Oncogene (1998) 16, 2687 ± 2693 1998 Stockton Press All rights reserved 0950 ± 9232/98 $12.00 http://www.stockton-press.co.uk/onc SHORT REPORT ANA, a novel member of Tob/BTG1 family, is expressed in the ventricular zone of the developing central nervous system Yutaka Yoshida1, Satoru Matsuda1, Naoko Ikematsu1, Junko Kawamura-Tsuzuku1, Johji Inazawa2, Hisashi Umemori1 and Tadashi Yamamoto1 Department of 1Oncology and 2Human Genome Center, The Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108, Japan Using a polymerase chain reaction-mediated cloning factors are implicated in the neural commitment and procedure, we have identi®ed a novel member, termed dierentiation (Lee, 1997). Then a question arises: how ANA (from Abundant in Nueroepithelium Area), of Tob/ are the extracellular signals transmitted to the nucleus BTG1 family of antiproliferative genes. Molecular in the cells? cloning and analysis of cDNAs revealed that the human The tob gene, together with the btg1 and pc3/tis21/ and mouse ANA encoded a protein of 252 amino acids. btg2 genes, forms a new antiproliferative gene family The amino-terminal half of ANA was homologous to the (Matsuda et al., 1996; Rouault et al., 1992, 1996; previously characterized antiproliferative gene products, Bradbury et al., 1991; Fletcher et al., 1991). Both the BTG1, PC3/TIS21/BTG2, and Tob. The human ANA BTG1 and Tob proteins suppress growth of NIH3T3 gene was localized at chromosome 21q11.2-q21.1. ANA cells when overexpressed (Matsuda et al., 1996; was expressed in a variety of tissues and cell lines, its Rouault et al., 1992). Overexpression of pc3 in expression being high in the ovary, testis, prostate, NIH3T3 and PC12 cells also leads to inhibition of thymus, and lung. Further analysis revealed that ANA their proliferation (Mantagnoli et al., 1996). Moreover, expression was high in the ventricular zone of the BTG2/TIS21/PC3 inactivation in ES cells leads to the developing central nervous system. Finally, overexpres- alteration of DNA damage-induced G2/M arrest sion of ANA impaired serum-induced cell cycle progres- (Rouault et al., 1996). However, the underlying sion from the G0/G1 to S phase. In conclusion, ANA is a mechanism(s) of the inhibition remains to be estab- fourth member of the Tob/BTG1 family that might play lished. roles in neurogenesis in the central nervous system. To further understand the function of the tob family genes, we searched for a new member of this family Keywords: Tob/BTG1 family; antiproliferative activity; using a PCR-mediated cloning procedure. Here we ventricular zone; cartilage show identi®cation and characterization of a new member of the tob family gene, termed ANA. Our data suggest that ANA is involved in the regulation of the development of the central nervous system. The In the developing mammalian central nervous system, molecular mechanism by which the cytoplasmic neural precursor cells dividing in the ventricular zone antiproliferative family protein is involved in neural determine their fate to become neurons or glial cells. commitment is discussed. The dierentiation of neural precursor is regulated, at least in part, by interactions between neural precursor Molecular cloning and characterization of a novel tob cells and their environments. For example, basic related gene ®bloblast growth factor (bFGF) participates in de®ning rostro-caudal identity of the neural tube We previously showed that the human tob mRNA is (Lumsden and Krumlauf, 1996). Sonic hedgehog 2.3 kb long. The tob cDNA probe also detected other (Shh) and members of the transforming growth mRNA species with weak signals in some cell lines factor-b (TGF-b) family in¯uence ventral and dorsal such as FL18 and Daudi cells (Matsuda et al., 1996), features of the development in the caudal neural tube, suggesting the presence of tob-related genes. To and recombinant Shh induces ¯oor plate cells and identify a novel tob-related gene(s), we employed the motor neurons in neural plate explants (McKay, 1997; reverse transcription and polymerase chain reaction Roelink et al., 1995; Marti et al., 1995). In addition to (RT ± PCR)-mediated cloning procedure. Using RNAs extracellular signals, several region- and cell-type- from FL18 cells and degenerated oligonucleotide speci®c transcription factors are involved in neural primers corresponding to the amino acid sequences development: the products of the Hox, Pax and POU WFPEKP and WVDPYE, which were highly con- genes are involved in the de®nition of speci®c regions served among Tob, BTG1 and PC3 (Matsuda et al., along the rostro-caudal axis of the developing nervous 1996), we obtained nearly 100 clones with insert of system (Kessel and Gruss, 1990; Graham et al., 1991), expected size. Nucleotide sequencing of the cloned and basic helix ± loop ± helix (bHLH) transcription fragments followed by homology search with the Genbank and PIR databases revealed that one of the fragments, a 