Publications by authors named "Satoru Miyagi"

50 Publications

Plant homeodomain finger protein 6 in the regulation of normal and malignant hematopoiesis.

Curr Opin Hematol 2020 07;27(4):248-253

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.

Purpose Of Review: Even though an increasing amount of sequencing data on the leukemia genome has highlighted a tumor-suppressive function for plant homeodomain finger protein 6 (PHF6), its role in the hematopoietic system remained elusive until recently. The purpose of this review is to describe the role of PHF6 in normal hematopoiesis and leukemogenesis based on recent findings from knockout mouse models.

Recent Findings: In a mouse model, the loss of Phf6 enhanced the bone marrow repopulating capacity of hematopoietic stem cells (HSCs) during serial transplantations without transforming hematopoietic cells, whereas donor mice, which lacked Phf6 expression in the hematopoietic system, did not show any apparent phenotypes in the steady-state. Mechanistically, Phf6 activates effectors in the tumor necrosis factor α (Tnfα) pathway. Therefore, a Phf6 deficiency attenuates the expression of the effectors and confers resistance against Tnfα-mediated growth inhibition to HSCs. Moreover, the loss of Phf6 promoted the development of leukemia induced by aberrant TLX3 expression or an active NOTCH mutation.

Summary: Phf6 restricts the self-renewal of HSCs by governing the Tnfα pathway. Phf6 fulfills a tumor-suppressive function, and its loss synergizes with leukemic lesions to promote the onset of hematological malignancies.
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http://dx.doi.org/10.1097/MOH.0000000000000588DOI Listing
July 2020

The KDM4/JMJD2 histone demethylases are required for hematopoietic stem cell maintenance.

Blood 2019 10 21;134(14):1154-1158. Epub 2019 Aug 21.

Biotech Research and Innovation Centre and.

KDM4/JMJD2 are H3K9- and H3K36-specific demethylases, which are considered promising therapeutic targets for the treatment of acute myeloid leukemia (AML) harboring MLL translocations. Here, we investigate the long-term effects of depleting KDM4 activity on normal hematopoiesis to probe potential side effects of continuous inhibition of these enzymes. Utilizing conditional / triple-knockout mice, we show that KDM4 activity is required for hematopoietic stem cell (HSC) maintenance in vivo. The knockout of the KDM4 demethylases leads to accumulation of H3K9me3 on transcription start sites and the corresponding downregulation of expression of several genes in HSCs. We show that 2 of these genes, and , are essential for the maintenance of hematopoietic cells. Taken together, our results show that the KDM4 demethylases are required for the expression of genes essential for the long-term maintenance of normal hematopoiesis.
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http://dx.doi.org/10.1182/blood.2019000855DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6776794PMC
October 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

Cholix toxin, an eukaryotic elongation factor 2 ADP-ribosyltransferase, interacts with Prohibitins and induces apoptosis with mitochondrial dysfunction in human hepatocytes.

Cell Microbiol 2019 08 14;21(8):e13033. Epub 2019 May 14.

Pulmonary Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland, USA.

Vibrio cholerae produced-Cholix toxin (Cholix) is a cytotoxin that ADP-ribosylates eukaryotic elongation factor 2, inhibiting protein synthesis, and inducing apoptosis. Here, we identified prohibitin (PHB) 1 and 2 as novel Cholix-interacting membrane proteins in immortalised human hepatocytes and HepG2 cells by Cholix immunoprecipitation assays. The expression level of PHB1 was decreased by Cholix after a 12hr incubation. Cholix-induced poly (ADP-ribose) polymerase (PARP) cleavage was significantly enhanced in PHB (PHB1 or PHB2) knockdown cells. In contrast, transiently overexpressed PHB in hepatocytes attenuated Cholix-induced Bax/Bak conformational changes and PARP cleavage. In addition, Cholix-induced reactive oxygen species production and accumulation of fragmented mitochondria were enhanced in PHB-knockdown cells. Furthermore, Cholix induced activation of Rho-associated coiled coil-containing protein kinase 1 (ROCK1), which was enhanced in PHB-knockdown cells, followed by actin filament depolymerisation and accumulation of tubulin in the blebbing cells. Inhibition of ROCK1 by siRNA or its inhibitor suppressed Cholix-induced PARP cleavage and reactive oxygen species generation. Our findings identify PHB as a new protein that interacts with Cholix and is involved in Cholix-induced mitochondrial dysfunction and cytoskeletal rearrangement by ROCK1 activation during apoptosis.
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http://dx.doi.org/10.1111/cmi.13033DOI Listing
August 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

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

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

Jmjd2/Kdm4 demethylases are required for expression of Il3ra and survival of acute myeloid leukemia cells.

