Altered chromatin architecture underlies progressive impairment of the heat shock response in mouse models of Huntington disease (original) (raw)
Systemic HSP990 administration activates the HSR in mouse brain tissue. To investigate the effects of HSP990 on the HSR in mouse brain in vivo, the expression levels of major HSPs were assessed in WT mouse cortex 20 hours after HSP990 administration. A single acute oral dose of HSP990 (12 mg/kg) significantly increased levels of HSP70 (2.7-fold), HSP25 (3.8-fold), and HSP40 (1.6-fold) relative to levels in vehicle-treated mice at 10 weeks of age (Figure 1, A and B). In contrast, the expression of HSP90 remained unchanged. In addition, no change in expression was observed in the UPR chaperones BiP and GRP94, which suggests that upregulation of stress proteins after HSP990 treatment is specific to the HSR arm of the chaperone network. A similar pattern of HSP expression was observed in striatum and cerebellum 20 hours after dosing (Supplemental Figure 1, A–D; supplemental material available online with this article; doi:10.1172/JCI57413DS1).
NVP-HSP990 elicits an HSR in mouse brain after acute oral administration. (A) Western blots of HSR and UPR proteins in 10-week-old WT mouse cortex 20 hours after treatment with HSP990 (12 mg/kg) or vehicle. (B) Fold upregulation of HSPs in HSP990-treated WT mice (black) was calculated relative to vehicle-treated WT mice (white) by densitometry. Values are mean ± SEM fold induction (n = 6 per treatment group). (C) Mouse cortices (WT) were harvested 0, 0.5, 1, 2, 4, and 8 hours after a single dose of HSP990 (12 mg/kg) or vehicle. Taqman RT-qPCR was used to determine the fold induction of HS genes relative to expression in the vehicle group 0 hours after dosing. Values (mean ± SEM) were calculated by the ΔCt method, normalized to the housekeeping gene Atp5b (n = 4 per treatment group). (D) Western blotting for HSF1 in cortices of 10-week-old WT mice harvested 1, 2, and 4 hours after an acute dose of HSP990 (12 mg/kg) or vehicle (n = 3 per treatment group). HSF1-P, hyperphosphorylated form of HSF1. Lanes were run on the same gel but were noncontiguous (white lines). **P < 0.01, ***P < 0.001, Student’s t test.
To determine the dynamics of HS gene induction, cortex was harvested 0, 0.5, 1, 2, 4, or 8 hours after treatment with HSP990 (12 mg/kg) or vehicle. Significant induction in Hspa1a/b (encoding HSP70; 11.3-fold), Hspb1 (encoding HSP25; 4.6-fold), and Dnajb1 (encoding HSP40; 2.4-fold) mRNA was observed as early as 1 hour after treatment (Figure 1C). Interestingly, although the magnitude of induction was markedly different between HS genes, the dynamics of induction were highly comparable, with all 3 HS genes presenting maximum induction 4 hours after HSP990 treatment.
The definitive mark of a bona fide HSR is activation of the transcription factor HSF1. The activation state of HSF1 was assessed in mouse brain regions 1, 2, and 4 hours after HSP990 treatment. Hyperphosphorylation of HSF1 correlates with its transcriptional activation and can be observed by Western blotting as a retardation of HSF1 migration during SDS-PAGE. Hyperphosphorylated HSF1 was observed in mouse cortex, striatum, and cerebellum 1, 2, and 4 hours after treatment (Figure 1D and Supplemental Figure 2, A and B), which suggests that HSP990 acts through HSF1 to induce an HSR in mouse brain tissue. We conclude that 1 acute oral dose of HSP990 at 12 mg/kg is able to efficiently induce a bona fide HSR in mouse brain in vivo.
