A novel CCAAT-binding protein necessary for adhesion-dependent cyclin A transcription at the G1/S boundary is sequestered by a retinoblastoma-like protein in G0.

Loss of adhesion leads to cell cycle arrest at the G1/S boundary in normal, adhesion-dependent, mesenchymal cells. This arrest is accompanied by the inability to produce cyclin A. Using deletional and mutational analysis of the cyclin A promoter, we have identified a CCAAT element that mediates the adhesion-dependent transcriptional activation of cyclin A in late G1 phase of the cell cycle. Specific binding of a novel 40/115-kDa heterodimeric protein complex, which we have named CBP/cycA, to this CCAAT element was detectable in growing but not in G0-arrested or nonadherent normal rat kidney fibroblasts. During G0 CBP/cycA appears to be present but sequestered by a retinoblastoma family member. These results suggest that expression of cyclin A, which controls cell cycle progression by adhesion at the G1/S boundary, is regulated by CBP/cycA and the phosphorylation status of the retinoblastoma protein or a retinoblastoma-related protein.

Cells of most tissues, with the exception of hematopoietic cells, require adhesion to a surface in order to grow. Nonadherent cells fail to proliferate despite the presence of growth factors (1). Transformation of cells by c-Ha-ras (2), v-src (3), c-sis (4), or the DNA tumor virus proteins SV40 large T antigen (5), adenovirus E1A (6), and human papillomavirus E7 (7) results in loss of adhesion dependence. This in vitro phenotype correlates well with tumorigenicity in vivo (8). Recently, it has been shown that the adhesion requirement in mesenchymal cells is likely to reflect a cell cycle checkpoint in the late G 1 phase of the cell cycle (9). Cells arrested by suspension failed to produce cyclin A (10). In addition, ectopic expression of cyclin A enabled the cells to bypass the adhesion requirement, implicating cyclin A as the major target of cell cycle control by the anchoragesignaling pathway. The adhesion signal itself appears to be mediated through adhesion plaques, since modulation of expression of components of adhesion plaques like integrins (11), vinculin (12), actinin (13), or focal adhesion kinase (14,15) affects the adhesion dependence of cells. However, little is known about the signal transduction pathway by which the adhesion signal exerts control of the cell cycle.
In this report, we have examined the mechanism by which the expression of cyclin A is regulated by cell adhesion. We show that the adhesion-dependent transcriptional activation of the cyclin A gene at the G 1 /S boundary is mediated by a CCAAT element in its promoter region. This activation is accompanied by binding of a 40/115-kDa heterodimeric protein complex to the CCAAT element. In addition, we demonstrate that during the G 0 phase of the cell cycle this novel trans-acting factor, which we have named CBP/cycA 1 (CCAAT-Binding Protein for cyclin A gene), seems to be sequestered and thereby held inactive by a retinoblastoma protein (Rb) family member.
Cell Culture, Cell Adhesion, and Transfection-Normal rat kidney (NRK) fibroblasts were synchronized at G 0 by incubation of 1 ϫ 10 6 cells per 10-cm dish for 3 days in serum-free Opti-modified Eagle's medium I (Opti-MEM I, Life Technologies, Inc.) containing 1 mg/ml bovine serum albumin and 0.5 g/ml gentamycin. After 48 h of incubation, cells were transiently transfected by lipofection. Ten micrograms of each reporter construct, mixed with 60 g of Lipofectin (Life Technologies, Inc.) in 2 ml of Opti-MEM I were left at room temperature for 10 min, after which 8 ml of Opti-MEM I was added. This mixture was added to a 10-cm dish and incubated at 37°C for 6 h. The medium was then replaced by serum-free medium as described above. Twenty-four h after the start of transfection, the cells were trypsinized and reseeded on 10-cm plastic tissue culture plates in (i) serum-free medium as described above, (ii) Dulbecco's modified Eagle's medium (DMEM) plus 5% fetal calf serum (FCS, HyClone Laboratories, Inc., Logan, UT) and 12 ng/ml recombinant human epidermal growth factor (rhEGF, Promega, Madison, WI), or (iii) on poly(2-hydroxyethylmethacrylate) (poly-HEMA)-coated dishes in DMEM containing 5% FCS and 12 ng/ml rhEGF plus 1.3% methylcellulose to prevent cell adhesion and aggregation. Twenty h after reseeding, cell lysates were prepared and luciferase assays were performed as described (17).