190-bp fragment, represented a novel Correspondence: T Yamamoto gene. To obtain the entire coding sequence of the gene, Received 23 June 1997; revised 18 December 1997; accepted 18 we screened an oligo-(dT) primed cDNA library December 1997 generated from Daudi cells and the murine testis in Expression of ANA in the neuroepithelium YYoshidaet al 2688 lZAPII. Nucleotide sequencing of the cDNA inserts (AATAAA), a poly (A) tail, and four ATTTA motifs. revealed a 756 bp open reading frame encoding a The ATTTA motif appears in the 3'-untranslated protein of 252 amino acids (Figure 1). The calculated regions of several immediate early genes and proto- molecular weight of the protein product was 28 970. oncogenes, and is thought to be responsible for rapid The gene for this protein was termed ANA from degradation of mRNAs (Shaw and Kamen, 1986). The Abundant in the Neuroepithelial Area. The 3'- homology between the mouse and human ANA genes untranslated region of mouse ANA was rich in A and was high: nucleotide sequence identity was 91%; and T nucleotides and contained a polyadenylation signal the amino acid sequence identity was 93% (Figure 1b). a b c d Figure 1 Cloning and sequence analyses of ANA.(a) The nucleotide sequence and its deduced amino acid sequence of the mouse ANA gene. The nucleotide and amino acid sequences are numbered on the left and right, respectively. The ATTTA motifs and polyA signal in the 3'-untranslated region are underlined by single and double lines, respectively. The mouse and human ANA cDNA sequences have been registered to the DDBJ, EMBL and GenBank databases with the accession number D83745 and D64110, respectively. (b) Sequence alignment of human and mouse ANA. Each vertical line and colon indicate an identical residue and a conservative substitution, respectively. (c) Schematic diagram illustrating the structure of Tob family proteins. The hatched boxes (sequences conserved among Tob family members), black box (nuclear localization signal), PQ (proline and glutamine rich region) and P (proline rich region) are indicated respectively. (d) Alignment of the amino-terminal half of ANA with the corresponding regions of the other members of Tob family. Dashes indicate amino acid residues identical to ANA. Oligonucleotide primers corresponding to the amino acid sequences WFPEKP and WVDPYE were synthesized (the sense primer and antisense primer are CATGAATTCA(T/C)TGGT(A/T)(T/C)CCNGA(A/G)AA(A/G)CC and CAAGAATTCTC(A/G)(A/T)ANGG(A/ G)TCNA(T/C)CCA, respectively). To synthesize cDNAs, total RNAs (10 mg) isolated from FL18 cells were incubated with 100 units of modi®ed reverse transcriptase of Molony murine leukemia virus (Superscript, BRL) following the manufacturer's protocol. The cDNA pool (0.5 ml) was PCR ampl®ed as described previously (Fujimoto and Yamamoto, 1994). PCR products were electrophoresed, checked by the expected size (about 190 bp), and ethanol-precipitated. The products were digested with EcoRI whose recognition sites were tagged to the PCR primers. After digestion, the products were subcloned into the EcoRI site of Bluescript SK-II (Stratagene). Screening of cDNA libraries were performed by using the newly isolated DNA insert with standard protocols (Sambrook et al., 1989) Expression of ANA in the neuroepithelium YYoshidaet al 2689 Inspection of the deduced amino acid sequence of the by staining for the ANA protein, and cells that entered ANA gene product (ANA) revealed its signi®cant the S phase were identi®ed by staining for BrdU homology at the amino-terminal half (residues 1 ± incorporation. While 64 out of 75 (85.3%) cells 115) to the corresponding portions of BTG1 (36% micro-injected with a vector containing the b- identical, Rouault et al., 1992), PC3 (38%, Bradbury et galactosidase cDNA incorporated BrdU, only 61 out al., 1991), Tob (31%, Matsuda et al., 1996), and the of 112 (54.5%) cells micro-injected with the ANA Xenopus B9 protein (57%, Genbank) (Figure 1c and d). cDNA showed BrdU incorporation (P50.05). Typical Therefore, we concluded that ANA is a member of data are shown in Figure 2. Since ANA-dependent Tob/BTG1 family antiproliferative proteins. Finally, inhibition of the cell cycle progression was leaky, we the ANA gene turned out to be the same gene as assumed that the elevated expression of ANA retarded recently reported btg3 (Gue henneux et al., 1997). the cell cycle progression through the G1 phase. Consistent with this notion, the degree of the apparent inhibition was aected by the length of BrdU ANA impairs cell cycle progression from the G /G to 0 1 incubation time (data not shown). Taken together we S phase conclude that the amino-terminal half of ANA, which To assess the ability of ANA to inhibit cell is conserved among Tob/BTG1 family members, is proliferation, we micro-injected the ANA expression inhibitory for cell cycle progression through the G1 plasmid into NIH3T3 cells that had been brought to phase.