Cyclin D1/cdk4 can interact with E2F4/DP1 and disrupts its DNA-binding capacity.

The E2F family of transcription factors regulate the expression of many growth-related genes in a cell cycle-dependent manner. These transcription factors can activate or, in conjunction with an Rb-related protein, repress transcription. E2F transcriptional activity is regulated at several different levels that are each linked to cell cycle progression. In many cell types, E2F4 and E2F5 are the predominant E2F species during G(0) and early G(1) and function primarily as repressors of E2F-regulated genes. In this study, co-immunoprecipitation techniques were used to demonstrate that cyclins D1, D2, and D3 are capable of interacting with E2F4, E2F5, and DP1. Overexpression of cyclin D1/cdk4 reduced E2F4-mediated transcription in a simple reporter gene assay and electrophoretic mobility shift analyses using nuclear extracts from transfected cells indicated that cyclin D1/cdk4 disrupts the DNA-binding ability of E2F4. Cell cycle analysis following stimulation of serum-starved 3T3 cells indicated that E2F4 undergoes changes in its phosphorylation pattern coincident with the synthesis of cyclin D1. Examination of a series of E2F4 deletion mutants indicated that a cyclin D1-binding site located close to the carboxyl terminus of E2F4 was critical for the disruption of DNA binding by cyclin D1/cdk4. These data support a model in which E2F4 DNA binding is abolished during mid-G(1) at the same time when E2F interactions with pRb-related proteins are disrupted by cyclin D1/cdk4.