Identification of a nuclear protein that constitutively recognizes the sequence containing a heat-shock element. Its binding properties and possible function modulating heat-shock induction of the rat heme oxygenase gene.
Heme oxygenase is an essential enzyme in heme catabolism, and also known as a 32-kDa heat-shock protein in rat. The rat heme-oxygenase gene promoter contains a functional heat-shock element (HSE) designated as HSE1 (-290 to -276 from the transcriptional initiation site), which consists of three copies of a 5-bp unit (5'-NGAAN-3';-->) in alternating orientation. Here we identified a putative HSE (-221 to -212), designated as HSE2, consisting of an inverted repeat of this 5-bp unit (<==>). Using transient expression assays, we show that HSE1 is sufficient to confer the heat-inducibility (a three fold to fourfold increase) on the reporter gene located downstream from the rat heme-oxygenase gene promoter, but HSE2 alone is not, suggesting that HSE2, a HSE of a tail-to-tail configuration, is not functional in vivo. However, the presence of both HSE1 and HSE2 in the promoter region increased the heat-mediated induction of the reporter-gene expression by more than 15-fold. Gel mobility-shift assays indicate that both HSE1 and HSE2 are recognized by activated heat-shock factor present only in heat-shocked rat glioma cells. Interestingly, the sequence containing HSE2 is also bound by a protein that is present in nuclear extracts prepared from either heat-shocked or non-shocked glioma cells, but this nuclear protein is unable to bind to HSE1. We suggest that a protein binding to the sequence containing HSE2 may be involved in transcriptional regulation of the rat heme oxygenase gene under thermal stress.
Okinaga
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Department of Applied Physiology and Molecular Biology, Tohoku University School of Medicine, Sendai, Japan.
Shibahara
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S
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D015342:DNA Probes; D004268:DNA-Binding Proteins / Q000378:metabolism*; D015971:Gene Expression Regulation, Enzymologic; D006367:HeLa Cells; D006360:Heat-Shock Proteins / Q000235:genetics*; D006419:Heme Oxygenase (Decyclizing) / Q000235:genetics*; D006358:Hot Temperature; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D051381:Rats; D012045:Regulatory Sequences, Nucleic Acid; D014158:Transcription, Genetic
D015342:DNA Probes; D004268:DNA-Binding Proteins; D006360:Heat-Shock Proteins; D009687:Nuclear Proteins; D006419:Heme Oxygenase (Decyclizing)