and defective erythropoiesis in fetal liver. Deletion of Ezh2 in adult BM, however, did not significantly compromise hematopoiesis, except for lymphopoiesis. Of note, Ezh2-deficient fetal liver cells showed a drastic reduction in trimethylation of histone H3 at lysine 27 (H3K27me3) accompanied by derepression of a large cohort of genes, whereas on homing to BM, they acquired a high level of H3K27me3 and long-term repopulating capacity. Quantitative RT-PCR revealed that Ezh1, the gene encoding a backup enzyme, is highly expressed in HSCs/progenitor cells in BM compared with those in fetal liver, whereas Ezh2 is ubiquitously expressed. These findings suggest that Ezh1 complements Ezh2 in the BM, but not in the fetal liver, and reveal that the reinforcement of PcG-mediated gene silencing occurs during the transition from proliferative fetal HSCs to quiescent adult HSCs.
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http://dx.doi.org/10.1182/blood-2011-03-340554DOI Listing
December 2011

The Hbo1-Brd1/Brpf2 complex is responsible for global acetylation of H3K14 and required for fetal liver erythropoiesis.

Blood 2011 Sep 13;118(9):2443-53. Epub 2011 Jul 13.

Department of Cellular and Molecular Medicine, Graduate School of Medicine, Graduate School of Pharmaceutical, Sciences, Chiba University, Chiba, Japan.

The histone acetyltransferases (HATs) of the MYST family include TIP60, HBO1, MOZ/MORF, and MOF and function in multisubunit protein complexes. Bromodomain-containing protein 1 (BRD1), also known as BRPF2, has been considered a subunit of the MOZ/MORF H3 HAT complex based on analogy with BRPF1 and BRPF3. However, its physiologic function remains obscure. Here we show that BRD1 forms a novel HAT complex with HBO1 and regulates erythropoiesis. Brd1-deficient embryos showed severe anemia because of impaired fetal liver erythropoiesis. Biochemical analyses revealed that BRD1 bridges HBO1 and its activator protein, ING4. Genome-wide mapping in erythroblasts demonstrated that BRD1 and HBO1 largely colocalize in the genome and target key developmental regulator genes. Of note, levels of global acetylation of histone H3 at lysine 14 (H3K14) were profoundly decreased in Brd1-deficient erythroblasts and depletion of Hbo1 similarly affected H3K14 acetylation. Impaired erythropoiesis in the absence of Brd1 accompanied reduced expression of key erythroid regulator genes, including Gata1, and was partially restored by forced expression of Gata1. Our findings suggest that the Hbo1-Brd1 complex is the major H3K14 HAT required for transcriptional activation of erythroid developmental regulator genes.
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http://dx.doi.org/10.1182/blood-2011-01-331892DOI Listing
September 2011

3-Deazaneplanocin A is a promising therapeutic agent for the eradication of tumor-initiating hepatocellular carcinoma cells.

Int J Cancer 2012 Jun 20;130(11):2557-67. Epub 2011 Aug 20.

Department of Medicine and Clinical Oncology, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8670, Japan.

Recent advances in stem cell biology have identified tumor-initiating cells (TICs) in a variety of cancers including hepatocellular carcinoma (HCC). Polycomb group gene products such as BMI1 and EZH2 have been characterized as general self-renewal regulators in a wide range of normal stem cells and TICs. We previously reported that Ezh2 tightly regulates the self-renewal and differentiation of murine hepatic stem/progenitor cells. However, the role of EZH2 in tumor-initiating HCC cells remains unclear. In this study, we conducted loss-of-function assay of EZH2 using short-hairpin RNA and pharmacological inhibition of EZH2 by an S-adenosylhomocysteine hydrolase inhibitor, 3-deazaneplanocin A (DZNep). Both EZH2-knockdown and DZNep treatment impaired cell growth and anchorage-independent sphere formation of HCC cells in culture. Flow cytometric analyses revealed that the two approaches decreased the number of epithelial cell adhesion molecule (EpCAM)(+) tumor-initiating cells. Administration of 5-fluorouracil (5-FU) or DZNep suppressed the tumors by implanted HCC cells in non-obese diabetic/severe combined immunodeficient mice. Of note, however, DZNep but not 5-FU predominantly reduced the number of EpCAM(+) cells and diminished the self-renewal capability of these cells as judged by sphere formation assays. Our findings reveal that tumor-initiating HCC cells are highly dependent on EZH2 for their tumorigenic activity. Although further analyses of TICs from primary HCC would be necessary, pharmacological interference with EZH2 might be a promising therapeutic approach to targeting tumor-initiating HCC cells.
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http://dx.doi.org/10.1002/ijc.26264DOI Listing
June 2012

Direct activation of STAT5 by ETV6-LYN fusion protein promotes induction of myeloproliferative neoplasm with myelofibrosis.

