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.
Fontes
J D
JD
Howard Hughes Medical Institute, Department of Medicine, University of California at San Francisco, 94143-0724, USA.
Jabrane-Ferrat
N
N
Peterlin
B M
BM
D000818:Animals; D001402:B-Lymphocytes; D019556:COS Cells; D002460:Cell Line; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000235:genetics*; D005802:Genes, MHC Class II / Q000235:genetics*; D017405:Herpes Simplex Virus Protein Vmw65 / Q000235:genetics; D008957:Models, Genetic; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011993:Recombinant Fusion Proteins
D004268:DNA-Binding Proteins; D017405:Herpes Simplex Virus Protein Vmw65; D011993:Recombinant Fusion Proteins; D004247:DNA