Characterization of a silencer regulatory element in the human interferon-gamma promoter.
Previous analysis of the human interferon-gamma (IFN-gamma promoter indicated that the region of DNA from -251 to -215 (designated here as BE (binding element)) possessed silencer activity, as deletion of this region caused an increase in promoter activity. Based on this finding, we have conducted a series of experiments to characterize BE function and analyze the binding proteins which interact with this region. Transient transfection assays in the Jurkat T cell line revealed that the BE region possesses silencer activity, which is orientation-dependent when reinserted 5' to the IFN-gamma core promoter. However, when the BE region was inserted in front of a heterologous promoter (thymidine kinase (TK)), a mild enhancer activity was observed. Utilizing the electrophoretic mobility shift assay, we have identified two major DNA-protein complexes (designated as S and E complexes) which interact with this region. Mutational analysis indicated that the silencer activity observed with the IFN-gamma promoter correlated with the S complex and the enhancer activity correlated with the E complex. Preliminary characterization of these two DNA-protein complexes has demonstrated the presence of multiple proteins in each complex. We have found that the S protein complex has a recognition sequence similar to the nuclear factor AP2, and we have identified the nuclear factor Yin-Yang 1 (YY1) as one of the proteins in the E complex.
Ye
J
J
Laboratory of Experimental Immunology, Program Resources Inc./DynCorp, NCI-FCRDC, Frederick, Maryland 21702-1201.
Ghosh
P
P
Cippitelli
M
M
Subleski
J
J
Hardy
K J
KJ
Ortaldo
J R
JR
Young
H A
HA
D001483:Base Sequence; D001665:Binding Sites; D015496:CD4-Positive T-Lymphocytes / Q000166:cytology; D002460:Cell Line; D004252:DNA Mutational Analysis; D004268:DNA-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D050981:Erythroid-Specific DNA-Binding Factors; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D006801:Humans; D007371:Interferon-gamma / Q000096:biosynthesis / Q000235:genetics*; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011485:Protein Binding; D014157:Transcription Factors / Q000302:isolation & purification; D014162:Transfection; D050986:YY1 Transcription Factor
D004268:DNA-Binding Proteins; D050981:Erythroid-Specific DNA-Binding Factors; D046911:Macromolecular Substances; D009687:Nuclear Proteins; D014157:Transcription Factors; D050986:YY1 Transcription Factor; C494698:YY1 protein, human; D007371:Interferon-gamma