E1a transactivation of the human HSP70 promoter is mediated through the basal transcriptional complex.
We have examined the promoter sequence requirements for E1a transactivation of the human HSP70 gene by using a transient cotransfection assay. A 5' deletion study has defined a basal transcription unit extending to -74 relative to the transcription initiation site which was fully E1a responsive. Further deletion, abolishing a CCAAT element at -67, drastically reduced basal and E1a-induced expression. A linker-scanner analysis has identified four functional elements within the basal transcription unit which may interact with CTF, SP1, TFIID, and an ATF/AP1-like factor. Sequences between -100 and -188 can partially compensate for mutations in these elements. No mutation specifically abolished E1a inducibility. Any reduction in absolute E1a-induced levels was accompanied by a corresponding reduction in absolute basal levels, thereby maintaining a constant relative fold induction. We conclude that E1a transactivation of the human HSP70 promoter does not require any single basal transcription element. We also examined an HSP70 promoter fragment, containing the CCAAT element at -67 and the purine-rich element at -54, out of its normal context by fusing it upstream of a transcriptionally inactive herpes simplex virus thymidine kinase deletion construct containing only the TATA box. The resulting chimeric promoter was fully E1a responsive. Mutagenesis of this promoter fusion demonstrated that the CCAAT element was essential for detectable basal and E1a-induced expression. Mutations in the purine-rich element resulted in an approximately 10-fold elevation in basal levels and rendered the promoter nonresponsive to E1a.
Williams
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GT
Department of Biochemistry, Molecular Biology, and Cellular Biology, Northwestern University, Evanston, Illinois 60208.
McClanahan
T K
TK
Morimoto
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RI
D017456:Adenovirus Early Proteins; D001483:Base Sequence; D002478:Cells, Cultured; D002678:Chimera; D002872:Chromosome Deletion; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics / Q000378:metabolism*; D005786:Gene Expression Regulation; D005822:Genetic Vectors; D006360:Heat-Shock Proteins / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D009856:Oncogene Proteins, Viral / Q000502:physiology*; D011401:Promoter Regions, Genetic; D012313:RNA / Q000235:genetics / Q000302:isolation & purification; D014157:Transcription Factors / Q000502:physiology*; D014158:Transcription, Genetic; D014162:Transfection
D017456:Adenovirus Early Proteins; D006360:Heat-Shock Proteins; D009856:Oncogene Proteins, Viral; D014157:Transcription Factors; D012313:RNA; D004247:DNA