Physical and Functional Sensitivity of Zinc Finger Transcription Factors to Redox Change

Abstract

Redox regulation of DNA-binding proteins through the reversible oxidation of key cysteine sulfhydryl groups has been demonstrated to occur in vitro for a range of transcription factors. The direct redox regulation of DNA binding has not been described in vivo, possibly because most protein thiol groups are strongly buffered against oxidation by the highly reduced intracellular environment mediated by glutathione, thioredoxin, and associated pathways. For this reason, only accessible protein thiol groups with high thiol-disulfide oxidation potentials are likely to be responsive to intracellular redox changes. In this article, we demonstrate that zinc finger DNA-binding proteins, in particular members of the Sp-1 family, appear to contain such redox-sensitive -SH groups. These proteins displayed a higher sensitivity to redox regulation than other redox-responsive factors both in vitro and in vivo. This effect was reflected in the hyperoxidative repression of transcription from promoters with essential Sp-1 binding sites, including the simian virus 40 early region, glycolytic enzyme, and dihydrofolate reductase genes. Promoter analyses implicated the Sp-1 sites in this repression. Non-Sp-1-dependent redox-regulated genes including metallothionein and heme oxygenase were induced by the same hyperoxic stress. The studies demonstrate that cellular redox changes can directly regulate gene expression in vivo by determining the level of occupancy of strategically positioned GC-binding sites.There is compelling evidence for both direct and indirect pathways for the regulation of gene expression by changes in cellular redox state. Hypoxic and hyperoxic stresses can activate or repress the transcription of certain genes by pathways that probably involve protein kinases (5,16,18,24,28,52,68,70,82,84). The response to severe oxidative stress may involve an additional effect in which redox-sensitive factors can be directly activated or inactivated through the oxidation of sulfhydryl residues. The binding of factors AP-1, Sp-1, Egr-1, NF-B, v-rel, c-myb, E2, IRE-BP, p53, and USF to nucleic acid is reduced or lost when critical cysteine residues are oxidized or alkylated (1, 3, 4, 33-35, 37, 48, 53, 60, 66, 78, 89). HoxB5, a member of the mammalian homeodomain gene family, is an example of a factor that is activated by oxidation (30). In the case of AP-1, a cellular DNA-repair protein that may regulate the redox equilibrium has been described previously (90). Oxidative inactivation of USF has been shown to correlate directly with transcriptional activity in an in vitro assay (60). It has been proposed that the reactive cysteines may constitute redox-sulfhydryl switches which directly regulate gene expression (35,60). In support of this, factor Sp-1 in rat liver appears to become progressively oxidized during aging, resulting in the reversible loss of binding activity by an in vitro assay (3). To date, there have been no reports to demonstrate that oxidizing agents or redox stresses can directly mediate transcription factor bindi...

Tumor Hypoxia and Gene Expression: Implications for Malignant Progression and Therapy

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Redox control of AP-1-like factors in yeast and beyond

“…Furthermore, a number of mammalian transcription factors bind DNA in a redox dependent manner. Regulation of these factors, which include NF1 (Bandyopadhyay and Gronostajski, 1994), Sp-1 (Wu et al, 1996), c-Mby (Myrset et al, 1993), p53 (Rainwater et al, 1995), erg-1 (Huang and Adamson, 1993) and NF-kB (Schreck et al, 1992), in most cases is thought to depend on the presence of critical cysteine residues. In an interesting parallel with yeast AP-1 redox dependent processes also regulate the mammalian AP-1 complex.…”

The repression of nuclear factor I/CCAAT transcription factor (NFI/CTF) transactivating domain by oxidative stress is mediated by a critical cysteine (Cys-427)

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