Genes Dev 2016 06 2;30(11):1278-88. Epub 2016 Jun 2.

Biotech Research and Innovation Centre (BRIC), University of Copenhagen, 2200 Copenhagen, Denmark; Centre for Epigenetics, University of Copenhagen, 2200 Copenhagen, Denmark; The Danish Stem Cell Center (DanStem), Faculty of Health and Medical Sciences, University of Copenhagen, 2200 Copenhagen, Denmark.

Acute myeloid leukemias (AMLs) with a rearrangement of the mixed-linage leukemia (MLL) gene are aggressive hematopoietic malignancies. Here, we explored the feasibility of using the H3K9- and H3K36-specific demethylases Jmjd2/Kdm4 as putative drug targets in MLL-AF9 translocated leukemia. Using Jmjd2a, Jmjd2b, and Jmjd2c conditional triple-knockout mice, we show that Jmjd2/Kdm4 activities are required for MLL-AF9 translocated AML in vivo and in vitro. We demonstrate that expression of the interleukin 3 receptor α (Il3ra also known as Cd123) subunit is dependent on Jmjd2/Kdm4 through a mechanism involving removal of H3K9me3 from the promoter of the Il3ra gene. Importantly, ectopic expression of Il3ra in Jmjd2/Kdm4 knockout cells alleviates the requirement of Jmjd2/Kdm4 for the survival of AML cells, showing that Il3ra is a critical downstream target of Jmjd2/Kdm4 in leukemia. These results suggest that the JMJD2/KDM4 proteins are promising drug targets for the treatment of AML.
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http://dx.doi.org/10.1101/gad.280495.116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4911927PMC
June 2016

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

SWI/SNF Subunits SMARCA4, SMARCD2 and DPF2 Collaborate in MLL-Rearranged Leukaemia Maintenance.

PLoS One 2015 16;10(11):e0142806. Epub 2015 Nov 16.

Biotech Research and Innovation Centre (BRIC), University of Copenhagen, Ole Maaløes Vej 5, 2200 Copenhagen, Denmark.

Alterations in chromatin structure caused by deregulated epigenetic mechanisms collaborate with underlying genetic lesions to promote cancer. SMARCA4/BRG1, a core component of the SWI/SNF ATP-dependent chromatin-remodelling complex, has been implicated by its mutational spectrum as exerting a tumour-suppressor function in many solid tumours; recently however, it has been reported to sustain leukaemogenic transformation in MLL-rearranged leukaemia in mice. Here we further explore the role of SMARCA4 and the two SWI/SNF subunits SMARCD2/BAF60B and DPF2/BAF45D in leukaemia. We observed the selective requirement for these proteins for leukaemic cell expansion and self-renewal in-vitro as well as in leukaemia. Gene expression profiling in human cells of each of these three factors suggests that they have overlapping functions in leukaemia. The gene expression changes induced by loss of the three proteins demonstrate that they are required for the expression of haematopoietic stem cell associated genes but in contrast to previous results obtained in mouse cells, the three proteins are not required for the expression of c-MYC regulated genes.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0142806PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4646637PMC
June 2016

Haploinsufficiency of the c-myc transcriptional repressor FIR, as a dominant negative-alternative splicing model, promoted p53-dependent T-cell acute lymphoblastic leukemia progression by activating Notch1.

Oncotarget 2015 Mar;6(7):5102-17

Department of Molecular Diagnosis, Graduate School of Medicine, Chiba University, Inohana, Chiba, Japan.

