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Nascent-protein ubiquitination is required for heat shock–induced gene downregulation in human cells

Abstract

Proteotoxic stress such as heat shock causes heat-shock factor (HSF)-dependent transcriptional upregulation of chaperones. Heat shock also leads to a rapid and reversible downregulation of many genes, a process we term stress-induced transcriptional attenuation (SITA). The mechanism underlying this conserved phenomenon is unknown. Here we report that enhanced recruitment of negative transcription elongation factors to gene promoters in human cell lines induces SITA. A chemical inhibitor screen showed that active translation and protein ubiquitination are required for the response. We further find that proteins translated during heat shock are subjected to ubiquitination and that p38 kinase signaling connects cytosolic translation with gene downregulation. Notably, brain samples of subjects with Huntington’s disease also show transcriptional attenuation, which is recapitulated in cellular models of protein aggregation similar to heat shock. Thus our work identifies an HSF-independent mechanism that links nascent-protein ubiquitination to transcriptional downregulation during heat shock, with potential ramifications in neurodegenerative diseases.

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Fig. 1: N-TEFs are enriched at chromatin upon heat shock.
Fig. 2: Active translation is required for stress-induced increase of N-TEFs at chromatin and SITA.
Fig. 3: Ubiquitination of nascent proteins is required for SITA.
Fig. 4: p38α kinase (MAPK14)-mediated signaling is required for SITA.
Fig. 5: Acute and chronic proteotoxic stress cause gene downregulation.

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Data availability

All the deep-sequencing data reported in this study are deposited in Gene Expression Omnibus and are available under accession number GSE112379. The mass spectrometry proteomics data have been deposited with the ProteomeXchange Consortium via the PRIDE partner repository with the data set identifier PXD012077. The source data for Figs. 2d, 3c, 4g and 5c,f are available online. Uncropped images of Figs. 1c,d, 2ac, 3a,b,fj, 4b,c,f and 5a,b,e are available with the paper online. All other data will be made available upon request.

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Acknowledgements

We thank Y.L. Dréan (University of Rennes), H. Kampinga (University of Groningen), R. Kopito (Stanford University), J.T. Lis (Cornell University), R. Paro (ETH), A. Shilatifard (Northwestern University), L. Sistonen (University of Turku), E. Trompouki (Max Planck Institute of Immunobiology and Epigenetics) and U. Wölfle (University of Freiburg) for sharing cell lines, protocols and plasmids; and J. Büscher, G. Mittler and sequencing and bioinformatics facilities for data acquisition and help with the analyses. We acknowledge critical discussions with P. Beli, U. Hartl, J. Palvimo, L. Sistonen and colleagues at the Max Planck Institute of Immunobiology and Epigenetics. Excellent technical assistance from S. Bares and A. Antonova is acknowledged. R.S. acknowledges financial support by the Max Planck Society, the German Research Foundation through the collaborative research center Medical Epigenetics, through the research grant SA 3190 (R.S.) and through Germany’s Excellence Strategy (CIBSS, EXC-2189, project ID 390939984).

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Contributions

R.S. conceived the project; R.S., F.A.G. and P.T. designed the study; F.A.G., P.T. and A.K. performed experiments and interpreted the results; F.A.G. conceived and performed experiments on nascent-protein ubiquitination during the revision process; B.H. performed all the computational analyses; F.A.G. and R.S. wrote the manuscript with input from all the authors.

Corresponding author

Correspondence to Ritwick Sawarkar.

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Integrated supplementary information

Supplementary Figure 1 Heat-shock-induced SITA in human cells quantified by RNA Pol II ChIP-seq and PRO-seq.

