Figures and data
The CC3 domain serves as the structural basis for full-length MORC2 dimerization.
(a) The domain organization of human MORC2FL, highlighting the ATPase module (GHL, CC1 and S5 domains, collectively termed NTD), the C-terminal domains (CTD) with coiled-coil regions (CC2 and CC3), CW-type zinc finger (CW), intrinsically disordered region (IDR, marked by a fresh green line by IUPRED2 prediction), nuclear localization signal (NLS), Tudor-chromodomain (TCD), and a 30-residues tail domain (IBD).
(b) Purification and characterization of full-length human MORC2 in HEK293F cell system. SDS-PAGE analysis of the purified protein demonstrates its purity and distribution.
(c) Static light-scattering (SLS) analysis shows that MORC2 forms dimers in the absence of ATP, with a molecular weight of 239 ± 1 kDa in standard buffer (mint green line) and 303 ± 2 kDa in the presence of 601 DNA (sunflower yellow line).
(d) SLS analysis reveals the oligomerization states of MORC2 truncations. Full-length MORC2 lacking CC3-IBD (1-900) is monomeric, while CC3-IBD (901-1032) and CC3 alone (901-1003) form stable dimers.
(e) Crystal structural of the CC3 dimer. Hydrophobic residues contributing to dimer stability (L911, L915, and F922) are highlighted using ball-and-stick representations. Top view of the hydrophobic core of the CC3 dimer. Layers of hydrophobic contacts stabilize the dimer interface, illustrated with paired residues (e.g., Layer 1: L911-L911’, Layer 2: L915-L915’, and Layer 3: F922-F922’).
(f) Additional SLS analysis confirms that fragments spanning residues 537-1032, 744-1032, and Trx-901-975 (CC3 truncation) adopt dimeric conformations.
MORC2 undergoes liquid-liquid phase separation (LLPS) under physiological conditions.
(a) SDS-PAGE analysis of EGFP-MORC2FL protein purified from HEK293F cells, showing protein concentration at 2.5 μM and 5 μM.
(b) Sedimentation assay shows the distribution of EGFP-MORC2FL (7.2 μM) between supernatant (S) and pellet (P) fractions under 1000 mM (control) and 150 mM NaCl, indicating its propensity for phase separation.
(c) EGFP-MORC2 (7.2 μM) undergoes phase separation into droplets in a buffer containing 150 mM NaCl, but not in 1000 mM NaCl buffer. Scaler bar: 2 μm.
(d) The proteins were examined in a buffer containing 150 mM NaCl, and phase separation was assessed by fluorescence microscopy with 488 nm excitation for EGFP. Quantification of droplet areas from (Fig. S2a), presented as violin plots with box plots indicating medians. Data are derived from ≥ 3 independent images and reflect the size distribution of phase-separated droplets. Data are presented as mean ± SEM; one-way ANOVA with Tukey’s post hoc test. ****p < 0.0001; *p < 0.05.
(e) Time-lapse FRAP imaging of EGFP-MORC2FL droplets formed in vitro, demonstrating fluorescence recovery over time. Scaler bar: 2 μm.
(f) Quantitative analysis of fluorescence recovery from (e), showing the dynamic properties of in vitro MORC2 droplets. Data represent mean ± SEM, with n ≥ 3 replicates.
(g) Immunostaining of endogenous MORC2 in HeLa cells revealed punctate localized within the nucleus, likely representing condensates involved in transcriptional regulation (white arrows). Nuclei are counterstained with DAPI to visualize chromatin-rich regions. Scaler bar: 5 μm.
(h) Live-cell imaging of transiently transfected EGFP-MORC2FL showed its assembly into dispersed, nearly spherical condensates in the nucleus, while stable expression of H2A-mCherry served to mark chromatin-enriched domains. Scaler bar: 10 μm.
(i) Time-lapse FRAP analysis of EGFP-MORC2FL condensates in the nuclei of transiently transfected HeLa cells revealed rapid fluorescence recovery within seconds, indicative of their dynamic, liquid-like nature. Scaler bar: 1 μm.
(j) Quantitative analysis of fluorescence recovery from (i), showing the dynamic properties of in vitro MORC2 droplets. Data represent mean ± SEM, with n ≥ 10 replicates.
(k) Sections of brain and spinal cord from endogenous EGFP-MORC2 chimeric mice show EGFP-MORC2 condensation distribution in NeuN-positive neurons. Scaler bar: 10 μm.
