COOPERATIVITY BETWEEN 2 NF-KAPPA-B COMPLEXES, MEDIATED BY HIGH-MOBILITY-GROUP PROTEIN-I(Y), IS ESSENTIAL FOR CYTOKINE-INDUCED EXPRESSION OFTHE E-SELECTIN PROMOTER

Cytokine-induced expression of the E-selectin gene requires the promot er binding and interaction of the transcription factors NF-kappa B and ATF. Here we have further analyzed the E-selectin promoter and reveal ed an additional region (nucleotides -140 to -105 [-140/-105]) which i s essential in controlling promoter activation by cytokines. We identi fied high-mobility-group protein I(Y) [HMG-I(Y)] interacting specifica lly at two sites within this region. We noted that one of the HMG-I(Y) -binding sites overlaps a sequence element (-127/-118) diverging at on ly one position from the NF-kappa B consensus binding sequence. This l ed us to ask whether the -127/-118 element represents a second functio nal NF-kappa B-binding site within the E-selectin promoter. Using spec ific antisera, me show that p50, p65, and, interestingly, RelB are com ponents of the complex interacting at this site. Mutational analysis o f the -127/-118 NF-kappa B site indicates that both NF-kappa B and HMG -I(Y) binding at this site are essential for interleukin-1 induction o f the promoter. We demonstrate that the binding affinity of the p50 su bunit of NF-kappa B to both NF-kappa B sites within the E-selectin pro moter is significantly enhanced by HMG-I(Y). In addition, an essential role for cooperative interaction between the two NF-kappa B complexes is shown by the requirement for both NF-kappa B sites to mediate E-se lectin promoter activation by interleukin-1 and p50/p65 expression. We conclude that HMG-IQ mediates binding of a distinct NF-kappa B comple x at two sites within the E-selectin promoter. Furthermore, a unique c ooperativity between these NF-kappa B complexes is essential for induc ed E-selectin expression. These results suggest mechanisms by which NF -kappa B complexes are involved in specific gene activation.