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EMBO Rep. Jul 2008; 9(7): 662–669.
Published online May 2, 2008. doi:  10.1038/embor.2008.70
PMCID: PMC2475325
Scientific Report

A transcription cofactor required for the heat-shock response


The Stress-responsive activator of p300 (Strap) is a transcription cofactor that has an important role in the control of DNA damage response through its ability to regulate p53 activity. Here, we report that Strap is inducible by heat shock and stimulates the transcription of heat-shock genes. A chromatin-associated complex involving heat-shock factor 1 (HSF1), Strap and the p300 coactivator assembles on the heat-shock protein 70 (hsp70) promoter, and Strap augments HSF1 binding and chromatin acetylation in Hsp genes, most probably through the p300 histone acetyltransferase. Cells depleted of Strap do not survive under heat-shock conditions. These results indicate that Strap is an essential cofactor that acts at the level of chromatin control to regulate heat-shock-responsive transcription.

Keywords: heat shock, transcription, Strap, HSF1, p300


DNA damage is one form of cellular stress that activates an evolutionarily conserved signalling pathway that culminates in a checkpoint response, usually resulting in DNA repair, cell-cycle arrest and apoptosis (Zhou & Elledge, 2000). The p53 tumour suppressor protein has a crucial role in the DNA damage response through regulating various target genes that instigate the checkpoint response (Bates & Vousden, 1996). The ability of p53 to activate transcription is controlled by coactivator complexes, involving p300/CBP (CBP for CREB-binding protein) proteins, which physically interact with the transcription activation domain (Barlev et al, 2001; Espinosa & Emerson, 2001). The histone acetyltransferase (HAT) in p300/CBP acetylates the chromatin of p53-responsive target genes, together with lysine residues in the p53 protein, to augment the p53 response (Gu et al, 1997; Ito et al, 2001).

p300/CBP proteins interact with various cofactors that influence transcriptional activity (Chan & La Thangue, 2001). JMY (junction-mediating and regulatory protein) and Strap (Stress-responsive activator of p300) are two cofactors that cooperate in regulating p53 activity in a pathway in which Strap is targeted and regulated by DNA damage-responsive kinases (Shikama et al, 1999; Demonacos et al, 2001; Coutts & La Thangue, 2005; Coutts et al, 2007). For example, Strap is phosphorylated by the phosphatidylinositol-3 kinase-like kinase ATM (ataxia-telangiectasia mutated), which enables it to undergo nuclear accumulation (Demonacos et al, 2004). As a consequence, Strap forms a p300-dependent coactivator complex that activates p53 transcription (Demonacos et al, 2001, 2004). Six tetratricopeptide repeats (TPR), which function as protein-binding modules and facilitate protein assembly, are instrumental in allowing Strap to influence p300 activity (Demonacos et al, 2001).

However, DNA damage is only one form of cellular stress and it is possible that certain components of stress response pathways are shared between different types of stress. During the heat-shock response, a group of chaperones—exemplified by heat-shock protein (Hsp)90 and Hsp70—control various cellular processes, including protein folding, assembling multi-component protein complexes, translocation across cellular compartments and targeting protein degradation to the proteasome (Nollen & Morimoto, 2002). As a consequence, the action of chaperones in response to heat shock is believed to maintain the balance between misfolded and native-state proteins.

The expression of Hsp70 and Hsp90 genes is regulated through a conserved transcription response involving heat-shock factor 1 (HSF1), which binds to heat-shock elements (HSEs) located in the promoters of target genes (Morimoto, 2002). Activation of HSF1 requires phosphorylation and trimerization, which then allows HSF1 to bind to the HSE (Morimoto, 2002). In turn, chaperone activity is influenced by a group of regulatory proteins, called co-chaperones, which physically bind to the Hsp70/Hsp90 complex (Morimoto, 1998).

