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Leukemia (1997) 11, 370–375 1997 Stockton Press All rights reserved 0887-6924/97 $12.00 Cloning of the mouse BTG3 gene and definition of a new gene family (the BTG family) involved in the negative control of the cell cycle ´ F Guehenneux1, L Duret2, MB Callanan3, R Bouhas1, S Hayette4, C Berthet1, C Samarut1, R Rimokh1, AM Birot1, Q Wang5, JP Magaud1,4 and JP Rouault1 ´ ´ ´ ´ ´ ´ Unite INSERM 453 affiliee au CNRS, 5Unite d’Oncologie moleculaire, Centre Leon Berard, Lyon; 2Laboratoire BGBP-UMR CNRS 5558, ´ ´ 3 UCLB, Villeurbanne; Groupe de recherches sur les lymphomes, Institut A Bonniot, La Tronche and; 4Laboratoire de Cytogenetique et ´ ´ ´ ˆ Cytogenetique Moleculaire, Hopital Edouard Herriot, Lyon, France 1 It is well known that loss of tumor suppressor genes and more generally of antiproliferative genes plays a key role in the development of most tumors. We report here the cloning of the mouse BTG3 gene and show that its human counterpart maps on chromosome 21. This evolutionarily conserved gene codes for a 30 kDa protein and is expressed in most adult murine and human tissues analyzed. However, we demonstrate that its expression is cell cycle dependent and peaks at the end of the G1 phase. This gene is homologous to the human BTG1, BTG2 and TOB genes which were demonstrated to act as inhibitors of cell proliferation. Its description allowed us to define better this seven gene family (the BTG gene family) at the structural level and to speculate about its physiological role in normal and tumoral cells. This family is mainly characterized by the presence of two conserved domains (BTG boxes A and B) of as yet undetermined function which are separated by a nonconserved 20–25 amino acid sequence. Keywords: BTG 1, 2, 3; PC3; TIS21; TOB; antiproliferative gene Introduction Cell cycle progression is tightly controlled by the sequential activation of proliferative and antiproliferative genes. Imbalance in this control system can lead to tumorigenesis. Initially, most cancers were thought to arise through inappropriate activation of oncogenes. However, increasing evidence indicated that loss of function of antiproliferative genes (tumor suppressor genes) represented a major route to tumor development. Extensive searches using diverse strategies have since led to the identification of a number of antiproliferative genes including for example P53 and RB1, and more recently the (CDK) cyclin-dependent kinase inhibitors.1,2 However, characterization of other physiologically important antiproliferative genes was also achieved using other cloning strategies (GAS genes, for instance).3 The molecular characterization of a chromosomal translocation observed in a lymphoid malignancy led us to clone a gene, BTG1, which acts as an inhibitor of cell proliferation.4,5 Sequence analysis revealed that BTG1 was highly homologous to the rat PC3 and mouse TIS21 genes;5,6 PC3 being expressed in the neuronal PC12 cell line induced to differentiate by the nerve growth factor7 and TIS21 expression induced by serum stimulation of starved fibroblastic cell lines.8 Recently, we cloned the human counterpart of the PC3/TIS21 gene, BTG2. We demonstrated that BTG2 expression is regulated by P53, involved in the negative regulation of the cell cycle and in response to DNA damage induced by different genotoxic agents.9 Recently, antiproliferative activity for PC3 was confirmed by another group.10 Finally, TOB, another gene homologous to BTG1 and 2, was ´ ´ Correspondence: J-P Rouault, INSERM U453, Centre Leon Berard, 28 ¨ rue Laennec, F-69373 Lyon Cedex 08, France Received 20 November 1996; accepted 11 December 1996 shown to be involved in the modulation of the biochemical growth signal transduced by the growth factor receptor ErbB2 and to exert an antiproliferative activity on NIH3T3 cells.11 Furthermore, consistent with a presumptive antiproliferative activity, a role for these genes in cellular differentiation is evident. BTG1 was implicated in myoblast differentiation12 and expression of PC3 is an early event observed during the differentiation of the neuronal precursors in the mouse.13 It is likely that BTG1, 2 and TOB constitute a novel family of functionally related genes involved in the regulation of cell proliferation/differentiation. In this paper, we describe the isolation/characterization of