HDAC6-ubiquitin interaction controls the duration of HSF1 activation after heat shock.
Bottom Line: Here we show that a full response to heat shock (activation of both HSP70 and HSP25) depends on the duration of HSF1 activation, which is itself controlled by HDAC6, a unique deacetylase known to bind monoubiquitin and polyubiquitin with high affinity.In cells expressing HDAC6 mutated in the ubiquitin-binding domain, the AAA ATPase factor p97/VCP mediates rapid inactivation of HSF1, precluding late activation of the HSP25 gene.In these cells, knockdown of p97/VCP rescues HSF1 from this rapid inactivation and restores HSP25 expression.
Affiliation: University Grenoble-Alpes, CRI INSERM, U823, Institut Albert Bonniot, La Tronche 38042, Grenoble Cedex 9, France.Show MeSH
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Mentions: We next compared the DNA-binding capacity of HSF1 to HSP70 and HSP25 gene promoters in the four cell lines. To this end, chromatin immunoprecipitation (ChIP) analysis was carried out with an anti-HSF1 antibody in both unstressed and stressed cells exposed or not to a period of recovery of 4.5–12 h at 37°C. Binding of HSF1 to both HSP70 and HSP25 gene promoters was then assessed with sets of primers surrounding the heat shock elements (HSEs) present in the HSP70 and HSP25 gene promoters (Figure 3A). HSF1 binding to the U6 gene promoter was also analyzed as a negative control. The efficacy of HSF1 immunoprecipitation was confirmed by Western blot analysis with an anti-HSF1 antibody (Supplemental Figure S3), and the specificity of binding was controlled by ChIP using an immunoglobulin G (IgG) antibody (Figure 3B). In wild-type cells, delayed binding of HSF1 to the HSP25 gene promoter was observed compared with that of the HSP70 gene promoter, demonstrating delayed action of HSF1 on the HSP25 gene. In wild-type cells, HSF1 binding to the HSP70 promoter was already observed in cells submitted to a 1-h heat shock, whereas, in contrast, HSF1 binding to the HSP25 promoter was detected only in cells submitted to a recovery period of 4.5 h at 37°C. Of interest, a twofold decrease in the binding ability of HSF1 to the HSP70 gene promoter was observed in the KO HDAC6 cell line. This observation indicates that despite a decrease in HSF1 binding to HSP70 gene promoter in KO HDAC6 cells, HSF1 phosphorylation and HSP70 activation are not affected, which suggests that full activation of HSP70 can occur despite a lower level of HSF1 binding to HSP70 promoter. This observation also holds for cells expressing Ubm and HDm HDAC6 mutants, for which less HSF1 binding to HSP70 gene promoter was also observed.
Affiliation: University Grenoble-Alpes, CRI INSERM, U823, Institut Albert Bonniot, La Tronche 38042, Grenoble Cedex 9, France.