To explore the interaction between different stress response pathways, we tested whether Strap participates in the heat-shock response. Here, we describe an unexpected yet crucial role of Strap in the heat-shock response. Under heat-shock conditions, Strap assembles with HSF1 and p300, and activates transcription through the HSE. A heat-inducible chromatin-associated complex involving HSF1, Strap and p300 coincides with enhanced chromatin acetylation in Hsp genes through a mechanism that probably involves the HAT activity of p300. Notably, cells depleted of Strap are compromised in their ability to survive under heat-shock conditions. Thus, Strap is a cofactor that participates in heat-shock-responsive transcription and facilitates cell survival under heat shock.


Strap stimulates the Hsp70 promoter

As Strap is a transcription cofactor, we considered that it might have a wider role in the cellular stress response, for example, in regulating heat-shock-inducible transcription. Therefore, we assessed the effect of Strap on the hsp70B promoter, which is heat-shock inducible through several HSEs (Voellmy et al, 1985; Wang et al, 2000). In a transfection-based reporter assay, Strap efficiently activated transcription driven by the hsp70B promoter (Fig 1A). Activation was dependent on the presence of HSEs because a reporter construct lacking the HSE could not be activated (Fig 1A). As expected, heat-shock-dependent activation of the hsp70B promoter was mediated through the HSE (Fig 1A).

Figure 1
Strap activates heat-shock protein genes. (A) Luciferase reporter assays were performed using a pGL3-luc vector (200 ng) containing hsp70B promoter (+HSE) and a control pGL3-luc vector (−HSE). Expression vectors encoding Strap (200 or ...

The ability of Strap to activate the hsp70B promoter reflected an increase in endogenous Hsp70-inducible protein and RNA because the expression of Strap increased the level of Hsp70 under heat shock (Fig 1B,C), which was mirrored by a concomitant increase in hsp70 RNA (Fig 1D). To confirm that endogenous Strap regulates hsp70 expression, we used a short interfering RNA (siRNA) that specifically depletes endogenous Strap (Fig 1E). Treating cells with Strap siRNA reduced the level of Hsp70, which was most evident in heat-shocked cells (Fig 1E). The effect was specific because lamin siRNA, used as the control siRNA (Coutts et al, 2007), did not affect Hsp70 induction (Fig 1E; supplementary Fig 1A online).

Strap and HSF1 exist in a heat-inducible complex

To investigate the mechanism by which Strap augments Hsp70 transcription, we tested whether Strap associates with HSF1 under heat-shock conditions and, thereafter, whether Strap exists in a chromatin-bound complex on the hsp70B promoter. An interaction was apparent between endogenous HSF1 and Strap (Fig 2A). Although the complex was present in normal cells, the level of Strap bound to HSF1 increased by about threefold on heat shock and, under heat-shock conditions, Strap associated with the heat-inducible phosphorylated form of HSF1 (Fig 2A). The interaction between Strap and HSF1 reflected a time-dependent increase in the steady-state level of Strap protein under heat-shock conditions, as shown by immunoblotting experiments (Fig 2Bi). As expected, DNA-damaging agents caused an induction of Strap (Fig 2Bi; Demonacos et al, 2004). To confirm that the regulation of ectopic Strap reflected physiological control, we investigated the response of Strap in several cell types (here the response in U2OS cells is shown). Endogenous Strap behaved similarly to the ectopic protein; the response was maximum after 120–150 min heat-shock treatment and resulted in a 2.5-fold increase in levels of Strap (Fig 2Bii).

Figure 2
Strap binds to heat-shock factor 1 and undergoes heat-shock-dependent chromatin association. (A) Whole-cell lysates from normal or heat-shocked (43°C, 1 h; HS) U2OS cells transfected with pcDNA3 or haemagglutinin (HA)-Strap (2 μg) were ...