Establishment of a well-tolerated HSP990 chronic dosing regimen. To evaluate whether pharmacological induction of the HSR might prove beneficial in the context of the HD mutation, we established an HSP990 dosing regimen that was well tolerated in mice. Compounds frequently show increased toxicity in WT and R6/2 mice at 4–6 weeks of age compared with older mice (39). Therefore, we assessed the ability of lower doses to induce an HSR and found that HSP70, HSP25, and HSP40 were effectively upregulated by a single 1.2 mg/kg dose in 4-week-old R6/2 mice, although a 7.2 mg/kg dose was required to elicit the same effect in mice of 8 weeks of age (Supplemental Figure 3, A and B). To ascertain the duration of HSP70 induction, 7.2 mg/kg HSP990 was administered to 8-week-old R6/2 mice, which were sacrificed 0, 4, 8, 12, 16, 24, 72, 96, and 120 hours after treatment (Supplemental Figure 3C). The level of HSP70 in the cortex peaked at 12 hours and returned to baseline by 120 hours. Therefore, we devised a dose escalation scheme whereby HSP990 was administered weekly: 1.2 mg/kg at 5 weeks of age, 2.4 mg/kg at 6 weeks, and 7.2 mg/kg thereafter.
Induction of the HSR with HSP990 improves phenotype and reduces aggregate load in R6/2 mice. We first assessed whether the induction of molecular chaperone levels via HSP990 treatment decreased the aggregate load in R6/2 mice. WT and R6/2 mice (n = 6) were treated with HSP990 or vehicle from 5 to 9 weeks of age, after which aggregate load was measured by Seprion ligand ELISA (7). Levels of aggregated huntingtin were significantly decreased in cortex, hippocampus, brain stem, and muscle, which was confirmed in the cortex by Western blotting and immunohistochemistry (Figure 2, A and B, and Supplemental Figure 4, A and B).
HSP990 treatment transiently improves rotarod performance and reduces aggregate load in R6/2 mice. (A) Seprion ligand ELISA was used to quantify aggregate load in tissues of R6/2 mice after treatment for 4 weeks with vehicle (green) or HSP990 (purple). Values were plotted as mean absorbance ± SEM (n = 6 per treatment group). (B) Western blotting and immunodetection with S830 was used to obtain visual representation of results in A. (C and D) Assessment of (C) body weight and (D) rotarod ability with age. (E) Brain weight in 9- and 14-week-old mice after vehicle or HSP990 treatment. Values are presented as mean ± SEM (n = 12 per group). (F–H) R6/2 brain tissues were harvested from 9-week-old satellite or end-of-trial mice (14 weeks). (F) 2B7-MW1 TR-FRET was used to determine levels of soluble exon 1 in mouse cortices after vehicle or HSP990 treatment. Values are presented as mean ± SEM for each group (n ≥ 4 per treatment group). (G and H) S830 Seprion ligand ELISA was used to measure aggregate load after HSP990 treatment at (G) 9 weeks (n = 4 per treatment group) and (H) 14 weeks of age (n ≥ 6 per treatment group). *P < 0.05, **P < 0.01, 1-way ANOVA (C and D) or Student’s t test (A, B, and E–H).
Mutant huntingtin has been proposed to form a spectrum of oligomeric species both in vitro and in vivo (7), with different huntingtin oligomers exhibiting different levels of cytotoxicity (40, 41). Changes in aggregate size and morphology can be observed as alterations in huntingtin antibody reactivity (40, 41) and/or by altered migration of aggregates through an agarose gel (42). To determine whether HSP990 treatment promotes the formation and/or removal of a specific subpopulation of huntingtin aggregates in R6/2 brain tissue, cortical protein lysates were subjected to agarose gel electrophoresis for resolution of aggregates (AGERA) followed by Western blotting and immunodetection with antibodies that have differential affinities for huntingtin aggregates depending on their conformation (40, 41). Immunoblotting with S830, 1C2, and 3B5H10 revealed aggregate smears greater than 500 kDa in size in R6/2, but not WT, cortical lysates (Supplemental Figure 4C). HSP990 treatment did not alter the size distribution of 1C2, 3B5H10, or S830 reactive aggregates in R6/2 mice. However, HSP990 treatment did result in a reduction in aggregate smear intensity that was comparable for all 3 huntingtin antibodies (Supplemental Figure 4C). These data suggest that HSP990 treatment leads to a uniform reduction in aggregate load rather than the reduction of a specific aggregate subpopulation. However, we cannot rule out the possibility that HSP990 treatment leads to subtle conformational changes that are not identifiable by these techniques.
