Central role of SUMOylation in the regulation of chromatin interactions and transcriptional outputs of the androgen receptor in prostate cancer cells

Abstract

The androgen receptor (AR) is pivotal in prostate cancer (PCa) progression and represents a critical therapeutic target. AR-mediated gene regulation involves intricate interactions with nuclear proteins, with many mediating and undergoing post-translational modifications that present alternative therapeutic avenues. Through chromatin proteomics in PCa cells, we identified SUMO ligases together with nuclear receptor coregulators and pioneer transcription factors within the AR's protein network. Intriguingly, this network displayed a significant association with SUMO2/3. To elucidate the influence of SUMOylation on AR chromatin interactions and subsequent gene regulation, we inhibited SUMOylation using ML-792 (SUMOi). While androgens generally facilitated the co-occupancy of SUMO2/3 and AR on chromatin, SUMOi induced divergent effects dependent on the type of AR-binding site (ARB). SUMOi augmented AR's pioneer-like binding on inaccessible chromatin regions abundant in androgen response elements (AREs) and diminished its interaction with accessible chromatin regions sparse in AREs yet rich in pioneer transcription factor motifs. The SUMOi-impacted ARBs divergently influenced AR-regulated genes; those associated with AR-mediated activation played roles in negative regulation of cell proliferation, while those with AR-mediated repression were involved in pattern formation. In conclusion, our findings underscore the pervasive influence of SUMOylation in shaping AR's role in PCa cells, potentially unveiling new therapeutic strategies.

Graphical Abstract

Figure 1.

Androgen-dependent protein network of chromatin-bound AR in VCaP cells. (A) Overview of AR chromatome identified with RIME and subdivided into functional groups by GO annotation. Protein sequence coverage percentages are shown as bars in polar coordinated plot and coloring by GO annotation. (B) Chromatome is categorized into androgen-induced (log₂[DHT/veh] > 0.5), and uninduced (log₂[DHT/veh] ≤ 0.5) fractions. DHT-dependency of AR chromatome in scatterplot with spectral counts normalized by AR as y axis. log₂[DHT/veh] = –8 and 8 are inputed values for chromatome members that are identified with vehicle only or with DHT only, respectively. DHT-induced members additionally highlighted red color. (C) AR DHT-dependent chromatome members subdivided into functional groups by their GO annotation. The size of the node represents the numbers of members in the group and coloring by annotation similarly to a. Examples of proteins shown for groups larger than 10 proteins, asterisks mark proteins that have association to prostatic disease in DisGeNet.

Figure 2.

Association of SUMO2/3 with chromatin-linked proteins in VCaP cells. (A) Overview of SUMO2/3 chromatome identified with RIME and subdivided into functional groups by GO annotation. Protein sequence coverage percentages are shown as bars in polar coordinated plot and coloring by GO annotation. (B) Chromatome is categorized into androgen-induced (log₂[DHT/veh] > 0.5), and uninduced (log₂[DHT/veh] ≤ 0.5) fractions and DHT-dependency plotted on scatter plot with SPCs normalized to SUMO2/3. DHT-induced members additionally highlighted red color. (C) SUMO2/3 chromatome members subdivided into functional groups by their GO annotation. Size of the node represents the numbers of members in the group and coloring by annotation similarly to (A). Examples of proteins are shown for groups larger than 30 proteins, SUMO2/3 chromatome members that are found also in the AR chromatome are in bold, others in italics with color matching to the node.

Figure 3.

Effect of SUMOi and androgen on SUMO2/3 landscape on the chromatin. (A) Heatmaps of SUMO2/3 and AR occupancy on chromatin, chromatin accessibility, and H3K27ac marks as normalized tag densities, divided to subcategories by androgen effect in SUMO2/3 signal (C1 = moderately changed – 3 < log₂(DHT/veh) < 3, C2 = markedly increased, log₂(DHT/veh) > 3 and C3 = markedly decreased, log₂(DHT/veh) < -3). For C1 sites only top 10k showed. (B–E) Box blots of tag densities in SUMO2/3, AR, ATAC and H3K27ac in clusters C1–3. Statistical significance for tag density boxplots calculated with One-way ANOVA with Bonferroni post hoc test and shown as asterisks with *P< 0.05, **P< 0.01 and ***P< 0.001.

Figure 4.

SUMOylation inhibition modifies AR occupancy on chromatin. (A) Heatmap of AR’s normalized tag densities on moderately SUMOi-affected ARBs (C4, – 3 < log₂[SUMOi/CTRL] < 3), clearly SUMOi-attenuated ARBs (C5, log₂[SUMOi/CTRL] < −3) and SUMOi-enhanced ARBs (C6, 3 < log₂[SUMOi/CTRL]) together with SUMO2/3 occupancy, chromatin accessibility and H3K27ac data. (B–E) Box plots of AR, SUMO2/3, ATAC and H3K27ac tag densities in clusters C4–C6. (F) Chromatin occupancy of FOXA1 and HOXB13 in the presence and absence of DHT and SUMOi in clusters C4–C6 as box plots of tag densities. Statistical significance for tag density box plots calculated with one-way ANOVA with Bonferroni post hoc test and shown as asterisks with *P< 0.05, **P< 0.01 and ***P< 0.001.

Figure 5.

Motif analysis of AR chromatin-binding sites differentially affected by SUMOi. (A) Fold enrichment bar plot for ARE, FOXA1, HOXB13 and ERG motifs for ARB clusters in Figure 4. (B) Comparison of log odd motif scores between ARB clusters of motifs in (C). Motif coverage in percentages from all ARBs within each cluster. (D) Number of AREs in each site within clusters. (E) Box blots of AR chromatin occupancy with and without SUMOi, depending on the number of AREs per site. Statistical significance for boxplots calculated with One-way ANOVA with Bonferroni post hoc test and shown as asterisks with *P< 0.05, **P< 0.01 and ***P< 0.001.

Figure 6.

Effect of SUMOi on AR-regulated gene expression and its association with SUMOi-affected AR chromatin-binding sites in VCaP cells. (A) The leftmost heatmap represents z-scores of SUMOi- and androgen-regulated DEGs in SUMOi dn-regulated clusters (C7–C9) and SUMOi up-regulated clusters (C10–C12). In the middle, there is information of whether each gene is DHT-regulated in control and/or after SUMOi. DHT-up-regulation is shown in red and DHT-dn-regulation in blue. On the right, gene-associated ARBs from SUMOi-altered clusters C5 and C6 are shown in green and purple, respectively. The color intensity indicates the number of ARBs within ±100 kb from TSS of each gene (B). Heatmaps and associations as in a but for SUMOi-unaffected DHT-regulated genes (C13). (C, D) Metascape pathway analysis for genes associating with SUMOi-attenuated ARBs (C5, in green) and SUMOi-enhanced ARBs (C6, in purple). Top5 enriched pathways shown as bar graph of adj. P-values for C5 and C6-shared pathways (topmost part of the bar plot), for pathways enriching significantly only with genes associating with C5 (middle part), for pathways enriching significantly only with genes associating with C6 (bottom part) in clusters C7–C12 (C) and C13 (D). Dashed line indicates significance threshold (adj. P-value) <0.05 after Benjamini–Hochberg multiple correction in-built in Metascape (28).