Interaction of BTG1 and p53-regulatedBTG2 Gene Products with mCaf1, the Murine Homolog of a Component of the Yeast CCR4 Transcriptional Regulatory Complex*

Both BTG1 and BTG2 are involved in cell-growth control. BTG2 expression is regulated by p53, and its inactivation in embryonic stem cells leads to the disruption of DNA damage-induced G2/M cell-cycle arrest. In order to investigate the mechanism underlying Btg-mediated functions, we looked for possible functional partners of Btg1 and Btg2. Using yeast two-hybrid screening, protein-binding assays, and transient transfection assays in HeLa cells, we demonstrated the physicalin vitro and in vivo interaction of both Btg1 and Btg2 with the mouse protein mCaf1 (i.e. mouse CCR4-associated factor 1). mCaf1 was identified through its interaction with the CCR4 protein, a component of a general transcription multisubunit complex, which, in yeast, regulates the expression of different genes involved in cell-cycle regulation and progression. These data suggest that Btg proteins, through their association with mCaf1, may participate, either directly or indirectly, in the transcriptional regulation of the genes involved in the control of the cell cycle. Finally, we found that box B, one of two conserved domains which define the Btg family, plays a functional role, namely that it is essential to the Btg-mCaf1 interaction.

BTG1 (B-cell translocation gene 1) 1 (1,2) and BTG2 (3) belong to a family which, in vertebrates, comprises at least seven members: BTG1, BTG2/TIS21/PC3, BTG3/TOB5, TOB, TOB4, B9.10, and B9. 15. Two short conserved domains (box A and box B), separated by a spacer sequence of 20 -25 nonconserved amino acids, define the signature of this family (4). Although little is known about the biological functions of the Btg family, it is likely that they constitute a group of function-ally related genes involved in the control of the cell cycle.
The following observations demonstrate the involvement of different members of this family in the negative circuits governing cell growth suppression. Both BTG1 and BTG2 are preferentially expressed in quiescent cells, and overexpression of these genes causes a decrease in the growth rate and clonability of NIH 3T3 cells (2). A negative correlation between BTG1 mRNA expression and cell proliferation has been observed in, for example, T lymphocytes, macrophages, and testis development (2,5,6). BTG2 is also implicated in the control of the cell cycle. In some models, such as T lymphocytes, 12-Otetradecanoylphorbol-13-acetate-treated mouse fibroblasts, nerve growth factor-stimulated PC12 cells, and neuron cell birthday in mouse embryo, its mRNA expression increases when cells cease to proliferate, or when they differentiate (3,(7)(8)(9). The putative rat BTG2 homolog was isolated as a gene induced by nerve growth factor in pheochromocytoma cells that had undergone the neural differentiation (8). The antiproliferative activity of this gene, called PC3 (for pheochromocytoma cell-3), and its involvement in cell-cycle regulation, were demonstrated more recently (10). It was also shown that overexpression of the Tob protein inhibits NIH 3T3 cell growth (11), and that its expression varies during amphioxus development (12). As for BTG3, its expression at the end of the G 1 phase has been detected in T lymphocytes (4).
We recently put forward evidence that BTG2 expression is up-regulated by p53 after DNA damage induced by genotoxic agents (3). It has also been suggested that BTG2 could be involved in the programmed cell death of PC12 cells (13,14).
However, these proteins exhibit no known functional signature, which makes it difficult to tell what their biological function may be. One notable observation was the association of Tob with the growth factor receptor ErbB2, which may modulate the signal elicited by epidermal growth factor. And it has been shown that both Btg1 and Btg2 interact with a proteinarginine N-methyltransferase (Prmt1), and modulate its activity positively (15). The importance of this result has been heightened by the fact that a molecular association has been found between Prmt1 and the Ifnar1 chain of the interferon type 1 receptor (16). It has also been shown that the prevention of Prmt1 expression impedes the usually observed arrest of cell growth by interferon (16). Prmt1 thus appears to be an important participant in this process.
In the present paper, we report the characterization of another Btg-associated protein, mCaf1. This characterization provides possible insights into the nature of others biochemical pathways that may be controlled by Btg1 and Btg2 and the role of Btg proteins in general.

