A novel G1-specific enhancer identified in the human heat shock protein 70 gene
| Autor: | Tomoko Narita, Hiroyoshi Ariga, Takahiro Taira, Sanae M.M. Iguchi-Ariga |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 1997 |
| Předmět: |
Transcription
Genetic Cyclin A Mutant Proliferating Cell Nuclear Antigen/genetics Polymerase Chain Reaction S Phase Mice L Cells G1 Phase Cyclins/genetics Cloning Molecular Luciferases Promoter Regions Genetic HSP70 Heat-Shock Proteins/biosynthesis L Cells (Cell Line) Cell Cycle Cyclins/biosynthesis 3T3 Cells Transfection Cell cycle Enhancer Elements Genetic Recombinant Fusion Proteins/biosynthesis Research Article Plasmids DNA Replication HSP70 Heat-Shock Proteins/genetics Recombinant Fusion Proteins Humans Biology Kinetics Cyclins Proliferating Cell Nuclear Antigen Prohibitins Genetics Animals HSP70 Heat-Shock Proteins Luciferase Enhancer Gene Luciferases/biosynthesis Promoter Molecular biology biology.protein Proliferating Cell Nuclear Antigen/biosynthesis |
| Zdroj: | Nucleic acids research. 25(10):1975-1983 |
| ISSN: | 0305-1048 |
| Popis: | Expression of the human heat shock protein 70 gene (hsp70) is induced by various kinds of stress and by oncogenes. In the absence of stress, hsp70 is mainly expressed in the G1and S phases of the cell cycle, but the elements contributing to cell cycle-dependent expression from the hsp70 promoter remain elusive. We have previously reported that two elements, named HSP-MYCA and HSP-MYCB, located approximately 200 bp upstream (-200) from the transcription start site (+1) of human hsp70, are important for initiation of DNA replication at the hsp70 locus. In this report we examine the effect of these two elements on transcriptional activity from the hsp70 promoter, especially in terms of cell cycle-dependent expression. Various segments of the hsp70 promoter region (up to -300) were linked to the luciferase gene and the constructs were transfected into mouse L cells to examine their transcriptional activity. A strong enhancer activity was defined in the HSP-MYCB element, but not in HSP-MYCA. Mutations introduced within HSP-MYCB abolished the transcriptional activation. In synchronized cells, pHB-Luc (a luciferase construct containing approximately 2.4 kb of the hsp70 promoter region) as well as endogenous hsp70 showed two peaks of expression; one in G1 and the other in the S phase. Site-directed mutagenesis of HSP-MYCB in pHB-Luc abolished the expression peak in G1, but not that in the S phase. To test promoter specificity, wild-type and mutant HSP-MYCB elements were then linked to the luciferase gene in combination with the hsp70 , the cyclin A or the PCNA promoter. Both in transient experiments and established cell lines, a strong peak of expression in mid-G1phase was observed with all the constructs containing wild-type HSP-MYCB, but not with the constructs containing the mutant sequence. These results suggest that the HSP-MYCB sequence is a G1-specific enhancer and is responsible for cell cycle-dependent expression of hsp70. |
| Databáze: | OpenAIRE |
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