We next used a set of established quantitative tests to determine whether prolonged HSP990 treatment might improve R6/2 behavioral phenotypes (36, 43, 44). Prior to treatment onset, female WT and R6/2 littermates (born over a 3-day period) were assorted into treatment groups matched for weight, grip strength, rotarod performance, and activity level, as previously described (43). WT and R6/2 mice were treated with HSP990 or vehicle (n ≥ 12 per treatment group) according to the dosing regimen devised above, and phenotypic parameters were measured from 5 weeks to 14 weeks of age. R6/2 vehicle- and HSP990-treated groups had well-matched CAG repeats (199 ± 3 and 200 ± 4, respectively; mean ± SD). In parallel, WT and R6/2 satellite groups (n = 4) were treated with HSP990 or vehicle from 5 to 9 weeks of age as in the initial dose-finding study.
Mice were weighed weekly from 4 to 14 weeks of age (Figure 2C). As expected, R6/2 mice weighed less than WT mice (F1,46 = 5.22; P = 0.027) and gained weight at a slower rate (F4,460 = 21.36; P < 0.001). Treatment with HSP990 had no effect on weight (F1,46 = 0.02; P = 0.888) or rate of weight gain (F4,460 = 0.94; P = 0.433) in WT mice, or on weight (F1,46 = 2.54; P = 0.118) or rate of weight loss (F4,460 = 1.18; P = 0.321) in R6/2 mice. Therefore, HSP990 was well tolerated but did not improve the weight loss phenotype in the R6/2 mice.
Rotarod performance, a sensitive indicator of balance and motor coordination, has been reliably shown to decline in R6/2 mice (43). Consistent with previous results, R6/2 rotarod performance was impaired (F1,47 = 58.38; P < 0.001) and deteriorated with age (F2,705 = 31.74; P < 0.001) (Figure 2D). Treatment with HSP990 did not modify the rotarod performance of WT mice (F1,47 = 0.11; P = 0.746) or how this changed with age (F2,705 = 0.24; P = 0.768). However, HSP990 improved the rotarod performance of R6/2 mice at 8 (F1,47 = 6.75; P = 0.012) and 10 (F1,47 = 4.60; P = 0.037) weeks of age, but the effect diminished by 12 weeks (F1,47 = 2.32; P = 0.134). The effect of HSP990 treatment on overall rotarod performance of R6/2 mice was close to statistical significance (F1,47 = 3.63; P = 0.063).
Forelimb grip strength was assessed at 4, 11, 12, and 13 weeks of age. Consistent with previous data, the grip strength of R6/2 mice was impaired (F1,48 = 64.09; P < 0.001) and deteriorated with age (F3,288 = 17.43; P < 0.001). Treatment with HSP990 had no overall effect on the grip strength of WT mice (F1,48 = 0.69; P = 0.410) or over the course of the experiment (F3,288 = 0.22; P = 0.866). HSP990 did not improve R6/2 grip strength (F1,48 = 0.575; P = 0.452) or its rate of decline (F3,288 = 0.12; P = 0.934) (Supplemental Figure 5).
Exploratory activity was assessed fortnightly from 5 to 13 weeks of age, as described previously (44). Mice were assessed for a period of 30 minutes for total activity, mobility, rearing, and unsupported rearing (Supplemental Figure 6 and Supplemental Table 1). The pattern of R6/2 hypoactivity was consistent with previous data (45), but HSP990 treatment had no effect on the activity measures of WT or R6/2 mice (Supplemental Table 1).
R6/2 brain weight decreases with disease progression (4). At 9 weeks of age, HSP990 treatment led to an increase in R6/2 brain weight (P = 0.029) (Figure 2E). In addition, soluble exon 1 huntingtin (46, 47), as measured by time-resolved Forster resonance energy transfer (TR-FRET), was increased in the cortex of treated mice (Figure 2F), and Seprion ligand ELISA showed that there was a trend toward a concomitant decrease in aggregation in the cortex (P = 0.061), hippocampus (P = 0.084), and brain stem (P = 0.062) (Figure 2G), consistent with the results obtained in the initial dose-finding study (Figure 2A). However, these effects were not maintained at 14 weeks of age (Figure 2H). In addition, AGERA revealed that aggregate size distribution and huntingtin antibody immunoreactivity were also unaltered at 14 weeks (Supplemental Figure 4D). Taken together with rotarod performance, which was improved at 8 and 10 but not 14 weeks of age, these data suggest the beneficial effects of chronic HSP990 treatment diminish with disease progression.