FUSE-binding protein (FBP)-interacting repressor (FIR) is a c-myc transcriptional suppressor. A splice variant of FIR that lacks exon 2 in the transcriptional repressor domain (FIRΔexon2) upregulates c-myc transcription by inactivating wild-type FIR. The ratio of FIRΔexon2/FIR mRNA was increased in human colorectal cancer and hepatocellular carcinoma tissues. Because FIRΔexon2 is considered to be a dominant negative regulator of FIR, FIR heterozygous knockout (FIR⁺/⁻) C57BL6 mice were generated. FIR complete knockout (FIR⁻/⁻) was embryonic lethal before E9.5; therefore, it is essential for embryogenesis. This strongly suggests that insufficiency of FIR is crucial for carcinogenesis. FIR⁺/⁻ mice exhibited prominent c-myc mRNA upregulation, particularly in the peripheral blood (PB), without any significant pathogenic phenotype. Furthermore, elevated FIRΔexon2/FIR mRNA expression was detected in human leukemia samples and cell lines. Because the single knockout of TP53 generates thymic lymphoma, FIR⁺/⁻TP53⁻/⁻ generated T-cell type acute lymphocytic/lymphoblastic leukemia (T-ALL) with increased organ or bone marrow invasion with poor prognosis. RNA-sequencing analysis of sorted thymic lymphoma cells revealed that the Notch signaling pathway was activated significantly in FIR⁺/⁻TP53⁻/⁻ compared with that in FIR⁺/⁺TP53⁻/⁻ mice. Notch1 mRNA expression in sorted thymic lymphoma cells was confirmed using qRT-PCR. In addition, flow cytometry revealed that c-myc mRNA was negatively correlated with FIR but positively correlated with Notch1 in sorted T-ALL/thymic lymphoma cells. Moreover, the knockdown of TP53 or c-myc using siRNA decreased Notch1 expression in cancer cells. In addition, an adenovirus vector encoding FIRΔexon2 cDNA increased bleomycin-induced DNA damage. Taken together, these data suggest that the altered expression of FIRΔexon2 increased Notch1 at least partially by activating c-Myc via a TP53-independent pathway. In conclusion, the alternative splicing of FIR, which generates FIRΔexon2, may contribute to both colorectal carcinogenesis and leukemogenesis.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4467136PMC
http://dx.doi.org/10.18632/oncotarget.3244DOI Listing
March 2015

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

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

shRNA screening identifies JMJD1C as being required for leukemia maintenance.

Blood 2014 Mar 5;123(12):1870-82. Epub 2014 Feb 5.

Biotech Research and Innovation Centre.

Epigenetic regulatory mechanisms are implicated in the pathogenesis of acute myeloid leukemia (AML) and acute lymphoid leukemia (ALL). Recent progress suggests that proteins involved in epigenetic control are amenable to drug intervention, but little is known about the cancer-specific dependency on epigenetic regulators for cell survival and proliferation. We used a mouse model of human AML induced by the MLL-AF9 fusion oncogene and an epigenetic short hairpin RNA (shRNA) library to screen for novel potential drug targets. As a counter-screen for general toxicity of shRNAs, we used normal mouse bone marrow cells. One of the best candidate drug targets identified in these screens was Jmjd1c. Depletion of Jmjd1c impairs growth and colony formation of mouse MLL-AF9 cells in vitro as well as establishment of leukemia after transplantation. Depletion of JMJD1C impairs expansion and colony formation of human leukemic cell lines, with the strongest effect observed in the MLL-rearranged ALL cell line SEM. In both mouse and human leukemic cells, the growth defect upon JMJD1C depletion appears to be primarily due to increased apoptosis, which implicates JMJD1C as a potential therapeutic target in leukemia.
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http://dx.doi.org/10.1182/blood-2013-08-522094DOI Listing
March 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

Role of SOX17 in hematopoietic development from human embryonic stem cells.

Blood 2013 Jan 20;121(3):447-58. Epub 2012 Nov 20.

