The DNA Binding Activity of C/EBP Transcription Factors Is Regulated in the G Phase of the Hepatocyte Cell Cycle

We have isolated the promoter of the rat C/EBPα gene and find a high degree of homology with the mouse gene, particularly in putative regulatory domains. Transactivation of this promoter by ectopic expression of rat C/EBPβ occurs through a C/EBP regulatory domain at position −170 to −195. An oligonucleotide corresponding to this domain binds to complexes expressed in rat liver that comprise C/EBPα-C/EBPβ heterodimers (αβ) as well as C/EBPβ complexed with itself and/or other unidentified nuclear factors (β1, β2, and β3). The DNA binding activity of these complexes changes both qualitatively and quantitatively following partial hepatectomy. Within 2-5 h postsurgery, the binding activity of the αβ complexes drops severalfold, reaching a nadir by 20 h. During the ensuing 3-8 days, as regeneration nears completion, this activity slowly returns to normal quiescent liver levels. Western blot analysis shows 3 major C/EBPα polypeptide species (42, 40, and 30 kDa), whose abundance in general parallels the decrease and recovery in DNA binding activity. In contrast to C/EBPα behavior, the DNA binding activity of the β complexes is transiently induced severalfold during the early G 1 period between 2 and 6 h posthepatectomy. The major C/EBPβ polypeptide is the 32-kDa LAP protein, whereas the LIP protein (21 kDa) is weakly expressed. Both remain essentially constant throughout the course of regeneration, suggesting that changes in DNA binding activity may reflect changes in the complexed proteins rather than the C/EBPβ polypeptides themselves. In primary hepatocyte cultures, under growth supporting conditions, in the absence of growth factors proliferation is negligible; C/EBPα is abundantly expressed at the outset, but is then extensively down-regulated. Epidermal growth factor causes further decay of C/EBPα polypeptides and DNA binding activity, and down-regulates C/EBPβ DNA binding activity as well. Addition of transforming growth factor β completely antagonizes the effects of epidermal growth factor on C/EBPβ activity, and partially overcomes the effect on C/EBPα. These results demonstrate that the DNA binding activity of C/EBPα and C/EBPβ complexes is regulated in the regenerating liver, and in hepatocyte cultures responding to growth factors that regulate their proliferation.