ASSEMBLY OF FUNCTIONAL REGULATORY COMPLEXES ON MHC CLASS-II PROMOTERSIN-VIVO

Regulatory factors that bind to the X box 1 to 5 (RFX1 to RFX5) and p3 6 interact with the X box in major histocompatibility class LI promote rs. RFX1 and RFX5 bind to DNA as a homodimer (RFX1) and heterodimer wi th p36 (RFX5:p36, the RFX complex), respectively. In this study, we ch aracterized the binding of RFX1 and the RFX complex to the X box in vi vo, and evaluated contributions of other proteins that bind to flankin g conserved upstream sequences (CUS: S, X, X2, and Y boxes) to these p rotein-DNA interactions. For this purpose, an intracellular DNA-bindin g assay was developed. Hybrid protein effectors between RFX1 and RFX5 and the activation domain of VP16 from the herpes simplex virus were c o-expressed with plasmid targets, which contained the isolated X box, X box and selected flanking CUS, or the entire DRA promoter. Whereas R FX1 bound better to isolated X boxes, the Y box selected for the bindi ng of the RFX complex and against the binding of RFX1 to the X box. Wi th proper spacing, S and X boxes stabilized the binding of both RFX1 a nd 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 f unctionally relevant proteins to conserved upstream sequences which re gulate class II transcription. (C) 1997 Academic Press Limited.