Journal: bioRxiv

Article Title: Heat Shock Factor 1 (HSF1) specifically potentiates c-MYC-mediated transcription independently of the canonical heat-shock response

doi: 10.1101/2022.02.22.481519

Figure Lengend Snippet: (A) Co-amplification of c-MYC and HSF1 in human cancers. Data are generated by the TGCA Research Network ( https://www.cancer.gov/tcga ). (B) Positive correlation between c-MYC and HSF1 mRNA levels in human cancers. Analyses were performed using the GEPIA2 web server . B2M: β-2-microglobulin. (C) The dual MYC reporter system, comprising an E-box element-driven SEAP plasmid and a CMV-driven Gaussia luciferase (GLuc) plasmid, were co-transfected with indicated plasmids into HEK293T cells for 48 hr (mean ± SD, n =3 independent experiments, One-way ANOVA). Cell lysates were immunoblotted. (D) Endogenous c-MYC activities were measured by the dual reporter system in HEK293T cells co-transfected with indicated plasmids (mean ± SD, n =3 independent experiments, One-way ANOVA). (E) Hsf1 was deleted in immortalized Rosa26-CreERT 2 ; Hsf1 fl/fl MEFs treated with and without 4-OHT for 7 days. c-MYC levels were detected by immunoblotting. (F) Top panel: schematic depiction of c-MYC-gDNA PLA technique. Middle panel: visualization of endogenous c-MYC binding to genomic DNAs by PLA (red) in immortalized Rosa26-CreERT2; Hsf1 fl/fl MEFs. Scale bars: 10µm. Lower panel: quantitation of c-MYC-gDNA binding by counting the numbers of PLA foci per nucleus (mean ± SD, n=98 nuclei, Mann Whitney test). (G) Quantitation of c-MYC-bound genomic DNA fragments following ChIP in immortalized MEFs (mean ± SD, n = 3 independent experiments, two-tailed Student’s t test).

Article Snippet: pBabe-HSF1-FLAG was a gift from Robert Kingston (Addgene plasmid#1948). pMSCV-HA-cMYCT58A was a gift from Scott Lowe (Addgene plasmid#18773). pCherry-HSP90alpha was a gift from Didier Picard (Addgene plasmid#108222). pCDNA3-2xHA-c-MYC was a gift from Martine Roussel (Addgene plasmid#74161). pLX304-LacZ-V5 was a gift from William Hahn (Addgene plasmid#42560). pBabe-LacZ, pBabe-HSF1 1-323 , and pBabe-HSF1 324-529 were described previously . pLX304-MAX-V5 (HsCD00440967) and pDONR221-GCN5 (HsCD00829789) vectors were purchased from DNASU plasmid depository. pLX304-LacZ-V5 and pLX304-GCN5-V5 vectors were co-transfected with packaging vector (delta VPR) and an envelope vector (VSV-G) into HEK293T packaging cells using TurboFect transfection reagent (Cat#R0531, ThermoFisher).

Techniques: Amplification, Generated, Plasmid Preparation, Luciferase, Transfection, Western Blot, Binding Assay, Quantitation Assay, MANN-WHITNEY, Two Tailed Test

Journal: bioRxiv

Article Title: Heat Shock Factor 1 (HSF1) specifically potentiates c-MYC-mediated transcription independently of the canonical heat-shock response

doi: 10.1101/2022.02.22.481519

Figure Lengend Snippet: (A) Quantitation of released genomic DNA fragments in the CUT&RUN experiments in immortalized MEFs (mean ± SD, n =2 biological replicates, two-tailed Student’s t test). (B) TSS plots of aligned CUT&RUN-seq reads following spike-in normalization (two biological replicates are combined). (C) Genomic distributions of CUT&RUN-seq peaks in Hsf1 WT and Hsf1 CKO MEFs. (D) Box plots of peak signals in Hsf1 WT and Hsf1 CKO MEFs. The box bounds the IQR divided by the median and the whiskers extend to the minimum and maximum values (Mann-Whitney U test). Left: all peaks (n=209,466 WT and 5,900 CKO); Right: peaks within promoters (n=18,859 WT and 4,090 CKO). (E) Venn diagram showing the overlaps of c-MYC target genes among different experiments. (F) Venn diagram showing the overlaps of c-MYC target genes identified by CUT&RUN-seq between Hsf1 WT and Hsf1 CKO MEFs. (G) Summary of c-MYC target genes encoding chaperones and co-chaperones. (H) Visualization of c-MYC binding to Hsp and Hsf1 genes

