Sumoylation is a post-translational modification of a protein and a ubiquitination-like modification. It involves the covalent attachment of a small ubiquitin-like modification (SUMO) to a lysine residue on the substrate. In mammals, three major SUMO isoforms have been identified, including SUMO1, SUMO2, and SUMO3. Similar to ubiquitination, SUMOsylation is catalyzed by a range of enzymes, including E1 SUMO-activating enzyme (SAE1 SAE2), a single E2-coupled enzyme (UBC9), and a limited E3 SUMO ligase. SUMO-based modification mediates localization and functional regulation of target molecules. At present, sumoylation modification plays a very wide role in cell biology and is also one of the hot spots of current research. The imbalance between sumoization and desumoization has been implicated in the occurrence and progression of a variety of diseases, including cancer, neurodegenerative diseases, heart disease, and innate immune diseases. Let's take a look at the literature:
Background:
Sumoylation regulates a large number of biological processes, and its inhibitors are currently being studied in clinical trials as anticancer drugs. Therefore, identifying new targets with site-specific sumoylation and determining their biological functions will not only provide new mechanistic insights into sumoylation signaling, but will also open the way for the development of new cancer strategies. The CW zinc finger 2 (MORC2) of the MORC family is a newly discovered chromatin remodeling enzyme that plays an emerging role in the DNA damage response (DDR), but its regulatory mechanism is unclear.
Main methods:
The SUMO-level of MORC2 was determined by in vivo and in vitro SUMO-ation methods. Overexpression and knockdown of SUMO-related enzymes were detected to detect their effects on MORC2 SUMOlation. Effect of dynamic morc2 sumoylation on the sensitivity of breast cancer cells to chemotherapeutic agents by in vitro and in vivo functional tests. Use immunoprecipitation, GST pull-down, MNASE, and chromatin segregation assays to explore the underlying mechanisms.
Main results:
Morc2 is modified by SUMO1 and SUMO2 3
To determine if Morc2 is modified by Sumoylation, the authors transiently transfected Flag-Morc2 into HEK293T cells and performed Sumoylation experiments using anti-Flag beads under denaturing conditions. Immunoblot analysis with anti-SUMO1 or anti-SUMO23 antibodies revealed that SUMO1 and SUMO23 were conjugated to MOR2 (Figure 1A). In addition, SUMO1 and SUMO2 3 binding to endogenous MORC2 was detected in MCF-7, T47D, and HEK293T cells (Figure 1B). Incubation with the Sumoylation inhibitor ML-792 results in a dose-dependent decrease in ectopically expressed FLAG-MORC2 (Figure 1C) in HEK293T cells and endogenous MORC2 (Figure 1D) in MCF-7 and T47D cells. Immunofluorescence staining showed that endogenous SUMO1 and SUMO2 3 colocalized with exogenous FLAG-MORC2 in HEK293T cells and with endogenous MORC2 in MCF-7 and T47D cells (Figs. 1E-F). These results suggest that in mammalian cells, all three SUMO isoforms are covalently bound to Morc2.
Since UBC9 is the only E2-conjugated enzyme required for Sumoylation, the authors then investigated whether MORC2 interacts with UBC9 by immunoprecipitation (IP) experiments. As shown in Figure 1G, Flag-MORC2 is immunoprecipitated by HA-UBC9 when HA-UBC9 and FLAG-MORC2 are co-expressed in HEK293T cells. Interactions between Morc2 and UBC9 at the endogenous protein level were detected in HEK293T cells (Figure 1H-I). In the presence of HA-UBC9, all three SUMO isoforms (GFP-tagged SUMO1, SUMO2, and SUMO3) bind to MORC2 (Figure 1J). Ectopic expression of HA-UBC9 was enhanced, while knockout of endogenous UBC9 by CRISPR Cas9 technology reduced the sumoylation of endogenous MORC2 in MCF-7 cells (Figure 1K-L). Taken together, these results suggest that Morc2 is a sumoylated protein.
