Differential Geminin Requirement for Proliferation of Thymocytes and Mature T Cells This information is current as Dimitris Karamitros, Panorea Kotantaki, Zoi Lygerou, of October 1, 2021. Henrique Veiga-Fernandes, Vassilis Pachnis, Dimitris Kioussis and Stavros Taraviras J Immunol 2010; 184:2432-2441; Prepublished online 27 January 2010; doi: 10.4049/jimmunol.0901983 http://www.jimmunol.org/content/184/5/2432 Downloaded from Supplementary http://www.jimmunol.org/content/suppl/2010/01/27/jimmunol.090198 Material 3.DC1 http://www.jimmunol.org/ References This article cites 54 articles, 25 of which you can access for free at: http://www.jimmunol.org/content/184/5/2432.full#ref-list-1 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision by guest on October 1, 2021 • No Triage! 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The Journal of Immunology Differential Geminin Requirement for Proliferation of Thymocytes and Mature T Cells Dimitris Karamitros,* Panorea Kotantaki,* Zoi Lygerou,† Henrique Veiga-Fernandes,‡,1 Vassilis Pachnis,x Dimitris Kioussis,‡ and Stavros Taraviras* Stem/progenitor cells coordinate proliferation and differentiation, giving rise to appropriate cell numbers of functionally specialized cells during organogenesis. In different experimental systems, Geminin was shown to maintain progenitor cells and participate in fate determination decisions and organogenesis. Although the exact mechanisms are unclear, Geminin has been postulated to influence proliferation versus differentiation decisions. To gain insight into the in vivo role of Geminin in progenitor cell division and differen- tiation,wehavegeneratedmicethatspecificallylackGemininincellsoflymphoidlineagethroughCre-mediatedrecombination.Tcells lacking Geminin expression upregulate early activation markers efficiently upon TCR stimulation in vitro and are able to enter the S phase of cell cycle, but show a marked defect in completing the cycle, leading to a large proportion of T cells accumulating in S/G2/M Downloaded from phases. Accordingly, T cells deficient in Geminin show a reduced ability to repopulate lymphopenic hosts in vivo. Contrary to expect- ations, Geminin deficiency does not alter development and differentiation of T cells in vivo. Our data suggest that Geminin is required fortheproliferationeventstakingplaceeitherinvitrouponTCRreceptoractivationorduringhomeostaticexpansion,butappearstobe redundantfortheproliferationanddifferentiationofthemajorityofprogenitorTcellpopulations. TheJournalofImmunology,2010, 184: 2432–2441. http://www.jimmunol.org/ regulated balance between proliferation and differentia- a multisubunit prereplicative complex that is essential for S-phase tion is fundamental for development, organogenesis, and onset (1). Geminin binds to the DNA licensing factor Cdt1 and in- A tissue homeostasis. To achieve this, cells must integrate hibits its function, ensuring that genome replication takes place only diverse extrinsic signals, remodel chromatin, and alter their tran- once per cell cycle (1), thereby maintaining genomic integrity (2). In scriptional profiles. Changes in chromatin organization and tran- addition, it has been shown that Geminin is expressed in neural scription must be coordinated with replication to establish or progenitor cells and is downregulated in fate-restricted precursor maintain the transcriptional profile of progeny cells. cells (3), although it participates in the control of cell fate specifi- Geminin has been implicated in proliferation-differentiation cation and progenitor cell maintenance during nervous system de- by guest on October 1, 2021 decisions through balanced interactions with multiple binding velopment through modulation of proliferation, transcription, and partners. Geminin participates in cell cycle control as a negative modification of chromatin structure (4). Ectopic overexpression of regulator of licensing, a process that precedes S-phase initiation Geminin in Xenopus embryos and Drosophila induces neural spe- and consists of the formation on origins of DNA replication of cific genes and hypertrophy of neural tissue, whereas Geminin has been shown to regulate the initiation of Sox2 expression during the establishment of the neural plate in vertebrate embryos (5–7). *Department of Pharmacology and †Department of Biology, Medical School, Uni- During eye development in Medaka, Geminin acts