Analysis of the DNA-binding Site for XenopusGlucocorticoid Receptor Accessory Factor
CRITICAL NUCLEOTIDES FOR BINDING SPECIFICITY IN VITROAND FOR AMPLIFICATION OF STEROID-INDUCED FIBRINOGEN GENE TRANSCRIPTION*
Abstract
In addition to the glucocorticoid receptor, DNA-binding proteins called accessory factors play a role in hormone activation of many glucocorticoid-responsive genes. Hormonal regulation of the γ-fibrinogen subunit gene from the frog Xenopus laevis requires a novel DNA sequence that binds a liver nuclear protein called Xenopus glucocorticoid receptor accessory factor (XGRAF). Here we demonstrate that the recognition site for XGRAF encompasses GAGTTAA at positions −175 to −169 relative to the start site of transcription. This sequence is not closely related to the binding sites for known transcription factors. The two guanosines make close contact with XGRAF, as shown by the methylation interference assay. Single-point mutagenesis of every nucleotide in the 9-base pair region from positions −177 to −169 showed an excellent correlation between ability to bind XGRAF in vitro and ability to amplify hormone-induced transcription from DNA transfected intoXenopus primary hepatocytes. Conversely, XGRAF had little or no effect on basal transcription of the γ-fibrinogen gene. Maximal hormonal induction also requires three half-glucocorticoid response elements (half-GREs) homologous to the downstream half of the consensus GRE. Interestingly, the XGRAF-binding site is immediately adjacent to the most important half-GRE. This close proximity suggests a new mechanism for activation of a gene lacking a conventional full GRE.
Footnotes
-
↵* This work was supported by NHLBI Grant RO1-HL39095 and Research Career Development Award HL02934 (to L. J. H.) and Post-doctoral and Pre-doctoral Training Grant HL07094 (to M. L. and R. N. W.) from the National Institutes of Health.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.
-
↵‡ Present address: Gladstone Inst. of Cardiovascular Disease, University of California, San Francisco, CA 94141.
-
↵§ To whom correspondence should be addressed: Dept. of Physiology, MA415 Medical Sciences Bldg., University of Missouri School of Medicine, Columbia, MO 65212. Tel.: 573-882-5373; Fax: 573-884-4276; E-mail: physljh{at}muccmail.missouri.edu.
- Received October 31, 1997.
- The American Society for Biochemistry and Molecular Biology, Inc.
