HSF1 phosphorylation establishes an active chromatin state via the TRRAP-TIP60 complex and promotes tumorigenesis

Abstract

Transcriptional regulation by RNA polymerase II is associated with changes in chromatin structure. Activated and promoter-bound heat shock transcription factor 1 (HSF1) recruits transcriptional co-activators, including histone-modifying enzymes; however, the mechanisms underlying chromatin opening remain unclear. Here, we demonstrate that HSF1 recruits the TRRAP-TIP60 acetyltransferase complex in HSP72 promoter during heat shock in a manner dependent on phosphorylation of HSF1-S419. TRIM33, a bromodomain-containing ubiquitin ligase, is then recruited to the promoter by interactions with HSF1 and a TIP60-mediated acetylation mark, and cooperates with the related factor TRIM24 for mono-ubiquitination of histone H2B on K120. These changes in histone modifications are triggered by phosphorylation of HSF1-S419 via PLK1, and stabilize the HSF1-transcription complex in HSP72 promoter. Furthermore, HSF1-S419 phosphorylation is constitutively enhanced in and promotes proliferation of melanoma cells. Our results provide mechanisms for HSF1 phosphorylation-dependent establishment of an active chromatin status, which is important for tumorigenesis.

Fig. 1. Identification of the co-activator TRRAP that interacts with HSF1.

a Relative enrichment of proteins identified in HSF1 ChIP preparations from heat-shocked cells. HeLa cells were untreated or treated with heat shock at 42 °C for 60 min, and HSF1-interacting proteins were identified by ChIP-MS. Thirty-one proteins highly enriched upon HS (difference of peptide numbers >3) are shown. Proteins related to histone modifications are indicated in red. b HSF1 interacts with TRRAP in the nucleus during heat shock. Cytoplasmic (Cyto) and nuclear (Nucl) extracts were prepared and complexes co-immunoprecipitated using anti-IgG or anti-HSF1 and subjected to immunoblotting. c Expression of HSP72 mRNA in TRRAP-KD cells during heat shock. Levels of HSP72 mRNA were quantified, and the levels relative to that in control SCR-treated cells are shown. Extracts of cells were subjected to immunoblotting. d Venn diagram of HSF1 and TRRAP ChIP-seq binding peaks in HeLa cells untreated (Cont.) or treated with heat shock (HS). Because the TRRAP antibody generates a low signal-to-noise ratio in ChIP assay, the limited numbers of TRRAP binding peaks were identified using ChIP-seq. e MA (log ratio versus abundance) plot of ChIP-seq binding intensities for HSF1 and TRRAP in control (R₁) and heat shocked (R₂) cells at the common binding peaks for HSF1-HS/TRRAP-HS (28 peaks). The number of reads in the peak regions after normalization for a given sample was counted and the M and A values of each peak were calculated and plotted, where M = log₂(R₂/R₁) and A = log₂(R₁ + R₂)/2. Dots with ratios (M) that increased during heat shock (−log₁₀P > 1) are indicated in red. f ChIP-seq binding profiles of HSF1 and TRRAP in control (Cont.) and heat-shocked (HS) cells. Normalized read numbers are shown and peaks are indicated in red. Norminal p-values were determined by by two-way ANOVA in c. Error bars indicate SEM (n = 3) in c. Experiments were repeated two times for b.

Fig. 2. Phosphorylated HSF1 at S419 recruits TRRAP to HSP72 promoter.

a, b Schematic representation of phosphorylation sites in hHSF1 and its mutants. DBD (red box), DNA-binding domain; HR (orange box), hydrophobic heptad repeat; DHR (light green box), downstream of HR-C (yellow box). Extracts of heat-shocked HSF1-null MEF cells expressing these mutants or GFP were co-immunoprecipitated using anti-TRRAP and subjected to immunoblotting. HeLa cells were treated with heat shock, and cell extracts were subjected to HSF1 immunoprecipitation and immunoblotting using antibody for HSF1 phospho-S419 or HSF1 (c). Some extracts were incubated without (−) or with lambda protein phosphatase (λPPase) (d). Blue arrows indicate the positions of HSF1-S419 phosphorylated bands. The intensity of the upper band was markedly enhanced during heat shock. e Alignment of amino acid sequences for the DHR domain containing S419 in human, mouse, chicken, lizard (Anolis sagrei, As), and frog (Xenopus tropicalis, Xt) HSF1. This domain is characterized by the heptad repeats of hydrophobic amino acids (black circles and triangles), which are conserved in vertebrate HSF family members (HSF1 to HSF4). A red circle indicates the position of HSF1-S419. f Cells, in which endogenous HSF1 was replaced with hHSF1-HA, S419A-HA or S419G-HA, were heat-shocked, and HSP72 mRNA levels were quantified. Extracts from these cells were subjected to immunoblotting. Occupancy of TRRAP, HSF1 (g), or Pol II (h) in cells expressing HSF1-S419 mutants. Cells were treated as described in f, and ChIP assay was performed. d, distal; p, proximal; and inter, intergenic. Norminal p-values were determined by one-way ANOVA, followed by Tukey-Kramer test in f–h. Error bars indicate SEM (n = 3) in f–h. Experiments were repeated two times for a–d.

