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Apoptosis Regulator Gimap4/IAN1 a Natural Hypomorphic Variant Of A Natural Hypomorphic Variant of the Apoptosis Regulator Gimap4/IAN1 Christine Carter, Carine Dion, Silke Schnell, W. John Coadwell, Margaret Graham, Lucy Hepburn, Geoffrey This information is current as Morgan, Amanda Hutchings, John C. Pascall, Heinz Jacobs, of September 26, 2021. J. Ross Miller and Geoffrey W. Butcher J Immunol 2007; 179:1784-1795; ; doi: 10.4049/jimmunol.179.3.1784 http://www.jimmunol.org/content/179/3/1784 Downloaded from References This article cites 36 articles, 18 of which you can access for free at: http://www.jimmunol.org/content/179/3/1784.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication by guest on September 26, 2021 *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2007 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology A Natural Hypomorphic Variant of the Apoptosis Regulator Gimap4/IAN11 Christine Carter,* Carine Dion,2* Silke Schnell,2† W. John Coadwell,* Margaret Graham,* Lucy Hepburn,* Geoffrey Morgan,* Amanda Hutchings,* John C. Pascall,* Heinz Jacobs,† J. Ross Miller,* and Geoffrey W. Butcher3* The Gimap/IAN family of GTPases has been implicated in the regulation of cell survival, particularly in lymphomyeloid cells. Prosurvival and prodeath properties have been described for different family members. We generated novel serological reagents to study the expression in rats of the prodeath family member Gimap4 (IAN1), which is sharply up-regulated at or soon after the stage of T cell-positive selection in the thymus. During these investigations we were surprised to discover a severe deficiency of Gimap4 expression in the inbred Brown Norway (BN) rat. Genetic analysis linked this trait to the Gimap gene cluster on rat chromosome 4, the probable cause being an AT dinucleotide insertion in the BN Gimap4 allele (AT(؉)). This allele encodes a Downloaded from truncated form of Gimap4 that is missing 21 carboxyl-terminal residues relative to wild type. The low protein expression asso- ciated with this allele appears to have a posttranscriptional cause, because mRNA expression was apparently normal. Spontaneous and induced apoptosis of BN and wild-type T cells was analyzed in vitro and compared with the recently described mouse Gimap4 (knockout. This revealed a “delayed” apoptosis phenotype similar to but less marked than that of the knockout. The Gimap4 AT(؉ (allele found in BN was shown to be rare in inbred rat strains. Nevertheless, when wild rat DNA samples were studied the AT(؉ http://www.jimmunol.org/ allele was found at a high overall frequency (ϳ30%). This suggests an adaptive significance for this hypomorphic allele. The Journal of Immunology, 2007, 179: 1784–1795. he Gimap/IAN proteins are a family of putative GTPases the anti-apoptotic wild-type Gimap5 protein. Consistent with this in- found in vertebrates. They have relatives in higher plants terpretation, prosurvival properties of human, rat, and mouse Gimap5 T but not in most other organisms studied (1). The impor- have been demonstrated in vitro (9–11). Gimap5 has a predicted tance of the Gimap/IAN GTPase family for T lymphocyte survival transmembrane domain that appears to direct its expression to the became evident from investigations of the rat lymphopenia (lyp) surface of mitochondria (a key site of apoptotic regulation) as well as gene. This recessive mutation causes severe peripheral T lym- to some other internal cell membranes (9, 10). by guest on September 26, 2021 phopenia when homozygous and is an essential susceptibility locus Rats, mice, and humans each express seven or eight Gimap in autoimmune models of both spontaneous diabetes mellitus (the genes clustered tightly on a single autosome. The predicted pro- BioBreeding diabetes prone (BB-DP)4 rat) and eosinophilic bowel teins that they encode share amino-terminal features consisting of disease (the PVG-RT1u,lyp/lyp rat) (2–4). Positional cloning of a GTPase domain and other sequence motifs leading to their clas- lymphopenia identified a single base pair deletion in the Gimap5 sification in the AIG1 family, a sequence-based category named gene. The predicted frameshift leads to a polypeptide product from after a protein involved in responses to bacterial pathogens in this gene that is truncated by about two-thirds compared with wild plants (12). The carboxyl-terminal features of the Gimap proteins type (5, 6). The lymphopenic phenotype observed in Gimap5-de- are more diverse, with some carrying obvious transmembrane do- ficient BB-DP rats is thought to result from the in vivo apoptosis mains. Given the profound impact of Gimap5-deficiency