Lysine 767 (K767) is the major sumoylation site in Morc2
To identify potential SUMOYLATION sites for MORC2, the authors used GPS (Group-Based Prediction System)-SUMO and JASSA (Joint Analyzer of SUMOYLATION Site and SIMS) programs to ** SUMOYLATION sites of MORC2. Narrowing down the potential sumoylation sites in Morc2 to lysine 767 (K767) and K827. To verify that Morc2 is SUMOtified at these two sites, the authors mutated K767 and K827 to non-SUMOlate residue arginine (R), respectively, and then transiently transfected wild-type (WT), K767R, or K827R mutant Flag-Morc2 with GFP-SUMO1, GFP-SUMO2, or GFP-SUMO3 into HEK293T cells, respectively. Sumoylation analysis with the shown antibodies showed a reduction in the modification of SUMO1 and SUMO2 3 in cells expressing K767R compared to the mutant corresponding to WT, while there was no reduction in the mutant Morc2 of K827R (Figure 2A-C). In addition, endogenous SUMO1 and SUMO2 3 bind to the K767R mutant Flag-MORC2 decreased compared to WT MORC2 (Figure 2D). These results suggest that K767 is the major SUMO site of Morc2.
Having a functional sim on the n-side of Morc2 is necessary for its effective sumoylation
SIMs are required for efficient sumoization of some SUMO substrates. Analysis of the Morc2 protein sequence using the GPS-SUMO and JASSA programs revealed the presence of two putative SIMs in MORC2, located in residues 144-148 and 413-417, called SIM1 and SIM2, respectively. To assess the functional importance of these two SIMs in Morc2 Sumoylation, the authors mutated the two SIM sequences by substituting alanine for hydrophobic residues (referred to here as SIM1mut and SIM2mut), respectively. Sumoylation experiments showed that flag-Morc2 Sim1mut, but not Sim2mut, reduced Morc2 Sumoylation (Fig. 2E-F).
Trim28 is involved in MORC2 SUMO modification as SUMO E3 ligase.
BioGrid analysis, a database of protein interactions, identified two putative SUMO E3 ligases, TRIM28 and ChromoBox 4 (CBX4). Although Flag-Morc2 was associated with endogenous CBX4 in HEK293T cells, ectopic expression of FLAG-CBX4 did not significantly affect the Sumoylation of Flag-Morc2, suggesting that CBX4 was not the Sumo E3 ligase of Morc2 Sumoylation.
Next, the authors investigated whether TRIM28 induces Morc2 sumoylation. IP and western blotting experiments showed that Flag-Morc2 was co-immunoprecipitated with HA-trim28 in HEK293T cells (Figure 3A). Immunofluorescence staining showed that colocalization of FLAG-MORC2 and HA-TRIM28 was also seen in the nuclei of MCF-7 cells (Figure 3B). In addition, an association between endogenous MORC2 and endogenous TRIM28 was detected in MCF-7 cells (Figure 3C-D).
To map the domains required for the interaction of Morc2 with TRIM28, the authors generated three MORC2 deletion and truncated structures in HEK293T cells and performed a Co-IP assay. As shown in Figure 3E-F, the N-terminus of MORC2 contains a conserved ATPase domain that is required for its interaction with TRIM28. Similarly, Morc2, which is missing residue 1-420 at its N-terminus, is unable to bind to TRIM28 (Figure 3G-H). These results suggest that the N-terminal atpase domain of MORC2 is critical for its interaction with TRIM28.
To determine whether TRIM28 is required for MORC2 SUMOYLATION, the authors ectopic expression of HA-TRIM28 and detection of MORC2 SUMOYLATION levels. As shown in Figure 3i, ectopic TRIM28 significantly enhances the sumoylation of WT, but has no effect on the K767R mutant Morc2. In addition, overexpression of TRIM28 had no significant effect on Morc2 protein levels. In addition, only overexpression of WT TRIM28, rather than its catalytically inactive C651A mutant, can effectively enhance the sumoylation of Morc2 (Figure 3J). In contrast, knockdown of TRIM28 by two independent shRNAs significantly reduced the sumoylation of ectopically expressed Flag-Morc2 in HEK293T cells (Figure 3K) and endogenous Morc2 in MCF-7 cells (Figure 3L). These results suggest that trim28 is the sumo E3 ligase required for Morc2 Sumoylation.
Senp1 is a Morc2 desumolating enzyme
Since Morc2 is modified by all three SUMO isoforms (Figure 1), the authors next investigated the potential role of Senp1-3 in the deSUMOlation of MORC2. To do this, FLAG-MORC2, HA-UBC9, and GFP-SUMO were transfected into HEK293T cells. The results showed that after co-transfection with Senp1 and Senp2, the sumoylation of Morc2 was attenuated, while Senp3 was not attenuated (Figure 4A). Immunofluorescence staining showed that HA-SENP1 co-localized with FLAG-MORC2 in the nucleus, while HA-SENP2 and HA-SENP3 did not colocalize (Figure 4B).