Plasmids-Plasmids pWt918 and pWt434 were generated by inserting a 918-or 434-bp PCR-amplified fragment of the human cyclin A promoter into the HindIII site of pSV0A-L⌬5Ј (18). PCR amplifications of the cyclin A promoter fragments were performed using K562 cell genomic DNA as a template and 5Ј-CTCGTCGACTTGTTGGTTGCA-CAGCTTGG-3Ј and 5Ј-CACAAGCTTGAATAGTCGTAGCCGCCGGT-3Ј (p2) for the 918-bp fragment and 5Ј-CTCGTCGACTTCGGACAGC-CTCGCTCACTA-3Ј and p2 for the 434-bp fragment as primers. The sequence for the PCR primers was based on the published sequence of the cyclin A promoter (19). The PCR reactions were carried out at 94°C for 60 s, 55°C for 90 s, and 72°C for 120 s for 35 cycles in 20 M Tris, pH 8.8, 2.6 mM MgCl 2 , 50 M KCl, 0.05% Tween 20, 0.05% Nonidet P-40, * This work was supported by National Institutes of Health Grant R37-CA42024. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 1 The abbreviations used are: CBP/cycA, CCAAT-binding protein for cyclin A gene; Rb, retinoblastoma protein; DMEM, Dulbecco's modified Eagle's medium; Opti-MEM I, Opti-modified Eagle's medium; FCS, fetal calf serum; rhEGF, recombinant human epidermal growth factor; polyHEMA, poly(2-hydroxyethylmethacrylate); NRK, normal rat kidney; EMSA, electrophoretic mobility shift assay; GST, glutathione Stransferase; GST101-249Tag, amino acids 101-249 of SV40 large T antigen fused to GST; GST-HPV E7, E7 protein from human papillomavirus fused to GST; PCR, polymerase chain reaction; bp, base pair(s); cdk, cyclin-dependent kinase; Wt, wild type. and 3% dimethyl sulfoxide. pWt929 was derived from pWt918 by PCR amplification using 5Ј-CACAAGCTTACCCGGCCAAAGAATAGTCG-TAGCCGCCGGT-3Ј as the 3Ј primer. pMut434 was generated using the unique site elimination mutagenesis kit (Pharmacia Biotech Inc.) according to manufacturer's instructions using 5Ј-CTCTGGGCGTCTTTa-aaTCGCCACGCTGGGC-3Ј as the mutagenic primer. pWt284, pWt225, and pWt184 were derived from pWt434 by PCR amplification of 284-, 225-, or 184-bp fragments spanning from Ϫ120, Ϫ61, or Ϫ20 to ϩ164 of the cyclin A promoter, respectively. These fragments were inserted into pSV0A-L⌬5Ј. Construction of pMut284 was performed in a two-step procedure. First, two PCR reactions were performed using 5Ј-CTCGTC-GACTGGTTTACCCTTCACTCGCCC-3Ј and 5Ј-AAAAAGTACTAGCG-CTCGCGGCCTTGACGTC-3Ј and 5Ј-AAAAAGTACTGGTCCATTTCA-ATAGTCGCGG-3Ј and p2 as primers and pWt434 as template. As a result of this reaction two PCR products were formed spanning from Ϫ120 to Ϫ60 and from Ϫ53 to ϩ164 of the cyclin A promoter. Primers were designed so that ligation of the two fragments at the Ϫ56 position resulted in the formation of a ScaI site, thereby creating a 4-bp mutation ((Ϫ60)-TTCATT-(Ϫ53) to (Ϫ60)-agtAcT-(Ϫ53)). The two PCR-amplified fragments were ligated, and the ligated product was PCR-amplified using the flanking primers. The product of the secondary PCR reaction was inserted into the HindIII site of pSV0A-L⌬5Ј, and the presence of the mutation was confirmed by digestion with ScaI and sequencing of the positive plasmids.
Immunoprecipitations-1.5 ϫ 10 6 G 0 -synchronized NRK cells were harvested 12 h after addition of DMEM supplemented with 5% FCS and 12 ng/ml rhEGF. Protein complexes were precipitated from cleared lysates by the addition of 1 g of anti-cyclin E antibody (M20, Santa Cruz Biotechnology, Santa Cruz, CA) as described (20).
