The promoter of the human major histocompatibility complex class II-associated invariant-chain gene (Ii) contains two NF-KB/Rel binding sites located at -109 to -118 (Ii KB-1) and -163 to -172 (Ii KB-2) from the transcription start site. We report here that the differential function of each of these NF-KB/Rel sites in several distinct cell types depends on cell-specific binding of NF-KB/Rel transcription factors. Ii KB-1 is a positive regulatory element in B-cell lines and in the li-expressing T-cell line, H9, but acts as a negative regulatory element in myelomonocytic and glial cell lines. In vivo protein-DNA contacts are detectable at Ii KB-1 in cell lines in which this site is functional as either a positive or negative regulator. Electrophoretic mobility supershift assays determine that members of the NF-KB/Rel family of transcription factors can bind to this site in vitro and that DNA-binding complexes that contain p50, p52, p65, and cRel correlate with positive regulation whereas the presence of p50 correlates with negative regulation. Ii KB-2 is a site of positive regulation in B-cell lines and a site of negative regulation in H9 T cells, myelomonocytic, and glial cell lines. In vivo occupancy of this site is observed only in the H9 T-cell line. Again

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