EXPERIMENTAL PROCEDURES
Yeast Expression Constructs-The Btg1 and Btg2 "bait" for the yeast two-hybrid system was based on the pPC62 yeast expression vector (17). pPC62 was cut with SalI and XbaI and ligated with SalI/XbaI BTG1 and BTG2 human cDNA, obtained by polymerase chain reaction, giving pPC62BTG1 and BTG2 encoding fusion proteins, consisting of the Gal4 DNA-binding domain fused to Btg1 and Btg2. Several BTG1 deletion mutants were also constructed as GAL4 fusions. These are shown in Fig. 1.
Library Screening-To identify genes encoding proteins that interact with Btg1, we used the two-hybrid system to detect interactions via the reconstitution of a functional transcription activator in yeast, with, as a recipient, the Y190 strain (18) which was transformed with pPC62BTG1. The Btg1-expressing yeast cells were subsequently transformed with the GAL4 transactivation domain-tagged 14.5-day-old mouse embryo cDNA library (17), following the protocol described by Durfee et al. (19), the only significant modification being the use of salmon sperm DNA as a carrier. After 4 days at 30°C on L/W/H amino acid-depleted SC medium, the transformants were tested for ␤-galactosidase activity using a yeast colony filter assay. Positive (blue) colonies were grown for 2 days to recover the prey plasmid. The electromax DH10B bacterial strain was used to recover the expression plasmids from the selected transformed yeast (17).
mCAF1 Open Reading Frame-The cDNA encompassing the entire mCAF1 open reading frame was cloned by reverse transcriptase-polymerase chain reaction. One g of NIH 3T3 total RNA was reversetranscribed using 100 ng of CAFB as a specific primer, following the Superscript 2 (Life Technologies, Inc.) protocol. 1/10 of this mixture was used as a template for polymerase chain reaction. Fifty picomoles of CAFB and CAFA oligos were added, and the reaction was carried out following Promega's instructions, at an annealing temperature of 50°C. The expected cDNA was recovered, digested with BamHI and BglII, cloned, and sequenced. Sequences of synthetic oligonucleotides used: oligo CAFA, 5Ј-caggatcccagtATGCCAGCAGCAACCGTA-3Ј and oligo CAFB, 5Ј-acagatctAAGAAGACTATTTCCTGTCATG-3Ј.
Bacterial Expression Constructs-To generate the bacterial expression vectors for BTG1 and BTG2, their full-length coding sequences were inserted into the pGEX-ET expression vector (Pharmacia) inframe with the glutathione S-transferase (GST) coding sequence.
Mammalian Reporter Plasmid-The pG4-TK-CAT reporter plasmid, which contains six GAL4 consensus elements upstream from the thymidine kinase (TK) promoter region, fused to the CAT gene, was provided by E. Manet (ENSL, Lyon, France) (20).
Mammalian Expression Constructs-All the mammalian expression constructs used were derivatives of the SV40 promoter-driven expression vector pSG5 (Stratagene). The plasmid pSG5flag was derived from pSG5 by insertion between the EcoRI and BamHI sites of an oligonucleotide containing the sequence of the flag peptide (IBI Flag system, Kodak) and a polylinker. The full-length mCAF1 cDNA were subcloned into the XhoI/SpeI sites of the pSG5flag vector to generate pSG5flagCAF1. pGALBTG1, pGALBTG2, pGALBTG1(1-96), and pGALCAF vectors were obtained by cloning the respective coding regions domain into Gal4polyII plasmid (21), in-frame with the yeast GAL4 DNA-binding domain coding sequence (amino acids 1-147), which contains GAL4 elements responsible for DNA binding, homodimerization, and nuclear localization. pVPBTG1, pVPBTG2, pVPBTG1(1-96), and pVPCAF constructs were obtained by cloning the respective coding regions in-frame with the VP16 activation domain coding sequence into the pSG-FNV vector, kindly provided by P. Jalinot (ENSL, Lyon). pSG-FNV (22) is a pSG5 derivative encoding the flag epitope fused to the SV40 nuclear localization signal and the VP16 activation domain (amino acids 403-479). The cloned products were verified by DNA sequencing, and the correct expression of all the proteins was checked.
