SUMO paralogue-specific functions revealed through systematic analysis of human knockout cell lines and gene expression data

Abstract

The small ubiquitin-related modifiers (SUMOs) regulate nearly every aspect of cellular function, from gene expression in the nucleus to ion transport at the plasma membrane. In humans, the SUMO pathway has five SUMO paralogues with sequence homologies that range from 45% to 97%. SUMO1 and SUMO2 are the most distantly related paralogues and also the best studied. To what extent SUMO1, SUMO2, and the other paralogues impart unique and nonredundant effects on cellular functions, however, has not been systematically examined and is therefore not fully understood. For instance, knockout studies in mice have revealed conflicting requirements for the paralogues during development and studies in cell culture have relied largely on transient paralogue overexpression or knockdown. To address the existing gap in understanding, we first analyzed SUMO paralogue gene expression levels in normal human tissues and found unique patterns of SUMO1-3 expression across 30 tissue types, suggesting paralogue-specific functions in adult human tissues. To systematically identify and characterize unique and nonredundant functions of the SUMO paralogues in human cells, we next used CRISPR-Cas9 to knock out SUMO1 and SUMO2 expression in osteosarcoma (U2OS) cells. Analysis of these knockout cell lines revealed essential functions for SUMO1 and SUMO2 in regulating cellular morphology, promyelocytic leukemia (PML) nuclear body structure, responses to proteotoxic and genotoxic stress, and control of gene expression. Collectively, our findings reveal nonredundant regulatory roles for SUMO1 and SUMO2 in controlling essential cellular processes and provide a basis for more precise SUMO-targeting therapies.

FIGURE 1:

SUMO paralogue expression levels vary among human cell lines and tissues. (A) SUMO1–3 mRNA expression (–log₁₀ RPKM) from 467 human cancer cell lines derived from six different tissues. P values were determined using a Student’s t test and are marked with an asterisk (* = 0.027; ** = 2.8e-6; *** < 1.6e-14). (B) SUMO1–4 mRNA expression from 61 human bone cancer cell lines. P values were calculated using a Student’s t test. The inlay shows data from CCLE for the U2OS cell line. SUMO5 expression was below levels of detection in all bone cancer cell lines. (C) Anatomical heatmaps of SUMO paralogue mRNA expression in normal human tissues. (D) Heatmap of SUMO1 paralogue mRNA expression in normal human tissues. Human data in C and D are from GTEx and are reported in TPM.

FIGURE 2:

Generation and validation of SUMO KO cell lines. (A) Schematic of CRISPR-Cas9 knockout strategy and sequencing results. (B) SUMO paralogue mRNA expression levels in WT and KO cells, measured by RNA-seq. (C) Immunoblots of WT and KO whole cell lysates show a significant reduction in SUMO1 and SUMO2/3 conjugated proteins in respective KO cells, with tubulin used as a loading control. (D) Immunofluorescence microscopy images reveal loss of SUMO1- or SUMO2/3-specific signal in respective KO cells. Tubulin costaining reveals changes in cellular morphology.

FIGURE 3:

SUMO2 has a unique role in regulating cellular morphology. (A) Percentage of fibroblast-like cells in the indicated cell lines. More than 2400 cells from three independent experiments were analyzed by immunofluorescence microscopy for each cell line. (B) SUMO2 reintroduction in S2KO+S2 cells and SUMO1 overexpression in S2KO+S1 cells were validated by immunoblotting using specific antibodies. Tubulin was used as a loading control. (C) Expression levels of SUMO2/3 and SUMO1 were assessed by immunofluorescence microscopy with specific antibodies and DAPI counterstaining. (D) Morphology of the indicated cell lines was analyzed by anti-tubulin immunofluorescence microscopy with DAPI counterstaining. Scale bars: 20 μm.

FIGURE 4:

Rescue of S2KO cells reveals paralogue-specific functions of SUMO2 in regulating U2OS cell morphology. (A) Quantitative shape analysis was performed using FIJI and Prism (see Materials and Methods) revealing changes in the average cellular aspect ratio, area, and circularity in the indicated cell lines. More than 200 cells from three independent experiments were analyzed for each cell line. Error bars represent SDs. P values were calculated by a Kruskal–Wallis test using Prism software; **** p ≤ 0.0001, *** p ≤ 0.001, * p ≤ 0.05, ns p > 0.05. (B) Aspect ratio, area, and circularity of WT, S2KO, and S2KO+S1 cell lines, analyzed as in A.

