Analysis of the DNA-binding site for Xenopus glucocorticoid receptor accessory factor. Critical nucleotides for binding specificity in vitro and for amplification of steroid-induced fibrinogen gene transcription.
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.
Li
M
M
Department of Physiology, University of Missouri School of Medicine, Columbia, Missouri 65212, USA.
Ye
X
X
Woodward
R N
RN
Zhu
C
C
Nichols
L A
LA
Holland
L J
LJ
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002478:Cells, Cultured; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D019175:DNA Methylation; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000378:metabolism*; D005260:Female; D005340:Fibrinogen / Q000096:biosynthesis*; D017930:Genes, Reporter; D005938:Glucocorticoids / Q000494:pharmacology*; D008099:Liver / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009687:Nuclear Proteins / Q000378:metabolism*; D009865:Oocytes / Q000502:physiology; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D011965:Receptors, Glucocorticoid / Q000378:metabolism*; D012044:Regression Analysis; D014158:Transcription, Genetic / Q000187:drug effects*; D014162:Transfection; D029867:Xenopus Proteins; D014982:Xenopus laevis
D017931:DNA Primers; D004268:DNA-Binding Proteins; D005938:Glucocorticoids; D009687:Nuclear Proteins; D011965:Receptors, Glucocorticoid; D029867:Xenopus Proteins; C111897:glucocorticoid receptor accessory factor, Xenopus; D005340:Fibrinogen; D004247:DNA