Transfections-The plasmids used for transfection were prepared by the alkaline lysis method, and purified by PEG/LiCl. HeLa cells were grown in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) supplemented with 10% (v/v) fetal calf serum, and seeded at 2.5 ϫ 10 5 cells/well in 6-well microtiter plates 8 h prior to transfection. The transfected DNA included 0.5 g of the pG4-TK-CAT reporter plasmid, 200 ng of the GAL4, and/or VP16 fusion vectors, 50 ng of the pCMV-LACZ control plasmid in 5 l of LipofectAMINE (Life Technologies, Inc.), and 1 ml of Opti-MEM (Life Technologies, Inc.). The amount of SV40 promoter transfected was kept constant, where necessary, by the addition of pSG5 to the transfection mixture. The transfected cells were washed and collected 48 h after transfection.
CAT Enzyme-linked Immunosorbent Assay-CAT enzyme-linked immunosorbent assays were performed using the Boehringer Mannheim CAT enzyme-linked immunosorbent assay kit, following the manufacturer's instructions. The transfected cells were lysed in 150 l of lysis buffer. The supernatants were assayed for CAT protein production and ␤-galactosidase activity. All transfection data were normalized by ␤-galactosidase assays, which was quantified by O-nitrophenit-␤-D-galactopyranoside assay using a standard linear curve. Reporter activity was expressed as the ratio of fold induction to the activity of the reporter vector alone. Each set of experiments was repeated at least three times, and similar results were obtained.
Immunoblot Analysis-For protein expression assays, 50 l of lysate of HeLa cells transfected as described was subjected to electrophoresis on a 12% polyacrylamide-SDS gel. The separated proteins were transferred to a nitrocellulose membrane (Schleicher & Schuell) by electroblotting. Equal amounts of protein were loaded into each lane, as measured by the Bradford assay and confirmed by red Ponceau staining of the transferred membrane. Gal4 fusion proteins were detected with an anti-GAL4 DNA-binding domain mouse monoclonal antibody (RK5C1, Santa Cruz Biotechnology, Inc.). The M2 monoclonal antibody (IBI Flag system, Kodak) was used to detect the VP16 fusion proteins. The membranes were then incubated with horseradish peroxidaseconjugated rabbit anti-mouse immunoglobulins. The proteins were visualized by means of the enhanced Amersham chemiluminescence kit following the manufacturer's instructions.
GST "Pull-down" Experiments-GST and GST fusion proteins were expressed in Escherichia coli DH5␣, purified on glutathione-Sepharose beads (Pharmacia), and quantified using the Bio-Rad method. SDS-PAGE and Coomassie staining were used to confirm the integrity of the full-length fusion proteins. For in vitro protein-protein interaction assays, 10 g of GST or GST fusion proteins were incubated for 1 h at room temperature with 50 l of glutathione-Sepharose beads in the binding buffer (20 mM Tris-HCl, pH 7.4, 100 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol, 1% milk, 10% glycerol), and packed in mini columns. 15 l of in vitro synthesized [ 35 S]methionine-labeled mCaf1 and luciferase were suspended in the binding buffer and passed through GST-, GST-Btg1-, and GST-Btg2-glutathione-Sepharose mini columns. After washing, retained proteins were eluted with 10 mM glutathione in 50 mM Tris-HCl, pH 8, analyzed on a 10% SDS-PAGE gel, and visualized by autoradiography.
Northern and Dot Blot Analysis-10 g of total cellular RNA obtained from Lovo (human colocarcinoma cell line) and from NIH 3T3 cells were resolved by agarose gel electrophoresis, transferred to a nitrocellulose membrane, and hybridized with full-length mCAF1 cDNA labeled by random priming. For the multiple tissue dot blot, a poly(A)ϩ RNA containing membrane (CLONTECH) was hybridized with the same labeled probe.