Article Snippet: pBabe-HSF1-FLAG was a gift from Robert Kingston (Addgene plasmid#1948). pMSCV-HA-cMYCT58A was a gift from Scott Lowe (Addgene plasmid#18773). pCherry-HSP90alpha was a gift from Didier Picard (Addgene plasmid#108222). pCDNA3-2xHA-c-MYC was a gift from Martine Roussel (Addgene plasmid#74161). pLX304-LacZ-V5 was a gift from William Hahn (Addgene plasmid#42560). pBabe-LacZ, pBabe-HSF1 1-323 , and pBabe-HSF1 324-529 were described previously . pLX304-MAX-V5 (HsCD00440967) and pDONR221-GCN5 (HsCD00829789) vectors were purchased from DNASU plasmid depository. pLX304-LacZ-V5 and pLX304-GCN5-V5 vectors were co-transfected with packaging vector (delta VPR) and an envelope vector (VSV-G) into HEK293T packaging cells using TurboFect transfection reagent (Cat#R0531, ThermoFisher).

Techniques: Quantitation Assay, Two Tailed Test, MANN-WHITNEY, Binding Assay

Journal: bioRxiv

Article Title: Heat Shock Factor 1 (HSF1) specifically potentiates c-MYC-mediated transcription independently of the canonical heat-shock response

doi: 10.1101/2022.02.22.481519

Figure Lengend Snippet: (A) Co-IP of FLAG-HSF1, HA-c-MYC, and V5-MAX from transfected HEK293T cells (representative images of three independent experiments). HC: heavy chain. (B) Endogenous c-MYC-HSF1 interactions were detected by PLA in HeLa cells using a rabbit anti-c-MYC (D3N8F) antibody and a mouse monoclonal anti-HSF1 (E-4) antibody. Scale bars, 10µm. (C) In vitro direct interactions between recombinant HSF1 and c-MYC/MAX dimers were detected by the Lumit™ immunoassay. The reactions without primary antibodies were set up as the blanks, which were subtracted (mean ± SD, n =3 independent experiments, two-tailed Student’s t test). (D) In vitro interactions between recombinant c-MYC and MAX proteins, with and without recombinant HSF1 proteins, were detected by the Lumit™ immunoassay (mean ± SD, n =3 independent experiments, two-tailed Student’s t test). (E) In vitro binding of recombinant c-MYC/MAX dimers to E-box oligos, with and without recombinant HSF1 proteins, was detected by ELISA (mean ± SD, n =3 independent experiments, two-tailed Student’s t test). (F) Lysates of HEK293T cells co-transfected with indicated plasmids for 3 days were treated with EtBr (400 µg/mL) on ice for 30 min. The interaction of FLAG-HSF1 with HA-c-MYC/V5-MAX was detected by co-IP (representative images of three independent experiments). (G) Lysates of HEK293T cells co-transfected with indicated plasmids for 3 days were treated with either 10 U of DNase I or RNase at 37 °C for 20 min, followed by co-IP (representative images of three independent experiments). (H) Schematic depiction of two possible models of DNA-dependent protein-protein interactions. (I) Endogenous c-MYC-HSF1 interactions were detected by brightfield PLA in HeLa cells, following treatment with or without EtBr (100 µg/mL) for 1 hr. Scale bars: 10µm.

Article Snippet: pBabe-HSF1-FLAG was a gift from Robert Kingston (Addgene plasmid#1948). pMSCV-HA-cMYCT58A was a gift from Scott Lowe (Addgene plasmid#18773). pCherry-HSP90alpha was a gift from Didier Picard (Addgene plasmid#108222). pCDNA3-2xHA-c-MYC was a gift from Martine Roussel (Addgene plasmid#74161). pLX304-LacZ-V5 was a gift from William Hahn (Addgene plasmid#42560). pBabe-LacZ, pBabe-HSF1 1-323 , and pBabe-HSF1 324-529 were described previously . pLX304-MAX-V5 (HsCD00440967) and pDONR221-GCN5 (HsCD00829789) vectors were purchased from DNASU plasmid depository. pLX304-LacZ-V5 and pLX304-GCN5-V5 vectors were co-transfected with packaging vector (delta VPR) and an envelope vector (VSV-G) into HEK293T packaging cells using TurboFect transfection reagent (Cat#R0531, ThermoFisher).

