Subunit Association and DNA Binding Activity of the Heterotrimeric Transcription Factor NF-Y Is Regulated by Cellular Redox

doi:10.1074/jbc.271.46.28784

Subunit Association and DNA Binding Activity of the Heterotrimeric Transcription Factor NF-Y Is Regulated by Cellular Redox*

From the Laboratory of Gene Regulation, The Picower Institute for Medical Research, Manhasset, New York 11030

^¶To whom correspondence should be addressed:
Laboratory of Gene Regulation, The Picower Institute for Medical Research, 350 Community Drive, Manhasset, NY 11030.
Tel.: 516-562-9432; Fax: 516-365-5090; E-mail: racurrie{at}picower.edu.

↵^‡ Present address: Department of Surgery and Biochemistry, Indiana University School of Medicine, Indianapolis, IN 46202.
↵^§ Present address: Cancer Center, Indiana University School of Medicine, Indianapolis, IN 46202.

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.

Footnotes

↵* This work was supported by USPHS Grant DK 47272 from the NIDDK (to R. A. C.). The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The human NF-YC and murine NF-YC sequences reported in this paper have been deposited in the GenBank database with accession numbers U62296 and U62297, respectively.
↵¹ The abbreviations used are:

DTT

dithiothreitol

ADF

adult T-cell leukemia-derived factor

BSO

buthionine sulfoximine

CDNB

1-chloro 2,4-dinitrobenzene

EMSA

electrophoretic mobility shift assay

GST

glutathione S-transferase

NEM

N-ethylmaleimide

PCR

polymerase chain reaction

SRE

serum response element

SRF

serum response factor

PAGE

polyacrylamide gel electrophoresis

MHC

major histocompatibility complex

CYP1A1

cytochrome P450-dependent monooxygenase.
- Received May 2, 1996.
- Revision received August 23, 1996.