[CANCER RESEARCH57, 5117-5121, November 15, 19971

ABSTRACT

Cells of most tissues, with the exception of hematopoietic cells, require

adhesion to an appropriate surface to grow. Cyclin A is needed for cellcycle progression at the Ge-S tI@1)SldOI1, W)d appearance of cydin A

mRNA and protein In late G, has been shown to be dependent onadhesion-initiated signals In normal rat kidney fibroblasts. Previously, wehave reported that the adhesion-dependent activation of cydlin A transcription in late G1 is mediated by CBPIcycA (CCAAT-bindlng protein forcydlin A gene), a novel CCAAT-blading protein Specific binding ofCBP/cycA, a Mr 30,000'40,0001115,000heterotrimeric protein complex, tothe CCAAT element of the cydlin A promoter was detectable in growingbut not in G0-arrested or nonadherent normal rat kidney cells. Here, wedemonstrate that the M@30'000M0'000 subunits of CBPkycA are Identicalwith NF-YA and NF-Y5, the two subunits of NF-Y. In addition, we showthat, aside from CBP/cycA, NF-Y itself also binds to the CCAAT elementof the cydlin A promoter. But, whereas the binding of CBP/cycA isadhesion and cell cycle dependent and correlates with the expression ofcydlin A in late G1 phase, NF-Y itself seems to bind in a cell cycle

independent manner.

INTRODUCTION

Loss of adhesion leads to cell cycle arrest at the G1-S boundary.Nonadherent cells fail to proliferate despite the presence of growthfactors. Transformation of cells by c-Ha-ras (1), v-src (2), c-sis (3), orthe DNA tumor virus proteins SV4O large T antigen (4) and humanpapillomavirus E7 (5) results in loss of adhesion dependency. This in

vitro phenotype correlates well with tumorigenicity in vivo (6). Recently, it has been shown that the adhesion requirement in mesenchymal cells is likely to reflect a cell cycle checkpoint in the late G1 phaseof the cell cycle (7). NRK3 cells arrested by suspension failed to

produce cyclin A (8). In addition, ectopic expression of cyclin A

enabled these cells to bypass the adhesion requirement, implicatingcyclin A as the major target of cell cycle control by the anchoragesignaling pathway. Expression of cycin A and activation of its associated kinase begin in late G@,shortly prior to S phase entry (9, 10).

By analyzing the cyclin A promoter, we have previously reportedthat control of cyclin A expression by adhesion is mediated at thetranscriptional level (11). The adhesion-dependent transcriptional activation of the cyclin A gene at the G1-S boundary is induced bybinding of a novel, Mr 30,000/40,0001115,000 heteromeric transacting factor, CBP/cycA (CCAAT-binding protein for cyclin A gene),to the CCAAT element of the cyclin A promoter. In NRK cells, thebinding of CBP/cycA to the CCAAT element of the cyclin A pro

moter was found to be adhesion and cell cycle dependent. Whereas

Received 5/5/97; accepted 9/12/97.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 with18 U.S.C. Section 1734 solely to indicate this fact.

I This work was supported by NIH Grant R37-CA42024. A. K. is the recipient of a

postdoctoral fellowship from the Deutsche Krebshilfe.2 To whom requests for reprints should be addressed, at McArdle Laboratory for

CancerResearch,UniversityofWisconsin,1400UniversityAvenue,Madison,WI53706.Phone: (608) 262-1275; Fax: (608) 262-2824; E-mail: fahl@oncology.wisc.edu.

3 The abbreviations used are: NRK, normal rat kidney; CBPIcycA, CCAAT-binding

protein for cyclin A gene; Rb, retinoblastoma protein; EMSA, electrophoretic mobilityshift assay; cdk, cyclin-dependent kinase; Mab, monoclonal antibody.

CBP/cycA was absent from nuclear extracts of G0-arrested or nonadherent cells, it could be detected in nuclear extracts from adherent,cycling NRK fibroblasts, thereby paralleling the expression of cyclinA. The absence of CBP/cycA during the G@phase of the cell cycleseems to be due to its sequestration by hypophosphorylated Rb orRb-related protein.

