E1a transactivation of the human HSP70 promoter is mediated through the basal transcriptional complex.

Abstract

We have examined the promoter sequence requirements for Ela 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 Ela responsive. Further deletion, abolishing a CCAAT element at -67, drastically reduced basal and Ela-induced expression. A linker-scanner analysis has identified four functional elements within the basal transcription unit which may interact with CTF, SPI, TFIID, and an ATF/APl-like factor. Sequences between -100 and -188 can partially compensate for mutations in these elements. No mutation specifically abolished Ela inducibility. Any reduction in absolute Ela-induced levels was accompanied by a corresponding reduction in absolute basal levels, thereby maintaining a constant relative fold induction. We conclude that Ela 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 Ela responsive. Mutagenesis of this promoter fusion demonstrated that the CCAAT element was essential for detectable basal and Ela-induced expression.Mutations in the purine-rich element resulted in an rendered the promoter nonresponsive to Ela.An important point at which gene expression is controlled is transcriptional initiation. The regulation of this step is mediated by DNA binding tr-ans-acting factors that select the site and modulate the rate of specific initiation (for reviews, see references 20 and 41). General transcription factors, such as TFIID, direct RNA polymerase II to the specific start site. The same types of general factors are probably required for specific initiation from most protein-coding genes. Promoter-specific factors bind to transcription elements that regulate the frequency of RNA chain initiation. One class of transcription elements, called upstream elements, is characterized by short sequences of approximately 10 base pairs (bp) that are usually located up to about 110 bp upstream from the RNA start site. These elements modulate transcription in a distance-dependent manner. The other class of transcription elements is enhancers, which are capable of modulating transcription independent of both orientation and distance. Regulation of promoter activity depends on both the number and the type of transcription elements present. The combinatorial organization of elements allows for many complex regulatory patterns with only a small number of transcription factors.Superimposed on the regulation conferred by protein-DNA interactions are the effects of transcription factors which do not bind to DNA. A well-studied example is the 289-amino-acid adenovirus Ela protein, which has ...

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