Figure 6. Post-HS nucleolar recovery of CBX proteins and epigenetic recovery depends on heat-shock protein activity.

(A) Percentage of cells with nucleolar accumulation of GFP-CBX2 during recovery at 37°C after HS (30 min, 44°C). Error bars indicate the mean ± range calculated from independent microscopical images (n = 2; total cell number 100–170) (B) Representative images of fixed K562 GFP-CBX2 cultured at 37°C after HS (30 min, 44°C) for indicated time intervals in the presence of 5 μM VER-155088 or DMSO. Scale bar represents 25 μm. (C) Percentage of K562 GFP-CBX2 cells with nucleolar accumulations. Cells are cultured at 37°C after HS (30 min, 44°C) in the presence of 5 μM VER-155088 or DMSO. Error bars indicate the mean ± range calculated from independent microscopical images (n = 2; total cell number 50–90). Statistical analysis was performed using Student’s t-test, *p<0.05, **p<0.01. Similar results were obtained in independent experiments. (D) Experimental design of thermotolerance experiment. (E) Representative images of K562 GFP-CBX2 cells fixed at indicated time points according to panel D. Scale bar represents 25 μm. (F) Quantification of percentage of K562 GFP-CBX2 cells with nucleolar accumulations at time points according to panel D. Error bars indicate the mean ± SD calculated from independent microscopical images (n = 5; total cell number 230–350). (G) ChIP-qPCR analyses of H2AK119ub, H3K27me3, GFP-CBX2 and endogenous EZH2 levels at the TCF21 locus in K562 GFP-CBX2 cells, either untreated or cross-linked at indicated time-points after a heat shock (30 min, 44°C). Error bars represent mean ± range (n = 2, independent replicates, *p<0.05 and **p<0.01). (H) Western blot analysis of H2AK119ub, H3K27me3, HSP70, DNAJB1, EZH2, CBX8 and β-ACTIN levels in K562 GFP-CBX2 cells samples isolated at indicated time points according to panel D. (I) Schematic representation of the effects of heat shock on PRC1/2 chromatin binding and epigenetic marks. Protein quality control in the nucleolus leads to refolding of Polycomb proteins, resulting in reinstallation of epigenetic modifications at Polycomb target genes.

Figure 6—figure supplement 1. GFP-CBX2, DNAJB1 and HSP70 show comparable HS-induced relocalization kinetics and post-HS GFP-CBX2 recovery is delayed upon HSP70 knockdown.

(A) Confocal images of K562 GFP-CBX2 cells (untreated, directly after HS [1 hr, 44°C], or 3 hr after HS) stained with anti-DNAJB1. White arrows indicate the nucleoli. Scale bar represents 10 μm. (B) Confocal images of K562 GFP-CBX2 cells (untreated, directly after HS [1 hr, 44°C], or 3 hr after HS) stained with anti-HSP70. White arrows indicate the nucleoli. Scale bar represents 10 μm. (C) Fluorescent images of HEK293T GFP-CBX2 cells, either untreated, directly after HS (30’, 44°C), 1 hr after HS, or 3 hr after HS. Scale bar represents 25 μm. (D) Quantification of post-HS (30’, 44°C) GFP-CBX2 recovery in control and HSP70 siRNA transfected HEK293T GFP-CBX2 cells. Error bars indicate the mean ± SD calculated from independent microscopical images (n = 5; total cell number 175–460). Statistical analysis was performed using Student’s t-test, *p<0.05, **p<0.01. (E) Western analysis of HEK293T GFP-CBX2 cells, transfected with control or HSP70 siRNAs, both untreated and 3 hr after HS (30’, 44°C), using HSPA1A/HSP70 and GAPDH antibodies.

Figure 6—figure supplement 2. HS-induced epigenetic changes are reversible and H2AK119ub recovery is not dependent on de novo protein synthesis.

(A) ChIP-qPCR analyses of GFP-CBX2, H2AK119ub, H3K27me3, H3K4me3 and H3K4me1 levels in untreated, heat shocked (1 hr, 44°C), and 24 hr post-HS K562 cells. Error bars are calculated on the basis of technical replicates. Error bars represent mean ± SD. (B) Western analysis of H2AK119ub, H3K27me3, and β-ACTIN in untreated and heat-shocked K562 cells (30 min, 44°C) treated with DMSO or 10 μg/ml cycloheximide (CHX; 1 hr pretreatment before HS). Heat-shocked cells were isolated at the indicated time points.