Br J Haematol 2011 Jun 15;153(5):589-98. Epub 2011 Apr 15.

Division of Haematology, Department of Clinical Cell Biology, Graduate School of Medicine, Chiba University, Chiba, Japan.

Myeloproliferative neoplasms (MPN), a group of haematopoietic stem cell (HSC) disorders, are often accompanied by myelofibrosis. We previously identified the fusion of the ETV6 gene to the LYN gene (ETV6-LYN) in idiopathic myelofibrosis with ins(12;8)(p13;q11q21). The introduction of ETV6-LYN into HSCs resulted in fatal MPN with massive myelofibrosis in mice, implicating the rearranged LYN kinase in the pathogenesis of MPN with myelofibrosis. However, the signalling molecules directly downstream from and activated by ETV6-LYN remain unknown. In this study, we demonstrated that the direct activation of STAT5 by ETV6-LYN is crucial for the development of MPN. ETV6-LYN was constitutively active as a kinase through autophosphorylation. ETV6-LYN, but not its kinase-dead mutant, supported cytokine-free proliferation of haematopoietic cells. STAT5 was activated in a JAK2-independent manner in ETV6-LYN-expressing cells. ETV6-LYN interacted with STAT5 and directly activated STAT5 both in vitro and in vivo. Of note, ETV6-LYN did not support the formation of colonies by Stat5-deficient HSCs under cytokine-free conditions and the capacity of ETV6-LYN to induce MPN with myelofibrosis was profoundly attenuated in a Stat5-null background. These findings define STAT5 as a direct target of ETV6-LYN and unveil the LYN-STAT5 axis as a novel pathway to augment proliferative signals in MPN and leukaemia.
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http://dx.doi.org/10.1111/j.1365-2141.2011.08663.xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3091948PMC
June 2011

A novel zinc finger protein Zfp277 mediates transcriptional repression of the Ink4a/arf locus through polycomb repressive complex 1.

PLoS One 2010 Aug 24;5(8):e12373. Epub 2010 Aug 24.

Department of Cellular and Molecular Medicine, Graduate School of Medicine, Chiba University, Chiba, Japan.

Background: Polycomb group (PcG) proteins play a crucial role in cellular senescence as key transcriptional regulators of the Ink4a/Arf tumor suppressor gene locus. However, how PcG complexes target and contribute to stable gene silencing of the Ink4a/Arf locus remains little understood.

Methodology/principal Findings: We examined the function of Zinc finger domain-containing protein 277 (Zfp277), a novel zinc finger protein that interacts with the PcG protein Bmi1. Zfp277 binds to the Ink4a/Arf locus in a Bmi1-independent manner and interacts with polycomb repressor complex (PRC) 1 through direct interaction with Bmi1. Loss of Zfp277 in mouse embryonic fibroblasts (MEFs) caused dissociation of PcG proteins from the Ink4a/Arf locus, resulting in premature senescence associated with derepressed p16(Ink4a) and p19(Arf) expression. Levels of both Zfp277 and PcG proteins inversely correlated with those of reactive oxygen species (ROS) in senescing MEFs, but the treatment of Zfp277(-/-) MEFs with an antioxidant restored the binding of PRC2 but not PRC1 to the Ink4a/Arf locus. Notably, forced expression of Bmi1 in Zfp277(-/-) MEFs did not restore the binding of Bmi1 to the Ink4a/Arf locus and failed to bypass cellular senescence. A Zfp277 mutant that could not bind Bmi1 did not rescue Zfp277(-/-) MEFs from premature senescence.

