Polycomb Group Gene mel-18 Regulates Early T Progenitor Expansion by Maintaining the Expression of Hes-1, a Target of the Notch Pathway This information is current as of September 29, 2021. Masaki Miyazaki, Hiroshi Kawamoto, Yuko Kato, Manami Itoi, Kazuko Miyazaki, Kyoko Masuda, Satoshi Tashiro, Hiroto Ishihara, Kazuhiko Igarashi, Takashi Amagai, Rieko Kanno and Masamoto Kanno J Immunol 2005; 174:2507-2516; ; Downloaded from doi: 10.4049/jimmunol.174.5.2507 http://www.jimmunol.org/content/174/5/2507 http://www.jimmunol.org/ References This article cites 54 articles, 17 of which you can access for free at: http://www.jimmunol.org/content/174/5/2507.full#ref-list-1 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! 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The Journal of Immunology Polycomb Group Gene mel-18 Regulates Early T Progenitor Expansion by Maintaining the Expression of Hes-1, a Target of the Notch Pathway1 Masaki Miyazaki,* Hiroshi Kawamoto,§ Yuko Kato,* Manami Itoi,¶ Kazuko Miyazaki,‡ Kyoko Masuda,ʈ Satoshi Tashiro,† Hiroto Ishihara,* Kazuhiko Igarashi,† Takashi Amagai,¶ Rieko Kanno,* and Masamoto Kanno2* Polycomb group (PcG) proteins play a role in the maintenance of cellular identity throughout many rounds of cell division through the regulation of gene expression. In this report we demonstrate that the loss of the PcG gene mel-18 impairs the expansion of the most immature T progenitor cells at a stage before the rearrangement of the TCR ␤-chain gene in vivo and in vitro. This impairment of these T progenitors appears to be associated with increased susceptibility to cell death. We also show that the Downloaded from expression of Hes-1, one of the target genes of the Notch signaling pathway, is drastically down-regulated in early T progenitors -isolated from mel-18؊/؊ mice. In addition, mel-18؊/؊ T precursors could not maintain the Hes-1 expression induced by Delta like-1 in monolayer culture. Collectively, these data indicate that mel-18 contributes to the maintenance of the active state of the Hes-1 gene as a cellular memory system, thereby supporting the expansion of early T progenitors. The Journal of Immunology, 2005, 174: 2507–2516. http://www.jimmunol.org/ he Polycomb group (PcG)3 genes were originally identi- protein complex that also contains M33, BMI-1, RAE-28, fied in Drosophila as a class of regulators responsible for RING1A, and RING1B (3, 4). This mammalian complex is similar T maintaining homeotic gene expression by contributing to to PRC1 in Drosophila, which is able to competitively inhibit the the cellular memory of somite identity throughout cell division. chromatin remodeling complex, SWI/SNF, and interacts with se- PcG genes are conserved from Drosophila to mammals, and their quence-specific, DNA-binding factors (Pipsquesk, Zeste, and protein products have been reported to localize to the nucleus as GAGA) and histone deacetylase (5–7). This PRC1 is shown to multimeric protein complexes. These proteins epigenetically main- collaborate with the ESC-E(Z) complex in regulation of gene ex- tain the repressed state of target genes through the modification of pression through epigenetic modification of chromatin structure (8, by guest on September 29, 2021 chromatin structure. In Drosophila, at least two types of PcG com- 9). Although several studies have recently described the silencing plexes, each with different properties, can be distinguished: Poly- mechanisms of PcG complexes, to our knowledge, it has rarely comb repressive complex 1 (PRC1) and ESC-E(Z) (1, 2). The been reported that the PcG gene is practically required for the mel-18 gene is a mammalian homologue of the Drosophila pos- maintenance of gene expression in the mammalian cell differenti- terior sex combs (Psc) gene, and its product is a member of a PcG ation system. It is well known that PcG genes play a significant role in the regulation of lymphocyte differentiation (10, 11). Mice deficient in Departments of *Immunology and †Biomedical Chemistry, Graduate School of Bio- medical Science, and ‡Department of Developmental Biology, Research Institute for the individual components of the PRC1-like complex, mel-18, Radiation and Medicine, Hiroshima University, Hiroshima, Japan; §Laboratory for bmi-1, rae-28, and m33, display SCID (12–15). Loss of function of Lymphocyte Development, RIKEN Research Center for Allergy and Immunology, Yokohama, Japan; ¶Department of Immunology and Microbiology, Meiji University bmi-1 causes a severe block of B cell development (12), and ʈ of Oriental