End-filling of an oligonucleotide duplex containing an MDBP site in the human hsp70 promoter inhibits protein·DNA complex formation

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.

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A cloned human CCAAT-box-binding factor stimulates transcription from the human hsp70 promoter

Molecular and Cellular Biology ◽

10.1128/mcb.10.12.6709-6717.1990 ◽

1990 ◽

Vol 10 (12) ◽

pp. 6709-6717

Author(s):

L S Lum ◽

L A Sultzman ◽

R J Kaufman ◽

D I Linzer ◽

B J Wu

Keyword(s):

Initiation Site ◽

Dependent Manner ◽

Hsp70 Gene ◽

Hsp70 Promoter ◽

Transcriptional Initiation ◽

Enhancer Binding Protein ◽

Binding Factor ◽

Ccaat Box ◽

Transcriptional Initiation Site ◽

Human Hsp70

The basal promoter of the human hsp70 gene is predominantly controlled by a CCAAT element at position -70 relative to the transcriptional initiation site. We report the isolation of a novel cDNA clone encoding a 114-kDa polypeptide that binds to the CCAAT element of the hsp70 promoter. Expression of this CCAAT-binding factor (CBF) cDNA activated transcription from cotransfected hsp70 promoter-reporter gene constructs in a CCAAT-dependent manner. CCAAT-binding factor shows no homology to the previously identified human CCAAT transcription factor or rat CCAAT/enhancer-binding protein.

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TATA-dependent and TATA-independent function of the basal and heat shock elements of a human hsp70 promoter

Molecular and Cellular Biology ◽

10.1128/mcb.10.4.1319-1328.1990 ◽

1990 ◽

Vol 10 (4) ◽

pp. 1319-1328

Author(s):

J M Greene ◽

R E Kingston

Keyword(s):

Heat Shock ◽

Consensus Sequence ◽

Simian Virus 40 ◽

Simian Virus ◽

Hsp70 Promoter ◽

Heat Shock Element ◽

Tata Element ◽

Sequence Elements ◽

Human Heat Shock Protein ◽

Human Hsp70

We have characterized the interactions between the TATA element and other sequence elements of a human heat shock protein 70 (hsp70) promoter by a mutational approach. Expression of a distal element of this promoter requires an intact TATA element in human cell lines. The hsp70 TATA element can be functionally replaced for this interaction by TATA elements from the simian virus 40 early and adenovirus EIIa promoters. The TATA element in this promoter therefore both determines the appropriate start site and determines strength by allowing function of the distal element. In contrast, three proximal upstream elements necessary for basal and heat-regulated transcription have no requirement either for a TATA element or for any other proximal element. The behavior of promoters multiply mutant in these proximal elements implies that these elements function independently. We examined the interaction between the heat shock element (HSE) and the TATA element as the distance between the two factor-binding sites was increased. It was necessary to create a mutant HSE with an extended consensus sequence in order for the HSE to function at a distance. Moving this extended HSE 500 bases upstream did not increase its dependence on the TATA element, suggesting that the TATA independence of this element is intrinsic to its function and is not determined by distance from the promoter.

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Binding of polyomavirus large T antigen to the human hsp70 promoter is not required for trans activation.

Molecular and Cellular Biology ◽

10.1128/mcb.6.9.3180 ◽

1986 ◽

Vol 6 (9) ◽

pp. 3180-3190 ◽

Cited By ~ 26

Author(s):

R E Kingston ◽

A Cowie ◽

R I Morimoto ◽

K A Gwinn

Keyword(s):

Protein Interactions ◽

Uninfected Cell ◽

T Antigen ◽

Antigen Binding ◽

Large T Antigen ◽

Antigen Binding Site ◽

Binding Interaction ◽

Hsp70 Promoter ◽

Early Proteins ◽

Human Hsp70

Polyomavirus large T antigen binds to two sites located between positions -110 and -170 of a human heat shock protein 70 (hsp70) promoter. Methylation interference studies show that binding for each site is determined by two GPuGGC pentanucleotide sequences. The specificity of this binding interaction is similar to that observed for large T binding to the viral genome. The existence of sequences that bind a viral protein in a cellular promoter raises the possibility that these sequences play a role in gene expression in an uninfected cell. We show that hsp70 large T antigen binding site 1 is capable of functioning as an upstream promoter element in cells that do not contain any viral T antigen. Genetic analysis of this effect suggests that a cellular factor exists that has a binding specificity that overlaps but is not identical to that of polyomavirus large T antigen. To determine whether binding of polyomavirus large T antigen can regulate expression of the intact human hsp70 promoter, we have introduced the promoter into mouse cells with plasmids that express the polyomavirus early proteins. These proteins stimulate the level of correctly initiated hsp70 transcripts, but surprisingly the degree of stimulation remains unchanged for promoter constructs in which the large T antigen binding sites have been deleted. These observations suggest that trans activation of the hsp70 promoter by the polyomavirus early proteins occurs through protein-protein interactions and not through sequence-specific DNA binding.

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

Molecular and Cellular Biology ◽

10.1128/mcb.9.6.2574 ◽

1989 ◽

Vol 9 (6) ◽

pp. 2574-2587 ◽

Cited By ~ 91

Author(s):

G T Williams ◽

T K McClanahan ◽

R I Morimoto

Keyword(s):

Transcription Initiation ◽

Promoter Sequence ◽

Initiation Site ◽

Transcription Unit ◽

Hsp70 Promoter ◽

Basal Transcription ◽

Induced Expression ◽

Transcriptional Complex ◽

Simplex Virus ◽

Human Hsp70

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.

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