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

To search for genes that promote hematopoietic development from human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs), we overexpressed several known hematopoietic regulator genes in hESC/iPSC-derived CD34(+)CD43(-) endothelial cells (ECs) enriched in hemogenic endothelium (HE). Among the genes tested, only Sox17, a gene encoding a transcription factor of the SOX family, promoted cell growth and supported expansion of CD34(+)CD43(+)CD45(-/low) cells expressing the HE marker VE-cadherin. SOX17 was expressed at high levels in CD34(+)CD43(-) ECs compared with low levels in CD34(+)CD43(+)CD45(-) pre-hematopoietic progenitor cells (pre-HPCs) and CD34(+)CD43(+)CD45(+) HPCs. Sox17-overexpressing cells formed semiadherent cell aggregates and generated few hematopoietic progenies. However, they retained hemogenic potential and gave rise to hematopoietic progenies on inactivation of Sox17. Global gene-expression analyses revealed that the CD34(+)CD43(+)CD45(-/low) cells expanded on overexpression of Sox17 are HE-like cells developmentally placed between ECs and pre-HPCs. Sox17 overexpression also reprogrammed both pre-HPCs and HPCs into HE-like cells. Genome-wide mapping of Sox17-binding sites revealed that Sox17 activates the transcription of key regulator genes for vasculogenesis, hematopoiesis, and erythrocyte differentiation directly. Depletion of SOX17 in CD34(+)CD43(-) ECs severely compromised their hemogenic activity. These findings suggest that SOX17 plays a key role in priming hemogenic potential in ECs, thereby regulating hematopoietic development from hESCs/iPSCs.
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http://dx.doi.org/10.1182/blood-2012-05-431403DOI Listing
January 2013

Ezh2 augments leukemogenicity by reinforcing differentiation blockage in acute myeloid leukemia.

Blood 2012 Aug 7;120(5):1107-17. Epub 2012 Jun 7.

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

EZH2, a catalytic component of the polycomb repressive complex 2, trimethylates histone H3 at lysine 27 (H3K27) to repress the transcription of target genes. Although EZH2 is overexpressed in various cancers, including some hematologic malignancies, the role of EZH2 in acute myeloid leukemia (AML) has yet to be examined in vivo. In the present study, we transformed granulocyte macrophage progenitors from Cre-ERT;Ezh2(flox/flox) mice with the MLL-AF9 leukemic fusion gene to analyze the function of Ezh2 in AML. Deletion of Ezh2 in transformed granulocyte macrophage progenitors compromised growth severely in vitro and attenuated the progression of AML significantly in vivo. Ezh2-deficient leukemic cells developed into a chronic myelomonocytic leukemia-like disease with a lower frequency of leukemia-initiating cells compared with the control. Chromatin immunoprecipitation followed by sequencing revealed a significant reduction in the levels of trimethylation at H3K27 in Ezh2-deficient leukemic cells, not only at Cdkn2a, a known major target of Ezh2, but also at a cohort of genes relevant to the developmental and differentiation processes. Overexpression of Egr1, one of the derepressed genes in Ezh2-deficient leukemic cells, promoted the differentiation of AML cells profoundly. Our findings suggest that Ezh2 inhibits differentiation programs in leukemic stem cells, thereby augmenting their leukemogenic activity.
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http://dx.doi.org/10.1182/blood-2011-11-394932DOI Listing
August 2012

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

Aldehyde dehydrogenase 1 is associated with recurrence-free survival but not stem cell-like properties in hepatocellular carcinoma.

Hepatol Res 2012 Nov 14;42(11):1100-11. Epub 2012 May 14.

Departments of Medicine and Clinical Oncology Cellular and Molecular Medicine General Surgery, Graduate School of Medicine, Chiba University, Chiba, Japan King's College Hospital, Institute of Liver Studies, Denmark Hill, London, UK.

Aim:   It has been reported that aldehyde dehydrogenase 1 A1 (ALDH1) could be not only a normal stem cell marker but also a cancer stem cell marker. ALDH1 expression could be a predictor of poor prognosis in a wide range of cancers. However, the role of ALDH1 in hepatocellular carcinoma (HCC) remains unclear.

Method:   We conducted loss-of-function assays for ALDH1 by using short-hairpin RNA in HCC cells and evaluated the correlation between ALDH1 expression and clinicopathological features based on immunohistochemical assessment of 49 primary HCC tissues.

Results:   Neither cell proliferation nor the anchorage-independent sphere formation ability of HCC cells were altered after ALDH1 knockdown. Flow cytometric analyses revealed that ALDH1 knockdown showed no remarkable change in the proportion of epithelial cell adhesion molecule (EpCAM)(+) tumor-initiating cells. Although non-tumor tissues in primary HCC samples diffusely and homogenously expressed ALDH1 at low levels, tumor tissues contained cells with high levels of ALDH1 expression at varying frequencies. Primary HCC samples were categorized as ALDH1-high or ALDH1-low based on the percentage of ALDH1-overexpressing cells. ALDH1-high HCC was characterized by low serum levels of α-fetoprotein (P < 0.01) and well-differentiated pathology (P = 0.03). Multivariate analysis showed that high ALDH1 expression was a favorable prognostic factor in recurrence-free survival of HCC (P = 0.02).