Conclusions/significance: Our findings implicate Zfp277 in the transcriptional regulation of the Ink4a/Arf locus and suggest that the interaction of Zfp277 with Bmi1 is essential for the recruitment of PRC1 to the Ink4a/Arf locus. Our findings also highlight dynamic regulation of both Zfp277 and PcG proteins by the oxidative stress pathways.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0012373PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2927437PMC
August 2010

Bmi1 promotes hepatic stem cell expansion and tumorigenicity in both Ink4a/Arf-dependent and -independent manners in mice.

Hepatology 2010 Sep;52(3):1111-23

Department of Cellular and Molecular Medicine, Graduate School of Medicine, Chiba University, Chiba, Japan.

Unlabelled: We previously reported that forced expression of Bmi1 (B lymphoma Moloney murine leukemia virus insertion region 1 homolog) in murine hepatic stem/progenitor cells purified from fetal liver enhances their self-renewal and drives cancer initiation. In the present study, we examined the contribution of the Ink4a/Arf tumor suppressor gene locus, one of the major targets of Bmi1, to stem cell expansion and cancer initiation. Bmi1(-/-) Delta-like protein (Dlk)(+) hepatic stem/progenitor cells showed de-repression of the Ink4a/Arf locus and displayed impaired growth activity. In contrast, Ink4a/Arf(-/-) Dlk(+) cells gave rise to considerably larger colonies containing a greater number of bipotent cells than wild-type Dlk(+) cells. Although Ink4a/Arf(-/-) Dlk(+) cells did not initiate tumors in recipient nonobese diabetic/severe combined immunodeficiency mice, enforced expression of Bmi1 in Ink4a/Arf(-/-) Dlk(+) cells further augmented their self-renewal capacity and resulted in tumor formation in vivo. Microarray analyses successfully identified five down-regulated genes as candidate downstream targets for Bmi1 in hepatic stem/progenitor cells. Of these genes, enforced expression of sex determining region Y-box 17 (Sox17) in Dlk(+) cells strongly suppressed colony propagation and tumor growth.

Conclusion: These results indicate that repression of targets of Bmi1 other than the Ink4a/Arf locus plays a crucial role in the oncogenic transformation of hepatic stem/progenitor cells. Functional analyses of Bmi1 target genes would be of importance to elucidate the molecular machinery underlying hepatic stem cell system and explore therapeutic approaches for the eradication of liver cancer stem cells.
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http://dx.doi.org/10.1002/hep.23793DOI Listing
September 2010

Dmap1 plays an essential role in the maintenance of genome integrity through the DNA repair process.

Genes Cells 2009 Nov 21;14(11):1347-57. Epub 2009 Oct 21.

Department of Cellular and Molecular Medicine, Graduate School of Medicine, Chiba University, Chuo-ku, Chiba, Japan.

Faithful control of cell cycle checkpoint and DNA repair contributes to prevent the cells from chromosomal instability and tumorigenesis. Dnmt1-associated protein 1 (Dmap1), a component of the NuA4 histone acetyltransferase complex, was originally identified as an interacting molecule with DNMT1 which co-localizes with PCNA at DNA replication foci. However, its role in cellular functions remains largely unknown. Here we show that Dmap1 knockdown in mouse embryonic fibroblasts (MEFs) lead to spontaneous double-strand breaks (DSBs), resulting in growth arrest because of p53-dependent cell cycle checkpoint activation. Deletion of both Dmap1 and p53 in MEFs synergized towards enhanced generation of DSBs, chromosomal abnormalities and tumor development in mice. Notably, we found that, on DNA damage, Dmap1 was recruited to the damaged sites to form complexes with gamma-H2AX and replication factors, including Pcna. Depletion of Dmap1 in MEFs abrogated the stable accumulation of Pcna at the DNA damaged sites. Furthermore, the re-introduction of Dmap1 mutants with a reduced capacity to bind Pcna failed to ameliorate the impaired DNA repair in Dmap1-depleted cells. These findings indicate that Dmap1 is required to involve Pcna in DNA repair. Together, Dmap1 plays a crucial role in DNA repair, and is indispensable for the maintenance of chromosomal integrity.
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http://dx.doi.org/10.1111/j.1365-2443.2009.01352.xDOI Listing
November 2009