Dimerization of MORC2 through its C-terminal coiled-coil domain enhances chromatin dynamics and promotes DNA repair
Decondesation of the highly compacted chromatin architecture is essential for efficient DNA repair, but how this is achieved remains largely unknown. Here, we report that microrchidia family CW-type zinc finger protein 2 (MORC2), a newly identified ATPase-dependent chromatin remodeling enzyme, is required for nucleosome destabilization after DNA damage through loosening the histone-DNA interaction. Depletion of MORC2 attenuates phosphorylated histone H2AX (γH2AX) focal formation, compromises the recruitment of DNA repair proteins, BRCA1, 53BP1, and Rad51, to sites of DNA damage, and consequently reduces cell survival following treatment with DNA-damaging chemotherapeutic drug camptothecin (CPT). Furthermore, we demonstrate that MORC2 can form a homodimer through its C-terminal coiled-coil (CC) domain, a process that is enhanced in response to CPT-induced DNA damage. Deletion of the C-terminal CC domain in MORC2 disrupts its homodimer formation and impairs its ability to destabilize histone-DNA interaction after DNA damage. Consistently, expression of dimerization-defective MORC2 mutant results in impaired the recruitment of DNA repair proteins to damaged chromatin and decreased cell survival after CPT treatment. Together, these findings uncover a new mechanism for MORC2 in modulating chromatin dynamics and DDR signaling through its c-terminal dimerization.
KeywordsDNA damage response Chromatin remodeling MORC2 Coiled-coil domain Dimerization
p53-binding protein 1
breast cancer type 1 susceptibility protein
DNA damage response
microrchidia family CW-type zinc finger protein 2
DNA is continuously challenged by both endogenous and exogenous genotoxic agents that induce a variety of DNA lesions. Inefficient or inaccurate repair of these genotoxic lesions is intimately implicated in genomic instability and carcinogenesis . To maintain genomic integrity, cells have developed a complex molecular network, termed the DNA damage response (DDR), to detect and repair damaged DNA in the context of chromatin . Naturally, chromatin is a highly condensed structure that presents a significant barrier to the ability of the DNA repair machinery to access and repair DNA lesions [3, 4, 5]. Consequently, damaged chromatin must become more accessible to enable DNA repair [5, 6]. Recent work highlights that ATP-dependent chromatin remodeling enzymes and histone modifying enzymes contribute to dynamic changes of chromatin in response to genotoxic stress [7, 8]. However, the underlying mechanisms remain largely unknown.
Microrchidia family CW-type zinc finger protein 2 (MORC2) is a member of the evolutionarily conserved nuclear protein superfamily, which contains an N-terminal catalytically active ATPase module, a central CW-type zinc finger (CW-ZF) domain, a C-terminal chromo-like domain, and four distinct coiled-coil (CC) domains [9, 10]. The ATPase module is composed of gyrase, Hsp90, histidine kinase, and MutL (GHKL) and S5-fold domains, which has been mechanistically linked to gene transcription and DNA repair by remodeling chromatin [11, 12, 13]. The CW-ZF domain is present in several chromatin-remodeling proteins  and acts as a histone methylation reader [15, 16]. In addition, the C-terminal chromo-like domain is commonly found in eukaryotic chromatin proteins and can recognize methylated peptides in histones and nonhistone proteins [17, 18, 19]. These structural features indicate that MORC2 is potentially implicated in chromatin-based processes. Indeed, emerging evidence shows that MORC2 regulates heterochromatin formation and epigenetic gene silencing through an association with human silencing hub (HUSH) complex [20, 21]. In addition, we recently demonstrated that MORC2 facilitates ATPase-dependent chromatin remodeling and efficient DNA repair . However, the detailed mechanism by which MORC2 regulates chromatin dynamics during DDR remains incompletely understood.
In this study, we report that MORC2 loosens histone-DNA interaction and promotes the accessibility of DNA repair machinery to DNA damage lesions, thus facilitating cell survival after DNA damage induced by DNA-damaging chemotherapeutic agent camptothecin (CPT). Moreover, the C-terminal coiled-coil domain of MORC2 is required for its dimerization and the noted biological functions in DDR. These findings provide a novel molecular link between MORC2 and chromatin dynamics in response to DNA damage. Given its emerging roles in tumor progression [22, 23, 24, 25] and in resistance to DNA-damaging radio- and chemotherapies [13, 25], MORC2 could be a good candidate target for cancer treatment.
