Mutational analysis of interferon (IFN) regulatory factors 1 and 2. Effects on the induction of IFN-beta gene expression.
Interferon (IFN) regulatory factor 1 (IRF-1) and IRF-2 are structurally similar but functionally distinct transcription factors that bind to the positive regulatory domains I and III (PRDI/III) within the human IFN-beta promoter. To begin structure-function analysis of IRF-1 and IRF-2, the regulatory potential of carboxyl-terminal deletion mutants was analyzed by co-transfection studies in human cells and was correlated with DNA binding capacity. Transcriptional repression by IRF-2 was contained within the first 125 amino-terminal amino acids and correlated directly with IRF-2 DNA binding; deletion to a protein of 100 amino acids resulted in loss of repression and IRF-2 DNA binding. Thus, the carboxyl terminus appears dispensible for trans-repression. Hybrid constructs which fuse the DNA binding domain of IRF-1 and IRF-2 to the trans-activation domain of NF-kappa B p65 were also generated; both IRF-1/p65 and IRF-2/p65 chimeras were strong transcriptional activators. IRF-2-mediated repression was also dominant over trans-activation by these fusion proteins. The trans-activation region of IRF-1 resides in the carboxyl terminus, primarily carboxyl-terminal to amino acid 250. Mutation of three potential casein kinase II phosphorylation sites within the IRF carboxyl terminus failed to identify an essential site that contributes to IRF-1 trans-activation potential.
Abe Stern Cancer Research Laboratory, Lady Davis Institute for Medical Research, Sir Mortimer B. Davis Jewish General Hospital Montreal, Quebec Canada.
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Lin
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Mustafa
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Nguyen
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Gewert
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Hiscott
D001483:Base Sequence; D001665:Binding Sites; D002478:Cells, Cultured; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D017353:Gene Deletion; D005786:Gene Expression Regulation; D006801:Humans; D050856:Interferon Regulatory Factor-1; D050836:Interferon Regulatory Factor-2; D016899:Interferon-beta / Q000096:biosynthesis / Q000235:genetics*; D008969:Molecular Sequence Data; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D012097:Repressor Proteins; D017384:Sequence Deletion; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic
D004268:DNA-Binding Proteins; C494257:IRF1 protein, human; C494229:IRF2 protein, human; D050856:Interferon Regulatory Factor-1; D050836:Interferon Regulatory Factor-2; D010750:Phosphoproteins; D012097:Repressor Proteins; D014157:Transcription Factors; D016899:Interferon-beta; D004247:DNA