Cloning of the Mouse BTG3 Gene and Definition of a New Gene

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 ´ ´ ´ ´ ´ ´ 1Unite INSERM 453 afﬁliee au CNRS, 5Unite d’Oncologie moleculaire, Centre Leon Berard, Lyon; 2Laboratoire BGBP-UMR CNRS 5558, ´ ´ UCLB, Villeurbanne; 3Groupe de recherches sur les lymphomes, Institut A Bonniot, La Tronche and; 4Laboratoire de Cytogenetique et ´ ´ ´ ˆ Cytogenetique Moleculaire, Hopital Edouard Herriot, Lyon, France

It is well known that loss of tumor suppressor genes and more shown to be involved in the modulation of the biochemical generally of antiproliferative genes plays a key role in the devel- growth signal transduced by the growth factor receptor ErbB2 opment of most tumors. We report here the cloning of the 11 mouse BTG3 gene and show that its human counterpart maps and to exert an antiproliferative activity on NIH3T3 cells. on chromosome 21. This evolutionarily conserved gene codes Furthermore, consistent with a presumptive antiproliferative for a 30 kDa protein and is expressed in most adult murine and activity, a role for these genes in cellular differentiation is evi- human tissues analyzed. However, we demonstrate that its dent. BTG1 was implicated in myoblast differentiation12 and expression is cell cycle dependent and peaks at the end of the expression of PC3 is an early event observed during the differ- G1 phase. This gene is homologous to the human BTG1, BTG2 entiation of the neuronal precursors in the mouse.13 It is likely and TOB genes which were demonstrated to act as inhibitors of cell proliferation. Its description allowed us to deﬁne better that BTG1, 2 and TOB constitute a novel family of func- this seven gene family (the BTG gene family) at the structural tionally related genes involved in the regulation of cell level and to speculate about its physiological role in normal proliferation/differentiation. In this paper, we describe the and tumoral cells. This family is mainly characterized by the isolation/characterization of a new BTG member, the mouse presence of two conserved domains (BTG boxes A and B) of BTG3 gene. Structural analysis of this new gene reveals a new as yet undetermined function which are separated by a non- common functional signature between the different members conserved 20–25 amino acid sequence. Keywords: BTG 1, 2, 3; PC3; TIS21; TOB; antiproliferative gene of the BTG family. We speculate on its possible physiological role in normal and tumoral cells.