Medicine, Kyoto, Japan; and Department of Immunology and Cell Bi- rae-28 deficiency reduces the generation of pre-B and immature B ology, Graduate School of Biostudies, Kyoto University, Kyoto, Japan cells from fetal liver (FL) hemopoietic progenitors (15). In T cell Received for publication August 6, 2004. Accepted for publication October 31, 2004. development, bmi-1 mutant mice exhibited impaired thymocyte The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance development at an immature stage (16). In mel-18 mutant mice, B with 18 U.S.C. Section 1734 solely to indicate this fact. cell maturation is arrested between the pro- and pre-B cell stages, 1 This work was supported by Grants-in-Aid for Science from the Ministry of Edu- and severe thymic atrophy is also observed (14). In mature resting cation, Culture, Sports, Science, and Technology of Japan. The authors have no fi- B cells, mel-18 negatively regulates B cell receptor-induced pro- nancial conflict of interest. liferation through the down-regulation of the c-Myc/cdc25 cascade 2 Address correspondence and reprint requests to Dr. Masamoto Kanno, Department of Immunology, Graduate School of Biomedical Science, Hiroshima University, (17). Th2 cell differentiation is also impaired in mel-18 mutant 1-2-3, Kasumi, Minami-ku, Hiroshima 734-8551, Japan. E-mail address: mkanno@ mice, and mel-18 is involved in the induction of GATA-3 under hiroshima-u.ac.jp Th2-skewed conditions (18). In contrast to the extensive analysis 3 Abbreviations used in this paper: PcG, Polycomb group; DL1, Delta-like-1; DN, double negative; DP, double positive; dpc, days postcoitum; ETP, enhancers of of the roles of mel-18 in both immature and mature B cell devel- Trithorax and Polycomb, early T progenitor; FB, fetal blood; FL, fetal liver; FT, fetal opment as well as in mature T cell function, much remains to be thymus; FTOC, FT organ culture; HOM-C, homeotic gene; HOS, high oxygen sub- mersion; HSC, hemopoietic stem cell; KSL, LinϪScaIϩc-Kitϩ cell; LinϪ, lineage investigated with regard to a potential role for mel-18 in thymocyte marker-negative; PI, propidium iodide; PRC1, Polycomb repressive complex 1; 7Rϩ, development. Therefore, we performed a comprehensive analysis LinϪSca-1ϩc-KitlowIL-7R␣ϩ; SCF, stem cell factor; siRNA, small interfering RNA; SP, single positive; TrxG, Trithorax group; TSt-4/DL1, TSt-4 stroma cell expressing of mel-18 function to clarify its role and the underlying mecha- Delta-like-1. nisms in the regulation of thymocyte development. Copyright © 2005 by The American Association of Immunologists, Inc. 0022-1767/05/$02.00 2508 mel-18 REGULATES EXPANSION OF EARLY T PROGENITORS The thymus is the major site of T cell differentiation and mat- B220-FITC and -biotin (RA3-6B2); anti-CD19-FITC and -biotin (1D3); uration. Thymocytes can be divided into four main populations: anti-Mac-1-FITC and -biotin (M1/70); anti-Gr-1-FITC and -biotin (RB6- CD4ϪCD8Ϫ double negative (DN), CD4ϩCD8ϩ double positive 8C5); Ter119-biotin; anti-NK1.1-FITC (PK136); anti-CD45.2-FITC (104); ϩ ϩ and anti-CD45.1-PE (A20). Anti-CD127-PE and -biotin (A7R34) and (DP), and CD4 or CD8 single positive (CD4SP or CD8SP) Ter119-FITC were purchased from eBiosciences. Biotinylated Abs were cells. DN cells can be further divided into four subpopulations revealed with streptavidin-CyChrome (BD Pharmingen). To analyze DN (DN1-DN4) according to their CD44 and CD25 expression pat- CD3Ϫ thymocytes, lineage marker-negative (LinϪ)ScaIϩc-Kitϩ cell terns. CD44ϩCD25Ϫ (DN1) thymocytes represent the earliest T (KSL), and common lymphoid progenitors in bone marrow and p-T cell in FL, all cells expressing lineage markers (CD4, CD8, CD3, B220, CD19, progenitors in the thymus. DN1 cells differentiate through a ϩ ϩ Ϫ ϩ Mac-1, Gr-1, Ter119, and NK1.1) were gated out of the analyses. FACS CD44 CD25 (DN2) stage into CD44 CD25 (DN3) cells, in analysis was performed on a FACSCalibur flow cytometer (BD Bio- which the TCR␤ rearrangement takes place. Those with a success- sciences), and data were analyzed using CellQuest software. For cell sort- ful TCR rearrangement at the ␤ locus receive a pre-TCR signal and ing, all cells were stained with biotinylated lineage markers, bound to differentiate through the CD44ϪCD25Ϫ (DN4) stage into DP cells streptavidin-magnetic beads and depleted of lineage-positive cells using MACS separation column (Miltenyi Biotec). The lineage-negative cells (19). During intrathymic T cell development, thymocytes are required were then stained with subsequent Abs as described above.