Nucleic Acids Research, 1993, Vol. 21, No. 16 3711-3717
© 1993
MOLECULAR BIOLOGY |
Two distinct NF-
B complexes differing in their larger subunit bind the E-selectin promoter B element
GLAXO Institute for Molecular Biology, Chemin des Aulx 1228 Plan-les-Ouates, Geneva, Switzerland
Received May 6, 1993. Revised June 29, 1993. Accepted June 29, 1993.
We have investigated the proteins binding the E-selectin promoter NF-
B element in its natural DNA context, using probes extending beyond the NF-B recognition decamer. In band shift assays, we detected two distinct NF-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-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 NFB- site. Although no specific sequence is required in this 3' extended DNA to bind the larger complex, an intact 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.
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