We have demonstrated that heat acclimation (AC) causes selective, long-lasting, transcriptional changes in cytoprotective and chromatin remodeling-associated genes, which maintain their AC transcriptome profile, despite the loss of the AC phenotype (Tetievsky et al. Physiol Genomics 34: 78–87, 2008). We postulated that AC memory involves upstream epigenetic information, which predisposes to rapid reacclimation (ReAC) and cytoprotective memory. Here we tested the hypothesis that posttranslational histone modifications are linked to this process. Rats subjected to AC (34°C for 2 or 30 days), deacclimation (DeAC; 24°C, 30 days), and ReAC (34°C, 2 days), and untreated controls were used. Histone H4 lysine acetylation and histone H3 acetylation and phosphorylation in the heat shock element (HSE) of the promoters of heat shock protein-70 (hsp70) and -90 (hsp90) genes were examined. Histone acetyltransferase recruitment of TIP60 (60-kDa histone acetyltransferase-interactive protein), the catalytic subunit of NuH4, was used to validate acetylation. Heat shock factor-1 (HSF-1)-HSE binding to the hsp70 and hsp90 genes was measured to confirm HSF-1 binding to euchromatin. Our results indicate that, while histone H3Ser10 phosphorylation occurred during the AC 2-day phase, AC constitutively elevated histone H4 acetylation in the HSE of hsp70 and hsp90 promoters. HSF-1-HSE binding was detected in the hsp70 gene throughout AC-DeAC-ReAC. The hsp90 gene lacked HSF-1 binding during DeAC, but resumed a high binding level upon ReAC. HSP-90 is a critical cytoprotective protein, and the HSF-1-hsp90 binding profile matched levels of this protein. We conclude that, while early histone H3 phosphorylation is probably required for subsequent histone H4 acetylation, the constitutively acetylated histone H4 and the preserved euchromatin state throughout AC-DeAC-ReAC predispose to rapid cytoprotective acclimatory memory.
- histone H4 acetylation
- histone H3 phosphorylation
- heat shock factor-1-heat shock element binding
- cytoprotective memory
humans and animals display a “within lifetime” ability to adjust their physiological mechanisms to overcome long-lasting shifts in ambient temperature. This process, called heat acclimation (AC), enhances endurance and tolerance to temperature extremes (13). The AC process also confers cross-tolerance to diverse stressors, inducing similar cytoprotective signaling pathways, of which cross-tolerance to ischemia-reperfusion insult in the heart (21) and to hyperoxia in the brain (1) are well documented (14). These AC benefits are temporary, yet the few published investigations on the time course of AC loss indicate that reacclimation (ReAC) is rapid upon return to a hot environment (26, 47). Understanding the time course of the decay and reacquisition of AC has theoretical and practical importance to human and animal populations at risk of thermal injury.
Using an experimental rat model, we showed that reinduction of the AC phenotype [even after 2-mo deacclimation (DeAC)] only takes 2 days, rather than the 30 days initially required to achieve acclimatory homeostasis, implying that AC involves memory (39). As in human studies, in our rat model the rapid return to the AC phenotype (26, 27, 31, 35, 38, 47) was noted in both thermoregulatory responses to heat stress and cytoprotective mechanisms (cross-tolerance). A genomewide approach (17) highlighted gene clusters, including several genes from the heat shock protein (HSP) family, their transcription factor heat shock factor-1 (HSF-1), chromatin remodelers, and genes linked with memory, that are altered during AC and maintain their altered expression throughout DeAC, despite the return of the DeAC phenotype to pre-AC/control physiological characteristics (39). This dichotomy led us to conclude that AC induces a long-lasting, transcriptional program that enables individuals who have undergone an initial AC session to achieve faster ReAC. Taken together, the reprogramming of chromatin remodelers and the uniform expression profile of gene clusters throughout DeAC/ReAC led us to hypothesize that the phenotypic plasticity seen in ReAC might be associated with upstream epigenetic information (42) generated in response to AC, predisposing to cellular acclimatory memory.
In a broad sense, the transfer of epigenetic information, such as instructions like “do not transcribe this gene” or “transcribe this gene if...”, is associated with chromatin remodeling (42) via molecular and biochemical processes that maintain the chromatin-DNA package in active or silent states. Posttranslational modifications (e.g., lysine acetylation, serine phosphorylation) of the NH2-terminal tails of histone proteins H3 and H4 that protrude from the nucleosome are the most common forms of chromatin remodeling (44–46). Histone acetylation and phosphorylation are associated with chromatin opening and the initiation of transcription (9, 20). Such modifications can be controlled by intracellular signaling [reviewed in Cohen and Yao (6)] and are likely to be important players in selective epigenetic tagging when environmental stressors are involved.