MORC2 weakens histone-DNA interaction in cells both under unstressed condition and after DNA damage
MORC2 promotes the recruitment of DNA repair proteins to damaged chromatin and decreases cellular sensitivity to DNA-damaging chemotherapeutic agents
MORC2 can homodimerize through its C-terminal coiled-coil domain
DNA damage enhances MORC2 dimerization
MORC2 dimerization is required for altered nucleosome stability after DNA damage and subsequent DNA repair signaling
Coiled-coil domain plays essential roles in protein assembly and molecular recognition. For instance, the coiled-coil domain of human ATR-interacting protein (ATRIP) contributes to self-dimerization in vivo, which is important for cellular response to replication stress and DNA damage . Dimerization of CtIP, a BRCA1- and CtBP-interacting protein, is mediated by an N-terminal coiled-coil motif . As MORC2 protein contains four distinct coiled-coil domains, we proposed that MORC2 may form a dimer. In support of this notion, co-IP and glutaraldehyde cross-linking assays demonstrated that MORC2 indeed forms a dimer (Fig. 4) and that the C-terminal coiled-coil domain is essential for MORC2 dimerization (Fig. 5). Unfortunately, we failed to map the specific sites responsible for MORC2 dimerization although we worked hard on this point (data not shown). Interestingly, we found for the first time that MORC2 dimerization is enhanced in response to DNA damage (Fig. 6). In contrast, growth factor EGF and CoCl2, a mimic of hypoxia, did not significantly affect dimer formation of MORC2. In addition, functional rescue experiments revealed that MORC2 dimerization is required for MORC2-mediated destabilization of histone and DNA interaction and subsequent DDR signaling (Fig. 7). Nevertheless, the functional and mechanistic roles for MORC2 dimerization in DDR remain to be explored.
In summary, findings presented here suggest that MORC2 can form a dimer through its C-terminal coiled-coil domain, which is regulated by DNA damage signaling and is implicated in DNA damage repair (Fig. 8). Given that MORC2 is upregulated in multiple types of human cancer  and contributes to cancer growth and progression [22, 23, 24, 25] as well as cellular response to ionizing radiation  and chemotherapeutic drugs , these findings broaden our understanding of the functional roles of MORC2 in DDR and highlight MORC2 as a potential target for cancer treatment.
Cell culture and reagents
HeLa, MCF-7, and HEK293T cell lines were obtained from the Bank of Type Culture Collection of Chinese Academy of Sciences (Shanghai, China). All of them were authenticated by short tandem repeat profiling and cultured in high-glucose DMEM media containing 10% fetal bovine serum (Gibco, Carlsbad, USA). All chemicals or regents were purchased from Sigma-Aldrich (St. Louis., USA) unless otherwise noted.
DNA constructs, transfection, and lentiviral infection
Myc-DDK-MORC2 expression vector was purchased from Origene (Rockville, USA). To generate Flag-tagged full-length MORC2 expression vectors, MORC2 cDNA was amplified by PCR and subcloned into the lentiviral vector pCDH-CMV-MCS-EF1-Puro (System Biosciences, Mountain View, USA). To generate HA-MORC2 and HA-MORC2 ∆C82 (deletion of C-terminal 82 amino acid), MORC2 cDNA was amplified by PCR and subcloned into either pCDH-CMV-MCS-EF1-Puro or pCDH-CMV-MCS-EF1-copGFP lentiviral vectors (System Biosciences). All constructs were verified by DNA sequencing (HuaGene Biotech, Shanghai, China). The primers used for molecular cloning and the expression constructs used in this study are listed in Additional file 1: Tables S1 and S2, respectively.
Transient plasmid transfection was carried out using Neofect DNA transfection reagent (TengyiBio, Shanghai, China) according to the manufacturer’s protocol. Knockout (KO) of MORC2 was performed using the CRISPR/Cas9 system  with LentiCas9-Blast and lentiGuide-Puro vectors (Addgene, Cambridge, USA). The short guide RNA (sgRNA) sequences targeting MORC2 are listed in Additional file 1: Tables S3. To re-express HA-MORC2 and HA-MORC2 ∆C82 in MORC2 KO cells, individual lentiviral expression vector, along with packaging plasmid mix, was transfected into HEK293T cells. After 48 h of transfection, the viruses in supernatant were collected and used to infect MORC2 KO cells. Sorting of GFP-positive cells was carried out by Fluorescence Activated Cell Sorting (FACS) and immunoblotting was used to further validate the positive cells.