FIGURE 5:

Cell cycle and nuclear body analysis of SUMO KO cells. (A) Overlay of WT, S1KO, and S2KO histograms from flow cytometry analysis. (B) Quantification of the percentage of WT, S1KO, and S2KO cells in G0/G1, S, G2/M, and >G2 cell cycle stages. Indicated p values were calculated using an ANOVA. (C) Immunofluorescence microscopy images using antibodies specific for PML and DAXX. (D) Quantification of PML-positive nuclear bodies (PML-NBs), DAXX-positive NBs, and PML-NB perimeter size estimates for each cell line; an average of 180 cells were analyzed for each cell line from two independent experiments. P values for each cell line as compared with WT were calculated using an unpaired Wilcoxon test (ns = not significant, ** p = 1 × 10^–8, and *** p < 2.2 × 10^–16).

FIGURE 6:

SUMO paralogues have nonredundant functions in stress responses. (A) Baseline linear readout of MTT assay signal for the indicated numbers of WT and KO cell lines. Simple linear regressions were calculated for each cell line: R² = 0.91 (WT), 0.96 (S1KO), 0.94 (S2KO), 0.81 (S2KO+S2), and 0.96 (S2KO+S1). (B) Cells were treated with the indicated doses of AZC for 72 h, and cell viability was determined using an MTT assay. (C) WT cells were treated with 8 µm EerI for the indicated times, and cell lysates were analyzed by immunoblotting with SUMO1- or SUMO2/3-specific antibodies. (D) Cells were treated with the indicated doses of EerI for 48 h, and cell viability was determined using an MTT assay. (E) Cells were treated with the indicated doses of HU for 72 h, and cell viability was determined using an MTT assay. Relative cell viability was calculated in B, C, and E as the fraction of MTT signal at each drug dosage compared with untreated control cells. (F) Cells were treated with (dashed line, +) or without (solid line, –) 700 µM HU for up to 4 d. Viable cells were counted at each time point and plotted. Error bars equal SDs, n = 3. (**** p ≤ 0.0001, *** p ≤ 0.001, * p ≤ 0.05, ns p > 0.05).

FIGURE 7:

SUMO1 and SUMO2 uniquely regulate gene expression. (A) Venn diagram showing the numbers of unique and overlapping up-regulated and down-regulated DEGs in the S1KO and S2KO cells, at two significance thresholds, FDR < 0.05 and FDR < 0.05 + Log₂ fold change (FC). (B) Volcano plots of unique and overlapping SUMO1 and SUMO2 KO cell DEGs. The horizontal dashed line represents FDR < 0.05, and the vertical dashed lines represent Log₂FC values of –2 and +2. (C) SUMO1 and SUMO2 gene expression values by qRT-PCR in WT, KO, and rescue cell lines. (D) Representative bar plots of Log₂FC expression values of up- and down-regulated genes, tested by qRT-PCR. (E) Heatmap summarizing SUMO2 KO and rescue cell line Log₂FC values for genes tested by RNA-seq and qRT-PCR (all graphs shown in Supplemental Figure 8).

FIGURE 8:

SUMO KO cell DEGs are localized throughout the genome. (A) Genomic locations of up- and down-regulated genes from the SUMO1 KO cells are mapped to their genomic loci. The gray plot below each chromosome represents gene density across each chromosome. The percent of DEGs per chromosome is labeled to the right of each chromosome: (# of DEGs on the chromosome/total # of DEGs) × 100. (B) Same as in A, but for SUMO2 KO cell DEGs. (C) Corresponding heatmap of histone gene expression as measured by RNA-seq.

FIGURE 9:

SUMO paralogues have opposing roles on gene expression. (A) GSEA results showing enriched gene sets for S1KO and S2KO cell lines as grouped into six broad categories. Abbreviations: Transc. = transcription, NES = normalized enrichment signal. (B) Cytoscape-STRING protein interaction networks for IFN-α, -γ, and collagen formation processes.