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Reversible phase separation of HSF1 is required for an acute transcriptional response during heat shock

Nature Cell Biology volume 24, pages 340–352 (2022)Cite this article

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Abstract

Heat-shock transcription factor 1 (HSF1) orchestrates the fast and vast cellular response to heat shock through increased expression of heat-shock proteins. However, how HSF1 rapidly and reversibly regulates transcriptional reprogramming remains poorly defined. Here by combining super-resolution imaging, in vitro reconstitution and high-throughput sequencing, we reveal that HSF1 forms small nuclear condensates via liquid–liquid phase separation at heat-shock-protein gene loci and enriches multiple transcription apparatuses through co-phase separation to promote the transcription of target genes. Furthermore, the phase-separation capability of HSF1 is fine-tuned through phosphorylation at specific sites within the regulatory domain. Last, we discovered that HSP70 disperses HSF1 condensates to attenuate transcription following the cessation of heat shock and further prevents the gel-like phase transition of HSF1 under extended heat-shock stress. Our work reveals an inducible and reversible phase-separation feedback mechanism for dynamic regulation of HSF1 activity to drive the transcriptional response and maintain protein homeostasis during acute stress.

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Fig. 1: HSF1 undergoes LLPS and forms small condensates during HS.
Fig. 2: HSF1 undergoes LLPS in vitro.
Fig. 3: Post-translational modifications regulate LLPS of HSF1.
Fig. 4: HSF1 compartmentalizes transcription apparatuses in LLPS condensates.
Fig. 5: Phase separation markedly enhances the chromatin binding of HSF1.
Fig. 6: Phase separation of HSF1 promotes HSP-gene transcription.
Fig. 7: HSP70 prevents HSF1 condensates from undergoing a gel-like phase transition under prolonged HS stress.

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Data availability

All of the data associated with the figures are provided as Supplementary Information. The human genome GRCh38/hg18 was used for the alignment of the Cut&Tag and RNA-seq dataset. The Cut&Tag and RNA-seq datasets generated in this study have been deposited to NCBI Gene Expression Omnibus under the accession numbers GSE192370 and GSE191134. All other data supporting the findings of this study are available from the corresponding authors on reasonable request. Source data are provided with this paper.

Code availability

Code for the analysis of STORM data has been deposited to Zenodo (https://zenodo.org/record/5804854#.YcgnNmjP0uU).

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Acknowledgements

We thank L. D. Lavis (HHMI, Janelia Research Campus) for providing the HaloTag ligand dyes JF549 and 646 (Janelia Fluor 549, 646) as well as T. Czerny and E. Riegel (University of Navarre) for providing us with the 6×HSE plasmid. We thank the National Center for Protein Sciences at Peking University in Beijing, China, for assistance with the microscopy imaging, flow cytometry and protein preparation. This work was supported by grants from the National Science Foundation of China (grant nos 21825401 (Y.S.), 31900898 (S.P.) and 82070301 (Y.Z.)), the China Postdoctoral Science Foundation (S.P.; grant nos 2019M660004 and 2019T120013) and the National Key R&D Program of China (Y.S.; grant no. 2017YFA0505300).

Author information

Author notes

  1. These authors contributed equally: Hongchen Zhang, Shipeng Shao.

Authors and Affiliations

  1. State Key Laboratory of Membrane Biology, Biomedical Pioneering Innovation Center (BIOPIC), School of Life Sciences, Peking University, Beijing, China

    Hongchen Zhang, Xiaotian Wang, Yizhi Qin & Yujie Sun

  2. National Biomedical Imaging Center, College of Future Technology, Peking University, Beijing, China

    Hongchen Zhang, Xiaotian Wang, Yizhi Qin & Yujie Sun

  3. Beijing Institute of Heart Lung and Blood Vessel Disease, Beijing Anzhen Hospital, Capital Medical University, Beijing, China

    Shipeng Shao & Yong Zeng

  4. MOE Key Lab for Cellular Dynamics, School of Life Sciences, University of Science and Technology of China, Hefei, China

