Subunit association and DNA binding activity of the heterotrimeric transcription factor NF-Y is regulated by cellular redox.
NF-Y is a heterotrimeric transcription factor that specifically recognizes a CCAAT box motif found in a variety of eukaryotic promoter and enhancer elements. The subunit association and DNA binding properties of the NF-Y complex were examined as a function of redox state using recombinant NF-YA, NF-YB, and NF-YC subunits. Reduction of NF-YB by dithiothreitol (DTT) was essential for reconstitution of specific NF-Y CCAAT box DNA binding activity in vitro. Approximately 30% of the Escherichia coli-derived NF-YB subunit existed as intermolecular disulfide-linked dimers. NF-YB mutants in which the highly conserved cysteine residues at positions 85 and 89 had been converted to serines existed only as monomers and did not require DTT for functional NF-Y DNA binding activity. DTT was required, however, for the functional association of NF-YC with wild-type NF-YB but not with the NF-YB cysteine mutants. The cellular redox factors Ref-1 and adult T-cell leukemia-derived factor stimulated the DNA binding activity of recombinant NF-Y in the absence of DTT. Cells treated with 1-chloro-2,4-dinitrobenzene, an irreversible inhibitor of thioredoxin reductase, exhibited reduced endogenous NF-Y DNA binding activity. Together these results suggest that the cellular redox environment of mammalian cells is an important posttranscriptional regulator of NF-Y subunit association and DNA binding activities.
Laboratory of Gene Regulation, The Picower Institute for Medical Research, Manhasset, New York 11030, USA. racurrie@picower.edu
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D000595:Amino Acid Sequence; D000818:Animals; D001704:Biopolymers; D022762:CCAAT-Enhancer-Binding Proteins; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D005033:Ethylmaleimide / Q000494:pharmacology; D005786:Gene Expression Regulation; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014157:Transcription Factors / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*
D001704:Biopolymers; D022762:CCAAT-Enhancer-Binding Proteins; D004268:DNA-Binding Proteins; D011994:Recombinant Proteins; D014157:Transcription Factors; D005033:Ethylmaleimide