a new BTG member, the mouse BTG3 gene. Structural analysis of this new gene reveals a new common functional signature between the different members of the BTG family. We speculate on its possible physiological role in normal and tumoral cells. Materials and methods Cells and culture conditions Peripheral blood lymphocytes (PBL) were obtained from the blood of normal volunteers by centrifugation on triosil Ficoll. Mitogen-stimulated lymphocytes were obtained by incubating PBL at a concentration of 2 × 106 cells/ml, in the presence of phytohemagglutinin (PHA; Welcome, Dartford, UK) diluted 1:200 in RPMI1640 medium supplemented with 20% fetal calf serum plus 0.03% L-glutamin, 100 mg/ml of penicillin G and 100 mg/ml of streptomycin sulfate. BTG3 mRNA level were then assessed at various times after PHA stimulation. RNA isolation Total cellular RNA was isolated from frozen samples by the acid guanidium thiocyanate-phenol-chloroform method. For Northern blot analysis, total RNA was size fractionated in formaldehyde–1.2% agarose gels and transferred into nylon filters. Probes a-32P-labeling, prehybridization, hybridization, and washing were carried out as previously described.4 Sequencing procedures and sequence analysis Overlapping deletions of DNA and cDNA cloned into Bluescript SK(−) (Stratagene, La Jolla, CA, USA) were obtained by the unidirectional exonuclease III digestion method (Erasea-base system; Promega, Madison, WI, USA). Deletion clones were sized on agarose gels, and inserts of the selected clones were sequenced by the dideoxy chain termination procedure The BTG antiproliferative gene family ´ F Guehenneux et al with Sequenase II (USB, Cleveland, OH, USA) as described by the manufacturer. Sequence similarity searches were performed at the NCBI BLAST Web server (http:// www.ncbi.nlm.nih.gov/BLAST/) using the BLASTP, BLASTN and BLASTX programs.14 Homologous sequences were aligned with the CLUSTALW program15 and phylogenetic trees were calculated according to the neighbour-joining method.16 Preparation and analysis of DNA and cDNA libraries A 10-day mouse embryo cDNA library cloned in lEXloxtm was purchased from NOVAGEN (Madison, WI, USA) and processed according to the manufacturer’s instructions. Approximately 800 000 clones were screened with the BTG1 (RIA) and TIS21 probes which were previously described.5–9 Hybridization of the filters was carried out using low stringency conditions (30% formamide, washing 3 × 20 mn at room temperature in 2 × SSC, 0.5% SDS). Inserts from the recombinant positive clones were characterized by partial sequencing. The mouse BTG3 genomic counterpart was isolated by screening an EMBL3 phage mouse genomic DNA library9 with a BTG3 cDNA probe. In vitro translation assay The BTG3 open reading frame (ORF) was subcloned into bluescript (SK−) and in vitro transcribed following standard procedures with T7 and T3 RNA pol (Boehringer, Mannheim, Germany). Specific mRNA were then translated using the ‘reticulocyte type I’ in vitro translation kit, (Boehringer Mannheim Biochemica) and the synthesized peptide was analyzed following the manufacturer’s instructions. Human chromosomal localization Human chromosome localization of the BTG3 gene was determined by Southern blot analysis of a human × rodent cell hybrid DNA panel purchased from BIOS laboratories (Newhaven, CT, USA) with a mouse BTG3 ORF probe. Results Several recombinant phage clones containing inserts different from BTG1 and TIS21 were isolated by low stringency screening of a 10-day mouse embryo cDNA library with BTG1 and TIS21 32P-labelled probes. Partial sequencing of the clones showed that they all code for the same protein. The corresponding gene was hence named BTG3. The longest clone – 1393 nucleotides (nt) – was sequenced on both strands as described in Materials and methods. The sequence exhibited an open reading frame coding for a peptide of 252 amino acids (aa), predicted molecular weight 28981, and a 455-nt long A + T rich 39 untranslated region. Finally, a polyadenylation site AATAAA (nt 1369) was found 25 bp upstream of the poly-A tail (Figure 1). Screening of a mouse genomic DNA library using a BTG3 cDNA probe and comparison of the cDNA and genomic DNA sequences revealed that the BTG3 gene contained five exons with typical splice donor and acceptor sites at the intron–exon junctions. The first exon is rich in G + C nucleotides. The