Serum response element binding protein (SRE BP) is a novel binding fac
tor present in nuclear extracts of avian and NIH 3T3 fibroblasts which
specifically binds to the cfos SRE within a region overlapping and im
mediately 3' to the CArG box. Site-directed mutagenesis combined with
transfection experiments in NIH 3T3 cells showed that binding of both
serum response factor (SRF) and SRE BP is necessary for maximal serum
induction of the SRE. In this study, we have combined size fractionati
on of the SRE BP DNA binding activity with C/EBP beta antibodies to de
monstrate that homodimers and heterodimers of p35C/EBP beta (a transac
tivator) and p20C/EBP beta (a repressor) contribute to the SRE BP comp
lex in NIH 3T3 cells. Transactivation of the SRE by p35C/EBP beta is d
ependent on SRF binding but not ternary complex factor (TCF) formation
. Both p35C/EBP beta and p20C/EBP beta bind to SRF in vitro via a carb
oxy-terminal domain that probably does not include the leucine zipper.
Moreover, SRE mutants which retain responsiveness to the TCF-independ
ent signaling pathway bind SRE BP in vitro with affinities that are ne
arly identical to that of the wild-type SRE, whereas mutant SRE.M, whi
ch is not responsive to the TCF-independent pathway, has a nearly 10-f
old lower affinity for SRE BP. We propose that C/EBP beta may play a r
ole in conjunction with SRF in the TCF-independent signaling pathway f
or SRE activation.