To determine whether senp1 mediates Morc2 desumoylation, the authors examined the interaction between senp1 and morc2. IP analysis showed that Senp1 interacted with Morc2 (Figure 4C). The interaction of senp1 and morc2 at the endogenous protein level was observed in MCF-7 cells (Figure 4D). In addition, SENP1 overexpression significantly reduced the sumoylation of ectopic expressed Morc2 in HEK293T cells and endogenous Morc2 in MCF-7 cells (Figure 4E-F). Ectopic expression of WT Senp1, rather than its catalytically inactive C603S mutant, effectively abolishes Sumoylation of Morc2 (Figure 4G). Similarly, deletion of Senp1 significantly induces Morc2 sumoylation in HEK293T (Figure 4H) and MCF-7 cells (Figure 4I).
After DNA damage, Morc2sumoylationLower
Since Sumoylation is associated with DNA damage response (DDR), the authors next measured whether DNA damaging agents affect Sumoylation of MORC2. After 2 h treatment of HEK293T cells with ADR (doxorubicin), Morc2 Sumoylation decreases in a dose-dependent manner.
The rapid loss of ADR-induced Morc2 Sumoylation was restored after HEK293T cell recovery (Figure 5A-B). These results suggest that the response of MORC2 Sumoylation to DNA damage is highly dynamic. To investigate the underlying mechanism of the dynamic changes of Morc2 Sumoylation in response to DNA damage, the authors tested whether ADR affects the interaction of Morc2 with TRIM28 or SENP1. As shown in Figure 5C, after 2 h of treatment with ADR, there was no significant change in protein levels of TRIM28 or SENP1, but the interaction of MORC2 with TRIM28 was significantly reduced, while Senp1 was not. Interestingly, the impaired interaction between Morc2 and TRIM28 was restored after the specified time of recovery (Figure 5C), while the association between Morc2 and Senp1 decreased slightly. Knockdown of TRIM28 significantly impaired the recovery of Morc2 Sumoylation after ADR treatment (Figure 5D). These data suggest that precisely regulated MORC2 Sumoylation mainly relies on the response of TRIM28 to DNA damage.
Previous studies have shown that MORC2 exerts ATPase-dependent chromatin remodeling activity in response to DNA damage. Next, the authors determined whether early MDR Morc2 desumoylation was associated with its chromatin remodeling activity. Treatment of WT MORC2-expressing cells with ADR enhances chromatin accessibility to MNase. In addition, this effect is enhanced in cells expressing Sumoylation-deficient Morc2 (K767R or SIM1mut) (Figure 5E-F). The authors applied chromatin segregation assays to identify different properties of chromatin. The anti-nuclease chromatin component is enriched with histone 3 lysine 9 trimethylation (H3K9Me3), a hallmark of heterochromatin formation, indicating highly condensed chromatin nucleosomes. As shown in Figure 5G-H, ADR treatment transfers H3K9Me3-rich nucleosomes from the C5 moiety to the C4 moiety, which is more sensitive to nucleases. This finding confirms the widely held view that damaged chromatin is relatively more accessible. The authors then tested whether morc2 sumoylation was involved in this process. The results of the study are consistent with those shown in Figure 5g. After adverse effects**, expression of WT Morc2 reduced the H3K9Me3-rich nucleosome chromatin in the C5 moiety. In addition, this effect was enhanced by Sumoylation-deficient Morc2 mutants (Figure 5i-j). Thus, the increase in chromatin accessibility dependent on Morc2 chromatin remodeling activity after genotoxic damage is enhanced by desumoylation, which further promotes DNA repair.