initiating codon of the longest deduced ORF (nt 193–950, 252 aa) is localized at the beginning of the second exon and fulfills the criteria for the eukaryotic start signal with an adenine in position −3.17 In agreement with the protein size predicted by sequence analysis, in vitro translation of this ORF generated a protein with a 30 kDa apparent molecular weight (Figure 2). Computer analysis of the amino acid sequence (PROSITE program) did not reveal any particular features except its homology with the other BTG protein. Like the other members of this family, BTG3 is evolutionarily conserved and zoo-blot analysis using a mouse probe shows that sequences homologous to BTG3 could be detected in human using normal stringency condition (data not shown). Northern blot analysis revealed that BTG3 expression is ubiquitous, a specific 1.6 kb transcript (size in accordance with the length of the cDNA) being detected with varying levels in most of the murine and human tissues analyzed (data not shown), except in homogeneous non-proliferating cell populations such as peripheral blood lymphocytes or serumstarved fibroblasts. The highest steady-state levels of the 1.6 kb transcripts are observed in cell lines whatever their origin. Furthermore, unlike BTG1 and 25–9 which are expressed early in the G1 phase of the cell cycle, it appeared that the highest level of BTG3 transcript was observed only after 24–48 h of stimulation of quiescent peripheral blood lymphocytes by PHA at the end of the G1 phase of the cell cycle, CDK1 being used as a marker of the G1–S transition18 (Figure 2). Taking advantage of the conservation of the BTG3 gene during evolution, assignment of the BTG3 locus to the human chromosome 21 was performed by Southern blot analysis of a panel of interspecies (rodent × human) somatic hybrid DNA using a mouse BTG3 cDNA probe (data not shown). Similarity searches allowed us to identify several homologues from different vertebrate species. To clarify the relationships between these genes, we calculated the multiple alignment of homologous proteins and derived a phylogenetic tree (Figure 3). To date, the BTG family comprises BTG1, BTG2, BTG3, TOB, PC3, TIS21, TOB4, B9.10 and B9.15 (see the accession numbers in Figure 3). During the preparation of this manuscript, the cDNA sequences of the human and mouse TOB5 genes have been deposited in databases (Yoshida Y, Matuda S, Yamamoto T, 1996, unpublished; see accession numbers in Figure 3). Sequence analysis showed that BTG3 and TOB5 are a single gene. The phylogenetic tree indicates that the human BTG2 gene is the counterpart of rat PC3 and mouse TIS21 genes. The closest relatives of BTG3 are the xenopus B9.10 and B9.15 genes. However, the length of the branch that links BTG3 to B9.10 and B9.15 suggests that they are paralogous. Hence, the mammalian counterpart(s) of xenopus B9.10 and B9.15 genes probably remain(s) to be identified. In conclusion, the BTG family comprises at least seven distinct genes in vertebrates: BTG1, BTG2 (TIS21, PC3), BTG3/TOB5, TOB, TOB4, B9.10 and B9.15. Three groups of proteins with similar lengths and specific conserved regions can be distinguished in this family: (1) BTG3, B9.10 and B9.15 (233–252 aa); (2) BTG1 and BTG2 (158–171 aa); and (3) TOB and TOB4 (344–360 aa) (Figure 4). Two short domains of respectively 22 (box A) and 20 amino acids (box B) conserved in all these proteins allowed us to define two signatures that characterize the BTG family (Figure 4). These two domains, separated by a spacer sequence of 20–25 non-conserved amino acids, are located in the first 120 residues of the BTG proteins. 371 The BTG antiproliferative gene family ´ F Guehenneux et al 372 Figure 1 Nucleotide sequence of the mouse BTG3 cDNA (Genbank accession number Z72000). The standard one-letter code is used for nucleotides and amino acids. The positions of the exons are indicated by arrows. The sequences of the conserved domains A and B are underlined. Bold type ATG and AATAAA indicate the initiation and the polyadenylation sites. The BTG antiproliferative gene family ´ F Guehenneux et al 373 Figure 2 (a) BTG3 expression after PHA stimulation of peripheral blood mononuclear cells. The Northern blot was successively hybridized to BTG3 (1.6 kb), CDK1 (1.6 and 2 kb) and S26 (0.7 kb) probes. S26 is an internal control that ascertained the equal loading of RNA in