Techniques: Co-Immunoprecipitation Assay, Transfection, In Vitro, Recombinant, Two Tailed Test, Binding Assay, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: Heat Shock Factor 1 (HSF1) specifically potentiates c-MYC-mediated transcription independently of the canonical heat-shock response

doi: 10.1101/2022.02.22.481519

Figure Lengend Snippet: (A) The expression of known c-MYC target genes was quantitated by qRT-PCR, following transient Gcn5 KD for 48 hr in immortalized MEFs (mean ± SD, n = 3 independent experiments, Two-way ANOVA). (B) Endogenous c-MYC transcriptional activities were measured by the dual reporter system in HEK293T cells transfected with indicated plasmids and siRNAs (mean ± SD, n = 3 independent experiments, One-way ANOVA). (C) Left panel: Endogenous c-MYC binding to gDNA binding was detected by PLA in immortalized MEFs stably expressing LacZ or GCN5 . Scale bars, 10 µm. Right panel: quantitation of these PLA foci per nucleus (mean ± SD, n≥100 nuclei, One-way ANOVA). (D) Quantitation of total RNAs extracted with immortalized MEFs stably expressing LacZ or GCN5 (mean ± SD, n = 3 biological replicates, One-way ANOVA). (E) Volcano plot of the differentially expressed genes due to Hsf1 KD. (F) Box-and-whisker plots of the abundance of DEGs in the control cells (n=1,640 or 1,269, Mann-Whitney U test). The box bounds the IQR divided by the median and the whiskers extend to the minimum and maximum values. (G) Visualization of DEGs in MEFs expressing different genes and siRNAs by clustering heatmaps (three biological replicates each group). (H) Seaborn correlation heatmap of gene expression among different experimental groups. (I) Venn diagram showing the overlaps among the c-MYC CUT&RUN-seq target genes, the DEGs following Hsf1 KD, and the DEGs rescued by GCN5 overexpression in immortalized MEFs. (J) Pathway enrichment analyses of the DEGs in immortalized MEFs following Hsf1 KD. (K) Heatmap visualization of the DEGs involved in the ribosome, proteasome, lysosome, and chaperone pathways (each data point represents the average of three biological replicates).

Article Snippet: pBabe-HSF1-FLAG was a gift from Robert Kingston (Addgene plasmid#1948). pMSCV-HA-cMYCT58A was a gift from Scott Lowe (Addgene plasmid#18773). pCherry-HSP90alpha was a gift from Didier Picard (Addgene plasmid#108222). pCDNA3-2xHA-c-MYC was a gift from Martine Roussel (Addgene plasmid#74161). pLX304-LacZ-V5 was a gift from William Hahn (Addgene plasmid#42560). pBabe-LacZ, pBabe-HSF1 1-323 , and pBabe-HSF1 324-529 were described previously . pLX304-MAX-V5 (HsCD00440967) and pDONR221-GCN5 (HsCD00829789) vectors were purchased from DNASU plasmid depository. pLX304-LacZ-V5 and pLX304-GCN5-V5 vectors were co-transfected with packaging vector (delta VPR) and an envelope vector (VSV-G) into HEK293T packaging cells using TurboFect transfection reagent (Cat#R0531, ThermoFisher).

Techniques: Expressing, Quantitative RT-PCR, Transfection, Binding Assay, Stable Transfection, Quantitation Assay, Whisker Assay, MANN-WHITNEY, Over Expression

Journal: bioRxiv

Article Title: Heat Shock Factor 1 (HSF1) specifically potentiates c-MYC-mediated transcription independently of the canonical heat-shock response

doi: 10.1101/2022.02.22.481519

Figure Lengend Snippet: (A) Co-IP of FLAG-HSF1, HA-c-MYC, and V5-GCN5 in transfected HEK293T cells (representative images of three independent experiments). (B) Endogenous c-MYC-GCN5 interactions were detected by PLA in HeLa cells. Scale bars, 10 µm. (C) Co-IP of endogenous c-MYC and GCN5, following transient Hsf1 KD in immortalized MEFs (representative images of three independent experiments). (D) In vitro binding of recombinant c-MYC proteins to individual HSF1 peptides immobilized on ELISA plates. Fold changes in binding are presented as a heatmap (n=3 independent experiments). (E) Visualization of interactions between transfected FLAG-HSF1 and endogenous c-MYC by PLA in HEK293T cells using a mouse monoclonal anti-FLAG antibody and a rabbit anti-c-MYC antibody. Scale bars, 10 µm. (F) c-MYC transcriptional activities were measured by the dual reporter system in HEK293T cells co-transfected with indicated plasmids (mean ± SD, n = 3 independent experiments, One-way ANOVA). (G) In vitro binding of recombinant GCN5 proteins to individual HSF1 peptides immobilized on ELISA plates. Fold changes in binding are presented as a heatmap (n=3 independent experiments). (H) Visualization of interactions between transfected FLAG-HSF1 and endogenous GCN5 by PLA in HEK293T cells. Scale bars, 10 µm. (I) Co-IP of endogenous c-MYC and GCN5 in HEK293T cells transfected with LacZ or FLAG-HSF1 (representative images of three independent experiments).

