Sequence-Specific DNA Binding by Glucocorticoid Receptor

Steroid hormone receptors can activate or repress transcription from responsive loci by binding to DNA. We have examined the mechanism of DNA binding by individually synthesizing the putative "zinc finger peptides" from the rat glucocorticoid receptor. Atomic absorption studies show that the peptides will bind zinc on an equimolar basis, and circular dichroism experiments demonstrate a significant alteration in secondary structure in the presence of zinc. The results from a series of experiments establish that metal ion is required for binding to DNA and that the amino-terminal zinc finger shows a significantly greater affinity for glucocorticoid response element-containing DNA over control DNA. These observations indicate that a single synthetic "zinc finger peptide" is able to bind to DNA in a sequence-specific manner. The ability of steroids to regulate transcription from specific genes has been intensively studied (1-3). In the case of glucocorticoids, the hormone binds to a cytosolic non-DNAbinding form of its specific receptor, which then translocates into the nucleus. This "activated" receptor is now able to bind to specific sites on chromatin and either enhance or repress transcription (1). With the isolation and sequencing of cDNA clones for the various steroid receptors, it has become apparent that they form a superfamily of transcription factors (4-7). The receptors share a common organizational plan, with the most conserved region of the molecules being a cysteine-rich DNA binding domain (8-10), designated region C. The experiments of several investigators have demonstrated that this conserved portion of the molecule is absolutely required for binding to DNA (11-13). Recent studies have demonstrated that this domain of the receptor, expressed in bacteria independent of flanking amino and carboxyl sequences, is sufficient to bind specifically to DNA (14). Analysis of the predicted protein sequence of region C revealed an intriguing homology with the DNA-binding transcription factor III A from Xenopus laevis (TFIIIA) (15). These findings led to the proposal that the receptor folds to form two subregions, CI and CII, that are analogous to the "zinc fingers" of TFIIIA and that interact with the DNA in a manner that is similar to the well-established binding of TFIIIA (16). Cloning and sequencing of the human estrogen receptor gene revealed that the DNA binding domain is encoded by two exons, one for each finger, raising the possibility that the fingers could be functionally distinct (17). Support for this idea has come from experiments that demonstrated that the amino-terminal finger, CI, can be experimentally exchanged between the estrogen receptor and glucocorticoid receptor, with corresponding changes in the binding specificity of the chimeric receptors (18). More recent experiments have demThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. ?1734 solely to indicate this fact. onstrated that changing the specificity of a steroid receptor may be accomplished by more subtle alterations (19-21). The alteration of two or three amino acids in the CI finger of either a glucocorticoid receptor or an estrogen receptor is sufficient to change the DNA sequence recognized by the mutant receptor (19). An implication from these experiments is that the carboxyl terminal finger, CII, is not involved in determining the specificity of the receptor DNA interaction but is necessary for tight binding to the cognate site. We have examined this question by chemically synthesizing as individual peptides the CI and CII "zinc fingers" from the glucocorticoid receptor. The peptides have been used in DNA binding studies with glucocorticoid response elements (GREs) and nonrelated response elements. The results of these experiments establish that individual finger peptides are capable of sequence-specific DNA binding in the presence of zinc. In addition we have synthesized a mutant CI peptide, CIM, in which the conserved Cys-440 is mutated to an alanine. This peptide will chelate zinc but is unable to bind DNA, suggesting that the precise coordination of the metal is important for DNA binding. EXPERIMENTAL PROCEDURES Synthesis of Peptides and Oligonucleotides. All peptides were prepared by solid-phase synthesis on an Applied Biosystems 430A automated peptide synthesizer (22). Completed peptides were released from the resin and deprotected by using hydrogen fluoride cleavage methods. The crude peptides were purified by gel filtration (Sephadex G-25, 10% HOAc) and or by semipreparative reverse-phase liquid chromatography (H20/CF3COOH to acetonitrile/CF3COOH gradient). Identity of the synthetic peptides were confirmed by amino acid analysis following hydrolysis with HCl (24 hr at 110?C under reduced pressure). The number of cysteine residues present in the peptides was confirmed by reaction with 5-5'-dithiobis(2-nitrobenzoic acid) [DTNB; Ellman's reagent]. Synthesis of Oligonucleotides. All oligonucleotides were prepared on an Applied Biosystems 380A DNA synthesizer and purified according to the manufacturer's instructions. Oligonucleotides were labeled with [y-32P]ATP (New England Nuclear; 6000 Ci/mmol; 1 Ci = 37 GBq) by using T4 polynucleotide kinase as described (23). Oligonucleotides were labeled to a specific activity of 1-4 x 104 cpm/fmol. Atomic Absorption Spectrophotometry. Zinc bound by the peptides was measured by atomic absorption spectrophotometry with a Perkin-Elmer atomic absorption spectrophoAbbreviations: GRE, glucocorticoid response element; ERE, estrogen response element; NFI, nuclear factor 1; MMTV, mouse mammary tumor virus. *To whom reprint requests should be addressed. tPresent address: Laboratory of Chemical Physics, Building 2, Room B2-08, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892.