Interferon induction of gene transcription analyzed by in vivo footprinting.
The promoters of two interferon-induced genes (the ISG54 and guanylate-binding protein [GBP] genes) have been analyzed in whole cells and in isolated nuclei by using a new genomic sequencing technique. The ISG54 gene contains an interferon-simulating response element (ISRE), earlier shown to be necessary and sufficient for alpha interferon (IFN-alpha) induction, that appeared complexed with proteins in both transcribing and nontranscribing cells. However, the extent of protection and hypersensitivity to DNase I or dimethyl sulfate within the ISRE region was changed upon transcriptional induction, suggesting the binding of different factors in different transcriptional states. In addition to the ISRE, the GBP gene needs a newly recognized DNA element, called the GAS, that partly overlaps the ISRE for full induction by either IFN-alpha or IFN-gamma. This GAS element was transiently protected against DNase I in the nuclei of interferon-treated cells but was not protected at later times when transcription reached maximal levels. Thus, the GAS-binding activity may be necessary only transiently for the initial assembly of a transcription initiation complex on the GBP promoter. Dimethyl sulfate methylation of genomic DNA performed on intact cells showed a characteristic sensitivity over the GAS that correlated with transcription levels and that persisted longer than did DNase I protection over the GAS. These results demonstrate the involvement of the GAS in IFN-alpha and -gamma induction of GBP and suggest the presence of an altered DNA conformation or a small protein in the major groove of the GAS associated with ongoing GBP transcription.
Mirkovitch
J
J
Rockefeller University, New York, New York 10021.
Decker
T
T
Darnell
J E
JE
D001483:Base Sequence; D002460:Cell Line; D004247:DNA; D003850:Deoxyribonuclease I / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000235:genetics*; D005786:Gene Expression Regulation; D006367:HeLa Cells; D006801:Humans; D016898:Interferon-alpha / Q000502:physiology*; D007371:Interferon-gamma / Q000502:physiology*; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D012045:Regulatory Sequences, Nucleic Acid; D013463:Sulfuric Acid Esters / Q000494:pharmacology; D016385:TATA Box; D014158:Transcription, Genetic
D016898:Interferon-alpha; D013463:Sulfuric Acid Esters; D007371:Interferon-gamma; D004247:DNA; D003850:Deoxyribonuclease I; D019204:GTP-Binding Proteins; C007482:dimethyl sulfate