Immunofluorescence Microscopy-HeLa cells were seeded on microscope slides in 6-well plates at 2.5 ϫ 10 5 cells per well. After 8 h the cells were transiently transfected by lipofection with 1 g of the pSG5flagCAF1 expression vector. Two days later, the cells were fixed with 4% paraformaldehyde for 15 min, and permeabilized in 0.1% Triton X-100 for 5 min. Nonspecific staining was blocked by a 30-min incubation with 0.2% gelatine in phosphate-buffered saline. Immunodetection was carried out using the M2 monoclonal antibody (IBI flag system, Kodak) at a 1:100 dilution, followed by a fluoresceine isothiocyanate-conjugated goat anti-mouse secondary antibody. The coverslips containing the stained cells were mounted on microscope slides and the immunofluorescence was recorded using a Zeiss Axioplan 2 microscope.

RESULTS
Interaction of mCaf1 with Btg1 and Btg2-In this study, we used the two-hybrid interaction system developed by Fields and Song (23). To screen for cDNA encoding proteins able to interact with Btg1, the Y190 pPC62BTG1 yeast strain was transformed with a 14.5-day-old mouse embryo cDNA library cloned in pPC51 (17). Out of 5 ϫ 10 5 transformants plated on an L/W/H amino acid-depleted SC medium, 100 grew. Two of these clones produced ␤-galactosidase, and they were further analyzed. One of them was found to encode a protein with a sequence identical to that of a homeotic protein (which will be described elsewhere). The second gene (clone 7.1) was found to be identical to the mCAF1 mouse gene. mCAF1 is the mouse homolog of the yCAF/POP2 yeast gene, which is involved in a number of transcription processes.
According to Draper (24), the 7.1 clone identified in the yeast two-hybrid system begins at nucleotide 71 (EMBL/GenBank accession number number U21855). The cDNA encompassing the entire mCAF1 open reading frame was cloned by reverse transcriptase-polymerase chain reaction using NIH 3T3 total RNA.
When tested using the two-hybrid system, the 7.1 clone product also interacted with the Btg2 fusion protein. Given that the Btg protein family is characterized by the presence of two conserved boxes separated by a spacer of relatively constant length, we looked at whether these boxes play a role in the association between the Btg proteins and mCaf1. Using a series of deletion mutants, encompassing or not the two conserved boxes, the region of the Btg1/mCaf1 association was mapped to box B. The results of this assay are summarized in Fig. 1B. All the pPC62-BTG1 derivative constructs containing box B interacted with the 7.1 clone product. Further deletions, including that of box B, completely abolished the association Btg1/mCaf1. In addition, when box B was fused to Gal4 DNAbinding domain, an interaction with the mCaf1 protein took place (Fig. 1B). These results suggest that the Btg-mCaf1 interaction is mediated by box B, one of the two conserved domains defining the BTG family, and that this domain has some functional significance.
Interaction of mCaf1 with Btg1 and Btg2 in Vitro-To verify that mCaf1 can interact directly with Btg1 and Btg2, we performed in vitro association assays with purified recombinant glutathione S-transferase fusion proteins. GST-Btg1, GST-Btg2, and GST alone, used as a control, were coupled to glutathione-Sepharose beads and incubated with [ 35 S]methionine-labeled mCaf1. As shown in Fig. 2B, the specific retention of mCaf1 was observed with the GST-Btg1 and GST-Btg2 beads, but not with the control GST beads. And the incubation of GST-Btg1 and GST-Btg2 with [ 35 S]methionine-labeled luciferase, used as a control, failed to show any specific interaction (Fig. 2B). These results point to a direct physical interaction of mCaf1 with both Btg1 and Btg2. It can be seen, however, that the interaction of mCaf1 with Btg1 was significantly weaker than with Btg2 (Fig. 2B).
Interaction of mCaf1 with Btg1 and Btg2 in Mammalian Cells-We next studied the interactions between mCaf1 and the Btg proteins in mammalian cells. Two-hybrid protein-protein interaction assays were performed in mammalian culture cells, with a reporter plasmid (pG4-TK-CAT) containing six GAL4 consensus elements upstream from the thymidine kinase (TK) promoter region fused to the CAT gene. Vectors were

FIG. 2. Physical interaction of mCaf1 with Btg1 and Btg2 in vitro.