HSP990-mediated HSP induction is impaired in HD mouse models. As the beneficial effects of HSP990 treatment were transient in R6/2 mice, we hypothesized that impairment of the HSR might occur with disease progression. Therefore, we investigated whether HSP990 treatment induced an HSR in the brains of R6/2 and/or HdhQ150/Q150 mice in late-stage disease. In our colonies, R6/2 mice and HdhQ150/Q150 homozygotes reached end-stage disease at approximately 15 weeks and 24 months of age, respectively. Treatment with HSP990 induced a 2.5-fold increase in HSP70 in cortical tissues from 12-week-old and 22-month-old WT mice 20 hours after dosing (Figure 3, A and B). In addition, both HSP25 (2.5-fold) and HSP40 (1.5-fold) levels were also significantly elevated. Interestingly, both R6/2 and HdhQ150/Q150 models showed markedly lower fold induction of all 3 HSPs in cortex (Figure 3, A and B), striatum (Supplemental Figure 7, A and C), cerebellum (Supplemental Figure 7, B and D), and muscle (Supplemental Figure 8, A and B). These data suggest that at late-stage disease, both HD mouse models may have an impaired HSR in both brain and periphery.
HSP upregulation is impaired in HD mouse models. (A) Representative Western blots of HSP70, HSP25, HSP40, and α-tubulin expression in WT, R6/2 or HdhQ150/Q150 (Hdh) cortex 20 hours after treatment with vehicle or HSP990 (12 mg/kg). (B) Immunoblotting and densitometry were used to calculate the expression levels of HSP70, HSP25, and HSP40 in mouse cortex relative to levels of α-tubulin. Fold expression of each chaperone was calculated for HSP990 relative to vehicle. Values are mean fold ± SEM (n ≥ 4 per treatment group). (C) Representative Western blots of HSP70, HSP25, HSP40, and α-tubulin in half brain of 4-, 8-, 10-, and 12-week-old WT or R6/2 mice 20 hours after treatment with vehicle or HSP990 (12 mg/kg). (D) Relative expression of HSP70, HSP25, and HSP40 at 20 hours after treatment with HSP990 (12 mg/kg) or vehicle, calculated relative to α-tubulin by densitometry. Fold expression relative to vehicle-treated 4-week-old WT mice was calculated for each chaperone and age, and values are mean ± SEM (n = 4). *P < 0.05, **P < 0.01, ***P < 0.001, Student’s t test.
HSR impairment is progressive and is pronounced in early-stage disease. In order to determine the relationship between HSR impairment and disease progression, WT and R6/2 mice were sacrificed 20 hours after an acute dose of vehicle or HSP990 (12 mg/kg) at 4, 8, 10, and 12 weeks of age. Given that HSR impairment had been observed in all brain regions studied, subsequent experiments were performed on half brains. As previously reported (19), we found significantly reduced levels of HSP70 and HSP40 in R6/2 compared with WT brain tissue by 12 weeks of age (Figure 3, C and D). This was not observed for HSP25, although interestingly, levels of HSP25 were generally lower in both WT and R6/2 mice at 4 weeks of age. WT mice showed consistent upregulation of HSP70 (≈3-fold), HSP25 (≈3-fold), and HSP40 (≈2-fold) in response to 12 mg/kg HSP990 treatment at all ages tested. At 4 weeks of age, induction of HSP70, HSP25, and HSP40 was extremely comparable between R6/2 and WT mice (Figure 3, C and D). However, upregulation of all 3 HSPs was compromised in R6/2 brain tissue by 8 weeks of age (HSP70, 1.7-fold; HSP25, 1.8-fold; HSP40, 1.3-fold), with impaired HSP upregulation continuing to 12 weeks despite equivalent levels of HSP990 reaching WT and R6/2 brain tissue 0.5, 1, 2, 4, and 24 hours after dosing (Supplemental Figure 9). These data suggest that HD mice are able to elicit an HSR early in disease, but this becomes impaired with disease progression.