Several proteins that specifically recognize CCAAT elements havebeen described, including CTF/NF-l (12), NF-Y (CP1 and CBF; Refs.13—15),CP2 (14), CBP (16), CIEBP (17), CBP/tk (18), and others.

Among those, NF-Y has been described to mediate the cell cycledependent transcriptional activation of the human thymidine kinasegene at the G1-S boundary (19). Chang and Liu (19) demonstrate thatthe binding of NF-Y to the CCAAT element of the human thymidinekinase promoter is serum dependent in IMR-90 fibroblasts. It has beensuggested by others that the cell cycle-dependent activation of cyclinA transcription in late G@phase might be mediated by NF-Y as well(20). These authors describe a constitutive, cell cycle-independentbinding of NF-Y to the CCAAT element of the cyclin A promoter insynchronized WI-38 cells. However, neither NF-Y nor any other

previously described CCAAT-binding protein is known to be mactivated by binding to Rb or a Rb family member.

The aim of this study was to further characterize CBP/cycA. To thisend, we have partially purified CBP/cycA by sequence-specific DNAaffinity chromatography. By the use of antibodies to NF-YA andNF-YB, the two subunits of NF-Y, for EMSA and Western blotanalysis, we have determined that the Mr 30,000/40,000 subunits ofCBP/cycA are identical with NF-YA and NF-YB. However, in contrastto CBP/cycA, the binding of NF-Y to the CCAAT element of thecyclin A promoter seems to be cell cycle independent. Hence, weconclude that CBP/cycA, consisting of NF-Y plus a third, Mr I 15,000subunit, and not NF-Y itself is responsible for the adhesion- and cellcycle-dependent transcriptional activation of the cyclin A gene at theG1-S boundary.

MATERIALS AND METHODS

Reagents. Antibodies against NF-YA (Mab YAla and a-CBF-B) andNF-Y5 were provided kindly by Drs. Diane Mathis (21) and Benoit deCrombrugghe (22), respectively. Antibodies against Rb (1F8), p107 (C-I 8),cyclin E (M20), and cdk2 (M-2) were obtained from Santa Cruz Biotechnology

(Santa Cruz, CA). The anti-cyclin A antibody was purchased from UpstateBiotechnology, Inc. (Lake Placid, NY).

Cell Culture and Cell AdhesIon. NRK fibroblastswere maintainedinDMEM plus 5% FCS (HyClone Laboratories, Inc., Logan, UT). HeLa cellswere cultured in RPM! 1640 plus 10% FCS. For cell cycle synchronization atGo, 1 X 106NRK cells per 10-cmdish were incubated for 3 days in serum-freeOpti-modified Eagle's medium I (Life Technologies, Inc., Gaithersburg, MD)containing 1 mg/mI BSA and 0.5 @g/m1gentamicin. After 72 h of incubation,the cells were trypsinized and reseeded on 10-cm plastic tissue culture platesin DMEM plus 5% FCS and 12 ng/ml recombinant human epidermal growthfactor (Promega, Madison, WI). Cells were harvested 0, 12, 18, and 24 h afterreseeding.

EMSA. NRK and HeLa cell nuclear extracts were prepared as describedelsewhere (23). For preparation of nuclear extracts from synchronized NRKfibroblasts, a micropreparation technique as described by Andrews and Fallerwas used (24). EMSA were performed according to the method of Costa er a!.

5117

CBP/cycA, a CCAAT.binding Protein Necessary for Adhesion-dependent Cyclin A

Transcription, Consists of NF-Y and a Novel Mr 115,000 Subunit'

Aiwin Kramer, Carsten-P. Carstens, Wyeth W. Wasserman, and William E. Fahl2III. Medizinische Klinik@ Klinikum Mannheim der Universität Heidelberg, 68305 Mannheim Germany [A. K.]: Department of Human Oncology, Universit-v of Wisconsin. Madison.Wisconsin 53792 (C-P. C.); and McArdle Laboratoryfor Cancer Research. University of Wisconsin, Madison, Wisconsin 53706 (W. W. W., W. E. F.]

