Cell cycle regulation of the E2F transcription factor involves an interaction with cyclin A

Maria Mudryj,” Stephen H. Devoto,’ Scott W. Hiebert,’ Tony Hunter,t Jonathon Pines,t and Joseph R. Nevins’ *Howard Hughes Medical Institute Section of Genetics Department of Microbiology and Immunology Duke University Medical Center Durham, North Carolina 27710 tThe Salk Institute La Jolla, California 92037 Summary We have examined E2F binding activity in extracts of synchronized NIH 3T3 cells. During the GO to Gl transi- tion, there is a marked increase in the level of active E2F. Subsequently, there are changes in the nature of EPF-containing complexes. A Gl-specific complex increases in abundance, disappears, and is then re- placed by another complex as S phase begins. Analy- sis of extracts of thymidine-blocked cells confirms that the complexes are cell cycle regulated. We also show that the cyclin A protein is a component of the S phase complex. Each complex can be dissociated by the ade- novirus ElA 12s product, releasing free E2F. The re- lease of E2F from the cyclin A complex coincides with the stimulation of an E2F-dependent promoter. We suggest that these interactions control the activity of E2F and that disruption of the complexes by El A con- tributes to a loss of cellular proliferation control. Introduction Signals that trigger cellular proliferation do so in part through the activation of new gene expression, the prod- ucts of which contribute to the subsequent changes in cell phenotype. Moreover, once cells re-enter the proliferative state, the expression of certain genes is tightly regulated as a cell progresses through the cell cycle. Thus, a defini- tion of the molecular events controlling these processes is critical for an understanding of the normal signaling events as well as the perturbations that occur when proliferation control is altered, such as during oncogenic transforma- tion. Particularly important is the identification of transcrip- tion factors that participate in cell cycle-dependent and proliferation-dependent gene control. Considerable effort has been focused on the very early events in the prolifera- tive response and has led to the identification of the serum response factor as a key participant in the activation pro- cess (Treisman, 1988, 1987; Gilman et al., 1988; Prywes and Roeder, 1988; Greenberg et al., 1987; Shaw et al., 1989). Less is known of the regulation of cell cycle-specific transcription. The best-studied case is the S phase regula- tion of histone gene transcription, which appears to involve at least two different transcription factors (Fletcher et al., 1987; La Bella et al., 1988, 1989). Other genes such as those encoding dihydrofolate reductase (Farnham and Schimke, 1985), thymidylate synthase (Nagarajan and Johnson, 1989) and the cyclins (Pines and Hunter, 1990) are also cell cycle regulated, but the underlying mecha- nisms controlling their expression are not clear. The cellular transcription factor E2F was originally iden- tified as a target for adenovirus El A-dependent transacti- vation of E2 transcription. The E2F factor is a critical com- ponent of the transcription apparatus of the viral E2 promoter and appears to be targeted in multiple ways to effect a stimulation of transcription. There is an ElA- dependent stimulation of the binding activity of the factor (Kovesdi et al., 1988; Bagchi et al., 1989; Raychaudhuri et al., 1990) as well as an alteration of the factor that induces cooperative binding to the E2 promoter (Hardy et al., 1989; Raychaudhuri et al., 1990). This latter effect is dependent on 19 kd product of the early viral E4 gene and involves a physical association of the E4 protein with E2F (Huang and Hearing, 1989; Neil1 et al., 1990; Marton 1990). In many cell types the E2F factor is not available for interaction with the E4 protein since it is already com- plexed to cellular factors (Bagchi et al., 1990). However, the ElA protein can dissociate these complexes, releasing a free E2F can then interact with the E4 protein (Bag- chi et al., 1990). This activity of ElA involves sequences within the protein that are also required for ElA to function as an oncogene (Bagchi et al., 1990; P. Raychaudhuri et al., submitted). Given the potential roleof E2Fdissociation in El A-mediated oncogenesis, we have now examined the cellular control E2F in greater detail. These experiments suggest that the formation of specific EPF-containing com- plexes, involving interactions with other cellular proteins, is regulated during the cell cycle. The formation of one these complexes during S phase correlates very well with the cell cycle-regulated synthesis of a protein known to associate with the ElA protein-cyclin A (Pines and Hunter, 1989). We demonstrate here that the S phase E2F,, complex contains the cyclin A protein. Thus, dissociation of E2F complexes by the ElA protein leads to a disruption of interactions that are under cell cycle control. Results Sequential Changes in E2F Binding Activity Following the Stimulation of Cell Proliferation An analysis of E2F binding activity extracts quiescent NIH 3T3 cells and that have been stimulated by serum addition is depicted Figure 1A. As we have observed previously (Mudryj et 1990), there is a sub- stantial increase in the level of active E2F (that is, that can bind to DNA) following serum addition. In this experiment, there is an approximately 5-fold increase in E2F levels, as measured by densitometry, in cells stimu- lated for 4 hrversus the quiescent cells. Since our previous experiments demonstrated that this increase still occurred