HSR impairment occurs at the level of transcription. To determine whether impairment of the HSR occurs at the level of transcription, 12-week-old WT and R6/2 mice were sacrificed 4 and 8 hours after treatment with vehicle or 12 mg/kg HSP990, and levels of HS gene mRNA were assessed by real-time quantitative PCR (RT-qPCR). An increase in HS gene mRNA after HSP990 treatment was found in both WT and R6/2 mice. However, R6/2 mice exhibited significantly lower fold induction of Hspa1a/b, Hspb1, and Dnajb1 mRNA compared with WT mice at both 4 and 8 hours after treatment (Figure 4A). This effect was also observed in HdhQ150/Q150 mice 2 hours after dosing (Figure 4B). Consistent with a progressive decline in the HSR in HD mice, impaired Hspa1a/b, Hspb1, and Dnajb1 mRNA production after HSP990 treatment was observed 4 hours after dosing in R6/2 mice at 8 weeks of age, but not at 4 weeks (Supplemental Figure 10, A and B). In contrast, basal levels of Hspa1a/b only decreased progressively in HD mice, whereas basal levels of Hspb1 or Dnajb1 were not dysregulated (Supplemental Figure 11, A and B). To determine the extent of this impaired induction, WT and R6/2 mice were dosed with vehicle or 12 mg/kg HSP990 (n ≥ 7 per treatment group), and striata were removed 4 hours after dosing. Expression profiling using Affymetrix microarrays (see Methods) showed that the fold induction in the expression of all major inducible HSPs was reduced in R6/2 compared with WT striatum (Table 1).
Impaired upregulation of HSPs occurs at the level of transcription. Taqman RT-qPCR of Hspa1a/b, Hspb1, and Dnajb1 was performed on half brains of 12-week-old WT and R6/2 mice 0, 4, or 8 hours after treatment with vehicle or HSP990 (12 mg/kg) (A), or on half brains of 22-month-old WT and HdhQ150/Q150 mice 2 hours after treatment with vehicle or HSP990 (B). (Due to the reduced availability of mice aged 22 months, HdhQ150/Q150 analysis was performed at 2 hours after dose to allow investigation of mRNA levels and chromatin architecture on the same samples.) Chaperone mRNA expression levels were normalized to the housekeeping gene Atp5b. Fold induction of each HS gene after HSP990 treatment was calculated relative to expression levels of WT vehicle groups 0 hours after treatment, and expressed as mean fold ± SEM (n = 4 per treatment group). *P < 0.05, **P < 0.01, ***P < 0.001, Student’s t test.
Induction of HS gene mRNA in striatum 4 hours after HSP990 treatment
HSF1 dissociation from HSP90, hyperphosphorylation, and nuclear translocation are not impaired in HD mice upon HSP990 treatment. Levels of HSF1 and HSP90 expression were not significantly different between 12-week-old WT and R6/2 or 22-month-old WT and HdhQ150/Q150 brain lysates (Figure 5, A–D). Therefore, the impaired HSR observed in HD mice is not simply caused by differences in the expression levels of HSF1 or HSP90. One possible explanation for the impairment is that upon HSP990 treatment, HSF1 cannot dissociate efficiently from its repressive HSP90 complex. To test whether this was the case, HSF1 was immunoprecipitated from 12-week-old WT and R6/2 brain tissue that had been harvested 2 hours after treatment with HSP990 or vehicle. Western blotting for HSP90 after HSF1 IP revealed that HSP90 coimmunoprecipitated with HSF1 in brain tissue from both WT and R6/2 vehicle-treated mice. This is consistent with previous findings (22), which suggests that the majority of inactive HSF1 is found sequestered in a repressive HSP90 complex. In contrast, 2 hours after treatment with HSP990, no HSP90 was detected after HSF1 IP from either WT or R6/2 brain tissue (Figure 5A). Therefore, HSF1 is able to efficiently dissociate from HSP90 in both WT and R6/2 mice.
