Dissection of functional NF-Y-RFX cooperative interactions on the MHC class II ea promoter

Transcription of major histocompatibility complex (MHC) class II genes depe nds upon the trimeric complexes RFX and NF-Y binding; to the conserved X-Y promoter elements. We produced and purified the RFX subunits from Escherich ia coli, reconstituted DNA-binding to the mouse Ea X box and dissected the interactions with NF-Y. RFX and NF-Y do not interact in solution, but make cooperative interactions in EMSA: a minimal NF-Y, composed of the evolution ary conserved domains, is sufficient and the RFXAP N-terminal half is expen dable. Altering the X-Y distance abolishes cooperativity, indicating that D NA imposes severe spatial constraints. When tested on a highly positioned n ucleosome, RFX binds DNA well and NF-Y does not increase its affinity furth er. Transfections of NF-Y subunits, but not RFX, in class II negative cells improves basal transcription and coexpression of the two activators has a synergistic effect, while modestly increasing CIITA-mediated activation. Th ese results show that interactions between the two trimers on DNA are key t o MHC class II expression. (C) 2000 Academic Press.