In yeasts, Hirota et al.(11) confirmed the stress (osmotic) specificity of chromatin tagging, which involves phosphorylation around the cAMP-response element and an activated protein kinase (SAPK) cascade. Using a similar principle, Rahman et al. (32) attributed the impact of chronic oxidative stress on proinflammatory responses in the lung to chromatin remodeling and signaling. With respect to thermally adverse environments, using chicks subjected to heat stress episodes during the critical period of postnatal thermoregulatory establishment, Kisliouk and Meiri (18) highlighted the specific epigenetic role of chromatin modifications at the Bdnf promoter in hypothalamic brain tissue in the acquisition of thermotolerance. Collectively, this compendium of evidence suggests that epigenetic mechanisms bridge environmental influences and gene expression, thereby affecting physiological responses to changing environmental conditions.
Studies are sparse concerning epigenetic mechanisms occurring during exposure to acute or chronic stress in adulthood or “within-life” long-term adaptive processes to environmental challenges such as AC. The phenomenon of “acclimation imprinting” is currently confined to physiological responses (26, 47). Nevertheless, analogies and perspectives can be drawn from several recent works on the epigenome of within-life psychological stress. Reul and Chandramohan (33) discovered that, in rats and mice, elevated and long-lasting phosphoacetylation of histone H3 occurs in neurons following exposure to physiological stress in a functional context, i.e., in those neurons involved in stress-related memory formation. The authors observed that the increase in histone H3 phosphoacetylation in dentate neurons after physiological stress causes a distinct and transient gene transcription response.
The finding that 1) heat stress induces selective histone H4 acetylation (41) and hsp70 transcription in a mammalian species, 2) the transcription factor HSF-1 is essential for directing histone H4 acetylation (but not histone H3 acetylation) of hsp70 chromatin (41), and 3) phosphorylation of H3Ser10 occurs in transcriptionally activated heat shock loci (25), together with our findings that hsps, Hsf1, and chromatin remodelers are included in gene clusters with altered expression throughout AC, DeAC, and ReAC, in tandem with the physiological loss of the AC phenotype on DeAC and reacquisition of AC and cross-tolerance on ReAC (39), provided the rational for our hypothesis that AC memory is linked to chromatin remodeling and substantiates the suitability of our model for this study.
The aim of this investigation, using the HSP system as a prototype, was to determine whether “AC memory” involves epigenetic machinery. To this end, we studied histone modifications at the heat shock element (HSE) binding site of the promoters of hsp70 and hsp90. These modifications may provide clues regarding the chromatin state. We studied the hearts of rats undergoing AC-DeAC-ReAC (34°C-24°C-34°C) to evaluate histone modifications by measuring 1) the levels of acetylated histones H4 and H3 and phosphorylated histone H3, 2) the level of TIP60 histone acetyltransferase (HAT) recruitment to the hsp70 and hsp90 promoters, and 3) the binding of HSF-1 to HSE of hsp70 and hsp90 as an indicator of the initiation of transcription.
Our data provide clear evidence that, throughout AC-DeAC-ReAC, cytoprotective memory is linked to AC-induced, constitutively elevated acetylation of histone H4 at the HSE of hsp70 and hsp90. In the hsp70 gene, HSF-1-HSE binding was detected in all experimental phases; the hsp90 gene, however, lacked HSF-1 binding during DeAC (normothermic temperature 24°C), but resumed a high binding level when the animals returned to the acclimating temperature. The HSF-1-hsp90 binding profile matched that of HSP-90, a critical cytoprotective protein (29). We conclude that the constitutively acetylated histone H4 and preserved euchromatin state found throughout AC-DeAC-ReAC lead to acclimatory memory, namely, a predisposition to prompt HSF-1-HSE binding and subsequent activation of the cytoprotective heat shock response (HSR).
MATERIALS AND METHODS
Animals and maintenance.