each lane.23 (b) SDS-PAGE analysis of the BTG3 ORF in vitro translation. +, sense BTG3 RNA; −, antisense BTG3 RNA. Figure 3 Phylogenetic tree of all the proteins from the BTG family. Genbank accession numbers: BTG1: X61123 (human); L26268 (rat), Z16410 (mouse), X64146 (chicken); BTG2/TIS/PC3: U72649 (human), M64292 (mouse), M60921 (rat); TOB: D38305 (human); B9.10; X73317; B9.15: X73316; BTG3: Z72000 (mouse); TOB5: D83745 (mouse), D64110 (human); TOB4: D64109 (human). Discussion In this paper we report the cloning of the mouse BTG3 gene and show that its human counterpart maps on chromosome 21. This evolutionarily conserved gene codes for a 30 kDa protein and is expressed in most adult murine and human tissues analyzed. However, we demonstrate that its expression is related to the cell cycle in PHA-stimulated human peripheral blood lymphocytes, the highest levels of BTG3 transcripts being observed at the end of the G1 phase of the cell cycle with a peak at the G1–S transition. In quiescent PBL, BTG3 mRNA is undetectable. BTG3 is therefore confirmed as a new member of the BTG family which already comprises at least three members (BTG1, 2 and TOB) involved in growth control and or differentiation. The most remarkable structural features of the BTG family reside in the existence of two highly conserved short domains (BTG boxes A and B) separated by a relatively constant (20–25 aa) stretch. This molecular organization is reminiscent to that of other proteins playing a major role in cell growth control such as the pocket protein family The BTG antiproliferative gene family ´ F Guehenneux et al 374 Figure 4 Comparison of the amino acid sequences of the predicted BTG protein family members. They were aligned with the CLUSTALW program. The standard one-letter code is used for the amino acids. (RB1, p107, p130) and the BCL2 protein family.19,20 It is likely that, as described for the pocket protein family, the BTG boxes are involved in protein–protein interactions. So far, it has been demonstrated that TOB binds to and probably modulates the ERB-2 receptor signal transduction11 and that BTG1 and TIS21 bind and activate a protein arginine methyl transferase (PRMT1), some substrates of which have already been characterized (Histone/hnRNP A1).21 Another point to be discussed is the timing of expression of BTG1, 2 and 3 during the cell cycle. In fact, BTG1 and 2 are expressed in early G1 and BTG3, as demonstrated in the present work, is expressed in late G1 before the entry of the cells in S phase. Although we do not know the exact function of the BTG/TOB family, we can assume that these genes control different checkpoints in G1 and could constitute the negative counterpart of the G1 cyclins which are also sequentially activated during this phase.1,2 In this respect, the activity of the BTG genes could be compared to that of the cyclin G1dependent-kinase inhibitors but we have never been able to demonstrate interaction of any of these genes with CDK or cyclins. The fact that BTG1 and 2 bind to and modulate the enzymatic activity of PRMT suggests that methylation of target proteins at different stages of the cell cycle may play a role in cell growth and or differentiation control.21 So far, phosphorylation and dephosphorylation of various substrates have been considered to play a central role in cell cycle regulation. However, recent works strongly suggest that other post-translational modifications such as histone acetylation may also be implicated in growth and differentiation control.22 Now, in the light of our results, it remains to be demonstrated that enzymatic systems involved in the transfer of methyl groups (protein methyltransferase and methylesterase) might participate in the regulation of homeostasis. Goals for future studies include characterization of the BTG proteins partners in order to define more precisely if they constitute the elements of a novel negative signalling pathway. With regards to their growth inhibiting activity, these genes could act as tumor suppressor genes and studies to find if they are inactivated in some solid tumors or malignant blood disorders would be justified. Acknowledgements This work was supported by grants from ARC (1391) and Ligue Nationale Contre le Cancer. FG is the recipient of a fellowship from Ligue Nationale Contre le Cancer. 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