HSF1 dissociates from HSP90, becomes hyperphosphorylated, and translocates to the nucleus upon HSP990 treatment. (A) Western blots of HSF1 and HSP90 after HSF1 IP from 12-week-old WT and R6/2 mouse brains 2 hours after treatment with vehicle or HSP990 (12 mg/kg). (B and C) Representative Western blots of HSF1 and HSP90 in (B) 12-week-old WT and R6/2 or (C) 22-month-old WT and HdhQ150/Q150 mouse half brains 2 hours after treatment with vehicle or HSP990 (12 mg/kg). α-Tubulin was used as a loading control. (D) Expression of HSP90 and HSF1 relative to α-tubulin, or phosphorylated HSF1 (HSF1-P) relative to unphosphorylated HSF1, in 12-week-old WT and R6/2 or 22-month-old WT and HdhQ150/Q150 mice. Densitometry values are mean ± SEM (n = 4 per treatment group). (E) Representative Western blots for HSF1 in nuclear and cytoplasmic fractions derived from 12-week-old WT and R6/2 mouse half brains 2 hours after treatment with vehicle or HSP990 (12 mg/kg). Purity of nuclear (N) and cytoplasmic (C) fractions was demonstrated by immunoblotting for α-tubulin and histone H3.
An alternative explanation for the impaired HSR is that upon HSP990 treatment, HSF1 hyperphosphorylation does not occur, and subsequently, HSF1 is less potent at inducing HS gene expression. Western blotting revealed that 2 hours after treatment with HSP990, HSF1 was hyperphosphorylated in brain tissues of 12-week- and 22-month-old WT, 12-week-old R6/2, and 22-month-old HdhQ150/Q150 mice (Figure 5, B–D). Furthermore, HSF1 hyperphosphorylation was observed as early as 30 minutes after treatment with HSP990 and continued to 8 hours after dosing in both WT and R6/2 brain tissue (Supplemental Figure 12). This suggests that the HSR impairment in HD mice is not simply caused by an alteration in the dynamics of HSF1 activation.
Finally, we investigated whether HSF1 can effectively translocate to the nucleus. Nuclear and cytoplasmic fractions were obtained from 12-week-old WT and R6/2 mouse brains harvested 2 hours after HSP990 treatment. HSF1 was not observed in the nuclear fractions of WT or R6/2 mice treated with vehicle; however, hyperphosphorylated HSF1 resided predominantly in the nuclear fraction of both WT and R6/2 brain tissue (Figure 5E).
Taken together, these findings demonstrate successful target engagement of HSP990 in the mouse brain and also suggest that upon HSP990 treatment, HSF1 is able to dissociate from its HSP90 repressor complex, become hyperphosphorylated, and translocate to the nucleus. Furthermore, the capacity of HSF1 to perform these functions is highly comparable in both WT and HD mouse brain tissue at late-stage disease.
Reduced HSF1 promoter binding and chromatin accessibility in R6/2 versus WT mice 2 hours after HSP990 treatment. Given the unimpaired nuclear translocation of hyperphosphorylated HSF1 (Figure 5), we reasoned that diminished binding of HSF1 to HS promoters may be the cause of impaired HSR (Figures 3 and 4). To investigate this possibility, SYBR green quantitative PCR (qPCR) using primers specific for HS gene promoters was coupled to HSF1 ChIP in mouse brains harvested 2 hours after vehicle or HSP990 treatment. HSF1 ChIP qPCR revealed a significant increase in HSF1 promoter binding 2 hours after HSP990 treatment in mouse brain tissue compared with tissue from vehicle-treated mice. However, the level of HSF1 binding at all 3 HS promoters after HSP990 treatment was significantly lower in the brains of 12-week-old R6/2 mice (Figure 6A) and 22-month-old HdhQ150/Q150 mice (Figure 6D).
Reduced HSF1 promoter binding and altered nucleosome landscapes are observed at HS loci in R6/2 mice, but do not correlate with chromatin accessibility. (A–E) Levels of HSF1 (A and D), RNA polymerase 2 (RNApol2), H3AcK9, H3AcK27 (B), and Tetra AcH4 (C and E) bound to HS promoters 2 hours after vehicle or HSP990 treatment (12 mg/kg) was determined by ChIP in 12-week-old WT and R6/2 (A–C) and 22-month-old WT and HdhQ150/Q150 (D and E) mouse half brains. Chromatin was immunoprecipitated, and SYBR green qPCR was performed on the resulting DNA with primers specific for the Hspa1b, Hspb1, and Dnajb1 promoters. Signal was normalized to 10% of the input for each sample. Values are mean ± SEM (n = 5 per treatment group). Black lines in A–E indicate mean signal obtained after pulldown with rabbit IgG alone (n = 2). (F) MNase digestion was performed on chromatin extracted from mouse brain tissue 2 hours after treatment with vehicle or HSP990. SYBR green was then performed using primers spanning the Hspa1b gene. Digested signal was normalized to undigested signal, and values are mean ± SEM (n = 4 per treatment group). *P < 0.05 vs. WT HSP990, #P < 0.05 vs. WT vehicle, Student’s t test.
