PURIFIED X2 BINDING-PROTEIN (X2BP) COOPERATIVELY BINDS THE CLASS-II MHC X-BOX REGION IN THE PRESENCE OF PURIFIED RFX, THE X-BOX FACTOR DEFICIENT IN THE BARE-LYMPHOCYTE-SYNDROME

The conserved X2 box sequence of MHC class II promoters is homologous to TRE/CRE elements, and is required for B cell expression and IFN-gam ma induction of MHC class It genes. The X2 binding protein (X2BP) was initially identified as a DNA-binding activity that specifically inter acts with the conserved X2 box sequence in both the MHC HLA-DRA and HL A-DRB promoters. To begin to demonstrate that X2BP is the X2 box facto r responsible for class II expression in B cells, we have purified X2B P to homogeneity from B cell nuclear extracts using DNA-affinity chrom atography. X-box DNA-affinity purification indicates that X2BP is most likely composed of two polypeptides of 120 kDa and 46 kDa. The 120-kD a protein was specifically cross-linked to an X-box probe by exposure to UV irradiation. The 46-kDa subunit of X2BP cross-reacted with anti- rat CREB polyclonal Abs but not to anti-human CREB Abs in Western anal ysis and supershift assays, indicating that it may be a novel member o f the ATF/CREB family. Purified X2BP interacted with purified RFX, a f actor that binds to the adjacent X1 box and is absent in some cell lin es that are mutant for MHC class II transcription. This interaction in creases the DNA-binding half-life of RFX from 5 to at least 60 min, su ggesting that X2BP functions in class II MHC gene expression by formin g a stable complex with RFX.