(l) Representative FRAP images from of FRAP experiments performed on fresh 250-μm brain slices of endogenous EGFP-MORC2 chimeric mice demonstrate the dynamic nature of endogenous MORC2 condensates. Data are presented as mean ± SEM, with n = 3 droplets analyzed. Scaler bar: 5 μm.
Multiple domains regulate phase separation of MORC2.
(a) Sequential deletion analysis revealed that the NTD is dispensable for condensate formation, as EGFP-tagged NTD alone failed to form condensates and was excluded from the nucleus. Fusion with a canonical nuclear localization signal (“PKKKRKV”) restored nuclear localization but did not rescue condensate formation. In contrast, the CTD alone was sufficient to form nuclear condensates. Deletion of the CW domain resulted in enlarged condensates in a subset of cells, while removal of CC2 or the TCD had no apparent effect on condensate formation. Strikingly, deletion of either the IDR or CC3 completely abolished nuclear condensate assembly. Deletion of the IBD markedly reduced the frequency of condensate formation. Loss of the intrinsic NLS led to cytoplasmic localization of MORC2; however, no condensates were detected in the cytoplasm under transient transfection conditions in HeLa cells. Together, these results define the IDR and CC3 as the minimal and essential elements for MORC2-mediated phase separation, and implicate the CW domain, IBD, and nuclear microenvironment in the fine-tuning of condensate assembly. Scaler bar: 10 μm.
(b) In vitro phase separation of Cy3-labeled CTDΔCW at concentrations from 1.25 μM to 20 μM in 150 mM NaCl buffer, visualized as spherical condensates. Scaler bar: 10 μm.
(c) Quantification of droplet areas from (b), displayed as violin plots with box plots indicating median values. Data are presented as mean ± SEM; one-way ANOVA with Tukey’s post hoc test. ****p < 0.0001, n.s. not significant.
(d) SDS-PAGE analysis of Cy3-CTDΔCW protein distribution between supernatant (S) and pellet (P) fractions after centrifugation at increasing concentrations (5 μM to 40 μM) in 150 mM NaCl buffer. At 40 μM, increasing the NaCl concentration to 300 mM or 500 mM reduced droplet formation, indicating salt sensitivity.
(e) Quantification of Cy3-CTDΔCW showing the percentage of soluble protein from densitometric analysis of S/P fractions in panel (d), based on three independent biological replicates.
Multivalent interactions between IDR and IBD drive MORC2 LLPS.
(a) Domain organization of MORC2 C-terminal regions analyzed by NMR titration, including the IDR, CC3, and IBD.
(b) 15N-labeled CC3 (residues 901–1003) shows no chemical shift upon titration with IDR (residues 593–735), indicating no detectable interaction.
(c) 15N-labeled IBD (residues 1004–1032) exhibits clear chemical shift perturbations (CSPs) upon IDR titration, confirming a direct, specific interaction.
(d-f) NMR titrations of 15N-labeled IBD with IDR subregions: IDRa (593–643), IDRb (644–694), and IDRc (695–735). IDRa induces clear CSPs (d), while IDRb (e) and IDRc (f) show minimal or no shifts, identifying IDRa as the primary IBD-binding segment.
(g) Sequence analysis of IDR subregions reveals that IDRa is enriched in proline and arginine residues, enabling electrostatic interactions. Sequence alignment of IBD across species highlights conserved residues involved in IDRa binding. A summary table lists representative residues from IDRa and IBD exhibiting CSPs in titration assays (marked with asterisks).
(h) Representative confocal images of HeLa cells expressing EGFP-tagged MORC2ΔIDRa, ΔIDRb, or ΔIDRc constructs. Scale bar: 10 μm.
(i) Quantification of droplet area per cell in (h). Deletion of IDRa significantly impairs condensate formation. n = 10 cells per condition. Data are presented as mean ± SEM; statistical analysis: one-way ANOVA with Tukey’s post hoc test. ***p < 0.001; n.s. not significant.
(j) Working model of MORC2 LLPS. CC3 dimerization serves as a structural scaffold, while weak, transient, but specific multivalent interactions between IDRa and IBD cooperatively promote condensate formation.
DNA binding enhances MORC2 phase separation and modulates its ATPase activity.
(a) Electrophoretic mobility shift assay (EMSA) showing that MORC2FL binds 25 nM 601 DNA, as evidenced by shifts in DNA mobility corresponding to protein-DNA complexes.
