Identification of a DNA binding site for the nuclear factor YY1 in the human GM-CSF core promoter.
It has been well documented that the repeated CATT(A/T) sequence, localized between -64 and -35 in the human GM-CSF promoter, is required for the promoter activity, and this region likely serves as a core recognition sequence for a cellular transcription factor. However, the transcription factor that interacts with this site was not identified. Here, we report that this element contains a binding site for the nuclear factor YY1, which has not been reported to play a role in the regulation of cytokine gene transcription. Results from transient transfection assays of the Jurkat T cell line revealed that this repeated CATT(A/T) element exhibited enhancer activity when linked to both the human IFN-gamma promoter and the TK promoter. Mutation of the YY1 binding site eliminated about 60% of the enhancer activity of the element. We have found that the YY1 binding site could form two specific DNA-protein complexes, A and B, with Jurkat nuclear proteins in the electrophoretic mobility shift assay and that the binding of these complexes correlates with the enhancer activity. UV cross-linking analysis revealed that the A complex is a multi-protein complex and in addition to YY1, other proteins are required for formation of the protein complex. Cotransfection assays with a YY1 expression vector revealed that overexpression of YY1 resulted in an inhibitory effect on the repeated CATT(A/T) element, indicating that in addition to YY1, cofactors also are required for the activator function of the A complex.
Laboratory of Experimental Immunology, DCT, NCI-FCRDC, MD 21702-1201.
J
J
Ye
H A
HA
Young
J R
JR
Ortaldo
P
P
Ghosh
D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004742:Enhancer Elements, Genetic; D050981:Erythroid-Specific DNA-Binding Factors; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009687:Nuclear Proteins / Q000378:metabolism; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D011401:Promoter Regions, Genetic; D013329:Structure-Activity Relationship; D014157:Transcription Factors / Q000378:metabolism*; D050986:YY1 Transcription Factor
D004268:DNA-Binding Proteins; D050981:Erythroid-Specific DNA-Binding Factors; D009687:Nuclear Proteins; D009838:Oligodeoxyribonucleotides; D014157:Transcription Factors; D050986:YY1 Transcription Factor; C494698:YY1 protein, human; D016178:Granulocyte-Macrophage Colony-Stimulating Factor