Article - Limited access to UNIGE
Other version: http://www.sciencedirect.com/science?_ob=MImg&_imagekey=B6WK7-45F511T-5G-X&_cdi=6899&_user=779890&_pii=S0022283600940...
Dissection of functional NF-Y-RFX cooperative interactions on the MHC class II Ea promoter
|Published in||Journal of Molecular Biology. 2000, vol. 302, no. 3, p. 539-552|
|Abstract||Transcription of major histocompatibility complex (MHC) class II genes depends upon the trimeric complexes RFX and NF-Y binding to the conserved X-Y promoter elements. We produced and purified the RFX subunits from Escherichia 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 evolutionary conserved domains, is sufficient and the RFXAP N-terminal half is expendable. Altering the X-Y distance abolishes cooperativity, indicating that DNA imposes severe spatial constraints. When tested on a highly positioned nucleosome, RFX binds DNA well and NF-Y does not increase its affinity further. 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. These results show that interactions between the two trimers on DNA are key to MHC class II expression.|
|Keywords||Animals — Binding Sites — CCAAT-Enhancer-Binding Proteins — Cell Line — Conserved Sequence/genetics — DNA/chemistry/genetics/metabolism — DNA-Binding Proteins/genetics/ metabolism — Genes, MHC Class II/ genetics — Kinetics — Mice — Mutation — Nuclear Proteins — Nucleic Acid Conformation — Nucleosomes/chemistry/genetics/metabolism — Precipitin Tests — Promoter Regions, Genetic/ genetics — Protein Binding — Recombinant Proteins/metabolism — Response Elements/genetics — Thermodynamics — Trans-Activators/genetics/metabolism — Transcription Factors/genetics/ metabolism — Transcription, Genetic/genetics — Transcriptional Activation/genetics — Transfection|