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CBP/cycA CONSISTS OF NF-Y AND A Mr I 15.000 SUBUNIT

(25). The sequence 5'-TAGCTFGGAACTAUCGAAGTAATGCGCAAATAGGAAC1TI'-3' was used as a random competitor where indicated. Forsupershift experiments, I @.tgof antibody was mixed with 8 @gof nuclear

extract protein from cycling or synchronized NRK cells and preincubated for30 mm at 4°C.Subsequently, the EMSA procedure was performed as usual.

Partial Purification of CBP/cycA. HeLa S-3 cells were grown in suspension culture in Joklik's medium plus 5% FCS, harvested at 3 X l0@ to 5 X l0@

cells/ml, and stored at —70°C at a concentration of —I .0 g of cellslml in 5 nmi

KH2PO4,25 mM KOH, 30 mM NaC1,0.5 mM MgCl2, 20 mM lactate, 5 mMglucose, 0.2 mM sorbitol, and 10% DMSO. All subsequent steps were per

formed at 4°Cunless indicated otherwise. Nuclear extracts were preparedaccording to the method of Dignam er a!. (23). Nuclear extracts were dialyzed

into buffer B [50 mM HEPES (pH 7.9), 12.5 mM MgC12, 1.0 mrsi EDTA, 17%

glycerol, 0. 1 MKCI, 1.0 mMDTT, and 0.5 mMphenylmethylsulfonyl fluoride]and passed through an 80-mI DEAE-cellulose column (Whatman, Inc.) alsoequilibrated in buffer B minus phenylmethylsulfonyl fluoride. The

flowthrough was loaded onto a 100-mIheparmn-Sepharosecolumn equilibratedin buffer A [50 mM HEPES (pH 7.4), 6 mM MgCl2, 0.2 m@i EDTA, 20%glycerol, 100m@iKCI,and 0.5 mMDli']. CBP/cycA activity was assayed hereand in subsequent purification steps by EMSA and was eluted in a 325—750mM KCI step in buffer A. After dialysis into buffer A, the fractions exhibiting

peak CBP/cycA activity were applied to an oligonucleotideaffinity column(2-mI bed volume) in the presence of a 200 p.g/ml sample volume of

poly(deoxyinosinic-deoxycytidylic acid)/poly(deoxyinosinic-deoxycytidylicacid). The oligonucleotide affinity column was prepared as described byKadonaga and Tjian (26) with cyanogen bromide-activated Sepharose (Pharmacia) and the annealed and ligated synthetic sequences 5'-GATC

CGAOCOC'ITFCATTGQTCCAmCG-3' and 5'-GATCCGAAATGGACCAATGAAAOCGCTCG-3', containing the CCAAT element of the cyclin Apromoter as oligonucleotides. Fractions eluting during a 100-1000 mM KC1gradient in buffer A were collected. Those fractions containing the peakCBP/cycA activity (600—900mM KCI) were adjusted to 50% glycerol andstored at —70°C.Proteins were resolved by SDS-PAGE (27) and visualized bysilver staining according to Heukeshoven and Dernick (28).

Immunoblotting. After partialpurificationof CBP/cycA from HeLa cellnuclear extracts, 4 p1 of the eluate from the heparmn column and 40 p1 of the

fractions eluted from the oligonucleotide affinity column were separated on

polyacrylamide gels and transferred to nitrocellulose membranes (Protean,

Schleicher & Schuell, Keene, NH) by semidry transfer for immunoblotting.Membranes were blocked by incubation with 5% dry milk in Tris-buffered

saline [10 mM Tris-HCI (pH 8.0) and 150 mtvi NaCI] and probed with I @g/ml

of the primary antibody in Tris-buffered saline for 1 h. After incubation witha horseradish peroxidase-conjugated antirabbit IgG secondary antibody (Promega, Madison WI), horseradish peroxidase activity was detected by enhancedchemiluminescence using the ECL kit (Amersham Corp., Arlington Heights,IL) following the manufacturer's instructions.

