In vivo protein-DNA interactions at the c-jun promoter: preformed complexes mediate the UV response.
Irradiation of cells with UV light triggers a genetic response, called the UV response, which results in induction of a set of genes containing AP-1-binding sites. The c-jun gene itself, which codes for AP-1-binding activity, is strongly (> 100-fold) and rapidly activated by UV. The UV induction of c-jun is mediated by two UV response elements consisting of AP-1-like sequences within its 5' control region. We have analyzed protein-DNA interactions in vivo at the c-jun promoter in noninduced and UV-irradiated HeLa cells. In vivo footprint analysis was performed by using dimethyl sulfate on intact cells and DNase I on lysolecithihin-permeabilized cells in conjunction with ligation-mediated polymerase chain reaction to cover about 450 bp of the c-jun promoter, including the transcription start sites. We find that this region does not contain methylated cytosines and is thus a typical CpG island. In uninduced cells, in vivo protein-DNA interactions were localized to an AP-1-like sequence (nucleotides [nt] -71 to -64), a CCAAT box element (nt -91 to -87), two SP1 sequences (nt -115 to -110 and -123 to -118), a nuclear factor jun site (nt -140 to -132), and a second AP-1-like sequence (nt -190 to -183). These results indicate that complex protein-DNA interactions exist at the c-jun promoter prior to induction by an external stimulus. Surprisingly, after stimulation of c-jun expression by UV irradiation, all in vivo protein-DNA contacts remained essentially unchanged, including the two UV response elements located at the AP-1-like sequences. The UV-induced signalling cascade leads to phosphorylation of c-Jun on serines 63 and 73 (Y. Devary, R.A. Gottlieb, T. Smeal, and M. Karin, Cell 71:1081-1091, 1992). Taken together, these data suggest that modification of the transactivating domain of DNA-bound c-Jun or a closely related factor may trigger the rapid induction of the c-jun gene.
Rozek
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Department of Biology, Beckman Research Institute of the City of Hope, Duarte, California 91010.
Pfeifer
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D001483:Base Sequence; D001665:Binding Sites; D002843:Chromatin / Q000648:ultrastructure; D004268:DNA-Binding Proteins / Q000378:metabolism*; D003850:Deoxyribonuclease I / Q000494:pharmacology; D003852:Deoxyribonucleoproteins / Q000737:chemistry; D005786:Gene Expression Regulation / Q000528:radiation effects*; D016758:Genes, jun; D006367:HeLa Cells; D006801:Humans; D066298:In Vitro Techniques; D008745:Methylation; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D016755:Proto-Oncogene Proteins c-jun / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism*; D014466:Ultraviolet Rays
D002843:Chromatin; D004268:DNA-Binding Proteins; D003852:Deoxyribonucleoproteins; D009838:Oligodeoxyribonucleotides; D016755:Proto-Oncogene Proteins c-jun; D014157:Transcription Factors; D003850:Deoxyribonuclease I