    Qiunan Ren & Shengqi Xiang

  5. State Key Laboratory of Protein and Plant Gene Research, Peking University, Beijing, China

    Yuxin Wang & Junyu Xiao

  6. School of Life Sciences, Peking University, Beijing, China

    Yuxin Wang & Junyu Xiao

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Contributions

H.Z., S.S. and Y.S. conceived and designed the experiments. H.Z. performed most of the experiments. S.S. performed the live-cell and STORM imaging. X.W. assisted with the identification of the IDR of HSF1. Y.Q. helped with the STORM imaging. Q.R. and S.X. performed the NMR experiments. Y.W. and J.X. helped with protein purification from HEK293F cells. H.Z. and S.S. analysed all of the data. H.Z., S.S., Y.Z. and Y.S. wrote the manuscript.

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Correspondence to Shipeng Shao or Yujie Sun.

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Nature Cell Biology thanks Bernd Bukau and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 HSF1 undergoes LLPS during HS.

(a) Prediction of intrinsically disordered residues in HSF1 protein by Predictor of Natural Disordered Regions (PONDR). The disordered region is shown by a green horizontal line. The domain organizations of HSF1 are shown at the bottom. (b) Fluorescence images of HeLa cells transfected with HSF1–EGFP under NHS and HS (42 °C, 0.5 h) conditions. Scale bars, 5 μm. (c) Frequency distribution of the number of HSF1 nSBs per cell. 2256 nSBs were analysed in 139 cells pooled across 3 independent experiments. (d) Roundness distribution of the HSF1 nSBs. 2256 nSBs were analysed in 139 cells pooled across 3 independent experiments. (e,f) FRAP recovery of nSBs formed by HSF1. Cells were heat-shocked for 0.5 h at 42 °C to induce phase separation and FRAP was performed after HSF1 droplets emerged. Whole droplets were bleached for the analysis of exchange between nucleoplasm and nSBs, while half droplets were bleached for the analysis of exchange within nSBs and exchange between nucleoplasm and nSBs. Data are presented as mean ± s.d. n = 16 droplets for whole, n = 16 droplets for half collected from 3 independent experiments. Scale bars, 1 μm (e). Fluorescence intensity tracks of the whole droplet and half droplet bleach (f). (g) Fluorescence images of HSF1 droplets under HS (42 °C, 0.5 h) condition before and after treatment with 10% 1,6-hexanediol for 1 min. Scale bars, 10 μm. (h) Inducing of HSF1 phase separation by Opto-Droplet in living cells under NHS condition. Representative fluorescence images showing the distribution of HSF1 before and after blue light activation. Scale bar, 10 μm. (i) Western blot showing slower migration of HSF1–HaloTag in the successful knock-in cells. GAPDH was used as a loading control. (j,k) Recruitment of HSF1 to LacO array can mediate the formation of a low-complexity domain (LCD) hub in living cells. Top, schematic for a LacO array (~256 LacO repeats) integrated into the genome of NIH3T3 cells. Bottom, HSF1-LacI-mCh formed an LCD hub after HS (42 °C, 0.5 h) when transiently expressed. Alternatively, LacI-mCh and HSF1-LacI-mCh were transiently expressed under NHS condition as controls. Scale bars, 5 μm (j). The mean fluorescence intensity at LacO foci in cells with different expression levels for the LacI-mCh (NHS), HSF1-LacI-mCh (NHS) and HSF1-LacI-mCh (HS) were measured (k). n = 22 cells for LacI, 21 cells for HSF1-LacI-NHS, and 38 cells for HSF1-LacI-HS pooled from 3 independent experiments. The data was smoothed in R using lm() function for linear smooths, and error bands represent the standard error of the smoothing. (l) EMSA showing that 1,6-hexanediol does not disrupt HSF1–DNA interaction. Images are representative of three independent experiments (b, e, g-j, l).

Source data

Extended Data Fig. 2 The formation of small condensates of HSF1 in the nucleoplasm in response to HS.