Fig. 3. TRRAP-TIP60 complex is responsible for histone acetylation at specific residues.

a Occupancy of pan-acetylated histone H3 and H4 in HSP72 promoter. ChIP assay was performed using HeLa cells treated as described in Fig. 2f. b Components of the TRRAP-TIP60 complex identified dominantly in heat-shocked cells. Nuclear extracts were prepared from cells overexpressing hTRRAP-3 × FLAG, and proteins co-immunoprecipitated with anti-FLAG were identified by MS. c Interaction between HSF1 and TIP60. Complexes co-immunoprecipitated using anti-HSF1 in nuclear extracts of heat-shocked cells were subjected to immunoblotting. d Occupancy of TIP60 in cells expressing HSF1-S419 mutants. ChIP assay was performed using cells treated as described in a. e Cells, in which components of HAT complexes were knocked down, were treated with heat shock. Levels of HSP72 mRNA were quantified, and relative levels are shown. f Occupancy of active chromatin marks in HSP72 promoter. ChIP assay was performed using untreated (Cont.) or heat-shocked (HS) cells, in which HSF1, TRRAP, TIP60, or p300 were knocked down. Norminal p-values were determined by one-way ANOVA, followed by Tukey-Kramer test in a and d–f. Error bars indicate SEM (n = 3) in a and d–f. Experiments were repeated two times for c.

Fig. 4. Acetylation-dependent histone H2B mono-ubiquitination by TRIM33.

Venn diagram of HSF1, TRIM33, and TRIM24 ChIP-seq binding peaks (a) and their binding profiles (b) in control (Cont.) and heat-shocked (HS) HeLa cells. Normalized read numbers are shown (green), and peaks are indicated in red. c Expression of HSP72 mRNA in TRIM33- or TRIM24-KD cells during heat shock. HSP72 mRNA levels relative to that in control SCR-treated cells are shown. d HSF1 interacts with TRIM33 and TRIM24 in the nucleus during heat shock. Cytoplasmic (Cyto) and nuclear (Nucl) extracts were prepared and complexes co-immunoprecipitated using anti-IgG or anti-HSF1 were subjected to immunoblotting. e Nuclear extracts were prepared from heat-shocked cells, in which HSF1, TRRAP, TIP60, or p300 was knocked down, and complexes co-immunoprecipitated using anti-HSF1 were subjected to immunoblotting. f HSF1 interacts with histone H3K18ac. Cells treated as described in e were incubated with the crosslinking reagent DSP. Nuclear extracts were prepared and co-immunoprecipitated complexes were subjected to immunoblotting. g TRIM33 occupancy in cells expressing HSF1-S419 phosphorylation site mutants. Cells were treated as described in Fig. 2f, and ChIP assay was performed. h TRIM33 occupancy in TRRAP- or TIP60-KD cells. Cells were treated as described in e. i TRIM33- and TRIM24-dependent H2BK120ub in HSP72 promoter. Cells, infected with adenovirus expressing shRNA for HSF1, TRRAP, TRIM33, or TRIM24, were heat-shocked, and ChIP assay was performed. j Occupancy of H2BK120ub and H3K4me3 in cells expressing a TRIM33 mutant. Cells, in which endogenous TRIM33 was replaced with the RING domain mutant of HA-hTRIM33, were heat-shocked, and ChIP assay was performed. Norminal p-values were determined by two-way ANOVA in c or by one-way ANOVA, followed by Tukey-Kramer test in g–j, Error bars indicate SEM (n = 3) in g–j. Experiments were repeated two times for d, e, and f.

Fig. 5. PLK1 phosphorylates HSF1-S419 and triggers histone modifications.

