Maximal serum stimulation of the c-fos serum response element requires both the serum response factor and a novel binding factor, SRE-binding protein.

We have previously reported on the presence of a CArG motif at -100 in the Rous sarcoma virus long terminal repeat which binds an avian nuclear protein termed enhancer factor III (EFIII) (A. Boulden and L. Sealy, Virology 174:204-216, 1990). By all analyses, EFIII protein appears to be the avian homolog of the serum response factor (SRF). In this study, we identify a second CArG motif (EFIIIB) in the Rous sarcoma virus long terminal repeat enhancer at -162 and show only slightly lower binding affinity of the EFIII/SRF protein for this element in comparison with c-fos serum response element (SRE) and EFIII DNAs. Although all three elements bind the SRF with similar affinities, serum induction mediated by the c-fos SRE greatly exceeds that effected by the EFIII or EFIIIB sequence. We postulated that this difference in serum inducibility might result from binding of factors other than the SRF which occurs on the c-fos SRE but not on EFIII and EFIIIB sequences. Upon closer inspection of nuclear proteins which bind the c-fos SRE in chicken embryo fibroblast and NIH 3T3 nuclear extracts, we discovered another binding factor, SRE-binding protein (SRE BP), which fails to recognize EFIII DNA with high affinity. Competition analyses, methylation interference, and site-directed mutagenesis have determined that the SRE BP binding element overlaps and lies immediately 3' to the CArG box of the c-fos SRE. Mutation of the c-fos SRE so that it no longer binds SRE BP reduces serum inducibility to 33% of the wild-type level. Conversely, mutation of the EFIII sequence so that it binds SRE BP with high affinity results in a 400% increase in serum induction, with maximal stimulation equaling that of the c-fos SRE. We conclude that binding of both SRE BP and SRF is required for maximal serum induction. The SRE BP binding site coincides with the recently reported binding site for rNF-IL6 on the c-fos SRE. Nonetheless, we show that SRE BP is distinct from rNF-IL6, and identification of this novel factor is being pursued.     

Regulation of the cfos serum response element by C/EBPbeta.

Serum response element binding protein (SRE BP) is a novel binding factor present in nuclear extracts of avian and NIH 3T3 fibroblasts which specifically binds to the cfos SRE within a region overlapping and immediately 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 fractionation of the SRE BP DNA binding activity with C/EBPbeta antibodies to demonstrate that homodimers and heterodimers of p35C/EBPbeta (a transactivator) and p20C/EBPbeta (a repressor) contribute to the SRE BP complex in NIH 3T3 cells. Transactivation of the SRE by p35C/EBPbeta is dependent on SRF binding but not ternary complex factor (TCF) formation. Both p35C/EBPbeta and p20C/EBPbeta bind to SRF in vitro via a carboxy-terminal domain that probably does not include the leucine zipper. Moreover, SRE mutants which retain responsiveness to the TCF-independent signaling pathway bind SRE BP in vitro with affinities that are nearly identical to that of the wild-type SRE, whereas mutant SRE.M, which is not responsive to the TCF-independent pathway, has a nearly 10-fold lower affinity for SRE BP. We propose that C/EBPbeta may play a role in conjunction with SRF in the TCF-independent signaling pathway for SRE activation.     

Sequences at the 3' side of the c-fos SRE mediate gene expression via an Sob1-dependent, TCF-independent pathway.

We previously described a 110-kDa tyrosine phosphoprotein, Sob 1, that regulates formation of the DNA binding complex Band A at the c-fos serum response element (SRE) during T cell activation. Using competition and mutant oligonucleotide analysis, we have determined that both the core CArG box of the c-fos SRE and the 3' sequences flanking the CArG box are necessary for stable Band A complex formation. Moreover, using transient transfection and reporter assays, we show that mutations affecting Band A complex formation in vitro also impaired serum induction of c-fos gene expression in vivo. Since mutation at this site has no effect on SRF binding, our results suggest that in combination with SRE/SRF, Sob 1-regulated factor(s) bind at the 3' side of SRE to form Band A, and this confers maximal serum induction of c-fos gene expression via the SRE.     

Cross-binding of factors to functionally different promoter elements in c-fos and skeletal actin genes.

A conserved 28-base-pair element in the skeletal actin promoter was sufficient to activate muscle-specific expression when placed upstream of a TATA element. This muscle regulatory element (MRE) is similar in structure to the serum response element (SRE), which is present in the promoters of the c-fos proto-oncogene and the nonmuscle actin genes. The SRE can function as a constitutive promoter element. Though the MRE and SRE differed in their tissue-specific expression properties, both elements bound to the same protein factors in vitro. These proteins are the serum response factor (SRF) and the muscle actin promoter factors 1 and 2 (MAPF1 and MAPF2). The SRF and MAPF proteins were resolved by chromatographic procedures, and they differed in their relative affinities for each element. The factors were further distinguished by their distinct, but overlapping, methylation interference footprint patterns on each element. These data indicate that the differences in tissue-specific expression may be due to a complex interaction of protein factors with these sequences.