Two distinct NF-{chi}B complexes differing in their larger subunit bind the E-selectin promoter {chi}B element

We have investigated the proteins binding the E-selectin promoter NF-χB element in its natural DNA context, using probes extending beyond the NF-χB recognition decamer. In band shift assays, we detected two distinct NF-χB 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-χB 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 NFχB- site. Although no specific sequence is required in this 3' extended DNA to bind the larger complex, an intact χB 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.