ELF-1 CONTRIBUTES TO THE FUNCTION OF THE COMPLEX INTERLEUKIN (IL)-2-RESPONSIVE ENHANCER IN THE MOUSE IL-2 RECEPTOR-ALPHA GENE

Lymphocytes regulate their responsiveness to IL-2 through the transcri ptional control of the IL-2R alpha gene, which encodes a component: of the high affinity IL-2 receptor. In the mouse IL-2R alpha gene this c ontrol is exerted via two regulatable elements, a promoter proximal re gion, and an IL-2-responsive enhancer (IL-2rE) 1.3 kb upstream. In vit ro and in vivo functional analysis of the IL-2rE in the rodent thymic lymphoma-derived, CD4(-)CD8(-) cell Line PC60 demonstrated that three separate elements, sites I, II, and III, were necessary for IL-2 respo nsiveness; these three sites demonstrate functional cooperation. Site III contains a consensus binding motif for members of the Ets family o f transcription factors. Here we demonstrate that Elf-1, an Ets-like p rotein, binds to site III and participates in IL-2 responsiveness. In vitro site III forms a complex with a protein constitutively present i n nuclear extracts from PC60 cells as well as from normal CD4(-)CD8(-) thymocytes. We have identified this molecule as Elf-1 according to a number of criteria. The complex possesses an identical electrophoretic mobility to that formed by recombinant Elf-1 protein and is super-shi fted by anti-Elf-1 antibodies. Biotinylated IL-2rE probes precipitate Elf-1 from PC60 extracts provided site III is intact and both recombin ant and PC60-derived proteins bind with the same relative affinities t o different mutants of site III. In addition, by introducing mutations into the core of the site III Ets-Like motif and comparing the corres ponding effects on the in vitro binding of Elf-1 and the in vivo IL-2r E activity, we provide strong evidence that Elf-1 is directly involved in IL-2 responsiveness. The nature of the functional cooperativity ob served between Elf-1 and the factors binding sites I and IT remains un resolved; experiments presented here however suggest that this effect may not require direct interactions between the proteins binding these three elements.