Dissection of functional NF-Y-RFX cooperative interactions on the MHC class II Ea promoter.
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.
Caretti
G
G
Dipartimento di Genetica e Biologia dei Microrganismi, Università di Milano, Via Celoria 26, Milano, 20133, Italy.
Cocchiarella
F
F
Sidoli
C
C
Villard
J
J
Peretti
M
M
Reith
W
W
Mantovani
R
R
D000818:Animals; D001665:Binding Sites; D022762:CCAAT-Enhancer-Binding Proteins; D002460:Cell Line; D017124:Conserved Sequence / Q000235:genetics; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D005802:Genes, MHC Class II / Q000235:genetics*; D007700:Kinetics; D051379:Mice; D009154:Mutation; D009687:Nuclear Proteins; D009690:Nucleic Acid Conformation; D009707:Nucleosomes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011233:Precipitin Tests; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism; D000071701:Regulatory Factor X Transcription Factors; D020218:Response Elements / Q000235:genetics; D013816:Thermodynamics; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic / Q000235:genetics; D015533:Transcriptional Activation / Q000235:genetics; D014162:Transfection
D022762:CCAAT-Enhancer-Binding Proteins; D004268:DNA-Binding Proteins; C083728:MHC class II transactivator protein; D009687:Nuclear Proteins; D009707:Nucleosomes; D011994:Recombinant Proteins; D000071701:Regulatory Factor X Transcription Factors; D015534:Trans-Activators; D014157:Transcription Factors; D004247:DNA