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 conserve d 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 o f these conserved upstream elements to respond to the presence of know n transcriptional activation domains. A series of HLA-DRA promoter rep orter constructions carrying Gal4 binding sites and CAL4 fusion protei n expression vectors were cotransfected into both wild type B cells an d mutant B cells representing the two RFX phenotypes. Regardless of RF X or class II phenotype, the activation domains of GAL4-BP16 and GAL4- E1a could synergistically stimulate expression of constructions contai ning both the X2 and Y boxes. GAL4-BP16- and GAL4-E1a-mediated express ion was inhibited by the presence of the X1 box in cells that containe d RFX. In mutant cells that lacked RFX, GAL4-VP16 activation was not i nhibited. In the RFX-positive class II mutant cell line RJ2.2.5, the X 1 box inhibitory activity could be overcome by separating the Gal4 sit e from the X1 box by two additional helical turns, suggesting that the RFX factor is bound at the X1 site and sterically interferes with act ivation. 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 m utant cells.