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HDAC6-ubiquitin interaction controls the duration of HSF1 activation after heat shock.

Pernet L, Faure V, Gilquin B, Dufour-Guérin S, Khochbin S, Vourc'h C - Mol. Biol. Cell (2014)

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.

View Article: PubMed Central - PubMed

Affiliation: University Grenoble-Alpes, CRI INSERM, U823, Institut Albert Bonniot, La Tronche 38042, Grenoble Cedex 9, France.

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The ZnF-UBP domain of HDAC6 is essential for the heat-induced transcriptional activation of the HSP25 gene. (A) The different cell lines analyzed in Figure 1 were submitted (+) or not (–) to heat shock (HS). After 8 h of recovery, total RNA fractions were prepared and analyzed by Northern blot with probes specific for HSP25 and HSP70 transcripts. A probe specific for the 18S transcripts was used as control. (B) Quantification based on two different Northern blots. Normalization calculated with the signals obtained with probe 18S. (C) Analysis of HSF1 migration profiles in the different cell lines submitted (HS) or not (NHS) to different heat-shock durations display similar kinetics of HSF1 hyperphosphorylation. Whole-cell extracts were analyzed by Western blot with an HSF1 antibody. Tubulin was used as loading control.
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Figure 2: The ZnF-UBP domain of HDAC6 is essential for the heat-induced transcriptional activation of the HSP25 gene. (A) The different cell lines analyzed in Figure 1 were submitted (+) or not (–) to heat shock (HS). After 8 h of recovery, total RNA fractions were prepared and analyzed by Northern blot with probes specific for HSP25 and HSP70 transcripts. A probe specific for the 18S transcripts was used as control. (B) Quantification based on two different Northern blots. Normalization calculated with the signals obtained with probe 18S. (C) Analysis of HSF1 migration profiles in the different cell lines submitted (HS) or not (NHS) to different heat-shock durations display similar kinetics of HSF1 hyperphosphorylation. Whole-cell extracts were analyzed by Western blot with an HSF1 antibody. Tubulin was used as loading control.

Mentions: We then sought to better characterize the mechanisms underlying the loss of HSP25 accumulation in cells expressing Ubm HDAC6 after heat shock. The putative effect of HDAC6 mutations on the level of HSP70 and HSP25 transcripts was therefore examined by Northern blot analysis in cells exposed or not to a 1-h heat shock followed by a recovery period of 8 h at 37°C. Quantification of HSP25 and HSP70 transcripts detected by Northern blot was performed on the basis of two independent Northern blot experiments. As shown in Figure 2, A and B, accumulation of HSP70 transcripts after heat shock was observed in all four cell lines. Of interest, although HSP25 transcripts were also detected in wild-type and knockout HDAC6 heat-shocked cells, as well as in cells expressing HDm HDAC6, no HSP25 transcripts were detected in cells expressing Ubm HDAC6. No HSP25 transcripts were detected across the entire time scale of the kinetics of recovery (Supplemental Figure S2). Taken together, Western and Northern blot analyses clearly indicate that absence of heat-induced accumulation of HSP25 in Ubm HDAC6– expressing cells results from a lack of heat-induced transcriptional activation of the HSP25 gene.


HDAC6-ubiquitin interaction controls the duration of HSF1 activation after heat shock.

Pernet L, Faure V, Gilquin B, Dufour-Guérin S, Khochbin S, Vourc'h C - Mol. Biol. Cell (2014)

The ZnF-UBP domain of HDAC6 is essential for the heat-induced transcriptional activation of the HSP25 gene. (A) The different cell lines analyzed in Figure 1 were submitted (+) or not (–) to heat shock (HS). After 8 h of recovery, total RNA fractions were prepared and analyzed by Northern blot with probes specific for HSP25 and HSP70 transcripts. A probe specific for the 18S transcripts was used as control. (B) Quantification based on two different Northern blots. Normalization calculated with the signals obtained with probe 18S. (C) Analysis of HSF1 migration profiles in the different cell lines submitted (HS) or not (NHS) to different heat-shock durations display similar kinetics of HSF1 hyperphosphorylation. Whole-cell extracts were analyzed by Western blot with an HSF1 antibody. Tubulin was used as loading control.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 2: The ZnF-UBP domain of HDAC6 is essential for the heat-induced transcriptional activation of the HSP25 gene. (A) The different cell lines analyzed in Figure 1 were submitted (+) or not (–) to heat shock (HS). After 8 h of recovery, total RNA fractions were prepared and analyzed by Northern blot with probes specific for HSP25 and HSP70 transcripts. A probe specific for the 18S transcripts was used as control. (B) Quantification based on two different Northern blots. Normalization calculated with the signals obtained with probe 18S. (C) Analysis of HSF1 migration profiles in the different cell lines submitted (HS) or not (NHS) to different heat-shock durations display similar kinetics of HSF1 hyperphosphorylation. Whole-cell extracts were analyzed by Western blot with an HSF1 antibody. Tubulin was used as loading control.
Mentions: We then sought to better characterize the mechanisms underlying the loss of HSP25 accumulation in cells expressing Ubm HDAC6 after heat shock. The putative effect of HDAC6 mutations on the level of HSP70 and HSP25 transcripts was therefore examined by Northern blot analysis in cells exposed or not to a 1-h heat shock followed by a recovery period of 8 h at 37°C. Quantification of HSP25 and HSP70 transcripts detected by Northern blot was performed on the basis of two independent Northern blot experiments. As shown in Figure 2, A and B, accumulation of HSP70 transcripts after heat shock was observed in all four cell lines. Of interest, although HSP25 transcripts were also detected in wild-type and knockout HDAC6 heat-shocked cells, as well as in cells expressing HDm HDAC6, no HSP25 transcripts were detected in cells expressing Ubm HDAC6. No HSP25 transcripts were detected across the entire time scale of the kinetics of recovery (Supplemental Figure S2). Taken together, Western and Northern blot analyses clearly indicate that absence of heat-induced accumulation of HSP25 in Ubm HDAC6– expressing cells results from a lack of heat-induced transcriptional activation of the HSP25 gene.

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.

View Article: PubMed Central - PubMed

Affiliation: University Grenoble-Alpes, CRI INSERM, U823, Institut Albert Bonniot, La Tronche 38042, Grenoble Cedex 9, France.

Show MeSH
Related in: MedlinePlus