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0085AD. Genes & Cell Death and Differentiation (1999) 6, 883 ± 889 ã 1999 Stockton Press All rights reserved 13509047/99 $15.00 http://www.stockton-press.co.uk/cdd WW domain-containing FBP-30 is regulated by p53 1 ,1 Valerie Depraetere and Pierre Golstein* Introduction 1 Centre d'Immunologie INSERM-CNRS de Marseille-Luminy, Case 906, 13288 Subtractive approaches might be appropriate to clone cell Marseille Cedex 9, France death signalling genes in systems where PCD is dependent * corresponding author: Dr. Pierre Golstein, Centre d'Immunologie INSERM- on RNA synthesis,1±6 whereas many of the genes encoding CNRS de Marseille-Luminy, Case 906, 13288 Marseille Cedex 9, France. the cell death machinery, which is thought to be constitutively tel: +33 491269468; fax: +33 491269430; e-mail: [email protected] expressed in all cells,7 would not be identified by such a procedure. The aim of the present study was to look for genes Received 24.3.99; revised 17.6.99; accepted 13.7.99 the expression of which is induced upon g-irradiation and may Edited by B. Osborne lead to activation of the cell death program. Thymocytes were chosen as a model because of their high sensitivity to g- irradiation, and because g-irradiation-induced death of Abstract thymocytes is dependent upon p53 expression and upon RNA and protein synthesis.3,4 A subtractive cloning approach A subtractive cloning approach was used to clone genes was developed to clone genes which are up-regulated in g- transcriptionally induced in thymocytes undergoing pro- irradiated- as compared to untreated-wild-type murine grammed cell death after g-irradiation. We thus identified thymocytes. Thirty-one known genes were identified in this about 60 upregulated genes. One of these genes encodes a screen, along with 28 new gene fragments. Some of the WW domain previously briefly reported by others, which former had previously been reported to be induced by g- defines this gene as FBP-30. cDNA sequencing showed FBP- irradiation, which validates our screening procedure. The 30 to be remarkably conserved in mammals. FBP-30 relevance of the other known genes to our screening will be expression, essentially restricted to T cells, was regulated briefly discussed. One gene, FBP-30, was studied in more by p53 since it increased (1) after g-irradiation in wild-type but detail. A 26 amino-acid proline-rich binding WW domain defining FBP-30 has been briefly reported.8 Importantly, WW not in p537/7 thymocytes and (2) in cells transfected with a domains have recently been shown to bind to phosphoserine- conditional temperature-sensitive p53 mutant, less than 1 h or phosphothreonine-containing proteins.9 We found the after shifting to permissive temperature. Upregulation of FBP- sequence of FBP-30 to be strikingly conserved in mammals, 30 expression thus depends upon p53 protein expression and suggesting that the sequence of the entire protein is important correlates with cell death induction in these systems. While for its function. In agreement with our cloning procedure, the kinetics of its induction are rapid, the observed increased expression of this gene was found to be increased after g- expression of FBP-30 in the presence of protein synthesis irradiation, moreover in a p53-regulated manner. inhibitors suggests that FBP-30 gene expression is indirectly regulated by p53, through downregulation of a labile inhibitor of FBP-30 expression. Results and Discussion Differential screening procedure Keywords: g-irradiation; p53; subtractive cloning; FBP-30; WW Representational Difference Analysis (RDA,10 CLONTECH) domain is a subtractive cloning approach based on the selective amplification by competitive PCR of cDNA fragments Abbreviations: ActD, actinomycin D; AIF, apoptosis inducing corresponding to genes which are expressed at a higher factor; BLAST, basic local alignment search tool; ConA, level in a given population as compared to another population. concanavalin A; CHX, cycloheximide; DR5, death receptor 5; est, We used RDA to subtract untreated wild-type thymocyte expressed sequence tags; Fas-L, Fas ligand; FBP-30, formin cDNA from irradiated thymocyte cDNA. The subtraction binding protein 30; FcgR, receptor for the constant fragment of products were cloned; the approximately 5000 correspond- gamma immunoglobulins; FCS, foetal calf serum; HMG, high ing clones were ordered on high density filters, and mobility group; IL-1b, interleukine 1 beta; IL2R, interleukine 2 successively hybridized with complex probes made out of receptor; Irr, irradiated; ld, limb deformity; LPS, lipopolysaccharide; total RNA isolated from either irradiated or untreated MAPKKK, mitogen activated kinase kinase kinase; oligodT, thymocytes by reverse transcription, in order to confirm their oligodeoxy thymidinetriphosphate; ORF, open reading frame; differential expression pattern. The hybridization signals were PCD, programmed cell death; PCR, polymerase chain reaction; quantified, normalized by comparison with a vector control PDGF-R, platelet derived growth factor