RESULTS

Antibodies against NF-YA and NF-YB Led to a Supershift ofCBP/cycA in EMSA. To test the hypothesis that the M@40'000 and30,000 subunits of CBP/cycA are identical with NF-YA and NF-YB,the two subunits of the CCAAT-binding transcription factor NF-Y,nuclear extracts from adherent, cycling NRK fibroblasts were incubated with antibodies against murine NF-YA (Mab YAla; Ref. 21)and@ (a-NF-YB rabbit serum; Ref. 21), respectively. After 30mm, EMSAs were performed using a 24-bp, CCAAT element-contaming cyclin A promoter fragment as a probe. Preincubation with

both of the antibodies led to a specific supershift of the CBP/cycAband (Fig. 1). The same result was obtained when an affinity-purifiedantibody to NF-YB (21) or an antibody to CBF-B (22), the rathomologue of NF-YA, was used (data not shown). In contrast, antibodiesto Rb, p107, cyclin A, cyclin E, and cdk2 did not induce asupershift of the CBP/cycA band (data not shown).

@‘@Aand NF-Y0 Are Enriched in NRK Cell Nuclear Extract

Fractions, Partially Purified for CBF/cycA. To furtherstrengthenthe conclusion that the two smaller subunits of CBP/cycA are match

CBP/cycA -.

Fig. 1. Supershift of CBP/cycA by antibodies to NF-YA (Mab YAla) and NF-YB(a-NF-Y8 rabbit serum) in an EMSA. Eight @&gof nuclear extract from adherent, cyclingNRK cells were mixed with I @gof antibody and incubated for 30 mm at 4°C.Subsequently, EMSAs were performed using a double-stranded, 24-bp, cyclin A promoterfragment (5'-CGAOCGC'ITI'CATTOGTCCA1TFC-3') as a probe.

ing NF-YA and NF-YB, CBP/cycA was partially purified from HeLa

cell nuclear extracts. After passage through a DEAE-cellulose columnand a heparin-Sepharose column, nuclear extract from HeLa S-3 cellsgrown in suspension culture was passed through an oligonucleotideaffinity column. As oligonucleotide, a 24-bp, CCAAT element-con

taming cyclin A promoter fragment was used. Fractions were elutedfrom the oligonucleotide affinity column with increasing concentrations of KC1. CBP/cycA activity, as assayed by EMSA, was elutedbetween 600 and 900 miti KCI (Fig. 2A). Fractions eluted between 600and 850 mM KC1 were pooled. After separation on a denaturingpolyacrylamide gel, NF-YA and NF-YB were detectable in the startingmaterial eluted from the heparin-Sepharose column as well as in thefractions eluted between 600 and 900 nmi KC1 from the oligonucleotide affinity column by immunoblotting (Fig. 2B). However, in afraction eluted at 300 misi KCI from the oligonucleotide affinitycolumn, which did not contain CBP/cycA activity in EMSA, NF-YAand@ could not be detected.

In Addition to CBP/cycA, NF-Y Itself Also Binds to the CCAATElement of the Cydlin A Promoter. Because it has been describedthat NF-Y binds in a cell cycle-independent manner to the CCAATelement of the cyclin A promoter in. GdG1- and G2-synchronizedWI-38 cells (20), nuclear extracts from synchronized NRK cells 0, 12,18, and 24 h after release from a G0 arrest were prepared. With thesenuclear extracts, EMSAs were performed using a 24-bp, CCAATelement-containing cyclin A promoter fragment as a probe (Fig. 3). In

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CBP/cycA CONSISTS OF NF-Y AND A Mr I 15.000 SUBUNIT

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Fig. 2. PartIal purification of CBP/cycA. A.CBP/cycAis presentin fractions,elutedbetween500 and 900 mM KC1 from a sequence-specific

DNA-affinity column. EMSAs were performed using a double-stranded, 24-bp, cyclin A promoterfragment (5'-CGAGCGCITFCAUGGTCCATfTC-3') as a probe.B, NF-YAand NF-YBcan bedetectedbyimmunoblottingin thestartingmaterialas well as in those fractions eluted from the oligo

nucleotide affinity column that contain CBP/cycAas demonstrated by EMSA. Mab YAla and a-

NF-Y@ rabbit serum were used as primary antibodies to NF-YA and NF-YB, res9@tiVely.

