Abstract
Tumor necrosis factor-alpha (TNFalpha) is a pivotal early mediator of host defenses that is essential for survival in infections. We previously reported that exposing macrophages to febrile range temperatures (FRT) (38.5-40 degrees C) markedly attenuates TNFalpha expression by causing abrupt and premature cessation of transcription. We showed that this inhibitory effect of FRT is mediated by an alternatively activated repressor form of heat shock factor 1 (HSF-1) and that a fragment of the TNFalpha gene comprising a minimal 85-nucleotide (nt) proximal promoter and the 138-nt 5'-untranslated region (UTR) was sufficient for mediating this effect. In the present study we have used an electrophoretic mobility shift assay (EMSA) to identify a high affinity binding site for HSF-1 in the 5'-UTR of the TNFalpha gene and have used a chromosome immunoprecipitation assay to show that HSF-1 binds to this region of the endogenous TNFalpha gene. Mutational inactivation of this site blocks the inhibitory effect of overexpressed HSF-1 on activity of the minimal TNFalpha promoter (-85/+138) in Raw 264.7 murine macrophages, identifying this site as an HSF-1-dependent repressor. However, the same mutation fails to block repression of a full-length (-1080/+138) TNFalpha promoter construct by HSF-1 overexpression, and HSF-1 binds to upstream sequences in the regions -1080/-845, -533/-196, and -326/-39 nt in EMSA, suggesting that additional HSF-1-dependent repressor elements are present upstream of the minimal -85-nt promoter. Furthermore, although mutation of the HSF-1 binding site in the minimal TNFalpha promoter construct abrogates HSF-1-mediated repression, the same mutation fails to abrogate repression of this construct by high levels of HSF-1 overexpression or exposure to 39.5 degrees C. This suggests that HSF-1 might repress TNFalpha transcription through redundant mechanisms, some of which might not require high affinity binding of HSF-1.
Highlights
Tumor necrosis factor-␣ (TNF␣) is a pivotal early mediator of host defenses that is essential for survival in infections
The 5Ј-untranslated region (UTR) sequence spanning ϩ30 to ϩ68 contains an array of four nGAAn elements, three of which would form a perfect heat shock response elements (HRE) if the “CT” sequence at nt ϩ57 and ϩ58 were inverted to “TC.” In our electrophoretic mobility shift assay (EMSA) analysis, we probed with oligonucleotides spanning Ϫ83/Ϫ43 and containing three nGAAn elements and the Sp-1 binding sequence, Ϫ15/ϩ5 containing one nGAAn and the transcription start site, ϩ30/ϩ68 and containing the imperfect HRE, and ϩ65/ϩ85 and ϩ101/ϩ121, each containing one nGAAn element
We showed that exposing Raw 264.7 cells to febrile range temperature (39.5 °C) activates the heat stress-activated transcription factor heat shock factor 1 (HSF-1), but HSF-1 in the 39.5 °C cell culture binds to its cognate DNA binding site, it fails to activate transcription from the HSP70 promoter
Summary
TNF␣, tumor necrosis factor ␣; LPS, bacterial endotoxin lipopolysaccharide; HSP, heat shock protein; HSF, expressed in response to infection and injury [1]. We showed that the heat stressactivated transcription factor, heat shock transcription factor 1 (HSF-1) is activated at febrile range temperatures (FRT) to an alternate DNA binding form that acts as a repressor of gene expression [15] and binds to the minimal temperature-responsive TNF␣ gene sequence (nt Ϫ85/ϩ138). The minimal temperature-responsive TNF␣ gene sequence (Ϫ85/ϩ138) was inhibited by HSF-1 overexpression, but this sequence did not contain a complete cognate HSF-1 bindheat shock factor; rHSF, recombinant HSF; HRE, heat shock response element; UTR, untranslated region; IL, interleukin; EMSA, electrophoretic mobility shift assay; FRT, febrile range temperature; nt, nucleotide(s); NF, nuclear factor; ChIP, chromosomal immunoprecipitation; STAT, signal transducers and activators of transcription. In the present study we have identified the high affinity binding site for HSF-1 in the minimal temperature-responsive TNF␣ gene sequence and showed that inactivating the site by mutation reverses the repression of this promoter fragment by HSF-1 overexpression
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