Electrophoretic Mobility Shift Assays (EMSA)-NRK and HeLa cell nuclear extracts were prepared as described elsewhere (21). EMSA were performed according to the method of Costa et al. (22). The sequence 5Ј-TAGCTTGGAACTATTCGAAGTAATGCGCAAATAGGAAC-TTT-3Ј was used as a random competitor where indicated. For preincubation with GST101-249Tag or GST-HPV E7 fusion protein, 25 g of nuclear extract protein from serum-depleted, G 0 -arrested NRK cells was mixed with 0.8 g of GST101-249Tag or 0.8 g of GST-HPV E7 fusion protein and incubated for 50 min at 4°C. For preincubation with cyclin E immunoprecipitates, 15 l of nuclear extract containing 25 g of protein from serum-depleted, G 0 -arrested NRK cells was mixed with 20 l of cyclin E immunoprecipitate bound to protein A-agarose beads in 20 l of kinase buffer (50 mM Tris-HCl, pH 7.4, 30 M ATP, 10 mM MgCl 2 , 1 mM dithiothreitol) and incubated for 30 min at 37°C. Subsequently, the EMSA procedure was performed as usual.
Two-dimensional UV Cross-linking-A 32-bp probe was prepared by primer extension with Klenow polymerase in the presence of 5-bromo-2Ј-deoxyuridine, [␣-32 P]dCTP, and dNTPs using annealed, partially complementary oligonucleotides with the sequences 5Ј-GGGGAAATG-GACCAATGAAAGCGCTCG-3Ј and 5Ј-GGGGGCGAGCGC-3Ј and purified over a G-50 NICK Column (Pharmacia Biotech Inc.). The radioactive probe was added to an EMSA binding mix containing 20 g of nuclear extract from either adherent, cycling NRK fibroblasts or HeLa cells, and the reaction components were separated in a non-denaturing gel. The position of the CBP/cycA band was determined with a Phos-phorImager. The band of the first dimension, non-denaturing gel was excised and exposed to 304 nm light for 10 min. The excised band was then laid in the well of a 4% stacking gel on a 6% SDS-acrylamide gel and overlaid with 2 ϫ SDS-loading buffer. The covalently linked protein-DNA complexes were then separated in the second dimension under denaturing conditions. The positions of the CBP/cycA complexes were determined using PhosphorImager analysis.
Ferguson Analysis of Native Protein-DNA Complex Size-Ferguson analysis of the CBP/cycA EMSA band was performed as described using native acrylamide gels ranging in acrylamide concentration from 4 to 10% (23).

RESULTS AND DISCUSSION
Addition of growth factors to adherent, G 0 -arrested NRK cells enabled their re-entry into the cell cycle and resulted in a 20-fold induction of transcription from the 929-bp cyclin A promoter (pWt929; Fig. 1, A and B). However, when cells were plated on polyHEMA-coated dishes in methylcellulose-containing medium to prevent cell adhesion and aggregation, only a 2-fold induction of luciferase activity was seen. This demonstrates that transcription initiated by the cyclin A promoter is only induced when the cells are attached to a substratum.
Deletions within the cyclin A promoter (Fig. 1A), which included potential binding sites for the transcription factors E2F (pWt918), AP1, and E2F (pWt434) or AP1, E2F, and Sp1 (pWt284), did not affect induction of the cyclin A promoter (Fig.  1, A and B). Similarly, when a 3-bp mutation was introduced into the remaining E2F site of pWt434 (pMut434), inducibility of the cyclin A promoter was not affected. The additional deletion of a CCAAT element and an ATF site (pWt184) decreased the inducibility of luciferase activity in adherent cells to Ͻ1% of pWt929. A small portion of this decrease resulted from loss of the ATF site (pWt225) (24 -26); however, inducibility of the cyclin A promoter in adherent cells was still retained after deletion of the ATF site. Only removal of the CCAAT element located at Ϫ56 to Ϫ52 resulted in complete ablation of inducibility. CCAAT elements have been shown to mediate cell cycledependent transcriptional activation of the human histone H1 (27), heat shock protein 70 (hsp70) (28), DNA polymerase ␣ (29), and thymidine kinase genes (30 -32) at the G 1 /S boundary. We therefore introduced a mutation into the CCAAT element of pWt284 (pMut284, Fig. 1A), which completely abolished cyclin A promoter inducibility (Fig. 1B). Thus, the CCAAT element is necessary for the adhesion-dependent activation of cyclin A expression at the G 1 /S boundary.