Article Snippet: pBabe-HSF1-FLAG was a gift from Robert Kingston (Addgene plasmid#1948). pMSCV-HA-cMYCT58A was a gift from Scott Lowe (Addgene plasmid#18773). pCherry-HSP90alpha was a gift from Didier Picard (Addgene plasmid#108222). pCDNA3-2xHA-c-MYC was a gift from Martine Roussel (Addgene plasmid#74161). pLX304-LacZ-V5 was a gift from William Hahn (Addgene plasmid#42560). pBabe-LacZ, pBabe-HSF1 1-323 , and pBabe-HSF1 324-529 were described previously . pLX304-MAX-V5 (HsCD00440967) and pDONR221-GCN5 (HsCD00829789) vectors were purchased from DNASU plasmid depository. pLX304-LacZ-V5 and pLX304-GCN5-V5 vectors were co-transfected with packaging vector (delta VPR) and an envelope vector (VSV-G) into HEK293T packaging cells using TurboFect transfection reagent (Cat#R0531, ThermoFisher).

Techniques: Co-Immunoprecipitation Assay, Transfection, In Vitro, Binding Assay, Recombinant, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: Heat Shock Factor 1 (HSF1) specifically potentiates c-MYC-mediated transcription independently of the canonical heat-shock response

doi: 10.1101/2022.02.22.481519

Figure Lengend Snippet: (A) ChIP-qPCR assays were performed to detect the acetyl-histone 3 (Lys9/Lys14) on c-MYC target or non-c-MYC target loci in immortalized MEFs (mean ± SD, n = 3 biological replicates, One-way ANOVA). (B) Left panel: the protein expression of fusion between HA-HSF1 324-529 and c-MYC was detected by immunoblotting. Right panel: the transcriptional activity of fusion proteins was measured by the dual reporter system (mean ± SD, n = 3 independent experiments, One-way ANOVA). (C) The proposed model of HSF1-mediated c-MYC activation. HSF1 helps recruit GCN5 to c-MYC, thereby promoting chromatin remodeling and potentiating the c-MYC-mediated transcription. (D) HSF1 regulates two distinct activation states of c-MYC. Without HSF1 association, the transcriptional activity of cellular c-MYC remains low, sustaining at a primary state; by contrast, HSF1 association renders c-MYC highly active, transiting to an advanced state. (E) HSF1 governs at least two discrete transcriptional programs. Upon its activation, either in the face of environmental stress or within malignant cells, HSF1 initiates the canonical PSR/HSR, a mechanism of action depending on HSE binding. By contrast, in the absence of environmental stress most cellular HSF1 remains repressed; however, some HSF1 associates with c-MYC and potentiates its mediated transcription, a mechanism of action independent of HSE binding.

Article Snippet: pBabe-HSF1-FLAG was a gift from Robert Kingston (Addgene plasmid#1948). pMSCV-HA-cMYCT58A was a gift from Scott Lowe (Addgene plasmid#18773). pCherry-HSP90alpha was a gift from Didier Picard (Addgene plasmid#108222). pCDNA3-2xHA-c-MYC was a gift from Martine Roussel (Addgene plasmid#74161). pLX304-LacZ-V5 was a gift from William Hahn (Addgene plasmid#42560). pBabe-LacZ, pBabe-HSF1 1-323 , and pBabe-HSF1 324-529 were described previously . pLX304-MAX-V5 (HsCD00440967) and pDONR221-GCN5 (HsCD00829789) vectors were purchased from DNASU plasmid depository. pLX304-LacZ-V5 and pLX304-GCN5-V5 vectors were co-transfected with packaging vector (delta VPR) and an envelope vector (VSV-G) into HEK293T packaging cells using TurboFect transfection reagent (Cat#R0531, ThermoFisher).