Introduction Materials and methods

Cell cycle progression is tightly controlled by the sequential Cells and culture conditions activation of proliferative and antiproliferative genes. Imbal- ance in this control system can lead to tumorigenesis. Initially, Peripheral blood lymphocytes (PBL) were obtained from the most cancers were thought to arise through inappropriate acti- blood of normal volunteers by centrifugation on triosil Fic- vation of oncogenes. However, increasing evidence indicated oll. Mitogen-stimulated lymphocytes were obtained by incu- that loss of function of antiproliferative genes (tumor sup- bating PBL at a concentration of 2 × 106 cells/ml, in the pres- pressor genes) represented a major route to tumor develop- ence of phytohemagglutinin (PHA; Welcome, Dartford, UK) ment. Extensive searches using diverse strategies have since diluted 1:200 in RPMI1640 medium supplemented with led to the identification of a number of antiproliferative genes 20% fetal calf serum plus 0.03% L-glutamin, 100 ␮g/ml of including for example P53 and RB1, and more recently the penicillin G and 100 ␮g/ml of streptomycin sulfate. BTG3 (CDK) cyclin-dependent kinase inhibitors.1,2 However, mRNA level were then assessed at various times after PHA characterization of other physiologically important antipro- stimulation. liferative genes was also achieved using other cloning strategies (GAS genes, for instance).3 The molecular characterization of a chromosomal translo- RNA isolation cation observed in a lymphoid malignancy led us to clone a gene, BTG1, which acts as an inhibitor of cell proliferation.4,5 Total cellular RNA was isolated from frozen samples by the Sequence analysis revealed that BTG1 was highly homolo- acid guanidium thiocyanate-phenol-chloroform method. For gous to the rat PC3 and mouse TIS21 genes;5,6 PC3 being Northern blot analysis, total RNA was size fractionated in expressed in the neuronal PC12 cell line induced to differen- formaldehyde–1.2% agarose gels and transferred into nylon tiate by the nerve growth factor7 and TIS21 expression filters. Probes ␣-32P-labeling, prehybridization, hybridiz- induced by serum stimulation of starved fibroblastic cell ation, and washing were carried out as previously lines.8 Recently, we cloned the human counterpart of the described.4 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 Sequencing procedures and sequence analysis induced by different genotoxic agents.9 Recently, antiproliferative activity for PC3 was confirmed by another group.10 Overlapping deletions of DNA and cDNA cloned into Finally, TOB, another gene homologous to BTG1 and 2, was Bluescript SK(−) (Stratagene, La Jolla, CA, USA) were obtained by the unidirectional exonuclease III digestion method (Erase- ´ ´ Correspondence: J-P Rouault, INSERM U453, Centre Leon Berard, 28 a-base system; Promega, Madison, WI, USA). Deletion clones ¨ rue Laennec, F-69373 Lyon Cedex 08, France were sized on agarose gels, and inserts of the selected clones Received 20 November 1996; accepted 11 December 1996 were sequenced by the dideoxy chain termination procedure The BTG antiproliferative gene family ´ F Guehenneux et al 371 with Sequenase II (USB, Cleveland, OH, USA) as described deduced ORF (nt 193–950, 252 aa) is localized at the begin- by the manufacturer. Sequence similarity searches were ning of the second exon and fulfills the criteria for the eukary- performed at the NCBI BLAST Web server (http:// otic start signal with an adenine in position −3.17 In agreement www.ncbi.nlm.nih.gov/BLAST/) using the BLASTP, BLASTN with the protein size predicted by sequence analysis, in vitro and BLASTX programs.14 Homologous sequences were translation of this ORF generated a protein with a 30 kDa aligned with the CLUSTALW program15 and phylogenetic apparent molecular weight (Figure 2). Computer analysis of trees were calculated according to the neighbour-joining the amino acid sequence (PROSITE program) did not reveal method.16 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 Preparation and analysis of DNA and cDNA libraries probe shows that sequences homologous to BTG3 could be detected in human using normal stringency condition (data ␭ A 10-day mouse embryo cDNA library cloned in EXloxtm not shown). was purchased from NOVAGEN (Madison, WI, USA) and pro- Northern blot analysis revealed that BTG3 expression is cessed according to the manufacturer’s instructions. Approxi- ubiquitous, a specific 1.6 kb transcript (size in accordance mately 800 000 clones were screened with the BTG1 (RIA) with the length of the cDNA) being detected with varying lev- and TIS21 probes which were previously described.5–9 els in most of the murine and human tissues analyzed (data Hybridization of the filters was carried out using low strin- not shown), except in homogeneous non-proliferating cell gency conditions (30% formamide, washing 3 × 20 mn at populations such as peripheral blood lymphocytes or serum- room temperature in 2 × SSC, 0.5% SDS). Inserts from the starved fibroblasts. The highest steady-state levels of the 1.6 kb recombinant positive clones were characterized by partial transcripts are observed in cell lines whatever their origin. Fur- sequencing. The mouse BTG3 genomic counterpart was iso- 5–9 lated by screening an EMBL3 phage mouse genomic DNA thermore, unlike BTG1 and 2 which are expressed early in library9 with a BTG3 cDNA probe. 