(b) EMSA highlighting the strong DNA-binding affinity of the CC1 domain, corroborating previous reports that identify this region as a key mediator of DNA interaction.
(c-f) EMSA results demonstrating the binding capacities of individual MORC2 domains to 25 nM 601 DNA: CC2 (c), IDR (d), TCD-CC3-IBD (e), and CC3-IBD (f). The IDR exhibits robust DNA-binding activity, CC2 and TCD-CC3-IBD show weaker interactions, and CC3-IBD alone fails to bind DNA. These findings identify CC1, CC2, IDR, and the TCD as DNA-binding domains of MORC2.
(g) Confocal imaging showing that 601 DNA promotes MORC2 phase separation. At 0.9 μM, EGFP-MORC2FL alone does not form droplets, but the addition of 25 nM 601 DNA induces distinct condensate formation. Scaler bar: 5 μm.
(h) Quantification of MORC2 condensate sizes with or without 601 DNA. Violin plots illustrate the distribution of droplet sizes, with box plots indicating median values. Data are presented as mean ± SEM; unpaired student t-test. ****p < 0.0001.
(i) Fluorescence microscopy showing Cy3-labeled CTDΔCW (1.25 μM) coalescing with 25 nM FAM-labeled 601 DNA. DNA is recruited into MORC2 droplets, forming distinct condensates. Scaler bar: 5 μm.
(j) In the presence of 10 nM 601 DNA, the ATP hydrolysis activity of MORC2FL is significantly enhanced. The N39A mutant, which is deficient in ATP binding, serves as a negative control; nevertheless, a slight but detectable increase in ATPase activity is observed upon DNA binding. ATP hydrolysis activity of MORC2FL is significantly increased in the presence of 10 nM 601 DNA. The N39A mutant, which is deficient in ATP binding, serves as a negative control. Data are presented as mean ± SEM; statistical analysis: one-way ANOVA with Tukey’s post hoc test. ****p < 0.0001.
MORC2 phase separation is required for its transcriptional regulatory activity.
(a) Schematic of the rescue experiment strategy. HeLa cells with CRISPR-Cas9-mediated MORC2 knockout were reconstituted with EGFP, EGFP-MORC2FL, or phase separation-deficient mutants EGFP-MORC2ΔCC3 and EGFP-MORC2ΔIDR.
(b) RNA-seq-derived read counts for the MORC2 gene in EGFP, EGFP-MORC2FL, EGFP-MORC2ΔCC3, and EGFP-MORC2ΔIDR expressing cells.
(c-d) Venn diagram illustrating the overlap between differentially expressed genes identified following EGFP-MORC2FL overexpression in this study and previously characterized MORC2-regulated targets.
(e) Volcano plots of RNA-seq read counts for EGFP-MORC2FL and EGFP-MORC2ΔCC3 overexpression compared to EGFP control. Significantly upregulated genes are shown in yellow, and significantly downregulated genes are shown in green, based on three biological replicates (fold change > 1.2 or < 0.83, p < 0.05).
(f) LLPS-deficient mutant of MORC2 fails to regulate these 18 downregulated genes despite comparable protein expression levels, supporting a functional role of MORC2 condensates in transcriptional regulation.
Pathogenic variants of MORC2 alter phase separation, DNA binding, and ATPase activity.
(a) Schematic illustration of pathogenic MORC2 mutations associated with Charcot-Marie-Tooth disease type 2Z (CMT2Z, red) and spinal muscular atrophy (SMA, blue), mapped onto the MORC2 domain structure.
(b) Quantification of HeLa cells transfected with WT MORC2 or nine pathogenic mutations exhibiting nuclear condensates was performed for each field of view. E236G and T424R shows the most pronounced enhancement of phase separation. n ≥ 10 fields. Data are presented as mean ± SEM; one-way ANOVA with Tukey’s post hoc test. ****p < 0.0001, **p < 0.01, n.s. not significant.
(c) ATPase activity of WT MORC2 and eight pathogenic variants, measured under revised conditions. S218L, F256L, and T424R exhibit significantly elevated activity. We were unable to purify MORC2 constructs bearing the E236G mutation from either HEK293F cells, suggesting that it may cause misfolding of the ATPase module. Data are presented as mean ± SEM; one-way ANOVA with Tukey’s post hoc test. ****p < 0.0001, **p < 0.01, n.s. not significant.
