THE DNA-BINDING ACTIVITY OF C EBP TRANSCRIPTION FACTORS IS REGULATED IN THE G(1) PHASE OF THE HEPATOCYTE CELL-CYCLE/

We have isolated the promoter of the rat C/EBP alpha gene and find a h igh degree of homology with the mouse gene, particularly in putative r egulatory domains. Transactivation of this promoter by ectopic express ion of rat C/EBP beta occurs through a C/EBP regulatory domain at posi tion -170 to -195. An oligonucleotide corresponding to this domain bin ds 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 i tself and/or other unidentified nuclear factors (beta 1, beta 2, and b eta 3). The DNA binding activity of these complexes changes both quali tatively 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 alp ha polypeptide species (42, 40, and 30 kDa), whose abundance in genera l parallels the decrease and recovery in DNA binding activity. In cont rast to C/EBP alpha behavior, the DNA binding activity of the beta com plexes is transiently induced severalfold during the early G(1) period between 2 and 6 h posthepatectomy. The major C/EBP beta polypeptide i s the 32-kDa LAP protein, whereas the LIP protein (21 kDa) is weakly e xpressed. Both remain essentially constant throughout the course of re generation, suggesting that changes in DNA binding activity may reflec t changes in the complexed proteins rather than the C/EBP beta polypep tides themselves. In primary hepatocyte cultures, under growth support ing conditions, in the absence of growth factors proliferation is negl igible; C/EBP alpha is abundantly expressed at the outset, but is then extensively down-regulated. Epidermal growth factor causes further de cay of C/EBP alpha polypeptides and DNA binding activity, and down-reg ulates C/EBP beta DNA binding activity as well. Addition of transformi ng growth factor beta completely antagonizes the effects of epidermal growth factor on C/EBP beta activity, and partially overcomes the effe ct on C/EBP alpha. These results demonstrate that the DNA binding acti vity of C/EBP alpha and C/EBP beta complexes is regulated in the regen erating liver, and in hepatocyte cultures responding to growth factors that regulate their proliferation.