CBP/cycA@

A

addition to CBP/cycA, a second, faster migrating DNA-protein cornplex was detectable. However, in contrast to CBP/cycA, the formationof this faster migrating complex was cell cycle independent, becauseit could be detected with nuclear extracts from G0-arrested NRK cellsas well as with nuclearextractsharvested12, 18, and24 h afterreleasefrom G@arrest. Detection of both DNA-protein complexes was abolished by self-competition in the presence of unlabeled 24-bp of thewild-type sequence (Fig. 4A). Neither nonspecific DNA (Fig. 4A) normutated 24-bp CCAAT competitor sequences (Fig. 4B) were able toblock formation of the complexes, indicating that the CCAAT elementof the cyclin A promoter forms specific complexes with two differenttrans-acting factors from nuclear extracts of synchronized NRK cells.Finally, like CBP/cycA, the faster migrating DNA-protein complexwas supershifted specifically by antibodies against NF-YA and NF-YB(Fig. 5).

Taken together, these results suggest that (a) the two low molecularweight subunits of CBP/cycA are identical with the A and B subunitsof NF-Y; (b) both, CBP/cycA and NF-Y itself, bind to the CCAATelement of the cyclin A promoter; and (c) in contrast to CBP/cycA, thebinding of NF-Y itself to the CCAAT element of the cyclin Apromoter seems to be independent of cell cycle progression.

CCAAT-binding protein, which we have named CBP/cycA (1 1), thatbinds to the CCAAT element of the cyclin A promoter.

Progression through the G1 phase of the cell cycle is controlled by

Rb and the Rb-related proteins p107 and p130 (3 1). Although underphosphorylated during G0 and the first half of 0@ , Rb becomesphosphorylated in the second half of the G, phase, thereby releasinga number of cellular proteins, including the transcription factor E2F,from their bound state to an active state (32, 33). Phosphorylation of

CBP/cycA -0V.—

‘@

DISCUSSION

The absence of growth factors or adhesion leads to cell cycle arrestin G0 or late G@phase, respectively (7). Addition of growth factors toG0-arrested fibroblasts brings the cells to an adhesion-responsiveportion of the cell cycle (8)' Fibroblasts can then pass the cell cycleadhesioncheckpointin the late01 phaseif cyclinA expressionisinduced in response to an adhesion signal. Cyclin A rnRNA andprotein nrc at low levels or absent in G@phase and appear abundantlyat the 01-S transition(29),Thiscell cycle-regulatedexpressionofcyclin A is due to a tight control over its promoter activity (30),Recently, we have demonstrated that the activation of cyclin A transcription in the late 01 phase of the cell cycle is mediated by a

Fig. 3. Cell cycle-independent formation of a second DNA-protein complex betweenthe CCAATelementof the cyclinA promoterand nuclearextracitfromaynchronizedNRK celIa(arrows).EMSA wereperformedUalfl@a double-stranded,24-bp,cyclinApromoterfragment(5'-CGAOCOC'l11'CA'l°fOQTCCA1TFC-3')asa probeandnuclearextracts from serum-supplemented, cycling. nonaynchronized (us) NRK cells or fromtynchronized NRK celIa harvested 0, 12, 18, and 24 h after Go arrest by serum depletion.