(a) Voronoï-based segmentation of HSF1 condensates. Zoomed images were displayed at the bottom. Each molecule has a polygon defined by its neighbouring molecules. Edges of Voronoï polygons are located equidistant from the nearest two molecules. When a new molecule is added, this bisector is cut by the bisectors computed between the old molecules and the new ones. Each new molecule was computed to plot the Voronoï diagram repeatedly until all the molecules were counted. Then a threshold was set twice the average localization density to choose the clusters of HSF1 molecules. (b,c) Representative fluorescence images of widefield and STORM showing the formation of small condensates in different cell lines under NHS (b) and HS (42 °C, 0.5 h) (c) conditions. Scale bars, 2 μm. Zoomed scale bars, 100 nm. (d-f) Cluster analysis of HSF1 in different cell lines under NHS and HS (42 °C, 0.5 h) conditions. Localization per cluster (d), cluster number per cell (e), and cluster size (f) were shown. Data are presented as mean ± s.d. Individual data points correspond to the average value for one cell. n = 19 cells for A549-HS, 27 cells for HEK293T-HS, 18 cells for MDA-MB-231-HS, 15 cells for U2OS-HS, 15 cells for A549-NHS, 12 cells for HEK293T-NHS, 15 cells for MDA-MB-231-NHS, 20 cells for U2OS-NHS pooled from 3 independent experiments. The paired two-tailed Student’s t-test was used to compare the data (e). Boxplots: 25th to 75th percentiles, median, 1.5×interquartile as whiskers (d). (g) Schematic of the single molecule tracking experiment. Using the HaloTag knock-in cells, HSF1 was labelled with JF549 and tracked using HILO illumination. Then the trajectory of each molecule was extracted and analysed. (h) The single molecule trajectory of WT HSF1 and LLPS-incompetent M3 under NHS and HS (42 °C, 0.5 h) conditions. The trajectories were colour-coded according to their diffusion coefficients. n = 10 cells for WT HSF1 under NHS, 10 cells for WT HSF1 under HS, 20 cells for M3 under NHS, 16 cells for M3 under HS pooled from 3 independent experiments. (i) The mean square displacement of HSF1 and LLPS-incompetent M3 under NHS and HS (42 °C, 0.5 h) conditions. (j) The distribution of diffusion coefficient of HSF1 and LLPS-incompetent M3 under NHS and HS (42 °C, 0.5 h) conditions. Images are representative of three independent experiments (b-c).

Source data

Extended Data Fig. 3 Measurement of endogenous HSF1 concentration and HSE valency in the promoter of HSP genes.

(a) Schematic diagram showing the measurement process of HSF1 concentration. A calibration curve was obtained using a serial dilution of JF549 dye to get the relationship between the fluorescence intensity and dye concentration. Under the same imaging condition, JF549 labelled HaloTag knock-in HSF1 cells were imaged to obtain the mean fluorescence intensity of the nucleus. (b) Representative images of JF549 dye of different concentrations displayed using the 16 colours lookup table. 9 fields of view were captured to measure the average fluorescence intensity under different concentrations. (c) Representative image of HSF1 in HaloTag knock-in cells labelled using JF549 dye. The image was shown using the 16 colours lookup table. Images are representative of three independent experiments. (d) Calibration curve between JF549 concentration and fluorescence intensity. Data are displayed as mean ± s.d. n = 9 fields of view pooled from 3 independent experiments. The mean fluorescence intensity of HSF1 in HeLa cells was measured in 501 cells. The ribbon region represents the standard error of the linear fitting. (e) Pie plot showing the number of TTCnnGAAnnTTC motif in HSF1 Cut&Tag peaks. (f) Pie plot showing the number of GAAnnTTC motif in HSF1 Cut&Tag peaks.

Source data

Extended Data Fig. 4 Both the regulatory and trimerization domains are required for HSF1 phase separation.

(a) Domain structure of HSF1 truncations. Each amino acid number of the domain boundaries was labelled. (b) Confocal microscopy images of HeLa cells transfected with various HSF1 truncations as indicated under NHS and HS (42 °C, 0.5 h) conditions. Scale bars, 5 μm. (c) Representative images of HSF1 mutants under HS (42 °C, 0.5 h) condition before and after treatment with 10% 1,6-hexanediol for 1 min. Scale bars, 5 μm. (d) Co-localization analysis of HSF1△RD and stress granule marker G3BP1 under NHS and HS (42 °C, 0.5 h) conditions. Scale bars, 5 μm. Zoomed scale bars, 0.5 μm. (e,f) Co-localization analysis of TDP43 (e) and EWSR1 (f) with stress granule marker G3BP1 under NHS and HS (42 °C, 0.5 h) conditions. Scale bars, 5 μm. All Images in (b-f) are representative of 3 independent experiments.