All experimental protocols were approved by the Ethics Committee for Animal Experimentation of The Hebrew University, Jerusalem, Israel. Male Rattus norvegicus (Sabra strain), initially weighing 80–90 g (3-wk-old), were fed Ambar laboratory chow with water ad libitum and held under light-dark cycled conditions (12:12 h). The animals were randomly assigned to the following groups: heat acclimated for 2 (AC2d) and 30 days (AC); deacclimated for 1 mo (DeAC); reacclimated for 2 days after the 1-mo deacclimation (ReAC), and control, normothermic (C) (13, 39) (Fig. 1). C and DeAC rats were held at an ambient temperature of 24 ± 1°C; AC was attained by continuous exposure to 34 ± 1°C and 30–40% relative humidity, as described in Ref. 13. Chromatin remodeling via histone (H4, H3) acetylation/phosphorylation, TIP60 recruitment, and binding of HSF-1 to the HSE of the promoters of hsp70 were assessed for each group (Fig. 1).
Immediately on the termination of each AC or normothermic period, the animals were weighed and euthanized by ketamine-xylazine (8.5 mg/100g body wt ketamine in 0.5% xylazine ip) anesthesia, followed by cervical dislocation. The hearts were removed, weighed, and mounted on a Langendorff perfusion system, retrogradely perfused for 2 min, with Krebs-Henseleit buffer containing (in mM) 120 NaCl, 4.7 KCl, 1.2 MgSO4, 1.2 KH2PO4, 1.25 CaCl2, 25 NaHCO3, and 11 glucose, at pH 7.4, and aerated with a mixture of 95% O2-5% CO2 at 37°C, with a perfusion pressure of 100 cmH2O. The left ventricle was then excised and stored at −70°C until further processing (16, 39).
Tissue preparation and chromatin immunoprecipitation.
Fractionation of chromatin was performed according to the protocol of Umlauf et al. (46), modified for cardiac tissue. Briefly, 250 mg of frozen heart muscle were minced on dry ice. Chromatin was solubilized and extracted using detergent lysis. The chromatin underwent further digestion using 10 units of micrococcal nuclease (MNase I, Worthington) added to 500 μl of soluble chromatin at 37°C for 20 min, to obtain fragments of 200–1,000 bp. The reaction was stopped by adding EDTA to a final concentration of 20 mM at 4°C. Fragment size was validated on an agarose gel. Chromatin fractions were precleared by adding 60 μl of protein G agarose/salmon sperm DNA (Upstate) to 1 ml of chromatin immunoprecipitation dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris·HCl, pH 8.1, and 167 mM NaCl) and incubated for 30 min at 4°C on a rotating platform. After agarose removal by centrifugation, 1% of the precleared chromatin was saved and used as input DNA. Antibodies directed against acetyl H3K9, acetyl H3K18, acetyl H3K23, or phosphor (P)-H3Ser10 (5 μl/sample, H3 antibodies kit C-9927, Cell Signaling Technology); acetyl lysines 5, 8, 12, and 16 of histone H4 (5 μg/sample 06–598, Upstate Biotechnology); anti-HSF-1 (10 μl/sample ab2923; Abcam); and anti-TIP60 (5 μg/sample SC-5727, Santa Cruz) were added to the chromatin samples and incubated for 12 h at 4°C on a rotating platform. For mock immunoprecipitation (background), normal rabbit IgG (4 μl/sample, Cell Signaling Technology) was used. (Different units for the antibodies are presented, as the companies used different quantification methods). Sixty microliters of protein G agarose/salmon sperm DNA were added to each immunoprecipitate and incubated at 4°C with rotation for ∼2 h, followed by three washes with low-salt buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris·HCl, pH 8.1, and 150 mM NaCl). The preparations were then washed again for 15 min with rotation with the following buffers: high salt (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris·HCl, pH 8.1, and 500 mM NaCl), LiCl [0.25 M LiCl, 1% Igepal-CA630, 1% deoxycholic acid (sodium salt), 1 mM EDTA, and 10 mM Tris, pH 8.1], and two separate washes with Tris-EDTA buffer (10 mM Tris·HCl, 1 mM EDTA, pH 8.0). The chromatin-DNA complexes were then eluted (0.1 M NaHCO3, 1% SDS) from agarose beads, and the DNA was purified using phenol-chloroform and precipitation in ethanol.
