The SET protein and the cell cycle inhibitor p21 Cip1 interact in vivo and in vitro. We identified here the domain 157 LIF 159 of p21 Cip1 as essential for the binding of SET. We also found that SET contains at least two domains of interaction with p21Cip1 , one located in the fragment amino acids 81-180 and the other one in the fragment including amino acids 181-277. SET and p21Cip1 co-localize in the cell nucleus in a temporal manner. Overexpression of SET blocks the cell cycle at the G 2 /M transition in COS and HCT116 cells. Moreover, SET inhibits cyclin B-CDK1 activity both in vivo and in vitro in both cell types. This effect is specific for these complexes since SET did not inhibit either cyclin A-CDK2 or cyclin E-CDK2 complexes. SET and p21Cip1 cooperate in the inhibition of cyclin B-CDK1 activity. The inhibitory effect of SET resides in its acidic C terminus, as demonstrated by the ability of this domain to inhibit cyclin B-CDK1 activity and by the lack of blocking G 2 /M transition when a mutated form of SET lacking this C terminus domain was overexpressed in COS cells. These results indicate that SET might regulate G 2 /M transition by modulating cyclin B-CDK1 activity.

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Transcriptional repressor complexes containing p130 and E2F4 regulate the expression of genes involved in DNA replication. During the G1 phase of the cell cycle, sequential phosphorylation of p130 by cyclin-dependent kinases (Cdks) disrupts these complexes allowing gene expression. The Cdk inhibitor and tumor suppressor p27Kip1 associates with p130 and E2F4 by its carboxyl domain on the promoters of target genes but its role in the regulation of transcription remains unclear. We report here that p27Kip1 recruits cyclin D2/D3–Cdk4 complexes on the promoters by its amino terminal domain in early and mid G1. In cells lacking p27Kip1, cyclin D2/D3–Cdk4 did not associate to the promoters and phosphorylation of p130 and transcription of target genes was increased. In late G1, these complexes were substituted by p21Cip1-cyclin D1–Cdk2. In p21Cip1 null cells cyclin D1–Cdk2 were not found on the promoters and transcription was elevated. In p21/p27 double null cells transcription was higher than in control cells and single knock out cells. Thus, our results clarify the role of p27Kip1 and p21Cip1 in transcriptional regulation of genes repressed by p130/E2F4 complexes in which p27Kip1 and p21Cip1 play a sequential role by recruiting and regulating the activity of specific cyclin–Cdk complexes on the promoters.