Extended Data Fig. 5 Cut&Tag reveals similar binding patterns of WT HSF1 under HS and M1 under NHS.

(a) Western blot showing the successful knockout of HSF1 gene in HeLa cells. Images are representative of 3 independent experiments. (b) Peak numbers of Cut&Tag signals under different conditions. (c) Pie chart showing the distribution of the indicated annotation feature among the Cut&Tag peaks of WT HSF1 under HS (left) or M1 under NHS (right) condition. (d,e) Summary of the most enriched motifs identified within the peaks in WT HSF1 under HS (d) and M1 under NHS (e) conditions. Motif enrichment was analysed using Homer and statistically determined by ZOOPS scoring (zero or one occurrence per sequence) coupled with the hypergeometric enrichment calculations. (f) Scatterplot showing correlation of global Cut&Tag signal of WT HSF1 under HS and M1 under NHS conditions. The coefficient of determination (R) is determined by two-sided Pearson correlation. The genome was divided into 10 kb bins. (g,h) The 1,6-hexanediol treatment causes a significant loss of signal in the Cut&Tag experiment. Scatterplot showing correlation of global Cut&Tag signal of WT HSF1 under HS + Hex vs. WT HSF1 under HS (g) or M1 under NHS + Hex vs. M1 under NHS (h) conditions. The coefficient of determination (R) is determined by by two-sided Pearson correlation. The genome was divided into 10 kb bins.

Source data

Extended Data Fig. 6 IGV showing LLPS enhanced chromatin occupancy at multiple HSP gene loci.

(a-f) Integrative genomics viewer (IGV) snapshots for the indicated Cut&Tag signal at the well-known HSP gene foci such as the HSP90AA1 (a), HSP90AB1 (b), HSPA6 (c), HSPB1 (d), HSPH1 (e), and IER5 (f). Samples from top to bottom are HSF1 WT (tracks 1-3), HSF1-M1 (tracks 4-5), HSF1-M3 (tracks 6-7), RNA pol II (tracks 8-9), BRD4 (tracks 10-11), and CYCT1 (tracks 12-13). Tracks 3 and 5 were treated with 1.5% 1,6-hexanediol for 30 mins before Cut&Tag was performed.

Extended Data Fig. 7 LLPS-incompetent HSF1 mutant M3 were unable to form small condensates during HS.

(a) Super-resolution imaging of HSF1 M3 under NHS and HS (42 °C, 0.5 h) conditions. Scale bars, 5 μm. Zoomed scale bars, 0.2 μm. Images are representative of 3 independent experiments. (b-d) Cluster analysis of HSF1 M3 single molecules under NHS and HS conditions. Individual data points correspond to the average value for one cell. Cluster size (b), localization per cluster (c), and cluster number per cell (d) were shown. Data are presented as mean ± s.d. (d). n = 9 cells of WT NHS, 11 cells of WT HS, 17 cells of M3 NHS, and 18 cells of M3 HS pooled from 3 independent experiments. The paired two-tailed Student’s t-test was used to compare the data (d). Boxplots: 25th to 75th percentiles, median, 1.5×interquartile as whiskers (c).

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Extended Data Fig. 8 RNA-seq analysis process of all samples.

(a) Bar plot showing the total read counts of the RNA-seq libraries. (b) Boxplot of transformed data of read counts. The regularized log (rlog) transformation implemented in the DESeq2 package was used to transform the data, as it effectively reduces mean-dependent variance. The transformation was done in the iDEP web tool (http://bioinformatics.sdstate.edu/idep/). Expression profiles of 17683 genes were used to generate the plot. Boxplots: 25th to 75th percentiles, median, 1.5× interquartile as whiskers. (c) Density plot showing the read counts vs. the expression level in the transformed data of each library. (d) PCA indicated the group of samples. (e) Gene set enrichment analysis (GSEA) shows that the differential expressed genes of M1-NHS vs. WT-NHS were not enriched in the cancer-specific target of the HSF1 gene set. The P-value was calculated by an empirical phenotype-based permutation test; the false discovery rate (q) is adjusted for gene set size and several hypotheses testing.