receptor; PI 3K, phospho- probe, and compared as described.11 The 400 clones which inositol 3 kinase; PLA2, phospholipase A2; RACE, rapid were confirmed to be differentially expressed by this ampli®cation of cDNA ends; RDA, representational difference technique and 10 clones which were below detection limits analysis; RT ± PCR, reverse transcription coupled polymerase were further studied. Since RDA produces small cDNA chain reaction; TGF, transforming growth factor; TNF, tumour fragments, pools (80 ± 90 clones) of these subtraction necrosis factor; XRCC, X-ray cross-complementing group products were used as probes to screen an irradiated- WW domain-containing FBP-30 is regulated by p53 V Depraetere and P Golstein 884 thymocyte phage cDNA library in order to obtain longer cDNA the immune system, some of which have also been clones corresponding to these RDA products. The latter involved in the control of cell death (IL2Rp35,20 FcgRII,21 clones were ordered on high density filters, screened by and IL1b). Group D consists of molecules, either successive hybridization with complex probes as before and mitochondrially encoded, or mitochondrially located. This cross-hybridized to screen for redundancy. Sixty non- might be related to an increase in respiration after an redundant clones were thus identified and sequenced at irradiation stress, or to the involvement of mitochondrial their 5' and/or 3' end. Thirty-one of them corresponded to products such as cytochrome c and AIF in the control of known genes (Table 1), 28 were found to be unknown, and cell death.22,23 Finally, group E includes diverse genes, one clone was studied in more details. This clone encodes a some of which have been involved in the control of cell protein, FBP-30, which includes a previously reported 26 death, such as Blimp-124 or TGFb6 which can potentialize amino-acid WW domain.8 p53-induced cell death.25 The region of Blimp-1 which is critical for its cell death inducing properties is a 69 amino acid proline-rich region.24 This is interesting in the light of Screening results the identification in our screen of FBP-30, a protein bearing The 31 known genes which were identified in our screen a WW domain known to interact with proline-rich regions. for genes induced in mouse thymocytes after g-irradiation Our screen has thus identified a number of candidate can be grouped in five categories (Table 1). Group A regulators of g-irradiation and p53 induced cell death, corresponds to genes known to be induced by g-irradiation outside of the known p53 regulated (such as c-myc)or or other DNA damaging stresses, such as XRCC-1, HMG-1 regulating (such as HMG-1 and TGFb) molecules. It did or c-myc,12 ± 14 and thus to some extent validates the not, however, include other p53 induced genes such as screening procedure. Group B genes encode phospholi- Bax, Fas, DR5, or genes identified by differential screening pases and kinases, which have been involved in some in fibroblasts expressing a thermosensitive p53 mutant instances of cell death or cell survival such as PI3kinase,15 allele.18,26 This is most likely due to the respective biases MAPKKK,16 PLA2.17 Phospholipases and kinases can thus of the differential screening (differential display) and be regulated at the transcriptional level upon cell death subtractive cloning (RDA) procedures, as well as to the induction.18,19 Group C includes molecules with functions in biological systems used. FBP-30 expression in untreated and in g-irradiated Table 1 Sequences identi®ed in the screen for irradiation-induced genes thymocytes A. DNA-damage-induced genes One of the clones identified in our subtractive cloning in g- XRCC1 irradiated thymocytes encodes a protein known as Formin HMG1 ATP-dependent RNA helicase Binding Protein-30 since it includes a Formin binding WW oxidative stress-induced protein domain. Only the 26 amino-acid sequence corresponding c-myc to this WW domain has been published.8 In line with the subtractive cloning, we found by Northern blot analysis that B. Signal transduction molecules PLA2 FBP-30 mRNA was up-regulated in thymocytes upon p56tek irradiation: FBP-30 mRNA, expressed at a basal level in MAPKKK untreated wild-type thymocytes, is detectably increased 1 h PI3kinase after irradiation, peaks 1 ± 2 h after irradiation to ten times the level of expression in untreated thymocytes, on C. Molecules of the immune system 41BB average, and declines thereafter (Figure 1). A single 4 kb FCgRII transcript could be detected (Figure 1). Increased expres- B7.2 sion of FBP-30 mRNA after irradiation was also confirmed TCRa/d by semi-quantitative RT ± PCR (not shown). FBP-30 IL2p35 expression was not increased by other death-inducing D. Mitochondrial molecules agents such as anti-Fas antibodies, anti-CD3 antibodies, mitochondrial DNA dexamethasone or methotrexate (not shown). Among adult cytp450 tissues, FBP-30 mRNA could be detected on Northern cytochrome oxidase blots in thymus and uterus, in a T cell hybridoma cytb (L16.2427), and also in ConA-activated but not LPS- E.
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