C/EBP alpha regulates formation of S-phase-specific E2F-p107 complexes in livers of newborn mice

We previously showed that the rate of hepatocyte proliferation in livers fr om newborn C/EBP alpha knockout mice was increased. An examination of cell cycle-related proteins showed that the cyclin-dependent kinase (CDK) inhibi tor p21 level was reduced in the knockout animals compared to that in wild- type littermates. Here we show additional cell cycle-associated proteins th at are affected by C/EBP alpha, We have observed that C/EBP alpha controls the composition of E2F complexes through interaction with the retinoblastom a (Rb)-like protein, p107, during prenatal liver development. S-phase-speci fic E2F complexes containing E2F, DP, cdk2, cyclin A, and p107 are observed in the developing liver. In wild-type animals these complexes disappear by day 18 of gestation and are no longer present in the newborn animals. In t he C/EBP alpha mutant, the S-phase-specific complexes do not diminish and p ersist to birth. The elevation of levels of the S-phase-specific E2F-p107 c omplexes in C/EBP alpha knockout mice correlates with the increased express ion of several E2F-dependent genes such as those that encode cyclin A, prol iferating cell nuclear antigen, and p107, The C/EBP alpha-mediated regulati on of E2F binding is specific, since the deletion of another C/EBP family m ember, C/EBP beta, does not change the pattern of E2F binding during prenat al liver development. The addition of bacterially expressed, purified His-C /EBP alpha to the E2F binding reaction resulted in the disruption of E2F co mplexes containing p107 in nuclear extracts from C/EBP alpha knockout mouse livers. Ectopic expression of C/EBP alpha in cultured cells also leads to a reduction of E2F complexes containing Rb family proteins, Coimmunoprecipi tation analyses revealed an interaction of C/EBP alpha with p107 but none w ith cdk2, E2F1, or cyclin A. A region of C/EBP alpha that has sequence simi larity to E2F is sufficient for the disruption of the E2Fp107 complexes. De spite its role as a DNA binding protein, C/EBP alpha brings about a change in E2F complex composition through a protein-protein interaction. The disru ption of E2F-p107 complexes correlates with C/EBP alpha-mediated growth arr est of hepatocytes in newborn animals.