Fig 1: NELF SUMOylation drives nuclear condensate formation(A) Fluorescence microscopy images of HeLa NELFA-GFP cells treated with vehicle or CDK9 inhibitor 5,6-Dichloro-1-ß-d-ribofuranosylbenzimidazole (DRB) under no heat shock conditions (NHS). Scale bar indicates 10 µm.(B) Fluorescence microscopy images of HeLa NELFA-GFP cells exposed to heat shock (HS) and treated with vehicle or ML-792. Scale bar indicates 10 µm.(C) Percentage of cells with NELFA condensates after heat shock in vehicle or ML-792 treated samples. Data quantification of (B). Error bars represent SD (n = 2 independent cell cultures). Asterisks denote p value <0.001 as calculated by two-tailed unpaired t test.(D) Fluorescence microscopy images of heat-shocked HeLa cells expressing NELFA-GFP. Cells were treated with siRNAs against SUMO E2 conjugating enzyme (UBC9), SUMO E3 ligases (PIAS1, PIAS4, and ZNF451), non-targeting siRNA (siNON), or left untreated. Scale bar indicates 10 µm.(E) Percentage of heat-shocked HeLa cells with NELFA condensates from experiment described in (D). Error bars represent SD (n = 2 independent replicates). Conditions compared to siNON. Asterisks denote p value of (***, 0.001; **, 0.01) as calculated by one-way ANOVA with Dunnett’s multiple comparison tests.(F) Western blot analyses of NELF after performing in vitro SUMOylation reactions using NELFA (top) and NELFC/D antibodies (bottom). Either immunoprecipitated full-length E3 ligase ZNF451 or a recombinant N-terminal fragment were used at two different concentrations (STAR Methods). Reactions lacking ATP or ZNF451 serve as negative controls.(G) Western blot analysis of ZNF451 and NELFE is shown for indicated fractions from HeLa cells exposed to heat shock (HS) or no heat shock (NHS). SNRNP70 and RPB3 are used as loading controls.(H) Effect of SUMO E2 (UBC9) depletion on HS-induced changes in RNA Pol II ChIP-seq occupancy in gene body regions of top 250 expressed genes. Regions from transcription start site (TSS) +1.5 kb to transcription end site (TES) –0.5 kb are shown. p value from Wilcoxon test is indicated (n = 2 independent cell cultures).(I) Effect of SUMO E3 ligase ZNF451 depletion on HS-induced changes in nascent transcript counts as quantified by SLAM-seq in HeLa cells. Significantly downregulated genes in siNON cells as calculated by differential gene expression analysis based on the negative binomial distribution (DESeq2) are used. The red line indicates no change in expression. The violin plot depicts median, interquartile range, and 95% confidence interval with white dot, black bar, and thin black line, respectively. p value from Wilcoxon test is indicated (n = 3 independent cell cultures).See also Figures S3–S5.