A novel complex between the p65 subunit of NF-?B and c-Rel binds to a DNA element involved in the phorbol ester induction of the human urokinase gene

The NF-?B subunits, p50 and p65, have extensive sequence homology with the c-rel proto-oncogene and the Drosophila morphogen dorsal. It has recently been shown that in vitro translated c-Rel can bind to DNA and form a complex with p50. However, the conditions for DNA binding of c-Rel in vivo and its DNA sequence specificity have not been established. Here we report the identification a novel heterodimeric complex that binds to a ?B-like, phorbol ester (TPA) responsive DNA sequence, 5?-GGGAAAGTAC-3?, in the 5? flanking region of the human urokinase (uPA) gene. This sequence was shown to bind two protein complexes, LC and UC. LC was indistinguishable from NF-?B as it reacted with antibodies recognizing the p50 subunit of NF-?B, and was shown by LTV crosslinking to contain the p50 and p65 subunits of NF-?B. UC, on the other hand, strongly reacted with anti-v-Rel, but not with the anti-p50 antibodies, and was shown by crosslinking to contain 75 kDa and 85 kDa protein - DNA adducts. The 75 kDa and the 85 kDa adducts could be immunoprecipitated only by anti-p65 and anti-c-Rel antibodies, respectively, showing that c-Rel formed a heterodimer with p65. Both protein complexes were present in inactive forms in HeLa cell cytosol, and their nuclear translocation was induced by TPA. DNA binding of UC and LC could, furthermore, be inhibited by I?B-?. These results are the first demonstration that the c-rel proto-oncogene product can bind DNA independently of the p50 NF-xB subunit, in a heterodimeric complex with the p65 subunit of NT-?B. In contrast to the ?B element of the ? light chain enhancer, the uPA DNA element was shown to bind both c-Rel -p65 and p50-p65 complexes. Therefore, we have termed this novel TPA responsive element RRBE, for Rel-Related proteins Binding Element.