C/EBPα Regulates Formation of S-Phase-Specific E2F-p107 Complexes in Livers of Newborn Mice

Abstract

We previously showed that the rate of hepatocyte proliferation in livers from newborn C/EBPα knockout mice was increased. An examination of cell cycle-related proteins showed that the cyclin-dependent kinase (CDK) inhibitor p21 level was reduced in the knockout animals compared to that in wild-type littermates. Here we show additional cell cycle-associated proteins that are affected by C/EBPα. We have observed that C/EBPα controls the composition of E2F complexes through interaction with the retinoblastoma (Rb)-like protein, p107, during prenatal liver development. S-phase-specific 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 the C/EBPα mutant, the S-phase-specific complexes do not diminish and persist to birth. The elevation of levels of the S-phase-specific E2F-p107 complexes in C/EBPα knockout mice correlates with the increased expression of several E2F-dependent genes such as those that encode cyclin A, proliferating cell nuclear antigen, and p107. The C/EBPα-mediated regulation of E2F binding is specific, since the deletion of another C/EBP family member, C/EBPβ, does not change the pattern of E2F binding during prenatal liver development. The addition of bacterially expressed, purified His-C/EBPα to the E2F binding reaction resulted in the disruption of E2F complexes containing p107 in nuclear extracts from C/EBPα knockout mouse livers. Ectopic expression of C/EBPα in cultured cells also leads to a reduction of E2F complexes containing Rb family proteins. Coimmunoprecipitation analyses revealed an interaction of C/EBPα with p107 but none with cdk2, E2F1, or cyclin A. A region of C/EBPα that has sequence similarity to E2F is sufficient for the disruption of the E2F-p107 complexes. Despite its role as a DNA binding protein, C/EBPα brings about a change in E2F complex composition through a protein-protein interaction. The disruption of E2F-p107 complexes correlates with C/EBPα-mediated growth arrest of hepatocytes in newborn animals.

ACKNOWLEDGMENTS

We thank T. Bilyeu and P. Iakova for excellent technical assistance and K. Faraj for the preparation of the manuscript.

This work was supported by NIH grants R01 GM55188-01 (N.A.T), K01 AG00766-01 (N.A.T), DK49285 (G.J.D), AG13663 (G.J.D), and AFAR grant A97161 (N.A.T).