Input and immunoprecipitated DNA amplification was detected by real-time PCR (qPCR; ABI Prism 7300 Sequence Detection System, Applied Biosystems). The reaction was carried out in triplicates, using SYBR Green PCR Master Mix (Invitrogen). The primers (Table 1) were designed using Primer Express 2.0 software (Applied Biosystems) to have an annealing temperature (melting temperature) between 58 and 60°C. The thermal profile of the real-time PCR reaction was as follows: 50°C for 2 min, 95°C for 10 min, followed by 40 cycles at 95°C for 15 s (denaturation) and 60°C for 60 s (annealing and extension). The results were analyzed using the ΔCt (change in cycle threshold) method, which reflects the difference in threshold for the target gene relative to that of β-actin in each sample. β-Actin is less prone to dynamic changes (28), and our laboratory's previous experiments showed that β-actin expression is unaltered by our AC protocols (24, 17). The amount of immunoprecipitated DNA in each sample is represented as signal relative to 1% of the total amount of input chromatin.
Transcription factor binding site prediction was performed with TFSearch version 1.3 (http://www.cbrc.jp/research/db/TFSEARCH.html). Genomic analyses were performed using the University of California Santa Cruz (UCSC) genome browser: http://genome.ucsc.edu.
For statistical analyses, we used commercially available computer software (SigmaStat 2.03). The treatment time points (C, AC2d, AC, DeAC, ReAC) were taken as the independent categorical variables, and individual animals or hearts were considered a random sample from the population. To compare the treatment groups, we used one-way ANOVA. For post hoc pairwise comparisons between the C (not acclimated) and the various treatment groups, Dunnett's test was applied, unless otherwise specified. Additional details are specified in Fig. 1–10 legends. Data are presented as means ± SE. Differences were considered significant at P ≤ 0.05.
To address our hypothesis that the epigenetic machinery is involved in faster ReAC, we performed chromatin immunoprecipitation assays to detect histone modifications at the promoters of hsp70 and hsp90 genes and the recruitment of histone acetylase TIP60 to the area of interest. Real-time PCR primers that amplified the HSE region [the binding site of HSF-1 (24)] clustered within 500 base pairs of the transcription start site (30) at the promoter regions of these genes were used for DNA analysis. To validate the open chromatin state of the HSE region, HSF-1 binding to the promoters was measured. As a negative control, qPCR analyses of immunoprecipitated TIP60 and HSF-1 was performed using primers that amplified a sequence within the 3′-untranslated region (3′-UTR) of hsp70 and hsp90 genes. Analysis of HSF-1 binding to the promoter region of rat dihydrofolate reductase, a gene that lacks HSE promoter elements and does not appear to bind HSF family (48), was performed as an additional negative control.
Histone modifications, TIP60 recruitment, and HSF-1 binding at the HSE site of the hsp70 promoter.
The results, presented in Fig. 2, show significantly elevated levels [increase (Δ) of ∼18%] of acetylated lysines 5, 8, 12, and 16 of histone H4-associated DNA at the HSE site of the HSP-70 promoter. This elevation was seen, not only in the long-term AC group, but also in the DeAC and ReAC groups. Because the vast body of evidence supports histone H3 involvement in the HSR in nonmammalian species and its role in stress memory (33), we screened for H3 acetylation/phosphorylation and found that, in contrast to histone H4, the level of acetylation of lysines 9, 18, and 23 of histone H3 did not differ among the groups (Fig. 3A). Nevertheless, following each heating session, i.e., AC2d and ReAC (Fig. 1), histone H3 phosphorylation was elevated at the serine 10 site associated with hsp70 by Δ35% and Δ25%, respectively (Fig. 3B).
Our initial analysis of the histone modifications in all experimental groups showed a significant elevation in the acetylation of histone H4 in AC, DeAC, and ReAC groups. Hence we measured the levels of acetyltransferase recruitment of TIP60 (60-kDa HAT-interactive protein), a catalytic subunit of the NuA4 HAT complex that is involved in transcriptional activation of selected genes, principally by acetylation of nucleosome histone H4 at the promoter region (3, 34, 49). The results presented in Fig. 4A show a marked increase in TIP60 acetyltransferase recruitment to the hsp70 promoter in the AC, DeAC, and ReAC groups (Δ95–133%), whereas the recruitment of TIP60 to 3′-UTR (Fig. 4B) was negligible.
HSF-1 binding to the HSE of the hsp70 gene (Fig. 5) was elevated in the AC2d, AC30, DeAC, and ReAC groups. As seen in Fig. 5, B and C, HSF-1 binding to the 3′-UTR of hsp70 and to the dihydrofolate reductase promoter was negligible.