HeLa cells were infected with adenovirus expressing shRNA for PLK1, CSNK1A1, NEK7 or SCR (a), or treated with BI6727 (PLK1 inhibitor), FR180204 (ERK1/2 inhibitor), AZD6244 (MEK1/2 inhibitor), or rapamycin (mTOR inhibitor) (b). These cells were then treated with heat shock. Cell extracts were subjected to HSF1 immunoprecipitation and immunoblotting using antibody for HSF1 phospho-S419 or HSF1. Blue arrows indicate the positions of HSF1-S419 phosphorylated bands. The intensity of the upper band was markedly enhanced during heat shock. c PLK1 interacts with HSF1 in the nucleus during heat shock. Cell extracts were prepared as described in Fig. 1b and subjected to immunoblotting. Cells, in which endogenous PLK1 was replaced with GFP, wild-type HA-hPLK1, or its kinase-dead mutant, were treated with heat shock. Nuclear extracts were prepared and complexes co-immunoprecipitated using anti-PLK1 (d) or anti-HSF1 (e) were subjected to immunoblotting. Cells, in which endogenous PLK1 was replaced with GFP, HA-hPLK1, or HA-hPLK1-K82R, were treated with heat shock. ChIP-qPCR of HSF1, TRRAP, TRIM33, and PLK1 (f), or H2BK120ub and histone H2B (g) was performed. Norminal p-values were determined by one-way ANOVA, followed by Tukey-Kramer test in f and g. Error bars indicate SEM (n = 3) in f and g. Experiments were repeated two times for a–e.

Fig. 6. HSF1-S419 phosphorylation maintains proteostasis capacity.

a TRRAP promotes cell survival. Cells were infected with adenovirus expressing shRNA for HSF1, TRRAP or SCR, and were heat-shocked at 45 °C for the indicated periods. Number of viable cells excluding trypan blue was counted. Extracts of cells were subjected to immunoblotting. b Phosphorylation of HSF1-S419 promotes cell survival. Cells, in which endogenous HSF1 was replaced with GFP, wild-type hHSF1-HA, or hHSF1-S419A mutants were heat-shocked at 45 °C for 4 h. Number of viable cells excluding trypan blue was counted, and MTT assay was performed. Extracts of cells were subjected to immunoblotting. c Phosphorylation of HSF1-S419 inhibits the accumulation of ubiquitylated proteins. Cells treated as described in b were heat-shocked at 45 °C for 4 h. The accumulation of insoluble ubiquitylated proteins was examined by immunoblotting using anti-Ub antibody and quantified. β-Actin levels in the soluble fraction were also shown. d TRRAP promotes refolding of the luciferase sensor protein during recovery from heat shock. HeLa-Fluc cells treated as described in a were heat-shocked at 42 °C for 2 h. These cells were then recovered at 37 °C for the indicated periods, and luciferase activity values were calculated and normalized to the value of control cells (100%). Extracts of cells were subjected to immunoblotting. e Phosphorylation of HSF1-S419 promotes refolding of the luciferase sensor protein. HeLa-Fluc cells treated as described in b were heat-shocked at 42 °C for 2 h. These cells were then recovered at 37 °C for 4 h, and luciferase activity values relative to that of control cells were estimated. Extracts of cells were subjected to immunoblotting. Norminal p values were determined by two-way ANOVA in a and d or by one-way ANOVA, followed by Tukey-Kramer test in b, c and e. Error bars indicate SEM (n = 4) in a and b, or (n = 3) in c, d, and e.

Fig. 7. HSF1 phosphorylation supports tumor formation.

a Equal amounts of total cell extracts from melanoma cell lines, HeLa cells (control and heat-shocked), and OUMS-36T-3F cells were subjected to HSF1 immunoprecipitation and immunoblotting. Relative levels of HSF1-S419 phosphorylation normalized by HSF1 protein levels are shown. b Phosphorylation of HSF1-S419 is induced by overexpression of PLK1 in OUMS-36T-3F cells. HSF1 immunoprecipitation and immunoblotting were performed as described in Fig. 5a. c, d Endogenous HSF1 was replaced with GFP, hHSF1-HA, S419A-HA, S419G-HA (c, d), or S326A/S419A-HA (d) in melanoma and OUMS-36T-3F cells. Cell extracts were subjected to immunoblotting. e MeWo and OUMS-36T-3F cells were treated as described in d. HSP72 mRNA levels were quantified and shown. f, g Tumor sizes and masses of melanoma cells expressing hHSF1 phosphorylation site mutants in athymic nude mice. The sizes (f) and masses (g) of tumors at indicated time points after injection were calculated until 22 days. Bars in g indicate mean values (n = 8). h Schematic model for establishing an active chromatin state in HSP72 promoter. PLK1 phosphorylates HSF1-S419 (1), which recruits the TRRAP-TIP60 complex. TIP60 and p300 are responsible for H3K18ac and H3K23ac, respectively (2, 3). TRIM33 and TRIM24, recruited to the promoter by interactions with HSF1 and the histone acetylation marks, are required for H2BK120ub (4). Norminal p values were determined by one-way ANOVA, followed by Tukey-Kramer test in a and e or by two-way ANOVA in c, d and f. Error bars indicate SEM (n = 3) in a and e, (n = 4) in c and d, or (n = 8) in f. Experiments were repeated two times for b.