(a) Heatmap showing normalized RNA Pol II ChIP-seq signal in gene body of all significantly downregulated genes upon heat-shock in HEK293 cells. Each row represents a gene. Individual replicates from Fig. 1a are shown to demonstrate reproducibility. Gene subset with high basal expression levels and highest transcriptional downregulation upon HS, listed in Supplementary Data Set 1, are indicated by blue vertical bar adjacent to the heat-map (‘Top Downregulated genes’). These genes are used for all the genomic analyses in this study unless otherwise stated. The high basal expression of these genes allows a better statistical assessment of SITA, as detailed in Methods. The rest of the downregulated genes are indicated with a red bar, and are also listed in Supplementary Data Set 1. (b) Top left panel: metaplot of RNA Pol II binding in promoter vicinity around transcriptional start sites (TSS) in NHS and HS HEK293 cells. Bottom left panel (in gray): metaplot showing RNA Pol II occupancy in gene body regions (TSS + 1.5 kb to TES – 0.5kb). y-axes indicate normalized ChIP-seq occupancy of RNA Pol II in reads per million (rpm). Right panels: Genome browser tracks showing ChIP-seq occupancy of RNA Pol II in NHS and HS HEK293 cells. Two representative genes - RABGGTB and RPS20 - are shown. In gray: RNA Pol II occupancy in gene body regions shown at a scale different than promoter region. The vertical scale indicates normalized read density in reads per million (rpm). Histograms on the right: quantification of gene body RNA Pol II occupancy for the depicted genes across experimental replicates (n = 5 independent experiments, error bars depict standard error of the mean). (c) Heatmap showing normalized RNA Pol II ChIP-seq signal in gene body of all significantly downregulated genes upon heat-shock in K562 cells. Each row represents a gene. Individual replicates from Supplementary Fig. 1d are shown to demonstrate reproducibility. Gene subset with high basal expression levels and highest transcriptional downregulation upon HS, listed in Supplementary Data Set 2, are indicated by blue vertical bar adjacent to the heat-map (‘Top Downregulated genes’). The rest of the downregulated genes are indicated with a red bar, and are also listed in Supplementary Data Set 2. (d) MA plot of RNA Pol II ChIP-seq occupancy in gene body in K562 cells with or without heat-shock. Genes with significant (red dots) or nonsignificant changes (gray dots) in gene-body RNA Pol II occupancy (p-value <0.01 as calculated by DESeq2 analysis, n = 2 independent experiments) are indicated. Blue dots: gene subset with high basal expression levels and highest transcriptional downregulation upon HS, listed in Supplementary Data Set 2 and used for the genomic analyses in Fig. 1e. (e) Heatmap of normalized gene body counts from Precision Run-On sequencing (PRO-seq) signal in HEK293 cells with or without heat-shock. Each row represents a gene. The set of genes and their vertical order in the heatmap is the same as in Supplementary Fig. 1a. Individual replicates are shown to demonstrate reproducibility. (f) Effect of heat-shock on downregulation of transcription in HEK293 (right panels) and K562 cells (left panels) as assessed in individual replicates of RNA Pol II ChIP-seq. The y-axis represents the ratio of ChIP-seq signal in gene body in HS over NHS HEK293 cells. Violin plots show population distribution of the data. Upper panels: all downregulated genes; lower panels: most downregulated genes. (g) The empirical cumulative distribution function (ECDF) plot of the distribution of gene body ratios (HS over NHS) of RNA Pol II ChIP-seq values in K562 and HEK cells. Dotted lines: examples showing that 50% of HEK293 and K562 downregulated genes have more than 40% and 47% reduction in their expression levels respectively.

Supplementary Figure 2 Nascent-transcript quantification at individual loci by RT-qPCR as an assay for SITA.

