The DNA binding activity of C/EBP transcription factor is regulated in the G1 phase of the hepatocyte cell cycle

Abstract

We have isolated the promoter of the rat C/EBP alpha 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/EBP beta 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/EBP alpha-C/EBP beta heterodimers (alpha beta) as well as C/EBP beta complexed with itself and/or other unidentified nuclear factors (beta 1, beta 2, and beta 3). 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 alpha beta 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/EBP alpha polypeptide species (42, 40, and 30 kDa), whose abundance in general parallels the decrease and recovery in DNA binding activity. In contrast to C/EBP alpha behavior, the DNA binding activity of the beta complexes is transiently induced severalfold during the early G1 period between 2 and 6 h posthepatectomy. The major C/EBP beta 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/EBP beta polypeptides themselves. In primary hepatocyte cultures, under growth supporting conditions, in the absence of growth factors proliferation is negligible; C/EBP alpha is abundantly expressed at the outset, but is then extensively down-regulated. Epidermal growth factor causes further decay of C/EBP alpha polypeptides and DNA binding activity, and down-regulates C/EBP beta DNA binding activity as well. Addition of transforming growth factor beta completely antagonizes the effects of epidermal growth factor on C/EBP beta activity, and partially overcomes the effect on C/EBP alpha. These results demonstrate that the DNA binding activity of C/EBP alpha and C/EBP beta complexes is regulated in the regenerating liver, and in hepatocyte cultures responding to growth factors that regulate their proliferation.