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 In vitro translation assay PHA at the end of the G1 phase of the cell cycle, CDK1 being 18 used as a marker of the G1–S transition (Figure 2). The BTG3 open reading frame (ORF) was subcloned into Taking advantage of the conservation of the BTG3 gene dur- bluescript (SK−) and in vitro transcribed following standard ing evolution, assignment of the BTG3 locus to the human procedures with T7 and T3 RNA pol (Boehringer, Mannheim, chromosome 21 was performed by Southern blot analysis of Germany). Specific mRNA were then translated using the ‘reti- a panel of interspecies (rodent × human) somatic hybrid DNA culocyte type I’ in vitro translation kit, (Boehringer Mannheim using a mouse BTG3 cDNA probe (data not shown). Biochemica) and the synthesized peptide was analyzed fol- Similarity searches allowed us to identify several homo- lowing the manufacturer’s instructions. logues from different vertebrate species. To clarify the relationships between these genes, we calculated the multiple alignment of homologous proteins and derived a phylogenetic Human chromosomal localization tree (Figure 3). To date, the BTG family comprises BTG1, BTG2, BTG3, TOB, PC3, TIS21, TOB4, B9.10 and B9.15 (see Human chromosome localization of the BTG3 gene was the accession numbers in Figure 3). During the preparation of determined by Southern blot analysis of a human × rodent cell this manuscript, the cDNA sequences of the human and hybrid DNA panel purchased from BIOS laboratories mouse TOB5 genes have been deposited in databases (Newhaven, CT, USA) with a mouse BTG3 ORF probe. (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 Results indicates that the human BTG2 gene is the counterpart of rat PC3 and mouse TIS21 genes. The closest relatives of BTG3 Several recombinant phage clones containing inserts different are the xenopus B9.10 and B9.15 genes. However, the length from BTG1 and TIS21 were isolated by low stringency screen- of the branch that links BTG3 to B9.10 and B9.15 suggests that ing of a 10-day mouse embryo cDNA library with BTG1 and 32 they are paralogous. Hence, the mammalian counterpart(s) of TIS21 P-labelled probes. Partial sequencing of the clones xenopus B9.10 and B9.15 genes probably remain(s) to be showed that they all code for the same protein. The corre- identified. In conclusion, the BTG family comprises at least sponding gene was hence named BTG3. The longest clone – seven distinct genes in vertebrates: BTG1, BTG2 (TIS21, PC3), 1393 nucleotides (nt) – was sequenced on both strands as BTG3/TOB5, TOB, TOB4, B9.10 and B9.15. Three groups of described in Materials and methods. The sequence exhibited proteins with similar lengths and specific conserved regions an open reading frame coding for a peptide of 252 amino can be distinguished in this family: (1) BTG3, B9.10 and B9.15 acids (aa), predicted molecular weight 28981, and a 455-nt (233–252 aa); (2) BTG1 and BTG2 (158–171 aa); and (3) TOB long A + T rich 3Ј untranslated region. Finally, a polyadenylation site AATAAA (nt 1369) was found 25 bp upstream of the and TOB4 (344–360 aa) (Figure 4). Two short domains of poly-A tail (Figure 1). Screening of a mouse genomic DNA respectively 22 (box A) and 20 amino acids (box B) conserved library using a BTG3 cDNA probe and comparison of the in all these proteins allowed us to define two signatures that cDNA and genomic DNA sequences revealed that the BTG3 characterize the BTG family (Figure 4). These two domains, gene contained five exons with typical splice donor and separated by a spacer sequence of 20–25 non-conserved acceptor sites at the intron–exon junctions. The first exon is amino acids, are located in the first 120 residues of the BTG rich in G + C nucleotides. The initiating codon of the longest proteins. 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 with a peak at the G1–S transition. In quiescent PBL, BTG3 mRNA is undetectable. BTG3 is therefore conﬁrmed as a new In this paper we report the cloning of the mouse BTG3 gene member of the BTG family which already comprises at least and show that its human counterpart maps on chromosome three members (BTG1, 2 and TOB) involved in growth control 21. This evolutionarily conserved gene codes for a 30 kDa and or differentiation. The most remarkable structural features protein and is expressed in most adult murine and human of the BTG family reside in the existence of two highly con- tissues analyzed. However, we demonstrate that its expression served short domains (BTG boxes A and B) separated by a is related to the cell cycle in PHA-stimulated human periph- relatively constant (20–25 aa) stretch. This molecular organi- eral blood lymphocytes, the highest levels of BTG3 transcripts zation is reminiscent to that of other proteins playing a major being observed at the end of the G1 phase of the cell cycle 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 Acknowledgements that, as described for the pocket protein family, the BTG boxes are involved in protein–protein interactions. So far, it has been This work was supported by grants from ARC (1391) and Ligue demonstrated that TOB binds to and probably modulates the Nationale Contre le Cancer. FG is the recipient of a fellowship ERB-2 receptor signal transduction11 and that BTG1 and TIS21 from Ligue Nationale Contre le Cancer. We thank M Billaud bind and activate a protein arginine methyl transferase for helpful discussion. (PRMT1), some substrates of which have already been characterized (Histone/hnRNP A1).21 Another point to be discussed is the timing of expression of References BTG1, 2 and 3 during the cell cycle. In fact, BTG1 and 2 are

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