(a) Primer validation for detecting nascent transcripts in HEK293 cells. 5,6-Dichlorobenzimidazole 1-β-D-ribofuranoside (DRB) is an inhibitor of Cdk9, and is used to rapidly block transcriptional elongation. DRB treatment reduces nascent transcripts, as shown recently (Palozola et al., 2017 (ref. 19)) and can be recapitulated in the shown quantification. Primer pairs were designed on exon-intron junction for indicated genes to capture the nascent unspliced transcript, similar to the recent study (Palozola et al., 2017 (ref. 19)). (b) Genome browser track view of HNRNPA2B1 gene showing no change in RNA Pol II occupancy during heat shock. This transcript is used as a normalizing control for all RT-qPCR quantification of nascent transcripts in this work, unless otherwise stated. Right: quantification of gene body RNA Pol II occupancy assessed by ChIP-seq (n = 5 independent experiments, error bars depict standard error of the mean). (c) Effect of HS on the nascent transcript levels of indicated genes in HEK293, K562 and SH-SY5Y cells. Value of 1.0 on y-axis indicates expression level of genes in NHS cells, shown by a dashed line. Error bars represent standard error of the mean (n = 3 independent experiments). (d) Effect of HS on the nascent transcript levels of indicated genes in T cell blasts derived from human primary PBMCs. Error bars represent standard error of the mean (n = 2 independent experiments).

Supplementary Figure 3 N-TEF enrichment at chromatin upon heat shock.

Western blot analyses of N-TEFs in total cell extracts and chromatin fractions of NHS and HS in different cell lines or by various methodologies as indicated. Histone H3 was used as loading control. (a) K562 and HCT116 cells. (b) HEK293 cells were either exposed to standard HS treatment as in the rest of this work (HS) or an acute HS treatment as previously reported, referred to as HS (acute) (Mahat et al., 2016 (ref. 7)). Detailed procedures can be found in the Methods. (c) Chromatin fractions from HEK293 cells were either obtained using a high salt-based protocol as in the rest of this work or using a low salt-based protocol, as in Henikoff, 2009 (ref. 53). (d and e) Chromatin extraction was performed using protocol described by Kustatscher et al., 2014 (ref. 54) (d), or with the protocol described in Tresini et al., 2015 (ref. 55) (e). (f) NELFA and NELFE enrichment at promoters of downregulated genes as detected by ChIP-seq. Histogram showing the ratio of HS to NHS occupancy at promoters (TSS +/- 500bp) for NELFA (left) and NELFE (right). Majority of genes have a higher peak upon HS (threshold of HS/NHS>1.3 showed by the vertical dotted line). (g) Metaplots of NELFA, NELFE and RNA Pol II binding around transcriptional start sites (TSS) of control genes in K562 cells with or without heat shock. y-axes indicate normalized ChIP-seq occupancy in reads per million (rpm). Lower right panel (in gray): metaplot showing RNA Pol II occupancy in gene body regions (TSS+1.5kb to TES-0.5kb). Control genes are defined as genes with no change in NELFE ChIP-seq signal upon heat shock in the region TSS +/-250bp. A list of these genes is available in Supplementary Data Set 6. (h) Effect of NELFE depletion on control genes as assessed by Pol II ChIP-seq occupancy in gene body. The y-axis represents the ratio of ChIP-seq signal in gene body in HS over NHS HEK293 cells treated with siRNA against NELFE (SiNELFE) or scrambled (SiScr). Violin plots show population distribution of the data. List of genes used for this analysis is available in Supplementary Data Set 7. (i) Effect of NELFE and PAF1 knockdown on nascent transcription as measured by RT-qPCR-based nascent transcript quantification in NHS and HS HEK293 cells. Value of 1.0 on y-axis indicates expression level of genes in NHS cells. Error bars represent standard error of the mean (n = 3 independent experiments).

Supplementary Figure 4 SITA of metabolic genes.