As equivalent levels of a hyperphosphorylated form of HSF1 were found to reside in the nucleus of both WT and R6/2 mice (Figure 5E), we postulated that reduced HSF1 promoter binding is most likely caused by either the DNA binding competency of HSF1 itself or the chromatin architecture and accessibility of HS promoters. In order to determine how the nucleosome landscape influences HSF1 promoter binding, we conducted further ChIP analysis at the Hspa1b promoter. It has recently been published that the nucleosome landscape can significantly affect inducible binding of HSF1 to target promoters; thus, we focused our analysis on histone marks that had shown the most robust effects on the inducible DNA binding ability of HSF1 (38).
ChIP analysis at the Hspa1b promoter revealed that significant levels of RNA polymerase 2, H3AcK9, H3AcK18, H3AcK27, H3triMeK4, and Tetra AcH4 were all associated with the Hspa1b promoter region in vehicle-treated mice (Figure 6, B and C, and Supplemental Figure 13). Upon treatment with HSP990, the levels of all these factors (except H3AcK18) increased at the Hspa1b promoter in both WT and R6/2 brain tissue (Figure 6, B and C, and Supplemental Figure 13, A and B).
The increase in the level of RNA polymerase 2 was significantly lower in R6/2 than WT brain tissue, consistent with reduced HSF1 binding (Figure 6A) and reduced mRNA production (Figure 4A). No difference in H3 acetylation was observed between WT and R6/2 mice treated with vehicle or HSP990 (Figure 6B and Supplemental Figure 13B). In contrast, significant hypoacetylation of histone H4 was observed at all 3 HS genes between WT and R6/2 mice, whether treated with HSP990 or vehicle (Figure 6C). An equivalent pattern of hypoacetylation was also observed when comparing WT with HdhQ150/Q150 mice, regardless of treatment (Figure 6E). Furthermore, this effect was not observed in 4-week-old mice (Supplemental Figure 14). We therefore conclude that in the brains of HD mice, HS loci become hypoacetylated with disease progression. This phenomenon is specific to histone H4 (a known modifier of HSF1 DNA binding activity) and correlates with the reduced ability of HSF1 to bind target promoters following HSP990 treatment in these mice.
We reasoned that H4 hypoacetylation may reduce chromatin accessibility across HS genes, thereby reducing the ability of HSF1 to bind promoters and subsequently impairing the HSR. To investigate whether this was the case at the Hspa1b promoter, we performed a SYBR green qPCR-coupled micrococcal nuclease (MNase) digestion assay on chromatin isolated from mouse brains 2 hours after treatment with vehicle or HSP990. The Hspa1b promoter region was hypersensitive to MNase treatment in all treatment groups (Figure 6F), which suggests that the promoter is equally accessible in both WT and R6/2 mice and that this accessibility does not change upon HSP990 treatment. To investigate the chromatin accessibility downstream of the promoter region, we performed SYBR green qPCR for regions spanning the transcription start site to the 3′ untranslated region (48). As was observed for the promoter region, the chromatin accessibility of all regions of Hspa1b was highly comparable between WT and R6/2 mice at 12 weeks of age. However, upon treatment with HSP990, WT mice — but not R6/2 mice — showed increased MNase digestion along the length of the gene (Figure 6F), a feature coupled to HSF1 promoter binding (48).
These data strongly suggest that there is reduced HSF1 promoter binding and subsequent nucleosome dissociation from the Hspa1b gene in R6/2 mice compared with WT littermates after treatment with HSP990. However, this does not appear to be due to the accessibility of the chromatin itself around the Hspa1b promoter, despite the presence of hypoacetylated H4 in HD mice (Figure 6, C and E). Tetra AcH4 has been suggested to be a strong modulator of HSF1 binding (38), and it is conceivable that the acetylation state of H4 could dictate the inducible binding of HSF1 to target DNA sequences independently of promoter accessibility.