To explore mechanisms for specificity of function within the family of E2F 
transcription factors, we have identified proteins that interact with individual 
E2F proteins. A two-hybrid screen identified RYBP (Ring1- and YY1-binding 
protein) as a protein that interacts specifically with the E2F2 and E2F3 family 
members, dependent on the marked box domain in these proteins. The Cdc6 promoter 
contains adjacent E2F- and YY1-binding sites, and both are required for promoter 
activity. In addition, YY1 and RYBP, in combination with either E2F2 or E2F3, 
can stimulate Cdc6 promoter activity synergistically, dependent on the marked 
box domain of E2F3. Using chromatin immunoprecipitation assays, we show that 
both E2F2 and E2F3, as well as YY1 and RYBP, associate with the Cdc6 promoter at 
G(1)/S of the cell cycle. In contrast, we detect no interaction of E2F1 with the 
Cdc6 promoter. We suggest that the ability of RYBP to mediate an interaction 
between E2F2 or E2F3 and YY1 is an important component of Cdc6 activation and 
provides a basis for specificity of E2F function.