To directly demonstrate an interaction between the CCAAT element of the cyclin A promoter and a CCAAT-binding protein, EMSA were performed using a 24-bp, CCAAT elementcontaining cyclin A promoter fragment as a probe (Fig. 2A). No

FIG. 1. Contribution of potential transcription factor binding sites to the adhesion-dependent activation of the cyclin A promoter.
A, schematics of the cyclin A promoter region in plasmids pWt929, pWt918, pWt434, pMut434, pWt284, pMut284, pWt225, and pWt184, which contain the luciferase gene as a reporter. B, synchronous, serum-depleted NRK cells were transiently transfected with the plasmids mentioned above using lipofection. Luciferase activity in cellular extracts from (i) serum-depleted, G 0 -arrested, (ii) serum-supplemented, nonadherent, and (iii) serum-supplemented, adherent NRK cells is presented normalized to G 0 -arrested NRK cells (n ϭ 3).

Novel CCAAT-binding Protein Induces Cyclin A Transcription 6580
sequence-specific DNA-protein complexes were detectable when nuclear extracts from serum-depleted, G 0 -arrested or serum-supplemented, nonadherent NRK cells were incubated with the probe. Formation of a sequence-specific DNA-protein complex was observed when nuclear extracts from serum-supplemented, adherent NRK cells or actively dividing HeLa cells grown in suspension were used. Formation of this DNA-protein complex failed to occur with either nuclear extract when the probe contained a mutant CCAAT element (Fig. 2B). We named the DNA-binding protein complex CBP/cycA. UV cross-linking experiments and a modified Ferguson analysis revealed that CBP/cycA from NRK cells is a 155-kDa heterodimeric protein complex with subunit sizes of approximately 40 and 115 kDa (Fig. 2C). Identical results were obtained when CBP/cycA from HeLa cells was used (data not shown). Several proteins that specifically recognize CCAAT elements have been described, including CTF/NF-1 (33), CP1 and CP2 (34), CBP (35), NF-Y (36), C/EBP (37), and CBP/tk (32), among others. Most of these are composed of two heterologous subunits (27,31,34). Our finding that CBP/cycA is a heterodimer with subunit sizes of approximately 40 and 115 kDa is consistent with these reports.
However, to our knowledge, no CCAAT-binding protein subunit of 115 kDa has been described.
As demonstrated, cyclin A transcription is suppressed by suspension of NRK cells. This can be explained by two mechanisms: either by an adhesion-responsive repressor element or by failure to activate transcription through positive regulatory elements at the G 1 /S boundary. We found no indication of a repressor element, since none of our reporter construct deletions abolished transcriptional repression in response to suspension. However, the transcriptional activity of the cyclin A promoter appears to be dependent upon binding of CBP/cycA to the CCAAT element. Since CBP/cycA was not found in nonadherent cells, we conclude that the transcriptional regulation of cyclin A by adhesion is at least in part mediated by CBP/cycA. This indicates that the adhesion signal regulates cell cycle progression prior to the transcriptional activation of cyclin A at the G 1 /S boundary. Progression through the G 1 phase of the cell cycle is controlled by Rb and Rb-related proteins (p107, p130). While underphosphorylated during G 0 and the first half of G 1 , Rb becomes phosphorylated by cyclin D-cyclin-dependent kinase (cdk) 4/cdk6 (38) and cyclin E-cdk2 (39) in the second half of the G 1 phase, thereby releasing a number of cellular proteins including the transcription factor E2F from its bound form to an active state (40). Adenovirus E1A (41), SV40 large T antigen (42), and human papillomavirus E7 (43) mimic these events by binding to hypophosphorylated Rb and displacing cellular proteins sequestered by Rb. Recently, it has been described that the transcriptional activation of the hsp70 gene in late G 1 by adenovirus E1A is mediated by a CCAAT-binding protein (28). In addition, it has been demonstrated that the Rb-related protein p107 is able to repress the activation of a CCAAT elementcontaining herpesvirus thymidine kinase promoter fragment at the G 1 /S boundary (44). This raises the question whether the FIG. 3. Displacement of CBP/cycA from binding to an Rb family member. A, displacement of CBP/cycA by preincubation of nuclear extracts from G 0 -arrested NRK cells with a GST fusion protein that contained the Rb binding domain of SV40 large T antigen (GST101-249Tag). Nuclear extracts from G 0 -arrested NRK cells were preincubated with GST101-249Tag for 50 min at 4°C. Then, an EMSA was performed using a double-stranded, 24-bp, cyclin A promoter fragment (5Ј-CGAGCGCTTTCATTGGTCCATTTC-3Ј) as a probe. The arrow indicates a DNA-protein complex with the same electrophoretic mobility as CBP/cycA bound to the probe. GST101-249Tag was incubated with the probe in the absence of nuclear extract as a control. B, displacement of CBP/cycA by phosphorylation of nuclear extracts from G 0 -arrested NRK cells by cyclin E-cdk2 complexes immunoprecipitated from NRK cells. Nuclear extracts from G 0 -arrested NRK cells were preincubated with immunoprecipitated cyclin E-cdk2 complexes from NRK cells for 30 min at 37°C. As a control, cyclin E-cdk2 immunoprecipitates were incubated with the probe in the absence of nuclear extract. FIG. 2. Formation of a specific complex between the CCAAT element of the cyclin A promoter and a 40/115-kDa heterodimeric protein. A, EMSA was performed using a doublestranded, 24-bp, cyclin A promoter fragment (5Ј-CGAGCGCTTTCATT-GGTCCATTTC-3Ј (Wt)) as a probe and nuclear extracts from (i) serumdepleted, G 0 -arrested, (ii) serum-supplemented, nonadherent, (iii) serum-supplemented, adherent NRK cells, and (iv) nuclear extracts from HeLa cells grown in suspension. The arrow indicates a specific complex formed between Wt probe and nuclear protein, which we designated CBP/cycA. B, EMSA using either Wt or a single base mutant, double-stranded, 24-bp, cyclin A promoter fragment (5ЈCGAGCGCTT-TCATgGGTCCATTTC-3Ј (Mut)) as a probe and nuclear extracts from serum-supplemented, adherent NRK cells. C, monomer sizes of CBP/ cycA. A 5-bromo-2Ј-deoxyuridine-substituted Wt probe was covalently linked to CBP/cycA by UV-crosslinking (UVX).

Novel CCAAT-binding Protein Induces Cyclin A Transcription 6581
adhesion-dependent interaction of CBP/cycA with the CCAAT element of the cyclin A promoter is regulated by association with an Rb family member. We therefore preincubated nuclear extracts from G 0 -arrested NRK cells with a GST fusion protein that contained the Rb binding domain of SV40 large T antigen (residues 101-249; GST101-249Tag) in order to release CBP/ cycA. As shown in Fig. 3A, this pretreatment led to the formation of a DNA-protein complex indistinguishable from CBP/ cycA in EMSA. The same result was obtained when GST-HPV E7 was used for preincubation (data not shown). This suggests that in NRK cells CBP/cycA is inactivated during G 0 by binding to Rb or an Rb family member. In contrast to fibroblasts, HeLa cells that are transformed by human papillomavirus 18 are able to grow in suspension. This adhesion-independent growth is dependent on the expression of human papillomavirus 18 E7 (45). Our finding that active CBP/cycA is present in HeLa cells grown in suspension but not in nonadherent NRK cells is consistent with these data. We have previously been able to show that the kinase activity associated with cyclin E, which is maximally expressed at the G 1 /S boundary (20), is regulated by adhesion and might therefore constitute an element of the anchorage-signaling pathway governing the expression of cyclin A. 2 Therefore, we attempted to release CBP/cycA from its sequestered state by phosphorylation of Rb and Rb-like proteins with cyclin E-cdk2 kinase. As shown in Fig. 3B, preincubation of nuclear extracts from G 0 -arrested NRK cells by cyclin E-cdk2 complexes immunoprecipitated from NRK cells resulted in the formation of a DNA-protein complex indistinguishable from CBP/cycA in EMSA. To our knowledge, CBP/cycA represents the first CCAAT-binding protein, which appears to be sequestered by an Rb family member.
In summary, we demonstrate that activation of cyclin A transcription at the G 1 /S boundary is dependent upon adhesion of mesenchymal cells to a surface. The transcriptional activation is mediated by a novel heterodimeric CCAAT-binding protein that we have named CBP/cycA. During the G 0 phase of the cell cycle, CBP/cycA seems to be sequestered and thereby kept inactive by Rb or an Rb-related protein. The adhesion signal results in the release of CBP/cycA from binding to an Rb family member and enables the induction of cyclin A expression and progression through the G 1 /S adhesion checkpoint. Our model predicts that the control of cell cycle progression by adhesion is exerted through regulation of the phosphorylation status of Rb family members.