Abstract
Analysis of the major histocompatibility complex class II gene promoter DRA has previously identified at least five cis-acting regions required for maximal expression. We have examined the DRA promoter for protein-DNA interactions in the intact cell, which may mediate transcriptional activation. Using in vivo genomic footprinting we identified interactions in B-cell lines at the octamer site and the Y, X1, and X2 boxes. Class II antigen expressing T-cell lines maintained contacts identical to B-cell lines, while class II-negative T-cell lines exhibited no interactions. In lymphoid cell lines, the octamer site is occupied and required for maximal expression. This is most likely due to the presence of the lymphoid-specific OTF-2 factor. In contrast, the class II-positive nonlymphoid glioblastoma cell line does not exhibit interactions at the octamer site despite the presence of the ubiquitous OTF-1 factor and an open binding site. Thus, the DRA promoter discriminates against OTF-1 activation at the level of DNA binding in the glioblastoma line. Interferon gamma induces class II expression in this glioblastoma cell line and, in parallel, up-regulates X1 and X2 box protein-DNA interactions, while all other interactions remain unchanged. These results suggest that interferon gamma functions on a poised promoter by altering weak, nonproductive interactions at the X boxes to strong interactions. These findings provide direct in vivo evidence to strongly suggest that the modulation of X1 and X2 interactions is an important constituent of the interferon gamma induction pathway.