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CBP/cycA CONSISTS OF NF-Y AND A M, I 15,000 SUBUNIT

Fig. 4. CCAAT-sequence specificity of a second DNA-protein

complex forming between the CCAAT element of the cycin Apromoter and nuclear extr@t from synchronized NRK cells. A,EMSAusingnuclearextractfromNRKcells,harvested12h afterGo ai@estby serum depletion. A double-stranded,24-bp. cycbn Apromoter fragment (5'-CGAGCGCI1TC@Q@TCCA1TI'C-3'(Wt)] was used as a probe and unlabeled self-competitor. Theunlabeled arrow indicates the specific complex that funned inaddition to CBP/cycA between Wt probe and the nuclearextr@:t. B, EMSA using Wt as a probe and nuclear extract fromNRK cells, harvested 12 h after Ge-arrest by serum depletion. Asingle-base mutant. double-stranded, 24-hp, cyclin A promoterfragment (5'-CGAGCGCTC@g@TCCATITC-3' (Mut)Jwas used as a competitor.Mut does not compete with the bindingof CBP/cycA or the second specific protein complex to Wt. Theunlabeled arrow indicates the specific complex that formed inaddition to CBP/cycA between Wt probe and the nuclear extract.

CBP/cycA —0-0

B

CBP/cycA@

A

Rb, and thereby control of cell cycle progression, is exerted by cyclinD-cdk4/6 (34) and cyclin E-cdk2 (35). Recenfly, we have shown thatin NRK cells CBP/cycA, like E2F, is inactivated during the G0 phaseby binding to Rb or a Rb family member (1 1). SV4O large T antigenand human papillomavirus E7 protein bind to hypophosphorylated Rband displace CBP/cycA from Rb binding. In addition, we were able todemonstrate that, whereas the expression of cyclin E is unaffected by

suspension, cyclin E-associated kinase is only active in adherent cells(36, 37). Hence, the adhesion signal results in the activation of thecyclin E-cdk2 complex. This results in the release of CBP/cycA frombinding to a Rb family member and enables the induction of cycin Aexpression and progression through the Ga-S adhesion checkpoint.

Consistent with our observations, Zerfass et a!. (38, 39) havepublished results indicating that in NIH 3T3 fibroblasts, the cyclin Agene promoter is activated by human papillomavirus E7 protein aswell as by adenovirus E1A protein. The cyclin A promoter activationby E7 is reported to be mediated by the promoter fragment —891—32.This finding is in agreement with our data, because CBP/cycA bindsto the CCAAT element located at —56to —52of the cyclin Apromoter. In contrast to our findings, however, Zerfass et a!. (38, 39)state that a variant E2F site at positions —37to —33mediates the cellcycle and adhesion regulation of the cycin A gene promoter (40, 41).

Zwicker and coworkers report a different model of transcriptionalregulation of the cyclin A gene (20). They propose a phase-specificrepression of transcriptional activation by the constitutive binding ofNF-Y to the CCAAT element at —56to —52.NF-Y, also called CP1or CBF, is a ubiquitous heteromeric metalloprotein composed of twosubunits, NF-YA and 1―@B' both contacting DNA directly. Eachsubunit alone cannot bind stably to the target sequence. The molecularweights of NF-YA and@ are Mr 40,000 and 32,000, respectively.

As described previously, CBP/cycA is a heteromeric protein complex. By UV cross-linking, we found that the transcription factorconsists of subunits with sizes of approximately Mr 40'000 and

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Fig. 5. Supershift of CBP/cycA and NF-Y by antibodies to NF-YA (Mab YAla) andNF-YB(a-NF-YB rabbit serum) in an EMSA. Eight @gof nuclear extract from synchronized NRK cells, harvested 12 h after G0 arrest by serum depletion, were mixed with 1 @gofantibodyandincubatedfor30mmat4°C.Subsequently,EMSASwereperformedusinga double-stranded, 24-bp, cyclin A promoter fragment (5'-CGAGCGClTFCATTGGTCCA'ITI'C-3') as a probe.

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CBP/cycA CONSISTS OF NF.Y AND A M, 115.000 SUBUNIT

15. Maity, S. N., Golumbek, P. T.. Karsenty, G., and de Crombrugghe, B. Selectiveactivation of transcription by a novel CCAAT binding factor. Science (WashingtonDC),241:582—585,1988.

16. Graves, B. J., Johnson, P. F., and McKnight, S. L. Homologous recognition of apromoter domain common to the MSV LTR and the HSV tk gene. Cell, 44: 565—576,1986.