A, Coomassie-stained GST, GST-Btg1, and GST-Btg2. GST fusion proteins (10 mg) bound to glutathione-Sepharose beads were washed and eluted with 10 mM glutathione, and 30% of the eluted proteins were fractionated on a 12% SDS-PAGE. B, 35 S-labeled fulllength in vitro translated mCaf1 and luciferase were suspended in binding buffer and passed through GST, GST-Btg1, and GST-Btg2glutathione-Sepharose mini columns. The beads were washed and eluted with 10 mM glutathione. The eluted proteins and 1/3 of input radiolabeled proteins were analyzed by 12% SDS-PAGE and visualized by autoradiography. Molecular size markers are given in kilodaltons.

FIG. 1. Detection of Btg1/mCaf1 interaction in yeast.
A, schematic representation of the two-hybrid system used to isolate the Btg1associated protein mCaf1. BTG1 cDNA was cloned in-frame with the GAL4 DNA-binding domain (BD) of the pPC62 vector. This construction was introduced into the Y190 yeast strain which harbors two reporter genes, HIS3 and LACZ, under the control of promoters containing Gal4-binding sites, upstream from GAL1 (UAS G ). The resulting yeast cells were then transformed with a GAL4-tagged activation domain (TAD) cDNA expression library (see "Experimental Procedures"). B, the Y190 yeast strain was transformed with BTG1 or mutated BTG1 cDNA cloned in-frame with the GAL4 BD of the pPC62 vector along with the 7.1 clone carrying the mCAF1 cDNA cloned in-frame with the GAL4 TAD of the pPC51 vector. The co-transformed Y190 yeast strain was grown in a L/W/H amino acid-depleted SC medium. A yeast colony filter assay was used to determine the ␤-galactosidase activity of the clones which came out positive in the preceding selection. Results are given for each individual transformation assay (Ϫ, no colony and thus no interaction; ϩ, colonies which were further tested for ␤-galactosidase activity). constructed to express both Btg1 and Btg2 fused to the DNAbinding domain (amino acids 1-147) of the yeast Gal4 transcription factor. A vector expressing mCaf1 fused to the VP16 activation domain (amino acids 413-490) was also produced. HeLa cells were transfected with the reporter plasmid alone or with chimera constructs so as to analyze the ability of Btg1 and Btg2 to associate with mCaf1.
Only the coexpression of either GalBtg1 or GalBtg2 with the VpCaf construct elicited a significant increase in the expression of the pG4-TK-CAT reporter (Fig. 3B), which indicates a strong association between Caf1 and both Btg1 and Btg2. The interactions between Caf1 and the Btg proteins were also analyzed in reciprocal combination: mCAF1 fused to a GAL4 DNA-binding domain (pGALCAF) and BTGs fused to the VP16 activation domain (pVPBTG). As expected (Fig. 3B), the concomitant expression of the GalCaf1 and VpBtg proteins produced a strong increase in the activity of the CAT reporter. The expression of fusion proteins in transfected cells were determined by Western blot analysis, using specific antibodies (Fig. 3C). None of the hybrid proteins, on their own, activated the expression of the CAT reporter gene (Fig. 3B). And indeed, mCaf1 and Btg proteins did not seem to be capable of stimulating transcription when tethered to multimerized DNA sites through a Gal4binding domain in HeLa cells. In contrast, LexACaf1 fusion can activate transcription from a LexA operator-controlled reporter gene in yeast (data not shown), as was also found by Draper et al. (24). Experiments aimed at elucidating this discrepancy are being currently carried out.
A Btg1 deletion mutant, Btg1(1-96) (see Fig. 1B), lacking box B, failed to produce a specific interaction with mCaf1 (Fig. 3B), in agreement with our yeast two-hybrid assays. These results indicate that Btg1 and Btg2 are indeed able to interact with mCaf1 in mammalian cells, and that box B is necessary to this interaction. However, in contrast with the results obtained with yeast, we did not find any evidence of an interaction between box B alone and mCaf1 in HeLa cells (data not shown).
