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/EBP␣ 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/EBP␣. We have observed that C/EBP␣ 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/EBP␣ 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/EBP␣ 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/EBP␣-mediated regulation of E2F binding is specific, since the deletion of another C/EBP family member, C/EBP␤, does not change the pattern of E2F binding during prenatal liver development. The addition of bacterially expressed, purified His-C/EBP␣ to the E2F binding reaction resulted in the disruption of E2F complexes containing p107 in nuclear extracts from C/EBP␣ knockout mouse livers. Ectopic expression of C/EBP␣ in cultured cells also leads to a reduction of E2F complexes containing Rb family proteins. Coimmunoprecipitation analyses revealed an interaction of C/EBP␣ with p107 but none with cdk2, E2F1, or cyclin A. A region of C/EBP␣ 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/EBP␣ brings about a change in E2F complex composition through a protein-protein interaction. The disruption of E2F-p107 complexes correlates with C/EBP␣-mediated growth arrest of hepatocytes in newborn animals.

CCAAT/enhancer-binding proteins are required for granulopoiesis independent of their induction of the granulocyte colony-stimulating factor receptor

“…C/EBP␣ may inhibit the G 1 /S transition via interaction with E2F or with cdk2. [57][58][59][60] Interaction with cdk2 occurs via the C/EBP␣ basic region, and this segment is retained in K␣ER. 60 Expression of p21 WAF1/CIP1 or p27 Kip1 in U937 cells induces monocytic markers.…”

C/EBPαp30 plays transcriptional regulatory roles distinct from C/EBPαp42

“…The C/EBPαp30, compared with the full-length C/ EBPαp42, lacks the N-terminal 117 amino acids, which is required by adipocyte differentiation, granulopoiesis [12,22] and C/EBPα-Rb, C/EBPα-p21 interactions [8][9][10]. In liver cells, the expression ratio of C/EBPαp42 vs C/EBPαp30 is related to hepatocyte development [23].…”

“…It initiates growth arrest by stabilizing p21, and by disrupting E2F transcriptional complexes during the G1 phase of the cell cycle [7][8][9][10][11][12]. It was suggested that C/EBPα might be a tumor suppressor gene.…”

“…The N-terminal part of C/EBPαp42, which is absent in C/EBPαp30, is required to bind retinoblastoma (Rb) and p21cdki [8][9][10]. Consequently, C/EBPαp30 lacks an antimitotic activity [23].…”

Sumoylation and the function of CCAAT enhancer binding protein alpha (C/EBPα)

“…This growth arrest is thought occur by three main mechanisms. First, C/EBPα can induce the expression of p21 waf1/cip1 , a CDK (cyclin-dependent kinase) inhibitor, and disrupt E2F transcriptional complexes during the G1 phase of the cell cycle [13][14][15][16]. Second, C/EBPα can mediate growth control through interaction with the cyclin-dependent kinases CDK2 and CDK4 [17].…”