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 gamma-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 into Xenopus primary hepatocytes. Conversely, XGRAF had little or no effect on basal transcription of the gamma-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.
Highlights
Steroid hormones, which include glucocorticoids and mineralocorticoids from the adrenal cortex and estrogens, progestins, and androgens from the gonads, regulate a vast array of physiological processes that are essential for development, differentiation, growth, metabolism, homeostasis, behavior, and reproduction in vertebrate organisms
No guanosines are present on the antisense strand within the putative Xenopus glucocorticoid receptor accessory factor (XGRAF)-binding site between positions Ϫ177 and Ϫ169, this experiment confirmed that the binding site does not extend upstream to position Ϫ182 or downstream to position Ϫ164, where the nearest guanosines are located
Correlation between XGRAF Binding to DNA in Vitro and Stimulation of Transcription—In Table I, the ability of DNA with single-point mutations at positions Ϫ177 to Ϫ169 to bind to XGRAF in the gel shift assay is classified as strong (ϩ) if half-maximal competition was achieved with Ͻ7-fold molar excess of mutated competitor over wild-type probe or as weak (Ϫ) if 20-fold or greater excess competitor was needed
Summary
Introduction of Point Mutations into Reporter Gene Constructs—All possible single-point mutations in the potential XGRAF-binding site in the ␥ gene upstream region were obtained by the following general strategy. 1) The ␥ gene sequence from positions Ϫ187 to ϩ41 was synthesized by the polymerase chain reaction (PCR) using a downstream primer with wild-type sequence and an upstream primer consisting of a mixed population of oligonucleotides with mutations in the XGRAF-binding region from positions Ϫ177 to Ϫ169. 2) The PCR products were inserted into a luciferase reporter vector, and the DNA was cloned by transformation into bacteria. 3) The nucleotide sequence of the ␥ DNA in individual clones was determined to ascertain which nucleotide(s) of the XGRAF region had been mutated. 1) The ␥ gene sequence from positions Ϫ187 to ϩ41 was synthesized by the polymerase chain reaction (PCR) using a downstream primer with wild-type sequence and an upstream primer consisting of a mixed population of oligonucleotides with mutations in the XGRAF-binding region from positions Ϫ177 to Ϫ169. The PCR amplification was carried out with the pLL␥Ϫ187 construct [24] as the template DNA (which contains ␥ gene DNA from positions Ϫ187 to ϩ41), the upstream primers described above, a downstream primer within the vector, and Pfu polymerase (Stratagene) following the protocol from the manufacturer. Data Analysis for Gel Shift Assays—The ability of XGRAF to bind ␥ DNA with mutations in the putative XGRAF-binding region was determined by competition gel shift assays, which contained a constant amount of radioactively labeled wild-type ␥ DNA and either no competitor or various concentrations of DNA with a single-point mutation. The total amount of XGRAF in the complex with radioactive DNA in the absence of any competitor (Fig. 1A, lane 2) was defined as 1.0, and the amount of XGRAF in the complex with radioactive DNA in the presence of a 100-fold molar excess of wild-type competitor was defined as zero
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