When GalCaf was coexpressed with VpCaf, basal promoter activity did not increase (data not shown), which suggests that the mCaf1 protein cannot form dimeric complexes. Likewise, the coexpression of GalBtg and VpBtg did not enhance the activity of the reporter gene, which indicates that the Btg proteins do not interact either in homo-and in heterodimeric complexes in HeLa cells (data not shown). This finding is in keeping with the results obtained in yeast by Lin et al. (15).
Expression of CAF1-The in vitro translation of mCAF1 mRNA derived from full-length cDNA produced a doublet with an apparent molecular mass of 31 kDa (see Fig. 2B, mCaf1 imput).
The subcellular localization of mCaf1 was examined by indirect immunofluorescence of HeLa cells transiently transfected with a plasmid expressing a flag epitope-tagged version of mCaf1. mCaf1 was detected in both the nucleus and the cytoplasm (Fig. 4, Ca).
Finally, the expression pattern of the CAF gene in various tissue types was studied. Northern blot analysis in Lovo-and NIH 3T3-derived total RNA revealed a 2.5-kilobase message, as well as one of 1.2 kilobases (Fig. 4A), although their structure remains unclear at present. Furthermore, a multiple human tissue dot blot analysis (CLONTECH) (Fig. 4B) did not revealed any CAF mRNA in nervous system-derived tissues, but showed variable levels of expression among the positive tissues. High levels of expression were found in the case of the stomach (C8), salivary gland (D7), thyroid gland (D6), kidney (E1), lung (F2), fetal thymus (G6), and fetal lung (G7). The same procedure revealed similar patterns of BTG1 and BTG2 expression (data not shown).

DISCUSSION
The ability of normal cycling cells to exit from the cell cycle in response to changes in environmental conditions (e.g. nutrient deprivation, growth-inhibiting factors, or high cell density) points to the existence of pathways that inhibit growth. The Btg family of proteins seem to be involved in the cell-cycle regulation network. In order to investigate the mechanism underlying the Btg-mediated functions, we looked for possible functional partners of Btg1 and Btg2. We identified a mouse protein, mCaf1, which binds to both Btg1 and Btg2. The physical in vitro interaction of Btg1 and Btg2 with mCaf1 was confirmed by GST pull-down experiments and a reciprocal set of experiments was carried out, using the Gal4 DNA-binding domain and the VP16 activation domain to demonstrate the functional and physical associations of mCaf1 with both Btg1 and Btg2 in transfected recipient HeLa cells. Interestingly, the specific interaction of Btg1 and Btg2 with mCaf1 occurs through a conserved domain of the Btg protein family, box B. And in fact Btg3, another member of the Btg family, also displayed a two-hybrid interaction with mCaf1 (data not shown). It may even be the case that all the members of the Btg family may be able to interact with mCaf1. The functional specificity of complexes formed by mCaf1 and the individual members of the Btg family could perhaps be achieved through the use of different downstream cellular targets.
A two-hybrid assay demonstrated that mCaf1, which is an evolutionarily conserved protein, interacts with the general transcriptional regulator CCR4 (24). mCAF1 is a homolog of yeast yCAF1/POP2, whose protein is a component of the CCR4 complex (24). Genetic analysis in yeast suggests that the CCR4 complex may perform multiple functions in transcription regulation. It also appears to be a key regulator in a numbers of cellular processes. It is required, for example, for the full derepression of ADH2 and other non-fermentative genes under glucose-derepressed conditions (25,26). ccr4 and ycaf1 mutations affect the expression of genes involved in maintaining chromatin structure (27,28) and in methionine biosynthesis (29). The CCR4 function occurs downstream from SPT6 and SPT10, as a post-chromatin remodeling event (30,31). And yCAF1 disruption display phenotypes and transcriptional defects very similar to those of CCR4. Thus CAF1/POP2 and CCR4 appear to operate along the same pathway in the yeast model.
