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J Mol Biol. 2000 Sep 22;302(3):539-52.

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

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1
Dipartimento di Genetica e Biologia dei Microrganismi, Università di Milano, Via Celoria 26, Milano, 20133, Italy.

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.

PMID:
10986117
DOI:
10.1006/jmbi.2000.4028
[Indexed for MEDLINE]

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