(a) Schematic diagram of the lower pathway of glycolysis from glyceraldehyde 3-phosphate to pyruvate and further to lactate formation. Enzymes that are downregulated upon HS are highlighted in red. GAPDH: glyceraldehyde-3-phosphate dehydrogenase; PGK: phosphoglycerate kinase; PGAM: phosphoglycerate mutase; ENO: enolase; PKM: pyruvate kinase; LDHA: L-lactate dehydrogenase. (b) Quantification of gene body RNA Pol II occupancy for genes encoding glycolytic enzymes in scramble and NELFE-depleted HEK293 cells with or without heat shock. Value of 1.0 on y-axis indicates expression level of genes in NHS cells. Error bars represent standard error of the mean (n = 2 independent experiments). (c) Genome browser tracks showing RNA Pol II ChIP-seq occupancy in gene-body regions of metabolic genes in NHS and HS HEK293 cells treated with siRNA against NELFE or scramble controls. The vertical scale indicates normalized read density in reads per million (rpm). (d) Mass spectrometry-based quantification of three metabolites shown in Supplementary Fig. 4a, which are reaction products of enzymes that are transcriptionally downregulated in heat-shock. Quantification was performed in scramble and NELFE-depleted HEK293 cells with or without heat-shock. Results are expressed as % decrease in each condition relative to NHS metabolite levels. Error bars represent standard error of the mean (n = 5 independent experiments).

Supplementary Figure 5 Heat-shock factor (HSF) is not required for stress-induced increase of NELFE at chromatin and consequent transcriptional downregulation.

(a) Nascent transcript quantification for indicated genes in HS and NHS HEK293 cells treated with siRNA against HSF1 and HSF2. Value of 1.0 on y-axis indicates expression level of genes in NHS cells, shown by a dashed line. Error bars represent standard error of the mean (n=2 independent experiments). (b) HSP70 transcription quantification to functionally validate HSF knockdown in the same samples as in Supplementary Fig. 5a. HS-induction of HSP70 transcription requires HSF. These data accompany Western blot analyses in Fig. 2a. (c) Western blot analysis of NELFE in chromatin fractions of HEK293 cells treated with specific inhibitors of HSF, namely KRIBB11 and Quercetin. Histone H3 was used as loading control. (d) RT-qPCR quantification of nascent transcripts of indicated genes in NHS and HS HEK293 cells treated with HSF inhibitors. Error bars represent standard error of the mean (n=3 independent experiments). (e) HSP70 transcription quantification to functionally validate HSF inhibition. (f) Western blot analysis of NELFE in total extracts and chromatin fractions of wild-type or HSF1and HSF2 double knockout mouse embryonic fibroblasts with or without heat shock. Histone H3 was used as loading control. (g) Representative blots of the small-molecule screen to identify regulators of stress-induced N-TEF recruitment to chromatin. Western blot analysis of NELFE in chromatin fractions of HEK293 cells treated with either translation inhibitor cycloheximide (left panel) or inhibitor of ubiquitin-activating enzyme E1 (right panel) NSC 624206 (Ub-E1 inhibitor). Histone H3 was used as loading control. (h) Western blot analysis of newly translated protein labeling. Cells were incubated for the indicated times with the amino acid analog AHA, an azide to label newly translated proteins. After click-chemistry reactions in total cell extracts to biotinylate azide-labeled new proteins, the biotinylated proteins were detected with a streptavidin-HRP conjugated reagent on western blot. Vimentin was used as a loading control. Cycloheximide incubation during AHA labeling completely abrogated its incorporation, confirming translation inhibition. Labeling strategy depicted in Fig. 3f. (i) Western blot analysis of HSP70 in total fractions of HEK293 cells treated with cycloheximide or vehicle. Ponceau staining was used as loading control. (j) RT-qPCR quantification of heat-shock genes HSP70, HSP60 and HSP90 in NHS and HS HEK293 cells treated with cycloheximide or vehicle. Error bars represent standard error of the mean (n = 2 independent experiments). (k) RT-qPCR-based nascent transcription quantification of indicated genes in NHS and HS cells treated with cycloheximide or vehicle. Top panel: HEK293 cells, lower panel: K562 cells. Error bars represent standard error of the mean (n = 3 independent experiments). (l) Effect of heat shock or cycloheximide on gene expression as assessed by RNA Pol II ChIP-seq occupancy in gene body. The y-axis represents the ratio of ChIP-seq signal in gene body in each condition over the NHS vehicle condition. Violin plots show population distribution of the data.