Moreover, yCaf1 associates with a yeast cell cycle-regulated protein kinase, Dbf2, which is also a component of the CCR4 complex (32). These results provided a possible link between the CCR4 complex and the regulation of the cell cycle. Other proteins, components of the NOT complex, have recently been identified as being part of the CCR4 complex, which is thought to affect transcription both positively and negatively (33). Because of the evolutionary conservation of Caf among eucaryotes, it seems likely that this complex is conserved between yeast and higher eucaryotes, and that the Btg proteins are also among its components. Btg proteins may play a role in modulating transcriptional activity within a multiprotein complex, or in regulating the DNA binding properties of this complex, possibly operating as a kind of molecular glue. Or they may act in a transient way to facilitate interactions between multiple proteins and DNA, in which case they would not be a structural part of the transcriptionally active multiprotein complex. Experiments are currently being carried out to determine whether (and if so, how) Btg1 and Btg2 exist as multimeric complexes, and to investigate their potential role in transcription regulation.
However, there remains the possibility that CCR4 is not involved in the mCaf1-Btg interaction. And in fact Draper et al. FIG. 3. Interaction of mCaf1 with Btg1 and Btg2 in the mammalian two-hybrid system. 200 ng of the indicated pGAL4 and pVP16 fusion expression plasmids were transiently co-transfected into HeLa cells with 0.5 g of a reporter gene containing six GAL4-binding sites upstream from a minimal TK promoter fused to the CAT gene. Total DNA was kept constant at 1 g. Cells were co-transfected as described under "Experimental Procedures." Reporter activity was normalized with ␤-galactosidase activity, and the data expressed as the ratio of fold induction to the activity of the reporter vector alone. Each set of experiments was repeated at least three times, and similar results were obtained. Bars indicate standard deviation of the mean of at least three independent transfections. A, schematic representation of the reporter and effector vectors. The indicated regions of the BTGs and the mCAF1 cDNA were cloned into a vector containing the GAL4 DNA-binding domain. Similarly, overlapping regions were inserted into a vector containing the VP16 activation domain. B, interaction between GalBtgs and VpCaf, and between GalCaf and VPBtgs, in the mammalian two-hybrid system. As a positive control, we used the pGALVP plasmid which encodes a protein consisting of the Gal4 DNA-binding domain fused to the Vp16 activation domain. C, expression of the Gal4 and VP16 fusion proteins expressed in HeLa cells transfected as described above. Lysates from transfected HeLa cells were subjected to Western blot analysis after SDS-PAGE, and then revealed by the indicated antibodies. (24) have observed that yCaf1 may recruit the transcriptional machinery by an independent mechanism that does not involve CCR4. In both models Btg1 and Btg2 play a role in the regulation of transcription, which is consistent with the fact that, in the two-hybrid assays, both Btg1 and Btg2 were found to interact with a homeodomain containing transcription factor (data not shown).
In addition to affecting the cell cycle similarly to the dbf2 and ccr4 mutations, the caf mutation suppresses the defect of rad 52-20, an allele of RAD52, a gene involved in UV sensitivity and DNA repair (34). Of particular interest in this regard is a recent sequence analysis study which indicates the presence of a proofreading exonuclease domain in the mCaf1 protein (35). And it is interesting to speculate that Btg2, whose expression is rapidly induced after genotoxic stress, and which is transcriptionally activated by p53, may contact a cellular protein involved in the control of cell cycle progression and DNA repair. With this in mind, along with the observation that BTG2 inactivation in embryonic stem cells leads to the disruption of DNA damage-induced G 2 /M arrest (3), it is reasonable to conjecture that Btg2 might participate, in association with mCaf1, in DNA repair processes, including replication-associated DNA repair.
Lin et al. (15) have recently shown that both Btg1 and Btg2 interact with a protein-arginine N-methyltransferase (Prmt1), and modulate its activity positively (15). And the importance of this result has been increased by the fact that Prmt1 has also been found to participate in a molecular association with the Ifnar1 chain of the interferon type 1 receptor (16). Given that Prmt1/Ifnar1 association is necessary for interferon-mediated growth arrest (16), it is likely that Btg1 and -2 can be involved in this process.
These observations indicate that Btg proteins may associate with other proteins at different stages in the cell cycle, and may play a variety of roles as intermediaries between signal transduction pathways and the final transcriptionally competent initiation complex. The study of the Btg-associated proteins, and their regulation, could bring to light new mechanisms which influence the cell cycle. The complete elucidation of these pathways is vital to the understanding of oncogenesis and tumor progression.