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.
Hooft van Huijsduijnen
R
R
GLAXO Insitute for Molecular Biology, Geneva, Switzerland.
Pescini
R
R
DeLamarter
J F
JF
D001483:Base Sequence; D015815:Cell Adhesion Molecules / Q000235:genetics*; D004247:DNA; D019040:E-Selectin; D004591:Electrophoresis, Polyacrylamide Gel; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D016328:NF-kappa B / Q000737:chemistry / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D014407:Tumor Cells, Cultured
D015815:Cell Adhesion Molecules; D019040:E-Selectin; D016328:NF-kappa B; D004247:DNA