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.