Techniques: Expressing, Western Blot, Activity Assay, Activation Assay, Binding Assay

Journal: bioRxiv

Article Title: Heat Shock Factor 1 (HSF1) specifically potentiates c-MYC-mediated transcription independently of the canonical heat-shock response

doi: 10.1101/2022.02.22.481519

Figure Lengend Snippet: (A) Co-amplification of c-MYC and HSF1 in human cancers. Data are generated by the TGCA Research Network ( https://www.cancer.gov/tcga ). (B) Positive correlation between c-MYC and HSF1 mRNA levels in human cancers. Analyses were performed using the GEPIA2 web server . B2M: β-2-microglobulin. (C) The dual MYC reporter system, comprising an E-box element-driven SEAP plasmid and a CMV-driven Gaussia luciferase (GLuc) plasmid, were co-transfected with indicated plasmids into HEK293T cells for 48 hr (mean ± SD, n =3 independent experiments, One-way ANOVA). Cell lysates were immunoblotted. (D) Endogenous c-MYC activities were measured by the dual reporter system in HEK293T cells co-transfected with indicated plasmids (mean ± SD, n =3 independent experiments, One-way ANOVA). (E) Hsf1 was deleted in immortalized Rosa26-CreERT 2 ; Hsf1 fl/fl MEFs treated with and without 4-OHT for 7 days. c-MYC levels were detected by immunoblotting. (F) Top panel: schematic depiction of c-MYC-gDNA PLA technique. Middle panel: visualization of endogenous c-MYC binding to genomic DNAs by PLA (red) in immortalized Rosa26-CreERT2; Hsf1 fl/fl MEFs. Scale bars: 10µm. Lower panel: quantitation of c-MYC-gDNA binding by counting the numbers of PLA foci per nucleus (mean ± SD, n=98 nuclei, Mann Whitney test). (G) Quantitation of c-MYC-bound genomic DNA fragments following ChIP in immortalized MEFs (mean ± SD, n = 3 independent experiments, two-tailed Student’s t test).

Article Snippet: pBabe-HSF1-FLAG was a gift from Robert Kingston (Addgene plasmid#1948). pMSCV-HA-cMYCT58A was a gift from Scott Lowe (Addgene plasmid#18773). pCherry-HSP90alpha was a gift from Didier Picard (Addgene plasmid#108222). pCDNA3-2xHA-c-MYC was a gift from Martine Roussel (Addgene plasmid#74161). pLX304-LacZ-V5 was a gift from William Hahn (Addgene plasmid#42560). pBabe-LacZ, pBabe-HSF1 1-323 , and pBabe-HSF1 324-529 were described previously . pLX304-MAX-V5 (HsCD00440967) and pDONR221-GCN5 (HsCD00829789) vectors were purchased from DNASU plasmid depository. pLX304-LacZ-V5 and pLX304-GCN5-V5 vectors were co-transfected with packaging vector (delta VPR) and an envelope vector (VSV-G) into HEK293T packaging cells using TurboFect transfection reagent (Cat#R0531, ThermoFisher).

Techniques: Amplification, Generated, Plasmid Preparation, Luciferase, Transfection, Western Blot, Binding Assay, Quantitation Assay, MANN-WHITNEY, Two Tailed Test

Journal: bioRxiv

Article Title: Heat Shock Factor 1 (HSF1) specifically potentiates c-MYC-mediated transcription independently of the canonical heat-shock response

doi: 10.1101/2022.02.22.481519

Figure Lengend Snippet: (A) Quantitation of released genomic DNA fragments in the CUT&RUN experiments in immortalized MEFs (mean ± SD, n =2 biological replicates, two-tailed Student’s t test). (B) TSS plots of aligned CUT&RUN-seq reads following spike-in normalization (two biological replicates are combined). (C) Genomic distributions of CUT&RUN-seq peaks in Hsf1 WT and Hsf1 CKO MEFs. (D) Box plots of peak signals in Hsf1 WT and Hsf1 CKO MEFs. The box bounds the IQR divided by the median and the whiskers extend to the minimum and maximum values (Mann-Whitney U test). Left: all peaks (n=209,466 WT and 5,900 CKO); Right: peaks within promoters (n=18,859 WT and 4,090 CKO). (E) Venn diagram showing the overlaps of c-MYC target genes among different experiments. (F) Venn diagram showing the overlaps of c-MYC target genes identified by CUT&RUN-seq between Hsf1 WT and Hsf1 CKO MEFs. (G) Summary of c-MYC target genes encoding chaperones and co-chaperones. (H) Visualization of c-MYC binding to Hsp and Hsf1 genes

