Assembly of functional regulatory complexes on MHC class II promoters in vivo

Regulatory factors that bind to the X box 1 to 5 (RFX1 to RFX5) and p36 interact with the X box in major histocompatibility class II promoters. RFX1 and RFX5 bind to DNA as a homodimer (RFX1) and heterodimer with p36 (RFX5:p36, the RFX complex), respectively. In this study, we characterized the binding of RFX1 and the RFX complex to the X box in vivo, and evaluated contributions of other proteins that bind to flanking conserved upstream sequences (CUS: S, X, X2, and Y boxes) to these protein-DNA interactions. For this purpose, an intracellular DNA-binding assay was developed. Hybrid protein effectors between RFX1 and RFX5 and the activation domain of VP16 from the herpes simplex virus were co-expressed with plasmid targets, which contained the isolated X box, X box and selected flanking CUS, or the entire DRA promoter. Whereas RFX1 bound better to isolated X boxes, the Y box selected for the binding of the RFX complex and against the binding of RFX1 to the X box. With proper spacing, S and X boxes stabilized the binding of both RFX1 and the RFX complex. The X2 box did not contribute significantly to the binding of either RFX1 or the RFX complex to the X box. Thus, complex protein-protein and protein-DNA interactions dictate the binding of functionally relevant proteins to conserved upstream sequences which regulate class II transcription.