Strap and HSF1 are chromatin associated, as both proteins were shown, by chromatin immunoprecipitation (ChIP) analysis, to be present in the chromatin of the hsp70B promoter (Fig 2C). Consistent with the interaction between Strap and HSF1, when Strap was expressed ectopically, the increased level of chromatin-bound Strap reflected a concomitant increase in the association of endogenous HSF1 with chromatin (Fig 2C; supplementary Fig 1C online), indicating that Strap augments the DNA-binding activity of HSF1. Endogenous Strap was present on the hsp70B promoter, which also increased under heat-shock conditions (Fig 2D).

To clarify whether Strap and HSF1 exist in a complex on chromatin, we performed a sequential ChIP analysis in which chromatin bound to HSF1 was re-immunoprecipitated with a Strap antibody (by using a haemagglutinin (HA) antibody against HA-Strap). Strap and HSF1 were found to coexist in a heat-inducible chromatin-associated complex (Fig 2E). Again, the increased level of Strap that resulted from expressing the ectopic protein correlated with enhanced HSF1 binding. To obtain further evidence that endogenous Strap influenced chromatin-associated HSF1, we performed a ChIP analysis with cells treated with Strap siRNA. Under conditions of Strap depletion (Fig 1E), there was a coincident reduction in chromatin-associated HSF1 (Fig 2F). Thus, the association between Strap and HSF1 is heat inducible, Strap and HSF1 exist in a chromatin-associated complex and Strap regulates the level of chromatin-bound HSF1.

Strap modulates the HAT activity of p300

As Strap interacts with p300 coactivators (Demonacos et al, 2001), we reasoned that p300 might be involved in heat-shock-dependent transcription. Moreover, as p300 is a HAT and because p300 HAT is required for activation by certain transcription factors (Ito et al, 2001), the ability of Strap to activate the hsp70 gene might involve p300 and altered chromatin acetylation.

Strap was present in p300 immunoprecipitates, and the level of Strap in the p300 complex was higher in heat-shocked cells compared with untreated cells (Fig 3A), which coincided with the presence of HSF1 (supplementary Fig 1B online). Consequently, we assessed whether Strap could affect the HAT activity of p300, by studying p300 in a U2OS cell line in which Strap expression was under conditional control by using the TET-ON expression system (Fig 3B). p300 was immunoprecipitated from cell extracts in the presence of increasing levels of Strap and, thereafter, p300 HAT was measured by monitoring the acetylation of core histones. p300 immunocomplexes showed an increased level of acetyltransferase activity when immunoprecipitated from cell extracts containing induced levels of Strap (Fig 3B,C), suggesting that Strap augments the HAT activity of p300.

Figure 3
Strap regulates p300 histone acetyltransferase. (A) Immunoprecipitation (IP) was performed in normal or heat-shocked (43°C, 1 h; HS) U2OS cells that stably express Flag-Strap (Demonacos et al, 2004). p300 immunocomplexes were immunoblotted by ...

Next, we tested whether Strap had a direct influence on p300 HAT by studying the effect of purified Strap on the acetylation of core histones. Wild-type Strap stimulated the HAT activity of recombinant p300 by about twofold (Fig 3D). By contrast, denatured Strap failed to affect p300 HAT and, similarly, a truncated Strap derivative (Demonacos et al, 2001) could not stimulate p300 HAT (Fig 3D). These results indicate that Strap can augment the HAT activity of p300.

Strap augments chromatin acetylation in Hsp genes

Given the heat-shock-dependent association between Strap and p300, we investigated whether p300 was present on the hsp70B promoter. ChIP analysis showed that the level of chromatin-bound p300 increased under heat shock in a manner that paralleled the increase in HSF1 binding (Fig 4A). We reasoned further that if Strap augments p300 HAT as part of the HSF1 complex, then we might expect that the acetylation of chromatin in heat-shock-responsive genes would increase in a Strap-dependent manner. We investigated this possibility by performing ChIP assays, in which chromatin bound to the hsp70B and hsp90β promoters was immunoprecipitated with an acetyl histone H4 antibody in the presence of increasing levels of Strap. The expression of Strap correlated with increased chromatin acetylation in the hsp70 and hsp90 promoters in heat-shocked cells (Fig 4B). Trichostatin A, which blocks histone deacetylase, did not enhance acetylation to a similar extent as in the presence of ectopic Strap (Fig 4B), indicating that altered HAT activity—rather than decreased histone deacetylase activity—was responsible for the increased acetylation. The presence of Strap therefore enhances the level of chromatin acetylation in Hsp genes.