Article Snippet: pBabe-HSF1-FLAG was a gift from Robert Kingston (Addgene plasmid#1948). pMSCV-HA-cMYCT58A was a gift from Scott Lowe (Addgene plasmid#18773). pCherry-HSP90alpha was a gift from Didier Picard (Addgene plasmid#108222). pCDNA3-2xHA-c-MYC was a gift from Martine Roussel (Addgene plasmid#74161). pLX304-LacZ-V5 was a gift from William Hahn (Addgene plasmid#42560). pBabe-LacZ, pBabe-HSF1 1-323 , and pBabe-HSF1 324-529 were described previously . pLX304-MAX-V5 (HsCD00440967) and pDONR221-GCN5 (HsCD00829789) vectors were purchased from DNASU plasmid depository. pLX304-LacZ-V5 and pLX304-GCN5-V5 vectors were co-transfected with packaging vector (delta VPR) and an envelope vector (VSV-G) into HEK293T packaging cells using TurboFect transfection reagent (Cat#R0531, ThermoFisher).

Techniques: Quantitation Assay, Two Tailed Test, MANN-WHITNEY, Binding Assay

Journal: bioRxiv

Article Title: Heat Shock Factor 1 (HSF1) specifically potentiates c-MYC-mediated transcription independently of the canonical heat-shock response

doi: 10.1101/2022.02.22.481519

Figure Lengend Snippet: (A) Co-IP of FLAG-HSF1, HA-c-MYC, and V5-MAX from transfected HEK293T cells (representative images of three independent experiments). HC: heavy chain. (B) Endogenous c-MYC-HSF1 interactions were detected by PLA in HeLa cells using a rabbit anti-c-MYC (D3N8F) antibody and a mouse monoclonal anti-HSF1 (E-4) antibody. Scale bars, 10µm. (C) In vitro direct interactions between recombinant HSF1 and c-MYC/MAX dimers were detected by the Lumit™ immunoassay. The reactions without primary antibodies were set up as the blanks, which were subtracted (mean ± SD, n =3 independent experiments, two-tailed Student’s t test). (D) In vitro interactions between recombinant c-MYC and MAX proteins, with and without recombinant HSF1 proteins, were detected by the Lumit™ immunoassay (mean ± SD, n =3 independent experiments, two-tailed Student’s t test). (E) In vitro binding of recombinant c-MYC/MAX dimers to E-box oligos, with and without recombinant HSF1 proteins, was detected by ELISA (mean ± SD, n =3 independent experiments, two-tailed Student’s t test). (F) Lysates of HEK293T cells co-transfected with indicated plasmids for 3 days were treated with EtBr (400 µg/mL) on ice for 30 min. The interaction of FLAG-HSF1 with HA-c-MYC/V5-MAX was detected by co-IP (representative images of three independent experiments). (G) Lysates of HEK293T cells co-transfected with indicated plasmids for 3 days were treated with either 10 U of DNase I or RNase at 37 °C for 20 min, followed by co-IP (representative images of three independent experiments). (H) Schematic depiction of two possible models of DNA-dependent protein-protein interactions. (I) Endogenous c-MYC-HSF1 interactions were detected by brightfield PLA in HeLa cells, following treatment with or without EtBr (100 µg/mL) for 1 hr. Scale bars: 10µm.

Article Snippet: pBabe-HSF1-FLAG was a gift from Robert Kingston (Addgene plasmid#1948). pMSCV-HA-cMYCT58A was a gift from Scott Lowe (Addgene plasmid#18773). pCherry-HSP90alpha was a gift from Didier Picard (Addgene plasmid#108222). pCDNA3-2xHA-c-MYC was a gift from Martine Roussel (Addgene plasmid#74161). pLX304-LacZ-V5 was a gift from William Hahn (Addgene plasmid#42560). pBabe-LacZ, pBabe-HSF1 1-323 , and pBabe-HSF1 324-529 were described previously . pLX304-MAX-V5 (HsCD00440967) and pDONR221-GCN5 (HsCD00829789) vectors were purchased from DNASU plasmid depository. pLX304-LacZ-V5 and pLX304-GCN5-V5 vectors were co-transfected with packaging vector (delta VPR) and an envelope vector (VSV-G) into HEK293T packaging cells using TurboFect transfection reagent (Cat#R0531, ThermoFisher).