on T lym- of mature thymocytes and T lymphocytes (7, 8) in the absence of phocyte survival, it is important to discover whether all of the Gimap genes are engaged in the regulation of apoptosis as has been suggested in a recent publication by Nitta et al., who also *Babraham Institute, Cambridge, United Kingdom; and †The Netherlands Cancer reported physical interactions between Gimap proteins and mem- Institute, Amsterdam, The Netherlands bers of the Bcl-2 protein family (11). In addition, it will be of Received for publication July 13, 2006. Accepted for publication May 11, 2007. interest to ascertain whether these proteins act separately or in a The costs of publication of this article were defrayed in part by the payment of page coordinated fashion to regulate lymphocyte survival. charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. We previously demonstrated interesting changes in the expres- 1 This work was supported by Competitive Strategic Grant funding from the Bio- sion of the Gimap genes during the development of rat T lympho- technology and Biological Sciences Research Council (BBSRC), BBSRC GAIN cytes (13). Among the most dramatic of these were the changes in Grant 202/GAN13085 (to G.W.B. and J.R.M.), and The Netherlands Cancer Institute the expression of Gimap4 (IAN1), previously reported in mice Grant SFN SFR 2.1.29 (to H.J.). S.S. was supported by a travel allowance from Boehringer Ingelheim Fonds. (14), that shows a substantial rise in expression at the thymic ϩ ϩ 2 C.D. and S.S. contributed equally to this study and are listed alphabetically. CD4 CD8 double positive to mature single positive transition. 3 Address correspondence and reprint requests to Dr. Geoffrey W. Butcher, Babraham Gimap4 lacks an obvious transmembrane domain and appears to Institute, Cambridge, U.K. E-mail address: [email protected] lie downstream of the TCR in an as yet ill-defined signaling path- 4 Abbreviations used in this paper: BB-DP, BioBreeding diabetes prone; BN, Brown way (14–16). Norway; DN, double negative; LN, lymph node; ORF, open reading frame; SNP, We raised Abs against Gimap4 to study its protein expression in single nucleotide polymorphism; UTR, untranslated region. rats and mice (Ref. 13 and current report). Using these reagents, Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00 we have encountered an unexpected deficiency in Gimap4 protein www.jimmunol.org The Journal of Immunology 1785 expression in the inbred Brown Norway (BN) rat, a strain used with monoclonal anti-actin (catalog no. A5441; Sigma-Aldrich) as a load- widely in immunological research as well as in other biomedical ing control. disciplines. We also report here functional comparisons of BN rat Real-time PCR T lymphocytes with those from mice carrying a targeted deletion in Gimap4, recently described by one of our laboratories (16). Quantitative differences in Gimap4 message levels were measured using comparative real-time PCR. Lymphocyte subpopulations were flow sorted as described above. RNA was prepared using TRIzol (Invitrogen Life Materials and Methods Technologies) and cDNA was made using SuperScript III reverse tran- Animals scriptase (Invitrogen Life Technologies). Methods are essentially as de- n u b u scribed previously (13, 19), including the use of two control genes, 6-phos- Rats of the strains BN, PVG-RT1 (BN), PVG-RT1 ,RT7 , PVG-RT1 ,lyp/ phofructokinase C (6PFK) and cirhin. Exon-specific primer pairs were lyp, BB-disease resistant (BB-DR)/Ed (which is also genetically lyp/lyp; designed to span an intron in genomic DNA such that any products from Ref. 17), and DA, as well as some backcross populations (see Results), contaminating genomic DNA could be identified and excluded on the basis were bred and maintained in specific pathogen-free conditions at the of size. Primers for Gimap4 were: 5Ј-GAGCAGCCATGAGCTTGGAAT- Babraham Institute. In unpublished analyses we have confirmed that the Ј Ј Ј u 3 and 5 -TCAACAGGGAACAGCATCCTTG-3 . PCR was performed PVG-RT1 ,lyp/lyp strain, which derives its mutant lyp gene from the Ed- using a SYBR Green kit in accordance with the manufacturer’s instructions inburgh subline of BB-DP rats (8, 17), carries the frameshift mutation in (Applied Biosystems), and samples were amplified using the Chromo4 the Gimap5/Ian5 gene described previously (5, 6). Rats of the strains LEW, system (Bio-Rad). A two-way ANOVA was applied to the data to deter- LOU/C, WKY, and WAG were purchased from Harlan UK. C57BL/6 mice mine the relative expression between BN and wild-type rats for each of the used for Western blotting experiments were bred at the Babraham Institute.
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