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.     

Phosphorylation-dependent formation of a quaternary complex at the c-fos SRE.

The rapid and transient induction of the human proto-oncogene c-fos in response to a variety of stimuli depends on the serum responses element (SRE). In vivo footprinting experiments show that this promoter element is bound by a multicomponent complex including the serum response factor (SRF) and a ternary complex factor such as Elk-1. SRF is thought to recruit a ternary complex factor monomer into an asymmetric complex. In this report, we describe a quaternary complex over the SRE which, in addition to an SRF dimer, contains two Elk-1 molecules. Its formation at the SRE is strictly dependent on phosphorylation of S-383 in the Elk-1 regulatory domain and appears to involve a weak intermolecular association between the two Elk-1 molecules. The influence of mutations in Elk-1 on quaternary complex formation in vitro correlates with their effect on the induction of c-fos reporter expression in response to mitogenic stimuli in vivo.     

A family of muscle gene promoter element (CArG) binding activities in Xenopus embryos: CArG/SRE discrimination and distribution during myogenesis.

The CArG box is an essential promoter sequence for cardiac muscle actin gene expression in Xenopus embryos. To assess the role of the CArG motif in promoter function during Xenopus development, the DNA-binding activities present in the embryo that interact with this sequence have been investigated. A family of four Embryo CArG box1 Factors (ECFs) was separated by a 2-step fractionation procedure. These factors were distinct from the previously described C-ArG box binding activity Serum Response Factor (SRF). ECF1 was the most prominent binding activity in cardiac actin-expressing tissues, and bound the CArG box in preference to a Serum Response Element (SRE). SRF was also detectable in muscle, but it bound preferentially to an SRE. The properties of ECF3 were similar to those of ECF1, but it was much less prominent in cardiac actin-expressing tissues. The properties of the two other factors were distinctive: ECF2 was of relatively low affinity and high abundance, whilst ECF4 bound non-specifically to ends of DNA. The binding activity (or activities) that interacted with the CArG box was found to be influenced by both the concentrations of the other CArG box binding activities and the sequence of the site. Although there was no evidence for a muscle-specific CArG box binding activity, the properties of ECF1 suggest that it could play a role in the expression of the cardiac actin gene during Xenopus development.     

Casein kinase II induces c-fos expression via the serum response element pathway and p67SRF phosphorylation in living fibroblasts.

Elevation of intracellular casein kinase II (CKII) levels through microinjection of purified CKII results in the rapid and transient induction of c-fos in quiescent rat embryo fibroblasts, and activation of quiescent cells by serum is accompanied by the nuclear relocation of endogenous CKII. The induction of c-fos by CKII is inhibited by coinjection of oligonucleotides corresponding to the sequence of the serum response element (SRE) present in the c-fos promoter, indicating that competitive displacement of positive factors from the endogenous c-fos SRE prevents c-fos induction by CKII. Furthermore, the expression of c-fos induced by either CKII injection or serum activation is also inhibited by microinjection of antibodies against the 67 kDa serum response factor (p67SRF) indicating the absolute requirement of p67SRF in this process. Finally, we show the specific phosphorylation of p67SRF in vivo following microinjection of CKII into quiescent cells. Together, these data strongly support that CKII induces c-fos expression through binding/activation of the phosphorylated p67SRF at the SRE sequence.     

Sp1 represses IL-2 receptor alpha chain gene expression

Sp1 is a DNA-binding protein that acts as a positive regulator of eukaryotic gene expression. The interleukin-2 receptor alpha chain (IL2R alpha) gene 5' regulatory region contains a single Sp1 consensus motif that overlaps a CArG box capable of binding serum response factor (SRF). The CArG box has previously been shown to be important for IL2R alpha gene expression. In this study, the results of competition experiments suggest that Sp1 and SRF compete for binding to the CArG region. Site-directed mutagenesis and transient transfection assays indicate that the IL2R alpha gene Sp1 serves the unusual role of repressing gene expression, most likely by competing for binding of nuclear factor(s) to the CArG box.