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Dissection of functional NF-Y-RFX cooperative interactions on the MHC class II Ea promoter

Caretti, G.
Cocchiarella, F.
Sidoli, C.
Peretti, M.
Mantovani, R.
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 AnimalsBinding SitesCCAAT-Enhancer-Binding ProteinsCell LineConserved Sequence/geneticsDNA/chemistry/genetics/metabolismDNA-Binding Proteins/genetics/ metabolismGenes, MHC Class II/ geneticsKineticsMiceMutationNuclear ProteinsNucleic Acid ConformationNucleosomes/chemistry/genetics/metabolismPrecipitin TestsPromoter Regions, Genetic/ geneticsProtein BindingRecombinant Proteins/metabolismResponse Elements/geneticsThermodynamicsTrans-Activators/genetics/metabolismTranscription Factors/genetics/ metabolismTranscription, Genetic/geneticsTranscriptional Activation/geneticsTransfection
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PMID: 10986117
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