17. Johnson, P. F., Landschulz, W. H., Graves, B. J., and McKnight, S. L. Identificationof a rat liver nuclear protein that binds to the enhancer core element of three animalviruses. Genes Dcv., I: 133—146,1987.

18. Good, L., Chen, J., and Chen, K. Y. J. Analysis of sequence-specific binding activityof cis elements in the human thymidine kinase gene promoter during G -S phasetransition. J. Cell. Physiol., 163: 636—644, 1995.

19. Chang,Z. F., and Liu, C. J. HumanthymidinekinaseCCAAT-bindingproteinisNF-Y, whose A subunit expression is serum-dependent in human IMR-90 diploidfibroblasts. J. Biol. Chem., 269: 17893—17898, 1994.

20. Zwicker, J., Lucibello, F. C.. Wolfraim, L. A., Gross, C.. Truss, M., Engeland, K., andMUller,R. Cell cycle regulation of the cyclin A, cdc25C and cdc2 genes is based ona common mechanism of transcriptional repression. EMBO J., 14: 4514—4522,1995.

21. Mantovani, R., Pessara, U., Tronche, F., Li, X. Y., Knapp, A. M., Pasquali, J. L.,Benoist, C., and Mathis, D. Monoclonal antibodies to NF-Y define its function inMHCclassII andalbumingenetranscription.EMBOJ.. II: 3315—3322,1992.

22. Maity, S. N., and de Crombrugghe, B. Biochemical analysis of the heteromericCCAAT-binding factor. J. Biol. Chem., 267: 8286—8292, 1992.

23. Dignam. J. D., Lebovitz. R. M., and Roeder, R. G. Accurate transcription initiation byRNA polymerase II in a soluble extract from isolated mammalian nuclei. NucleicAcidsRca.,11:1475—1489,1983.

24. Andrews, N. C., and Faller, D. V. A rapid micropreparation technique for extractionof DNA-binding proteins from limited numbers of mammalian cells. Nucleic AcidsRes., 19: 2499, 1991.

25. Costa, R. H., Lai, E., Grayson, D. R., and Darnell, J. E., Jr. The cell-specific enhancerof the mouse transthyretin (prealbumin) gene binds a common factor at one site anda liver-specific factor(s) at two other sites. Mol. Cell. Biol., 8: 81—90,1988.

26. Kadonaga, J. T., and Tjian, R. Affinity purification of sequence-specific DNA bindingproteins. Proc. NatI. Acad. Sci. USA, 83: 5889—5893, 1986.

27. Laemmli, U. K. Cleavage of structural proteins during assembly of the head ofbacteriophage T4. Nature (Lond.), 227: 680—685, 1970.

28. Heukeshoven, J., and Dernick, R. Simplified method for silver staining of proteins inpolyacrylamide gels and the mechanism of silver staining. Electrophoresis, 6: 103—112,1985.

29. Lucibello, F. C., Sewing, A., Brusselbach, S., Burger, C., and MUller,R. Deregulationof cyclins Dl and E and suppression of cdk2 and cdk4 in senescent human fibroblasts.J. CellSci.,105: 123—133,1993.

30. Henglein, B., Chenivesse, X@,Wang, J., Eick, D., and Bréchot,C. Structure and cellcycle-regulated transcription of the human cyclin A gene. Proc. NatI. Acad. Sci. USA,91: 5490—5494, 1994.

31. Weinberg, R. A. The retinoblastoma protein and cell cycle control. Cell, 81: 323—330,1995.

32. Chen, P. L., Scully, P., Shew, J. Y., Wang, J. Y. J., and Lee, W. H. Phosphorylationof the retinoblastoma gene product is modulated during the cell cycle and cellulardifferentiation. Cell, 58: I 193—1198, 1989.

33. DeCaprio, J. A., Ludlow, J. W., Lynch, D., Furukawa, Y., Griffin, J., Piwnica-Worms,H., Huang, C. M., and Livingston, D. M. The product of the retinoblastoma susceptibility gene has properties of a cell cycle regulatory element. Cell, 58: 1085—1095,1989.