Histone modifications, TIP60 recruitment, and HSF-1 binding at the HSE site of the hsp90 promoter.
Both hsp70 and hsp90 are controlled by HSF-1. Due to the fact that HSP-90 has a different expression profile than inducible HSP-70 on DeAC (39), we also assessed chromatin modifications at the HSE of hsp90.
An assessment of chromatin modifications at the HSE of hsp90, depicted in Fig. 6, shows significantly elevated levels (Δ ∼69%) of acetylated lysines 5, 8, 12, and 16 of histone H4-associated DNA at the HSE, not only in the long-term AC group, but also in the DeAC (63%) and ReAC (44%) groups. In contrast, the level of acetylation of lysines 9, 18, and 23 of histone H3 did not differ between the groups (data not shown). Significant elevations in phosphorylation at the serine 10 site of histone H3 were measured at the onset of AC, i.e., in the AC2d group (Fig. 7).
As shown in Fig. 8A, TIP60 acetyltransferase recruitment was markedly increased in the AC2d, AC, DeAC, and ReAC groups (Δ ∼130–80%), while TIP60 binding at the 3′-UTR was negligible. Binding of HSF-1 to the HSE of the hsp90 gene was elevated in the AC2d, AC30, and ReAC groups (Fig. 9), confirming constitutive chromatin opening in these groups. Interestingly, no significant binding was measured in the DeAC group. Similar to hsp70, binding to 3′-UTR of the hsp90 gene (Fig. 9B) was insignificant, thus confirming the specificity of HSF-1 to HSE at the hsp90 promoter.
Prior studies demonstrated that AC imprints selective, long-lasting, transcriptional changes in cytoprotective and chromatin remodeling-associated genes, with clusters of genes maintaining their AC transcriptome profile, while the physiological phenotype resembles controls (40). These findings led us to hypothesize that AC memory is linked to stored upstream epigenetic information that predisposes to rapid ReAC and cytoprotective memory. The results of the present investigation indicate that the constitutively acetylated histone H4 and the preserved euchromatin state throughout AC-DeAC-ReAC are causally related to AC memory, favoring prompt HSF-1-HSE binding and subsequent rapid activation of the cytoprotective arsenal. The maintenance of an active chromatin state at the HSE of the hsp70 and hsp90 genes throughout the AC, DeAC, and ReAC regimen is associated with 1) constitutive binding of HSF-1 to the hsp70 promoter and greater HSP-70 reserves at normothermic temperatures (40), and 2) a rapid resumption of the acclimated phenotype when reexposed to acclimating conditions. One of the mechanisms allowing the rapid resumption of cytoprotection is the renewed binding of HSF-1 to the HSE at the hsp90 promoter, leading to the replenishment of HSP-90 stores.
To the best of our knowledge, this is the first study demonstrating persistent environmental temperature effects (AC) on the epigenome of a mammalian species in early adulthood.
Histone modifications at the hsp70 promoter.
Histone modifications, such as acetylation and phosphorylation, determine the functional state of chromatin (e.g., Refs. 42–44), and, therefore, these posttranslational changes found in specific histones may be a sign of transcriptional activity.
In Drosophila, elevated H3Ser10 has been found in transcriptionally activated heat shock loci (17, 25) in the chromosome puffs of heat-shocked larval Drosophila salivary glands. Nowak and Corces (25) also demonstrated that the loci, which were histone H3 phosphorylated after heat shock, were histones H3- and H4-acetylated before heat shock, suggesting that a gene locus can be acetylated, even if it is not actively transcribed. Labrador and Corces (19) provided evidence that P-H3Ser10 is essential in the transcriptional induction of hsp70, irrespective of the acetylation state, raising the hypothesis that P-H3Ser10 is required to establish a recognition site (conserved arginine 164 in GCN5) for subsequent acetylation (19). In mammalian cells, Solomon et al. (36), using immunoprecipitation of in vivo cross-linked histone H4-containing hsp70 chromatin fragments, and Thomson et al. (41) showed that only histone H4 (and not H3) acetylation of hsp70 chromatin is associated with the heat shock-induced changes in the hsp70 gene.