antagonistically versity of Patras, Rio, Patras, Greece; and ‡Division of Molecular Immunology and xDivision of Molecular Neurobiology, Medical Research Council/National Institute to Six3 homeobox-containing transcription factor, regulating the for Medical Research, The Ridgeway, London, United Kingdom balance between proliferation and differentiation. Thus, Geminin 1Current address: Immunobiology Unit, Instituto de Medicina Molecular, Faculdade overexpression leads to the reduction in eye size, exhibiting reduced de Medicina de Lisboa, Lisboa, Portugal. numbers of proliferating cells and premature induction of neuronal Received for publication June 24, 2009. Accepted for publication December 23, marker expression (8). Furthermore, it inhibits Hox transcription 2009. factor function through direct and polycomb-mediated interactions, This work was supported by research funding from the Association for International determining axial patterning during chicken embryogenesis (9). Cancer Research, United Kingdom (to S.T.) and Leukemia Research Foundation (to D. Kioussis). D. Karamitros was supported by a European Molecular Biology Orga- Interestingly, Geminin directly binds to Brg1, the catalytic subunit nization short-term fellowship (ASTF 26.00-2007) and Lady Tata Memorial Trust of the SWI/SNF complex, and reduces the ability of helix-loop- studentship. P.K. was supported by a European Molecular Biology Organization helix–type transcription factors to activate downstream target genes, short-term fellowship (ASTF 60.00-04) and a Federation of European Biochemical Societies summer fellowship. suggesting a role of Geminin in the maintenance of neuronal pro- Address correspondence and reprint requests to Dr. Dimitris Kioussis, Division of genitor cells in an undifferentiated state (10). Recently, Geminin has Molecular Immunology, Medical Research Council/National Institute for Medical Re- been shown to be involved in sustaining hematopoietic stem cells, search, The Ridgeway, London NW7 1AA, United Kingdom or Dr. Stavros Taraviras, expanding its potential role in other systems (11). Consistent with an Department of Pharmacology, Medical School, University of Patras, 26504 Rio, Patras, Greece. E-mail addresses: [email protected] and [email protected] essential role in proliferation and differentiation, mice lacking The online version of this article contains supplemental material. Geminin expression are dying during the first days of embryogen- Abbreviations used in this paper: 7-AAD, 7-aminoactinomycin D; CFSE, carboxy- esis, revealing no formation of inner cell mass (12, 13). fluorescein diacetate succinimidyl ester; DN, double-negative; DP, double-positive; We have used T cell differentiation as a model to address the role of ES, embryonic stem; Foxp3, forkhead box p3; LCR, locus control region; LN, lymph Geminin in developmental processes that require a regulated balance node; PDBU, phorbol 12,13-dibutyrate; SP, single-positive; WT, wild-type. between proliferation and differentiation. During immune system de- Copyright Ó 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00 velopment and maturation, undifferentiated hematopoietic precursor www.jimmunol.org/cgi/doi/10.4049/jimmunol.0901983 The Journal of Immunology 2433 cells originating in the bone marrow enter the thymus and give rise to allele, and DNA was amplified under the following conditions: 94˚C for 45 s, lineage-committed cells destined to become T cells (14, 15). De- 62.7˚C for 45 s, and 72˚C for 1 min (primer location is indicated in the Gem veloping thymocytes are classified into four major populations based Dex3-4 allele, Fig. 1A). Verification of the pGKNeo/tk cassette removal was established via a PCR that uses primers that anchor the neo gene (59- on the expression of two coreceptor molecules, CD4 and CD8. Double- cctgcgtgcaatccatcttgttca-39) and exon 3 of the mouse geminin gene (59-gca- negative (DN) thymocytes, lacking both CD4 and CD8 expression, are gaaggctggatcatcttcagcg-39), and DNA was amplified for 30 cycles (95˚C for the earlier progenitors and can be further subdivided into four sub- 45 s, 65˚C for 45 s, and 72˚C for 120 s). CD2Cre and PC3Cre transgenes were populations, DN1–4, based on the expression of CD44 and CD25 also detected by PCR as previously described (24, 25). To analyze excision efficiency in GemDex3-4 mice, genomic DNAwas prepared from tail, thymus, markers (16),