Figure 4
Strap augments chromatin acetylation in Heat-shock protein genes. (A) U2OS cells were treated with or without heat shock (43°C, 1 h; HS) and aliquots of chromatin were immunoprecipitated with p300, HSF1 or control antibodies. The PCRs were performed ...

Strap is required for cell survival during heat-shock

The results described above indicate that Strap has a crucial role in regulating the transcription programme induced by heat shock. To establish whether this role was functionally important in cell survival during the heat-shock response, we reduced the level of endogenous Strap through siRNA-dependent depletion (Fig 1E) and then measured the level of cell survival under lethal heat-shock conditions (45°C for 90 min; Shamovsky et al, 2006; Westerheide et al, 2006). Flow cytometric analysis showed that introducing Strap siRNA into cells growing under normal culture conditions caused an increase in the G2/M population relative to the control siRNA lamin treatment (Fig 5A). As expected from previous reports (Kuhl & Rensing, 2000), heat-shock treatment caused an increase in the G2/M population, and this effect was similar in both the mock and siRNA lamin treatments (Fig 5A). However, Strap siRNA-treated cells that had been subjected to heat shock underwent a significant increase in the level of apoptosis compared with cells that underwent control treatment (Fig 5A). Cells depleted of Strap therefore have impaired survival under heat-shock conditions.

Figure 5
Strap is required for survival under heat shock. (A) U2OS cells were treated with control siRNA lamin, siRNA Strap or mock treated for 72 h and then subjected to lethal heat shock (45°C, 90 min) and recovered at 37°C for 16 h before being ...


Strap influences heat-shock-induced transcription

We have obtained evidence that Strap regulates the transcription of Hsp genes. Specifically, ectopic Strap augmented hsp70 and hsp90 expression and, conversely, the depletion of endogenous Strap reduced hsp70 expression. Moreover, Strap is present in the chromatin of hsp70 and hsp90, activates transcription from the Hsp70 promoter and formed a chromatin-bound protein complex with HSF1. In fact, increased levels of Strap correlated with HSF1 binding and, conversely, depleting Strap caused a coincident reduction in chromatin-associated HSF1. Strap therefore has an important role in regulating HSF1 activity and Hsp gene transcription during heat shock. This is supported by the effect of Strap depletion on cell viability, where Strap is required for cell survival under heat shock.

At a mechanistic level, Strap enhanced chromatin acetylation of the Hsp70 and Hsp90 genes, suggesting a mechanism that might account for the ability of Strap to facilitate transcriptional activation. p300 co-immunoprecipitated with Strap and HSF1, and therefore is a candidate for mediating increased acetylation. Previous reports have suggested an association between Hsp70/Hsp90 and HSF1 (Abravaya et al, 1992; Thomson et al, 2004), and a role for p300 in the control of Hsp transcription (Li et al, 1998), and it has generally been regarded that the Hsp70/Hsp90 chaperone provides a feedback loop involved in downregulating HSF1-dependent transcription (Morimoto, 1998). From these results, it is possible that Strap participates in regulating this feedback loop, perhaps through its ability to recruit p300 and influence HSF1 activity (Fig 5B).

Strap integrates transcription with cellular stress

Strap participates in the cellular response to DNA damage (Demonacos et al, 2001, 2004). Under DNA damage, Strap drives the assembly of a coactivator complex containing p300 and several other cofactors (Demonacos et al, 2001). In fact, Strap acts to assemble a functionally competent transcription-regulating coactivator complex (Demonacos et al, 2001). DNA damage signalling kinases—most notably ATM—regulate Strap activity and, in turn, influence the assembly of the coactivator complex (Demonacos et al, 2004).