34. Kato, J., Matsushime, H., Hiebert, S. W., Ewen, M. E., and Sherr, C. J. Direct bindingof cyclin D to the retinoblastoma gene product (pRb) and pRb phosphorylation by thecyclin D-dependent kinase, CDK4. Genes 0ev., 7: 331—342,1993.

35. Hinds, P. W., Mittnacht, S., Dulic, V., Arnold, A., Reed, S. I., and Weinberg, R. A.Regulation of retinoblastoma protein functions by ectopic expression of humancyclins. Cell, 70: 993—1006, 1992.

36. Carstens, C. P., Kramer, A., and Fahl, W. E. Adhesion-dependent control of cyclinEicdk2 activity and cell cycle progression in normal but not in Ha-ras transformedNRK cells. Exp. Cell Rca.. 229: 86—92,1996.

37. Carstens, C. P., Kramer, A., and Fahl, W. E. Regulation of cyclins by adhesion:elements in the anchorage-signalling pathway. Radiat. Oncol. Invest., 3: 307—314,1996.

38. Zerfass, K., Schulze, A., Spitkovsky, D., Friedman. V., Henglein, B., and JansenDUrr,P. Sequential activation of cydin E and cyclin A gene expression by humanpapillomavirus type 16 El through sequences necessary for transformation. J. Virol..69: 6389—6399, 1995.

39. Zerfass, K.. Spitkovsky, D., Schulze, A., Joswig, S., Henglein, B., and Jansen-Dtirr,P. AdenovirusE1AactivatescyclinA genetranscriptionin the absenceof growthfactors through interaction with p107. J. Virol., 70: 2637—2642, 1996.

40. Schulze, A., Zerfass, K.. Spitkovsky, D., Middendorp, S., Berges, J., Helm, K.,Jansen-DUrr,P., and Henglein, B. Cell cycle regulation of the cyclin A gene promoteris mediated by a variant E2F site. Proc. Natl. Acad. Sci. USA, 92: I 1264—11268,1995.

41. Schulze, A., Zerfass-Thome, K., Berges. J., Middendorp, S., Jansen-Durr, P., andHenglein, B. Anchorage-dependent transcription of the cyclin A gene. Mol. Cell.Biol., 16: 4632—4638, 1996.

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115,000. Additional experiments demonstrated the existence of athird, approximately Mr 30,000 subunit.

In this work, we have demonstrated that the Mr 40'000 and 30,000subunits of CBP/cycA are identical with NF-YA and NF-YB, the twosubunits of the CCAAT-binding transcription factor NF-Y. In addition, the results described here confirm our previous finding that thebinding of CBP/cycA to the CCAAT element of the cyclin A promoter is cell cycle dependent. Moreover, we show that not onlyCBP/cycA but also NF-Y itself binds to this CCAAT element. Incontrast to CBP/cycA, however, the binding of NF-Y itself is muchweaker and not cell cycle regulated.

When a mutation is introduced into the CCAAT element of thecyclin A promoter, the adhesion-dependent promoter inducibility atthe G1-S boundary is abolished completely in transfection experiments with cyclin A promoterlluciferase reporter constructs (1 1).Because the adhesion-dependent binding of CBP/cycA to the CCAATelement of the cyclin A promoter in late G1 coincides with thetranscriptional activation of the cyclin A gene, our findings supportthe hypothesis that CBP/cycA and not NF-Y itself is responsible forthe adhesion-dependent activation of cyclin A expression.

ACKNOWLEDGMENTS

We thank Dr. Janet Mertz for HeLa cell nuclear extract and valuabletechnical advice. We are grateful to Drs. Diane Mathis and Benoit de

Crombrugghe for providing us with antibodies to NF-YA (Mab YAla anda-CBF-B) and NF-YB, respectively.

REFERENCES

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1997;57:5117-5121. Cancer Res   Alwin Krämer, Carsten-P. Carstens, Wyeth W. Wasserman, et al.  

115,000 SubunitrMand a Novel Adhesion-dependent Cyclin A Transcription, Consists of NF-Y CBP/cycA, a CCAAT-binding Protein Necessary for

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