2 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-kappaB element in its natural DNA context, using probes extending beyond the NF-kappaB recognition decamer. In band shift assays, we detected two distinct NF-kappaB 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-kappaB 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-kappaB-site. Although no specific sequence is required in this 3' extended DNA to bind the larger complex, an intact kappaB 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.