Class II MHC transcriptional mutants are defective in higher order complex formation

Human class II MHC genes are regulated in part by a series of conserved upstream sequence elements termed the X1, X2, and Y boxes. Class II MHC transcriptional mutant B cell lines have been defined that differ with regard to the presence of RFX, an X1 box DNA-binding activity. To further characterize these mutant cell lines, we tested the ability of these conserved upstream elements to respond to the presence of known transcriptional activation domains. A series of HLA-DRA promoter reporter constructions carrying Gal4 binding sites and GAL4 fusion protein expression vectors were cotransfected into both wild type B cells and mutant B cells representing the two RFX phenotypes. Regardless of RFX or class II phenotype, the activation domains of GAL4-VP16 and GAL4-E1a could synergistically stimulate expression of constructions containing both the X2 and Y boxes. GAL4-VP16- and GAL4-E1a-mediated expression was inhibited by the presence of the X1 box in cells that contained RFX. In mutant cells that lacked RFX, GAL4-VP16 activation was not inhibited. In the RFX-positive class II mutant cell line RJ2.2.5, the X1 box inhibitory activity could be overcome by separating the Gal4 site from the X1 box by two additional helical turns, suggesting that the RFX factor is bound at the X1 site and sterically interferes with activation. This was not the case in wild type B cells, suggesting that a stable higher order complex forms in wild type cells and not in the mutant cells.