Supplementary Figure 6 Ubiquitination and translation during heat shock.

(a) Western blot analyses of K48-linked protein ubiquitination in several human cell lines and T cell blasts derived from human primary PBMCs with or without heat-shock. POLR2C was used as loading control. (b) Western blot analysis of K48-linked protein ubiquitination in total extracts of HEK293 cells with or without heat shock treated with vehicle or translation inhibitors cycloheximide, puromycin or anisomycin. POLR2C was used as loading control. (c) Effect of heat shock or Ub-E1 inhibitor on gene expression as assessed by RNA Pol II ChIP-seq occupancy in gene body. The y-axis represents the ratio of ChIP-seq signal in gene body in each condition over the NHS vehicle condition. Violin plots show population distribution of the data. (d) Western blot analysis of total free ubiquitin levels in HEK293 cells. Cells were treated with cycloheximide or vehicle and subjected to heat-shock. Free ubiquitin is shown. POLR2C was used as loading control. (e) Western blot analysis of total free ubiquitin levels in HEK293 cells. Cells were treated with Ub-E1 inhibitor or vehicle and subjected to heat-shock. Free ubiquitin is shown. POLR2C was used as loading control. (f) Western blot analysis of newly translated proteins from HEK293 cells treated with Ub-E1 inhibitor or vehicle and subjected to heat-shock. The labeling of nascent proteins is depicted in Fig. 3f. Biotinylated proteins were detected with a Streptavidin-HRP conjugated reagent. Vimentin was used as a loading control. Heat shock causes a decrease in total translation amount.

Supplementary Figure 7 SITA in an acute model of heat shock.

(a and c) Western blot analyses of K48-linked protein ubiquitination in extracts from HeLa cells exposed to acute HS at 42 °C for the indicated times. Vimentin was used as loading control. (b, d and e) Nascent transcript quantification of the indicated genes. Value of 1.0 on y-axis indicates expression level of NHS cells. Error bars represent standard error of the mean (at least n = 3 independent experiments). (f) Western blot analysis of NELFE in total extracts and chromatin fractions of HeLa cells subjected to acute heat-shock at 42 °C for the indicated times. Histone H3 was used as loading control.

Supplementary Figure 8 p38α kinase (MAPK14)-mediated signaling is required for SITA.

(a) Western blot analyses of MAPK14 in total extracts and chromatin fractions of HEK293 cells with or without heat shock treated with p38 inhibitor. Histone H3 was used as loading control. (b) Top: Schematic representation of how the Pausing Index was calculated. Bottom: The empirical cumulative distribution function (ECDF) plot of the pausing index of RNA Pol II ChIP-seq values in HEK293 cells treated with p38 inhibitor or vehicle subjected or not to heat-shock. (c) Genome browser tracks showing RNA Pol II ChIP-seq occupancy in a representative gene RASL10B in NHS and HS HEK293 cells treated with p38 inhibitor or vehicle. The vertical scale indicates normalized read density in reads per million.

Supplementary Figure 9 Proteasome inhibition does not lead to SITA.