Conclusion:   Our findings show that ALDH1 expression has little association with stem cell-like features in HCC cells. ALDH1 might function as a differentiation marker rather than a stem cell marker in HCC.
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http://dx.doi.org/10.1111/j.1872-034X.2012.01028.xDOI Listing
November 2012

Lethal myelofibrosis induced by Bmi1-deficient hematopoietic cells unveils a tumor suppressor function of the polycomb group genes.

J Exp Med 2012 Mar 20;209(3):445-54. Epub 2012 Feb 20.

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

Polycomb-group (PcG) proteins form the multiprotein polycomb repressive complexes (PRC) 1 and 2, and function as transcriptional repressors through histone modifications. They maintain the proliferative capacity of hematopoietic stem and progenitor cells by repressing the transcription of tumor suppressor genes, namely Ink4a and Arf, and thus have been characterized as oncogenes. However, the identification of inactivating mutations in the PcG gene, EZH2, unveiled a tumor suppressor function in myeloid malignancies, including primary myelofibrosis (PMF). Here, we show that loss of another PcG gene, Bmi1, causes pathological hematopoiesis similar to PMF. In a mouse model, loss of Bmi1 in Ink4a-Arf(-/-) hematopoietic cells induced abnormal megakaryocytopoiesis accompanied by marked extramedullary hematopoiesis, which eventually resulted in lethal myelofibrosis. Absence of Bmi1 caused derepression of a cohort of genes, including Hmga2, which is an oncogene overexpressed in PMF. Chromatin immunoprecipitation assays revealed that Bmi1 directly represses the transcription of Hmga2. Overexpression of Hmga2 in hematopoietic stem cells induced a myeloproliferative state with enhanced megakaryocytopoiesis in mice, implicating Hmga2 in the development of pathological hematopoiesis in the absence of Bmi1. Our findings provide the first genetic evidence of a tumor suppressor function of Bmi1 and uncover the role of PcG proteins in restricting growth by silencing oncogenes.
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http://dx.doi.org/10.1084/jem.20111709DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3302226PMC
March 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

Forced expression of the histone demethylase Fbxl10 maintains self-renewing hematopoietic stem cells.

Exp Hematol 2011 Jun 12;39(6):697-709.e5. Epub 2011 Apr 12.

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

Objective: The methylation status of histones changes dramatically depending on cellular context and defines cell type-specific gene expression profiles. Histone demethylases have recently been implicated in this process. However, it is unknown how histone demethylases function in the maintenance of self-renewing hematopoietic stem cells (HSCs).

Materials And Methods: We profiled the expression of histone demethylase genes in mouse hematopoietic cells and listed genes preferentially expressed in HSCs. We analyzed the impact of a selected gene by transducing CD34(-)c-Kit(+)Sca-1(+)lineage marker(-) (CD34(-)KSL) HSCs using retroviral system followed by in vitro methylcellulose colony assays and in vivo competitive repopulation assays.

Results: We found that F-box and leucine-rich repeat protein 10 (Fbxl10, also known as Jhdm1b or Kdm2b), is highly expressed in CD34(-)KSL HSCs. Fbxl10 encodes a demethylase specific to the histone H3 mono/di-methylated at lysine 36 (H3K36me1/me2) and forms complexes with polycomb-group proteins, essential regulators of HSCs. Forced expression of Fbxl10 in HSCs expanded numbers of colony-forming cells with multilineage differentiation potential in culture and prevented exhaustion of the long-term repopulating potential of HSCs following serial transplantation. Fbxl10 tightly repressed the expression of cyclin-dependent kinase inhibitor genes, including Ink4a, Ink4b, and Ink4c, through direct binding to their promoters and gene bodies and demethylation at H3K36. Increased levels of mono-ubiquitylation of H2A at target loci also suggested the collaboration of Fbxl10 with polycomb-group proteins.

Conclusions: Our findings implicate Fbxl10 in the maintenance of self-renewal capacity of HSCs, thus highlight a role of histone demethylation for the first time in the epigenetic regulation of HSCs.
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http://dx.doi.org/10.1016/j.exphem.2011.03.008DOI Listing
June 2011

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

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