Two distinct NF-kappa B complexes differing in their larger subunit bind the E-selectin promoter kappa B element.

We have investigated the proteins binding the E-selectin promoter NF-kappa B element in its natural DNA context, using probes extending beyond the NF-kappa B recognition decamer. In band shift assays, we detected two distinct NF-kappa B complexes using nuclear extracts from several cytokine-induced cells. Subunit-specific antisera as blockers of complex formation plus DNA-protein cross-linking experiments revealed the faster migrating form to contain the NF-kappa B p50 plus p65 subunits. In contrast, the slower migrating form is composed of p50 plus the p65-related p75 protein. We show as the crucial determinant in generation of the larger complex the presence of more than five basepairs extra DNA sequence downstream of the NF-kappa B-site. Although no specific sequence is required in this 3' extended DNA to bind the larger complex, an intact kappa B binding site is. This may be explained by a requirement for activated p50 as part of this complex. The potential for a regulatory role for the p75 containing complex on the E-selectin promoter is discussed.     

A novel complex between the p65 subunit of NF-kappa B and c-Rel binds to a DNA element involved in the phorbol ester induction of the human urokinase gene.

The NF-kappa B subunits, p50 and p65, have extensive sequence homology with the c-rel proto-oncogene and the Drosophila morphogen dorsal. It has recently been shown that in vitro translated c-Rel can bind to DNA and form a complex with p50. However, the conditions for DNA binding of c-Rel in vivo and its DNA sequence specificity have not been established. Here we report the identification a novel heterodimeric complex that binds to a kappa B-like, phorbol ester (TPA) responsive DNA sequence, 5'-GGGAAAGTAC-3', in the 5' flanking region of the human urokinase (uPA) gene. This sequence was shown to bind two protein complexes, LC and UC. LC was indistinguishable from NF-kappa B as it reacted with antibodies recognizing the p50 subunit of NF-kappa B, and was shown by UV crosslinking to contain the p50 and p65 subunits of NF-kappa B. UC, on the other hand, strongly reacted with anti-v-Rel, but not with the anti-p50 antibodies, and was shown by crosslinking to contain 75 kDa and 85 kDa protein-DNA adducts. The 75 kDa and the 85 kDa adducts could be immunoprecipitated only by anti-p65 and anti-c-Rel antibodies, respectively, showing that c-Rel formed a heterodimer with p65. Both protein complexes were present in inactive forms in HeLa cell cytosol, and their nuclear translocation was induced by TPA. DNA binding of UC and LC could, furthermore, be inhibited by I kappa B-alpha.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of an NF-kappa B binding site in the bovine leukemia virus promoter.

Although the mechanism by which bovine leukemia virus (BLV) induces neoplastic transformation of the host B cells is unknown, it is likely that critical interactions between cellular DNA-binding proteins and the virus are involved. We have used DNase I protection (footprinting) assays to construct a map of protein-DNA interactions on the 5' long terminal repeat of BLV. In addition to the three cyclic AMP response elements previously reported, we have also found an NF-kappa B binding site between -118 and -70 nucleotides upstream of the RNA start site. This site binds several members of the kappa B family of proteins, including p49, p50, and p65, in both footprint and electrophoretic mobility shift assays and functions as an enhancer element when inserted upstream of the chloramphenicol acetyltransferase gene. NF-kappa B may be a critical nuclear binding protein that regulates both viral replication and key cellular genes in BLV-infected B cells.     

Structure and function of the enhancer 3'' to the human A gamma globin gene.

An enhancer is located immediately 3' to the A gamma globin gene. We have used DNase I footprinting to map the sites of interaction of nuclear proteins with the DNA sequences of this enhancer. Eight footprints were discovered, distributed over 600 base pairs of DNA. Three of these contain a consensus binding site for the erythroid specific factor GATA-I. Each of these GATA-1 sites had an enhancer activity when inserted into a reporter plasmid and tested in human erythroleukemia cells. Other footprints within the enhancer contained consensus binding sequences for the ubiquitous, positive regulatory proteins AP2 and CBP-1. An Sp1-like recognition sequence was also identified. Synthetic oligonucleotides encompassing two of the footprints generated a slowly migrating complex in gel mobility shift assays. The same complex forms on a fragment of the human gamma globin gene promoter extending from -260 to -200. The DNaseI footprint of this protein complex with the enhancer overlapped a sequence, AGGAGGA, found within the binding site for a protein that interacts with the chicken beta globin promoter and enhancer, termed the stage selector element. We propose that this complex of proteins may be involved in the human gamma globin promoter-enhancer interaction.