Subunit association and DNA binding activity of the heterotrimeric transcription factor NF-Y is regulated by cellular redox

Harikrishna Nakshatri, Poornima Bhat-Nakshatri, R. Alexander Currie

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Abstract

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.

Original languageEnglish
Pages (from-to)28784-28791
Number of pages8
JournalJournal of Biological Chemistry
Volume271
Issue number46
DOIs
StatePublished - 1996

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Oxidation-Reduction
Transcription Factors
Dithiothreitol
Association reactions
DNA
Cysteine
Thioredoxin-Disulfide Reductase
Dinitrochlorobenzene
Disulfides
Dimers
Serine
Escherichia coli
Monomers
Cells

ASJC Scopus subject areas

  • Biochemistry

Cite this

Subunit association and DNA binding activity of the heterotrimeric transcription factor NF-Y is regulated by cellular redox. / Nakshatri, Harikrishna; Bhat-Nakshatri, Poornima; Currie, R. Alexander.

In: Journal of Biological Chemistry, Vol. 271, No. 46, 1996, p. 28784-28791.

Research output: Contribution to journalArticle

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