Techniques: Co-Immunoprecipitation Assay, Transfection, In Vitro, Recombinant, Two Tailed Test, Binding Assay, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: Heat Shock Factor 1 (HSF1) specifically potentiates c-MYC-mediated transcription independently of the canonical heat-shock response

doi: 10.1101/2022.02.22.481519

Figure Lengend Snippet: (A) The expression of known c-MYC target genes was quantitated by qRT-PCR, following transient Gcn5 KD for 48 hr in immortalized MEFs (mean ± SD, n = 3 independent experiments, Two-way ANOVA). (B) Endogenous c-MYC transcriptional activities were measured by the dual reporter system in HEK293T cells transfected with indicated plasmids and siRNAs (mean ± SD, n = 3 independent experiments, One-way ANOVA). (C) Left panel: Endogenous c-MYC binding to gDNA binding was detected by PLA in immortalized MEFs stably expressing LacZ or GCN5 . Scale bars, 10 µm. Right panel: quantitation of these PLA foci per nucleus (mean ± SD, n≥100 nuclei, One-way ANOVA). (D) Quantitation of total RNAs extracted with immortalized MEFs stably expressing LacZ or GCN5 (mean ± SD, n = 3 biological replicates, One-way ANOVA). (E) Volcano plot of the differentially expressed genes due to Hsf1 KD. (F) Box-and-whisker plots of the abundance of DEGs in the control cells (n=1,640 or 1,269, Mann-Whitney U test). The box bounds the IQR divided by the median and the whiskers extend to the minimum and maximum values. (G) Visualization of DEGs in MEFs expressing different genes and siRNAs by clustering heatmaps (three biological replicates each group). (H) Seaborn correlation heatmap of gene expression among different experimental groups. (I) Venn diagram showing the overlaps among the c-MYC CUT&RUN-seq target genes, the DEGs following Hsf1 KD, and the DEGs rescued by GCN5 overexpression in immortalized MEFs. (J) Pathway enrichment analyses of the DEGs in immortalized MEFs following Hsf1 KD. (K) Heatmap visualization of the DEGs involved in the ribosome, proteasome, lysosome, and chaperone pathways (each data point represents the average of three biological replicates).

Article Snippet: pBabe-HSF1-FLAG was a gift from Robert Kingston (Addgene plasmid#1948). pMSCV-HA-cMYCT58A was a gift from Scott Lowe (Addgene plasmid#18773). pCherry-HSP90alpha was a gift from Didier Picard (Addgene plasmid#108222). pCDNA3-2xHA-c-MYC was a gift from Martine Roussel (Addgene plasmid#74161). pLX304-LacZ-V5 was a gift from William Hahn (Addgene plasmid#42560). pBabe-LacZ, pBabe-HSF1 1-323 , and pBabe-HSF1 324-529 were described previously . pLX304-MAX-V5 (HsCD00440967) and pDONR221-GCN5 (HsCD00829789) vectors were purchased from DNASU plasmid depository. pLX304-LacZ-V5 and pLX304-GCN5-V5 vectors were co-transfected with packaging vector (delta VPR) and an envelope vector (VSV-G) into HEK293T packaging cells using TurboFect transfection reagent (Cat#R0531, ThermoFisher).

Techniques: Expressing, Quantitative RT-PCR, Transfection, Binding Assay, Stable Transfection, Quantitation Assay, Whisker Assay, MANN-WHITNEY, Over Expression

Journal: bioRxiv

Article Title: Heat Shock Factor 1 (HSF1) specifically potentiates c-MYC-mediated transcription independently of the canonical heat-shock response

doi: 10.1101/2022.02.22.481519

Figure Lengend Snippet: (A) Co-IP of FLAG-HSF1, HA-c-MYC, and V5-GCN5 in transfected HEK293T cells (representative images of three independent experiments). (B) Endogenous c-MYC-GCN5 interactions were detected by PLA in HeLa cells. Scale bars, 10 µm. (C) Co-IP of endogenous c-MYC and GCN5, following transient Hsf1 KD in immortalized MEFs (representative images of three independent experiments). (D) In vitro binding of recombinant c-MYC proteins to individual HSF1 peptides immobilized on ELISA plates. Fold changes in binding are presented as a heatmap (n=3 independent experiments). (E) Visualization of interactions between transfected FLAG-HSF1 and endogenous c-MYC by PLA in HEK293T cells using a mouse monoclonal anti-FLAG antibody and a rabbit anti-c-MYC antibody. Scale bars, 10 µm. (F) c-MYC transcriptional activities were measured by the dual reporter system in HEK293T cells co-transfected with indicated plasmids (mean ± SD, n = 3 independent experiments, One-way ANOVA). (G) In vitro binding of recombinant GCN5 proteins to individual HSF1 peptides immobilized on ELISA plates. Fold changes in binding are presented as a heatmap (n=3 independent experiments). (H) Visualization of interactions between transfected FLAG-HSF1 and endogenous GCN5 by PLA in HEK293T cells. Scale bars, 10 µm. (I) Co-IP of endogenous c-MYC and GCN5 in HEK293T cells transfected with LacZ or FLAG-HSF1 (representative images of three independent experiments).

