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.     

Structure of the murine serum amyloid A gene family. Gene conversion

Serum amyloid A (SAA) is an apolipoprotein produced by the liver in response to inflammation; the levels of SAA mRNA and SAA protein increase at least 500-fold within 24 h. We have obtained clones of all three genes and pseudogene that make up the murine SAA gene family. Two of the genes have 96% sequence homology over their entire length, including introns and flanking sequences 288 base pairs (bp) 5' and 443 bp 3' to the genes: an overall length of 3215 bp. The sharp boundaries between homologous and nonhomologous sequences and the absence of interspersed repeated sequences there suggest that conversion has occurred between these two genes. The homologous regions are bounded by short inverted repeats containing alternating purine and pyrimidine residues, as described for other gene conversion units. The third SAA gene has evolved separately, although all are closely linked on chromosome 7. Comparison of the upstream regions of the SAA genes with those of the rat fibrinogen genes, whose expression is also induced by inflammation, reveals sequences common to all six genes which are very improbable on a random basis.     

Constitutive monocyte-restricted activity of NF-M, a nuclear factor that binds to a C/EBP motif.

In a search for monocyte-specific nuclear factors, we analyzed in human cells the promoter of the chicken myelomonocytic growth factor, a gene that, in the chicken, is expressed in myeloid and myelomonocytic cells. Reporter gene constructs were active in monocytic Mono Mac 6 cells and in monoblastic THP-1 cells but not in the hematopoietic stem cell line K562. When a region with homology to the sequence recognized by CAAT enhancer-binding proteins (C/EBP) was inactivated by site-directed mutagenesis, the reporter activity was reduced by a factor of 10. Multimers of this region, termed F, in front of a heterologous promoter were active in Mono Mac 6 and THP-1 cells but not in K562 cells, WIL2 B cells, BT20 mammary carcinoma cells, MelJuso melanoma cells, or SK-Hep-1 hepatoma cells. Gel shift analysis with the F oligonucleotide identified DNA-binding activity in monocytic Mono Mac 6, monoblastic THP-1, and myelomonocytic HL60 cells. No binding was detected in myelomonocytic RC2A cells, in myeloid KG-1 cells, or in the hematopoietic stem cell line K562. Furthermore, a panel of solid tumor cell lines, representing various tissues, were also negative. Stimulation by PMA could not induce this binding factor in any of the negative cell lines. Analysis of primary cells (granulocytes, T cells, monocytes, and alveolar macrophages) revealed binding activity only in monocytes and macrophages. This DNA-binding factor, termed NF-M, was found to consist of two molecules, of 50 and 72 kDa, as determined by affinity cross-linking. Binding of NF-M was competed by the region F oligonucleotide and by the C/EBP motif from the albumin enhancer but not by an AP-2 motif. These data suggest that NF-M is a member of the C/EBP family of nuclear factors. The monocyte-restricted activity of NF-M suggests that this nuclear factor may be involved in regulation of monocyte-specific genes.