Morc2 Sumoylation aids in DNA repair
The complete Morc2 sumoylation cycle is critical for cell survival in genotoxic stress responses. Conjugated SUMO molecules have been reported to exert a wide range of effects on substrates in various biological processes, altering substrate interactions and regulating their functions. The authors then investigated whether Morc2 Sumoylation altered its interactions and functionally contributed to DNA repair. The authors identified the Sumoylated Morc2 interacting group using Co-IP analysis combined with LC-MS MS. The functions of protein kinases CSK21, CHD4 and XRCC5 were found to be closely related to DNA repair. To validate these results, the authors performed an IP analysis and found that Morc2 Sumoylation significantly increased Morc2 interactions with CSK21 and CHD4, while Sumoylation-deficient Morc2 mutants greatly disrupted these interactions even in the presence of Sumo overexpression (Figure 6A). Treatment of MCF-7 and T47D cells with the Sumoylation inhibitor ML-792 20 reduces the Sumoylation of Morc2, thereby significantly reducing the interaction of Morc2 with CSK21 and CHD4 (Figure 6B). Next, the authors tested whether the restoration of MORC2 Sumoylation after ADR treatment could increase the affinity of Sumo-MORC2 to bind to CSK21 and CHD4, thereby enhancing DNA repair.
The authors first tested whether CSK21 is a Morc2 Sumoylation binding effect. The attenuation of MORC2's interaction with CSK21 was restored after ADR release (Figure 6C), but ML-792 treatment significantly impaired binding recovery (Figure 6C). Therefore, the deletion of Morc2 Sumoylation reduces the recruitment of CSK21 and may not be conducive to the further activation of downstream factors. The authors treated WT and K767R mutant Morc2 cells with ADR for 2 h and released them within the indicated time, and activation of DNA-PKCs was observed. Figure 6D-E shows that WT MORC2 significantly increases DNA-PKCs phosphorylation of serine 2056 compared to the K767R mutant, but does not affect its total protein levels. Immunofluorescence staining confirmed this result (Figure 6F-G). The above data suggest that Morc2 Sumoylation may promote efficient DNA repair in part through CSK21-induced activation of DNA-PKCs.
Morc2 Sumoylation is required for cells to survive the action of DNA damaging agents
Next, the authors investigated whether Sumoylation mediates the biological function of Morc2 in the DNA repair process, thereby affecting cell survival under genotoxic stress. To eliminate the potential effects of endogenous Morc2, the authors knocked out endogenous Morc2 in MCF-7 and T47D cells, and then infected with recombinant WT, Sumoylation-deficient mutants K767R, and Sim1mut Morc2 by lentivirus (Figure 7A). The cells were treated with DNA damaging agent ADR for 30 minutes and recovered for 24 hours, and the level of phosphorylated histone H2ax (H2AX) on Ser139 was detected by immunofluorescence staining. The long-term presence of H2AX after DNA damage indicates a defect in DNA repair. As shown in Figure 7B-C, after 24 h of recovery, cells expressing K767R and Sim1mut mutant Morc2 had higher levels of H2ax foci than WT Morc2-expressing cells, suggesting that Sumoylation-deficient mutants lead to inefficient clearance of DNA lesions. Colony formation experiments showed that WT MORC2 reduced the sensitivity of cells to ADR (Figure 7D-E) and another DNA damaging agent, MMS (Figure S10A-B), while K767R or SIM1mut mutants did not. The CCK-8 assay showed that Sumoylation of Morc2 had a similar effect on the sensitivity of cells to ADR (Figure 7F). These data suggest that morc2 sumoylation is required for cell survival in genotoxic stress responses.
To verify whether MORC2 Sumoylation plays an important role in the resistance of breast cancer cells to ADR, the authors established a xenograft tumor model from human LM2-4175 cells. Morc2-deleted LM2-4175 cells are injected subcutaneously into nude mice with stably recombinant WT or K767R MORC2. Mice were intraperitoneally injected with 3 mg kg ADR to monitor the tumor growth of xenografts. As shown in Figure 7G-H, tumors expressing K767R mutant MORC2 were smaller and lighter in weight than tumors expressing WT MORC2 after ADR treatment, indicating that MORC2 K767R mutant cells were more sensitive to ADR.
In summary, the results of this study suggest that at K767, Morc2 is modified by SUMO1 and SUMO2 3, and this event is precisely regulated by SUMO E3 ligases TRIM28 and SENP1. In addition, dynamically regulated Sumoylated Morc2 is essential for chromatin remodeling and DNA repair of DNA damage and drives chemoresistance in breast cancer. Therefore, interfering with the SUMO of MORC2 with SUMO inhibitors is a potential strategy to reverse MORC2-driven chemoresistance.
Protein translation