Article Snippet: pBabe-HSF1-FLAG was a gift from Robert Kingston (Addgene plasmid#1948). pMSCV-HA-cMYCT58A was a gift from Scott Lowe (Addgene plasmid#18773). pCherry-HSP90alpha was a gift from Didier Picard (Addgene plasmid#108222). pCDNA3-2xHA-c-MYC was a gift from Martine Roussel (Addgene plasmid#74161). pLX304-LacZ-V5 was a gift from William Hahn (Addgene plasmid#42560). pBabe-LacZ, pBabe-HSF1 1-323 , and pBabe-HSF1 324-529 were described previously . pLX304-MAX-V5 (HsCD00440967) and pDONR221-GCN5 (HsCD00829789) vectors were purchased from DNASU plasmid depository. pLX304-LacZ-V5 and pLX304-GCN5-V5 vectors were co-transfected with packaging vector (delta VPR) and an envelope vector (VSV-G) into HEK293T packaging cells using TurboFect transfection reagent (Cat#R0531, ThermoFisher).

Techniques: Co-Immunoprecipitation Assay, Transfection, In Vitro, Binding Assay, Recombinant, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: Heat Shock Factor 1 (HSF1) specifically potentiates c-MYC-mediated transcription independently of the canonical heat-shock response

doi: 10.1101/2022.02.22.481519

Figure Lengend Snippet: (A) ChIP-qPCR assays were performed to detect the acetyl-histone 3 (Lys9/Lys14) on c-MYC target or non-c-MYC target loci in immortalized MEFs (mean ± SD, n = 3 biological replicates, One-way ANOVA). (B) Left panel: the protein expression of fusion between HA-HSF1 324-529 and c-MYC was detected by immunoblotting. Right panel: the transcriptional activity of fusion proteins was measured by the dual reporter system (mean ± SD, n = 3 independent experiments, One-way ANOVA). (C) The proposed model of HSF1-mediated c-MYC activation. HSF1 helps recruit GCN5 to c-MYC, thereby promoting chromatin remodeling and potentiating the c-MYC-mediated transcription. (D) HSF1 regulates two distinct activation states of c-MYC. Without HSF1 association, the transcriptional activity of cellular c-MYC remains low, sustaining at a primary state; by contrast, HSF1 association renders c-MYC highly active, transiting to an advanced state. (E) HSF1 governs at least two discrete transcriptional programs. Upon its activation, either in the face of environmental stress or within malignant cells, HSF1 initiates the canonical PSR/HSR, a mechanism of action depending on HSE binding. By contrast, in the absence of environmental stress most cellular HSF1 remains repressed; however, some HSF1 associates with c-MYC and potentiates its mediated transcription, a mechanism of action independent of HSE binding.

Article Snippet: pBabe-HSF1-FLAG was a gift from Robert Kingston (Addgene plasmid#1948). pMSCV-HA-cMYCT58A was a gift from Scott Lowe (Addgene plasmid#18773). pCherry-HSP90alpha was a gift from Didier Picard (Addgene plasmid#108222). pCDNA3-2xHA-c-MYC was a gift from Martine Roussel (Addgene plasmid#74161). pLX304-LacZ-V5 was a gift from William Hahn (Addgene plasmid#42560). pBabe-LacZ, pBabe-HSF1 1-323 , and pBabe-HSF1 324-529 were described previously . pLX304-MAX-V5 (HsCD00440967) and pDONR221-GCN5 (HsCD00829789) vectors were purchased from DNASU plasmid depository. pLX304-LacZ-V5 and pLX304-GCN5-V5 vectors were co-transfected with packaging vector (delta VPR) and an envelope vector (VSV-G) into HEK293T packaging cells using TurboFect transfection reagent (Cat#R0531, ThermoFisher).

Techniques: Expressing, Western Blot, Activity Assay, Activation Assay, Binding Assay