The studies described here indicate that Strap takes on a similar role during the heat-shock response, by interacting with HSF1 and recruiting p300. Strap therefore has a conserved role in regulating coactivator function during the cellular response to various forms of stress. The different roles assigned to Strap might reflect distinct stress-responsive signalling pathways, and it will be interesting to clarify the nature of the signals that influence Strap during the heat-shock response.


Plasmids and expression vectors. pcDNA3, Flag-p300, HA-Strap, pET28 Strap(wt) and the Strap deletion mutant TPR1–3 (1–205) were cloned into either HA-tagged pcDNA3 or pET28 vectors. A firefly luciferase reporter vector containing the hsp70B promoter (372 bp containing two HSEs) region was as described previously (Wang et al, 2000).

Cell culture and transfection. U2OS cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum at 37°C with 5% CO2. Transfections were carried out by using Gene Juice (Novagen, Wisconsin, WI, USA) as described in the protocol. Inducible Strap stable cell lines, which were generated previously by using the TET-ON gene expression system (Demonacos et al, 2004), were grown and induced by the addition of doxycyclin (1 μg/ml) for 36 h. Except for Figs 2B, ,5A,5A, heat-shock treatment was performed at 43°C for 1 h (Zou et al, 1998). In Fig 2B, cells were heat shocked at 43°C for the indicated periods of time, followed by 4–6 h recovery at 37°C (Beere et al, 2000). In Fig 5A, lethal heat-shock treatment was performed at 45°C for 90 min, followed by a recovery period of 16 h at 37°C (Shamovsky et al, 2006; Westerheide et al, 2006).

Supplementary information is available at EMBO reports online (http://www.emboreports.org).

Supplementary Material

Supplementary Information


We thank the Medical Research Council, Cancer Research UK, the Association for International Cancer Research and the European Union for supporting this research. D.X. was in receipt of an Overseas Research Scholarship and a University Studentship. We are grateful to A. Coutts for comments and R. Williams for help in preparing the manuscript.


The authors declare that they have no conflict of interest.