As discussed above, the two histone modifications presented here, histone H3 phosphorylation (serine 10) and histone H4 acetylation (lysines 5, 8, 12, and 16), play a role in the regulation of heat stress-induced hsp70 transcription. Histone H3 phosphorylation is also important in stress memory (33). Hence, using the available data (7, 25, 40), we can examine the histone modifications detected in our protocol and determine whether these combined modifications are involved in acclimatory cytoprotective memory. In this investigation, we found elevated histone H3 phosphorylation at the serine 10 site in both AC2d and ReAC groups; i.e., this modification occurred at the onset of each AC regimen, when the animal experiences maximum strain (15). In contrast, high acetylation levels were detected in histone H4 after 30 days of AC, as well as in the DeAC and ReAC groups. The results also imply that acetylation occurs during short-term AC.
The detection of elevated histone H4 acetylation at lysines 5, 8, 12, and 16 in the AC, DeAC, and ReAC groups and elevated histone H3-phosphorylation at Ser10 (both associated with the hsp70 promoter region) following AC2d and ReAC, but not during DeAC, implies that the histone H4 modification is ambient-temperature independent, whereas histone H3-phosphorylation modification only occurs on exposure to the higher acclimating temperature. Histone H3 acetylation levels, in contrast, were similar in all experimental groups.
Our present data not only confirm the essential role of histone H3 phosphorylation in hsp70 induction upon transfer to a hot environment, but also imply a constitutive histone H4 acetylation effect in the production (via transcription) of sustained elevated HSP-72 reserves, independent of ambient temperature (23, 39). Due to the fact that histone H3 phosphorylation is a transient response, its contribution to the subsequent histone H4 acetylation seen in our study is irrefutable. Furthermore, this conclusion agrees with other reports (4, 5), demonstrating that histone acetylation may require a phosphorylated histone H3 at the inducible gene locus. To confirm the findings of high histone H4 acetylation in the AC, DeAC, and ReAC groups, we measured the recruitment of a specific histone H4 HAT (TIP60, the catalytic subunit of NuH4) to the hsp70 promoter (34, 49). Previous studies provided evidence that this complex is involved in the activation of transcriptional programs associated with repairing DNA strand breaks and apoptosis upon environmental stress (2, 37). We found a strong correlation between the levels of histone H4 acetylation and the recruitment of TIP60 to the hsp70 promoter.
Validation of hsp70 euchromatin state.
Histone modifications cannot provide a full understanding of transcriptional activity. In our experiment, designed to address HSF-1 binding to the HSE of hsp70, the detection of constitutive HSF-1 binding to HSE in the “nonstressed” DeAC group, possibly allowing high transcription and translation 30 days post-AC (39), agrees with several studies demonstrating that HSF-1 can bind constitutively to the hsp70 promoter under long-term nonstressful conditions. Similar mechanisms induce high levels of the hsp70 transcriptome in species evolutionarily adapted to desert conditions by maintaining large HSP-70 protein reserves vs. matched species inhabiting temperate areas with limited HSP-70 reserves (8, 50).
The question, how do histone H3 phosphorylation and histone H4 acetylation at the HSE site of the hsp70 promoter work together to promote transcriptional memory remains. According to Cheung et al. (4), the synergistic coupling of histone H3 phosphorylation and acetylation suggests that histone H3 phosphorylation can affect the efficiency of subsequent acetylation. Therefore, our data imply that the increased p-H3Ser10 during AC2d might define a particular recognition locus that is primed for HSF-1 binding and further hsp70 transcription, as described in the literature (51). Thomson et al. (41), using HSF-1 −/− fibroblasts, demonstrated that HSF-1 is an essential factor for directing inducible histone H4 acetylation at the hsp70 promoter upon heat stress. This finding led us to suggest that HSF-1 binding during AC2d and AC recruits histone H4 acetylation to the locus during DeAC. Our observation of a significant correlation between HSF-1-histone H4 acetylation only seen during AC, DeAC, and ReAC (r = 0.78, P < 0.0003) confirms constitutive chromatin opening during the 30-d DeAC and 2-day ReAC periods, resulting in elevated transcriptional events. Hence, long-term histone H4 acetylation might promote an open chromatin state and constitutive binding of HSF-1 to the hsp70 promoter, leading to the high transcription and translation of the gene long after the initial AC period, as well as when AC conditions are resumed.
Histone modifications at the hsp90 promoter.
