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.

Dissection of functional NF-Y-RFX cooperative interactions on the MHC class II Ea promoter / G. Caretti, F. Cocchiarella, C. Sidoli, J. Villard, M. Peretti, W. Reith, R. Mantovani. - In: JOURNAL OF MOLECULAR BIOLOGY. - ISSN 0022-2836. - 302:3(2000 Sep), pp. 539-552.

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

G. Caretti
Primo
;
R. Mantovani
Ultimo
2000

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.
MHC class II; NF-Y; Nucleosome; RFX; Transcription
Settore BIO/18 - Genetica
set-2000
Article (author)
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/2434/154945
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