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Extended Data Fig. 9 Phase separation of HSF1 specifically promotes transcription of HSP genes during HS but not cancer-specific genes.

(a) Left: fluorescence images of HSF1 (S326D) purified from NHS E. coli in the droplet formation assay. HSF1 purified from HS (42 °C, 1 h) human HEK293F cells was used as a positive control. Scale bars, 2 μm. Right: fluorescence images of HSF1 (S326D)-EGFP in HeLa cells under the NHS condition. HSF1–EGFP transfected HaLa cells under HS (42 °C, 0.5 h) were used as a positive control. Scale bars, 5 μm. Images are representative of three independent experiments. (b) Western blot showing successful RNAi of HSF1 in HEK293T, MDA-MB-231, A549, and U2OS cells. GAPDH was used as a loading control. Images are representative of three independent experiments. (c-f) RT–qPCR analysis of RBM23 mRNA expression in HSF1 RNAi HEK293T (c), MDA-MB-231 (d), A549 (e), and U2OS (f) cells transfected with various HSF1 mutants. Data are presented as mean ± s.d. P-values were determined using a paired two-tailed Student’s t-tests. n = 4 biologically independent samples. (g-j) RT–qPCR analysis of HSPA1A (HSP70) mRNA expression in HSF1 RNAi HEK293T (g), MDA-MB-231 (h), A549 (i), and U2OS (j) cells transfected with various HSF1 mutants. Data are presented as mean ± s.d. P-values were determined using a paired two-tailed Student’s t-tests. n = 4 biologically independent samples.

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Extended Data Fig. 10 Negative regulation of HSF1 phase separation during HS is HSP70-specific.

(a) Representative images of HSP70 FL, HSP70-N, and HSP70-C phase separation. Recombinant HSP70 WT (10 μM), HSP70 N (10 μM), and HSP70 C (10 μM) were added to phase separation buffer with 200 mM NaCl in the presence (top) or absence (bottom) of 10% Dextran-2000. Scale bars, 2 μm. Images are representative of 3 independent experiments. (b) HSP70 FL (8 μM) was added to pre-formed HSF1-mCh (4 μM) droplets, the HSF1 droplets were dissolved rapidly in vitro. Scale bars, 2 μm. Images are representative of 3 independent experiments. (c) Representative images of HSF1-mCh (4 μM) with indicated concentration of HSP70 FL without ATP (left) and with 5 mM ATP (right). Scale bars, 2 μm. Images are representative of 3 independent experiments. (d) Analysis of HSF1 droplet size with different HSP70 concentrations (4 μM or 8 μM) with or without ATP. n = 2397/HSF1 + 4 μM HSP70-WO ATP (droplets/group), 0/HSF1 + 8 μM HSP70-WO ATP, 820/HSF1 + 4 μM HSP70-With ATP, 768/HSF1 + 8 μM HSP70-With ATP pooled across 3 independent experiments. Boxplots: 25th to 75th percentiles, median, 1.5×interquartile as whiskers. (e) Representative images of HSF1-mCh (4 μM) with the 8 μM HSP40 and HSP90 in vitro. Scale bars, 2 μm. Images are representative of 3 independent experiments.

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Supplementary information

Supplementary Information

Supplementary Figs. 1,2.

Reporting Summary

Supplementary Tables

Supplementary Table 1, Plasmids used in this work. Supplementary Table 2, HSP70 (HSPA1L) RNA FISH probe sequence. Supplementary Table 3, DNA Oligopaint probes for DNAJB1, HSPA2, HSP90AA1 and GAPDH.

Supplementary Video 1

Single-molecule movie of HSF1 under NHS and HS conditions.

Supplementary Video 2

Single-molecule movie of HSF1 (M3) under NHS and HS conditions

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Zhang, H., Shao, S., Zeng, Y. et al. Reversible phase separation of HSF1 is required for an acute transcriptional response during heat shock. Nat Cell Biol 24, 340–352 (2022). https://doi.org/10.1038/s41556-022-00846-7

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