  • Abravaya K, Myers MP, Murphy SP, Morimoto RI (1992) The human heat shock protein hsp70 interacts with HSF, the transcription factor that regulates heat shock gene expression. Genes Dev 6: 1153–1164 [PubMed]
  • Barlev NA, Liu L, Chehab NH, Mansfield K, Harris KG, Halazonetis TD, Berger SL (2001) Acetylation of p53 activates transcription through recruitment of coactivators/histone acetyltransferases. Mol Cell 8: 1243–1254 [PubMed]
  • Bates S, Vousden KH (1996) p53 in signaling checkpoint arrest or apoptosis. Curr Opin Genet Dev 6: 12–18 [PubMed]
  • Beere HM, Wolf BB, Cain K, Mosser DD, Mahboubi A, Kuwana T, Tailor P, Morimoto RI, Cohen GM, Green DR (2000) Heat-shock protein 70 inhibits apoptosis by preventing recruitment of procaspase-9 to the Apaf-1 apoptosome. Nat Cell Biol 2: 469–475 [PubMed]
  • Chan H-M, La Thangue NB (2001) p300/CBP proteins: HATs for transcriptional bridges and scaffolds. J Cell Sci 114: 2363–2373 [PubMed]
  • Coutts AS, La Thangue NB (2005) The p53 response: emerging levels of co-factor complexity. Biochem Biophys Res Commun 331: 778–785 [PubMed]
  • Coutts AS, Boulahbel H, Graham A, La Thangue NB (2007) Mdm2 targets the p53 transcription co-factor JMY for degradation. EMBO Rep 8: 84–90 [PMC free article] [PubMed]
  • Demonacos C, Krstic-Demonacos M, La Thangue NB (2001) A novel TPR-motif co-factor contributes to p300 activity in the p53 response. Mol Cell 8: 71–84 [PubMed]
  • Demonacos C, Krstic-Demonacos M, Smith L, Xu D, O'Connor P, Jansson M, La Thangue NB (2004) A new effector pathway links ATM kinase with the DNA damage response. Nat Cell Biol 6: 968–976 [PubMed]
  • Espinosa JM, Emerson BM (2001) Transcriptional regulation by p53 through intrinsic DNA/chromatin binding and site-directed cofactor recruitment. Mol Cell 8: 57–69 [PubMed]
  • Gu W, Shi XL, Roeder RG (1997) Synergistic activation of transcription by CBP and p53. Nature 387: 819–823 [PubMed]
  • Ito A, Lai CH, Zhao X, Saito S, Hamilton MH, Appella E, Yao TP (2001) p300/CBP-mediated p53 acetylation is commonly induced by p53-activating agents and inhibited by MDM2. EMBO J 20: 1331–1340 [PMC free article] [PubMed]
  • Kuhl NM, Rensing L (2000) Heat shock effects on cell cycle progression. Cell Mol Life Sci 57: 450–463 [PubMed]
  • Li Q, Herrler M, Landsberger N, Kaludov N, Ogryzko VV, Nakatani Y, Wolffe AP (1998) Xenopus NF-Y pre-sets chromatin to potentiate p300 and acetylation-responsive transcription from the Xenopus hsp70 promoter in vivo. EMBO J 17: 6300–6315 [PMC free article] [PubMed]
  • Morimoto RI (1998) Regulation of the heat shock transcriptional response: cross talk between a family of heat shock factors, molecular chaperones, and negative regulators. Genes Dev 12: 3788–3796 [PubMed]
  • Morimoto RI (2002) Dynamic remodeling of transcription complexes by molecular chaperones. Cell 110: 281–284 [PubMed]
  • Nollen EA, Morimoto RI (2002) Chaperoning signaling pathways: molecular chaperones as stress-sensing ‘heat shock' proteins. J Cell Sci 115: 2809–2816 [PubMed]
  • Shamovsky I, Ivannikov M, Kandel ES, Gershon D, Nudler E (2006) RNA-mediated response to heat shock in mammalian cells. Nature 440: 556–560 [PubMed]
  • Shikama N, Lee C-W, Delavaine S, France J, Lyon W, Krstic-Demonacos M, La Thangue NB (1999) A novel co-factor for p300 that regulates the p53 response. Mol Cell 4: 365–376 [PubMed]
  • Thomson S, Hollis A, Hazzalin CA, Mahadevan LC (2004) Distinct stimulus-specific histone modifications at hsp70 chromatin targeted by the transcription factor heat shock factor-1. Mol Cell 15: 585–594 [PubMed]
  • Voellmy R, Ahmed A, Schiller P, Bromley P, Rungger D (1985) Isolation and functional analysis of a human 70,000-dalton heat shock protein gene segment. Proc Natl Acad Sci USA 82: 4949–4953 [PMC free article] [PubMed]
  • Wang X, Asea A, Xie Y, Kabingu E, Stevenson MA, Calderwood SK (2000) RSK2 represses HSF1 activation during heat shock. Cell Stress Chaperones 5: 432–437 [PMC free article] [PubMed]
  • Westerheide SD, Kawahara TL, Orton K, Morimoto RI (2006) Triptolide, an inhibitor of the human heat shock response that enhances stress-induced cell death. J Biol Chem 281: 9616–9622 [PubMed]
  • Zhou BB, Elledge SJ (2000) The DNA damage response: putting checkpoints in perspective. Nature 408: 433–439 [PubMed]
  • Zou J, Guo Y, Guettouche T, Smith DF, Voellmy R (1998) Repression of heat shock transcription factor HSF1 activation by HSP90 (HSP90 complex) that forms a stress-sensitive complex with HSF1. Cell 94: 471–480 [PubMed]

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