(a) Western blot analyses of total free ubiquitin levels. HEK293 cells were pretreated with Ub-E1 inhibitor or vehicle and incubated for 2 h with L-arginine or its analog L-canavanine. Free ubiquitin is shown. POLR2C was used as loading control. (b) Western blot analysis of newly translated proteins from HEK293 cells treated with Ub-E1 inhibitor or vehicle and incubated for 2 h with L-arginine or its analog L-canavanine. The labeling of nascent proteins is depicted in Fig. 3f. Biotinylated proteins were detected with a Streptavidin-HRP conjugated reagent. Vimentin was used as a loading control. (c) Western blot analyses of NELFE in total extracts and chromatin fractions of HEK293 cells with or without proteasome inhibitor Bortezomib (Bort). Histone H3 was used as loading control. (d) ChIP-qPCR measuring NELFE occupancy at indicated gene promoters in HEK293 cells incubated with Bortezomib or vehicle. ‘NO PEAK’ primer set amplifies genomic region not expected to bind NELFE and acts as a negative control. The y-axis indicates the amount of immunoprecipitated DNA relative to starting input material. Error bars represent standard error of the mean (n = 2 independent experiments). (e) Effect of Bortezomib on the nascent transcript levels of indicated genes in HEK293 cells. Value of 1.0 on y-axis indicates expression level of genes in vehicle-incubated cells, shown by a dashed line. Error bars represent standard error of the mean (n = 2 independent experiments).

Supplementary Figure 10 SITA in cells with Huntingtin misfolding.

(a and b) Effect of transient overexpression of HTT in neuronal SH-SY5Y cells (a) and HEK293 cells (b). Top: Fluorescence microscopy images of cells overexpressing GFP-tagged variants of either HTT25Q or HTT103Q. White arrow indicates HTT aggregate. Bottom: RT-qPCR-based nascent transcript quantification in cells expressing mutant HTT103Q. Value 1.0 on y-axis represents normalized expression levels of genes in cells expressing wild-type HTT25Q. Error bars represent standard error of the mean (n = 2 independent experiments for a and 3 replicates for b). (c) HTT 103Q or HTT 25Q transient overexpression in neuronal SH-SY5Y cell line. Left: Western blotting with antibody against K48-linked protein ubiquitination in total extracts and NELFE in chromatin fractions. POLR2C was used as loading control. Right: Corresponding densitometry immunoblot quantification. Enrichment of NELFE at chromatin in HTT 103Q-expressing cells relative to HTT 25Q-expressing cells. Error bars represent standard error of the mean (n = 2 independent experiments). (d) Western blot analysis of K48-linked protein ubiquitination in proteins translated during HTT expression or total proteins of HEK293 cells induced for 48 h to express either soluble HTT25Q or aggregating HTT119Q and labeled with AHA for the last 1h before lysis. Vimentin and biotinylated proteins were used as a loading control. (e) Densitometry immunoblot quantification of experiment depicted in Fig 5e. Enrichment of NELFE at chromatin after induction of cells harboring inducible variants of HTT relative to uninduced cells. Error bars represent standard error of the mean (n = 3 independent experiments). (f and g) Normalized expression levels in brain samples of healthy controls in comparison with Huntington’s disease subjects for genes encoding metabolic enzymes indicated in Supplementary Fig. 4a (f) and genes encoding chaperones (g). Statistical significance determined by Student’s t-test against Healthy controls is indicated by asterisks. *: p < 0.5; **: p < 0.05 and ***: p < 0.005.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–10 and Supplementary Tables 1–4

Reporting Summary

Supplementary Dataset 1

Differentially expressed genes assessed from HEK293 RNA Pol II ChIP-seq.

Supplementary Dataset 2

Differentially expressed genes assessed from K562 RNA Pol II ChIP-seq.

Supplementary Dataset 3

Comparison of differentially expressed genes between HEK293 and K562.

Supplementary Dataset 4

HS-induced changes in the chromatin proteome.

Supplementary Dataset 5

Blots in main figures shown as uncropped images.

Supplementary Dataset 6

Control genes used for analyzing NELF ChIP-seq and Pol II ChIP-seq in Supplementary Fig. 3g.

Supplementary Dataset 7

Control genes used for analyzing RNA Pol II ChIP-seq in Supplementary Fig. 3h.

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Aprile-Garcia, F., Tomar, P., Hummel, B. et al. Nascent-protein ubiquitination is required for heat shock–induced gene downregulation in human cells. Nat Struct Mol Biol 26, 137–146 (2019). https://doi.org/10.1038/s41594-018-0182-x

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