In addition to HSP-70, HSF-1-HSE binding controls other HSP species involved in the HSR, including hsp90. HSP-90 is not only a critical component of the HSR and protein quality control, it is also a master regulator, controlling hubs in homeostatic signal transduction and regulating chromatin structure and gene expression (29). In our AC-mediated cross-tolerance model, Bromberg (3a) and Horowitz demonstrated the role of HSP-90 in the trafficking of the hypoxia inducible transcription factor-1α [an essential component in cardioprotection (15, 22)] to the nucleus. Due to the fact that HSP-90 protein levels in our AC-DeAC-ReAC protocol dropped during DeAC (loss of AC), whereas HSP-70 levels remained elevated (39), we examined chromatin modifications in the HSE of hsp90. We only found increased H3Ser10 phosphorylation in the AC2d group, yet the higher levels of histone H4 acetylation established during AC2d remained throughout the AC, DeAC, and ReAC protocol. Surprisingly, HSF-1 binding to HSE of hsp90 was detected in AC2d, AC, and ReAC, but not in the DeAC phase, despite histone H4 acetylation at that phase. These data are congruent with Zhao et al. (51) and Uffenbeck and Krebs (45), who used an HSF-1-HSP-82 activation model in yeasts and showed that, following heat stress, rapid transient histone H4 acetylation is required for subsequent HSF-1 binding, induction of the DNase I hypersensitive site, and nucleosomal displacement. Zhao et al. (51) also demonstrated that the reassembly of nucleosomes upon a decrease in temperature is very rapid. Although we only examined constitutive features, the temperature effects on chromatin remodeling described by Zhao et al. (51) allow certain deductions. The data suggest the importance of constitutive histone H4 acetylation when exposed to acclimating temperatures, as well as HSF-1-HSE binding in the hsp90 gene. The lack of HSF-1-HSE binding during DeAC may partially explain the decrease in HSP-90 levels at this phase (39).
In contrast to long-term AC, when AC homeostasis has been achieved, AC2d is a transient, acute, and stressful state, where H3Ser10 phosphorylation induces HSF-1 binding. Neither HSP-70 nor HSP-90 was elevated (39) at this phase. This phenomenon is known to occur in the hsp72 gene of acclimating animals, where recruitment of adequate mRNA to establish greater HSP-70 reserves precedes the translation of the protein (24).
Heat AC predisposes to cytoprotective memory: support of concept.
The loss of AC and cytoprotection, despite the preservation of augmented HSP-70 reserves, agrees with previous investigations [showing that HSP-70 alone is essential but insufficient to render ischemic cardioprotection by thermal preconditioning or thermotolerance (10, 12)]. The findings concur with our laboratory's early studies showing that AC-mediated cross-tolerance and cytoprotection is the outcome of cross-talk between constitutively reprogrammed cytoprotective networks (16). Tetievsky et al. (39), profiling HSP-70, HSP-90, and BCLxL during AC-DeAc-ReAC, demonstrated that HSP-90 does not remain upregulated on DeAC. Given the important role of HSP-90 in the control of many client proteins and its relevance to our model by chaperoning hypoxia-inducible transcription factor-1α to the nucleus, the data presented here support the hypothesis that the return of HSP-90 to control levels during DeAC abolishes cytoprotection, contributes to the dichotomy between the molecular and physiological phenotype, and pinpoints HSF-1-HSE binding at hsp90 as essential to protection.
In sum, the profile of chromatin remodeling at the HSE of the promoter site of two hsp genes provides a conceptual model (Fig. 10) of the evolution of AC memory. We suggest the following chain of events. 1) At the onset of AC (AC2d), histone H3 phosphorylation switches on HSF-1 binding at the HSE with subsequent histone H4 acetylation at the HSE of both hsp70 and hsp90 genes. This occurs in a temperature-dependent manner. 2) The acetylation persists throughout DeAC and ReAC, resulting in constitutive HSF-1 binding to the hsp70 promoter, irrespective of transitions in ambient temperatures. In contrast, HSF-1 binding to hsp90 is temperature dependent. There is no HSF-1 binding to hsp90 in DeAC, despite elevated TIP60 activation and histone H4 acetylation. Due to the fact that HSF-1-hsp90 binding requires elevated temperatures, the increased histone H4 acetylation may facilitate the rapid resumption of HSF-1 binding, hsp90 transcript translation, and the reformation of a cytoprotective milieu upon ReAC.
No conflicts of interest, financial or otherwise, are declared by the author(s).
This study was supported by Israel Science Foundation Grant 321/06.
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