CDF-1, A NOVEL E2F-UNRELATED FACTOR, INTERACTS WITH CELL-CYCLE-REGULATED REPRESSOR ELEMENTS IN MULTIPLE PROMOTERS

The cdc25C, cdc2 and cyclin A promoters are controlled by transcriptio nal repression through two contiguous protein binding sites, termed th e CDE and CHR, In the present study we have identified a factor, CDF-1 , which interacts with the cdc25C CDE-CHR module, CDF-1 binds to the C DE in the major groove and to the CHR in the minor grove in a cooperat ive fashion in vitro, in a manner similar to that seen by genomic foot printing, In agreement with in vivo binding data and its putative func tion as a periodic repressor, DNA binding by CDF-1 in nuclear extracts is down-regulated during cell cycle progression. CDF-1 also binds avi dly to the CDE-CHR modules of the cdc2 and cyclin A promoters, but not to the E2F site in the B-myb promoter, Conversely, E2F complexes do n ot recognize the cdc25C CDE-CHR and CDF-I is immunologically unrelated to all known E2F and DP family members. This indicates that E2F- and CDF-mediated repression is controlled by different factors acting at d ifferent stages during the cell cycle, While E2F-mediated repression s eems to be associated with genes that are up-regulated early (around m id G(1)), such as B-myb, CDE-CHR-controlled genes, such as cdc25C, cdc 2 and cyclin A, become derepressed later, Finally, the fractionation o f native nuclear extracts on glycerol gradients leads to separation of CDF-1 from both E2F complexes and pocket proteins of the pRb family. This emphasizes the conclusion that CDF-1 is not an E2F family member and points to profound differences in the cell cycle regulation of CDF -1 and E2F.