Antibodies, immunoblotting, immunoprecipitation, and immunofluorescence
The detailed information for primary antibodies is provided in Additional file 1: Table S4. For immunoblotting analysis, cells were lysed in the modified RIPA buffer (50 mM Tris-HCl, pH 7.4, 1% NP-40, 0.25% sodium deoxycholate, 1 mM EDTA and 150 mM NaCl) containing 1 × protease inhibitor cocktail and 1 × phosphatase inhibitor cocktail (Bimake, Houston, USA). Proteins were quantified using the bicinchoninic acid assay (Yeasen, Shanghai, China), resolved by SDS-PAGE, and transferred onto PVDF membrane (Millipore, Billerica, USA). Antibody detection was carried out using enhanced chemiluminescence (Yeasen). The immunoblotting data was quantified using Image J software, and the expression levels of proteins were normalized to those of corresponding controls in each panel.
For immunoprecipitation (IP) analysis, cells were lysed in NP-40 lysis buffer (50 mM Tris-HCl, pH 8, 150 mM NaCl, 0.5% NP-40, 10% glycerol, 2 mM MgCl2, and 1 mM EDTA), and total 1–2 mg of exogenously expressed proteins were incubated anti-Flag or anti-HA magnetic beads (Bimake) overnight at 4 °C to pull down the protein-antibody complex. The resulting complexes were subjected to immunoblotting analysis.
For immunofluorescent staining, cells were fixed in 4% paraformaldehyde, permeabilized in 0.1% Triton X-100, and blocked in 5% normal goat serum in PBST. Cells were incubated with primary antibodies, washed three times in PBST, and then incubated with the appropriate secondary antibody conjugated with 555-Alexa (red) or 488-Alexa (green) (Cell Signaling Technology, Danvers, USA), respectively. DNA staining was conducted using fluoroshield mounting medium with DAPI (Abcam, Cambridge, USA). Microscopic analyses were performed using a Leica SP5 confocal laser scanning microscopy (Leica Microsystems, Buffalo Grove, USA).
Salt solubilization assays
Salt solubilization assays were performed as described previously [27, 28]. Briefly, nuclei were isolated from 2 × 106 cells using hypotonic lysis and were incubated in non-denaturing extraction buffers (20 mM Tris-Cl, pH 7.6, 5% glycerol) supplemented with 100 to 500 mM NaCl in the presence of protease inhibitors for 5 min. Cell nuclei were harvested by centrifugation at 700 g for 25 min. Salt soluble fractions were obtained by centrifugation, resolved by SDS-PAGE, and analyzed by immunoblotting using the indicated antibodies.
Glutaraldehyde cross-linking assays
Cells were lysed with the NP40 buffer and quantified as described above. Then, total cell lysates were cross-linked with 0.05% (w/v) glutaraldehyde (Sigma) on ice for 5 min and terminated by adding 1 M glycine for 15 min at room temperature. After that, the protein samples were analyzed by 8% SDS-PAGE and immunoblotting with the indicated antibodies.
Protein purification from mammalian cells
Purification of Flag-MORC2 from HEK293T cells was performed as described previously . Briefly, full-length Flag-MORC2 was transfected into HEK293T cells. After 48 h of transfection, cells were lysed in the modified RIPA buffer containing 1 × protease inhibitor cocktail and 1 × phosphatase inhibitor cocktail (Bimake). The clarified supernatant was subjected to pull-down assays using anti-Flag affinity gel (Bimake), and the enriched proteins were eluted using an elution buffer containing 1 mg/ml 3× Flag peptide, 25 mM Tris, pH 8.0, and 100 mM NaCl. The purified protein was used for chemical cross-linking assays.
Cell viability and clonogenic survival assays
For cell viability assays, cells were seeded in 96-well plates (1000 cells/well) in triplicate and cell viability was analyzed using Cell Counting Kit-8 (CCK-8) kit (Dojindo Laboratories, Kumamoto, Japan). For clonogenic survival assays, cells were seeded in 6-well plates (1000 cells/well) in triplicate and cultured under normal growth conditions for 1–2 weeks. Colonies were stained with 1% crystal violet and counted using an inverted microscope.
All data are presented as the mean ± standard deviation from at least three independent experiments. The Student’s t-test was used for assessing the difference between individual groups and p ≤ 0.05 was considered statistically significant.
We sincerely acknowledge members in the Li laboratory for their technical assistance and helpful advices.
HYX, TMZ, and SYH performed experiments and analyzed data. ZMS and DQL supervised the entire project. HYX was a major contributor in writing the manuscript. All authors read and approved the final manuscript.
This work is supported, in whole or in part, by the National Natural Science Foundation of China (No. 81572584, 81772805, and 81972461), the National Key R&D Program of China (No. 2017YFC0908400 and 2018YFE0201600), the Program for Professor of Special Appointment (Eastern Scholar) at Shanghai Institutions of Higher Learning (No. 2013–06), and the Science and Technology Innovation Action Plan of Shanghai Municipal Science and Technology Commission (No. 16JC1405400).
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The authors declare that they have no competing interests.
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