(d) Fluorescence polarization (FP) analysis of DNA binding affinities to the 601 DNA sequence. All protein variants were evaluated under uniform assay conditions, including those pertinent to phase separation. Dissociation constants (Kd) were determined by fitting the FP data to a log(agonist) vs. response model and are reported as mean ±SEM: WT (98 ± 1 nM), R252W (51 ± 2 nM), Q400R (145 ± 26 nM), D466N (49 ± 6 nM), S87L (107 ± 24 nM), S218L (92 ± 10 nM), F256L (69 ± 2 nM), R266A (63 ± 5 nM), and T424R (242 ± 13 nM). Several variants, including R252W, D466N, F256L, and R266A, demonstrated increased binding affinity relative to WT, while others, such as T424R and Q400R, exhibited markedly reduced binding.
(e) Proposed model summarizing the impact of DNA binding and phase separation on MORC2-mediated chromatin remodeling. DNA promotes MORC2 condensate formation through electrostatic interactions, which enhances its ability to compact chromatin and regulate transcriptional silencing.
Statistics of X-ray Crystallographic Data Collection and Model refinement
Summary of MORC2 protein constructs used in this study, including expression systems, extinction coefficients, A260/280 ratios, and corresponding experimental applications.
Full-length MORC2 is a dimeric protein, with dimerization mediated by its C-terminal CC3 domain.
(a) Negative-stain electron microscopy revealed that MORC2FL particles purified from HEK293F cells appeared poorly resolved and exhibited considerable morphological heterogeneity. Scaler bar: 100 nm.
(b) Structural prediction of MORC2FL using AlphaFold3 under a two-copy modeling condition revealed the presence of an extended disordered region at the C-terminus and suggested a potential dimerization interface within this region.
(c) Summary of theoretical and measured molecular weight of MORC2 constructs, including MORC2FL, MORC2ΔCC3-IBD, CC3-IBD, CC3, and CC3 mutants. Data are derived from recombinant protein purification and static light-scattering (SLS) analysis.
(d) AlphaFold3-based modeling of MORC2 with the N-terminal domain removed revealed structural features of the C-terminus, including extensive disorder and a putative dimerization interface.
(e) Molecular model of the CC3 dimer highlights hydrophobic core residues (I908, L911, L915, F921, F922, F951, Y954). Key intermolecular distances include I908-I908’ (3.4 Å), L911-L915’ (3.7 Å), F922-F922’ (3.9 Å) and L958-L958’ (4.0 Å), supporting dimer stability.
(f) Molecular model of the CC3 dimer formation interface show the additional hydrophobic interactions.
(g) Point mutations in key residues (L911, L915, F922) affect dimerization. Additional mutants (I908Q, F951Q, Y921A, Y954A, L958Q) show no significant change in molecular weight compared to WT, confirming their minimal impact on dimer formation. SLS analysis shows molecular weight distributions, with WT CC3-IBD forming dimers (∼ 60 kDa) as indicated by red curves, while mutants exhibit higher molecular weights indicative of aggregation. Constructs were tagged with a Trx tag (14 kDa) for solubility.
In vitro and in vivo evidence that MORC2 undergoes LLPS.
(a) Concentration gradients of EGFP-MORC2FL proteins (0.9, 1.8, 3.6, and 7.2 μM) were analyzed for in vitro phase separation. The proteins were examined in a buffer containing 150 mM NaCl, and phase separation was characterized by fluorescence microscopy with 488 nm excitation for EGFP. Scaler bar: 2 μm.
(b) SDS-PAGE analysis of EGFP-MORC2FL in sedimentation-based assays. Protein distribution between supernatant (S) and pellet (P) fractions is shown for increasing concentrations (0.9, 1.8, 3.6, and 7.2 μM).
(c) Time-lapse imaging of EGFP-MORC2FL phase separation over 120 s highlights dynamic droplet fusion. Scaler bar: 2 μm.
(d) Schematic of CRISPR-Cas9 strategy for generating MORC2 knockout (KO) HeLa cells. sgRNA targeting sequences and protospacer adjacent motifs (PAM) are highlighted. Genomic sequencing confirms insertion-deletion mutations introduced at target sites in MORC2 allelic genes.
(e) Immunostaining images of WT and MORC2 KO HeLa cells validate the specificity of the custom-made MORC2 antibody. Scaler bar: 5 μm.
(f) Generation of endogenous EGFP-MORC2 chimeric mice. Genomic sequencing confirms insertion-EGFP mutations introduced at target sites in MORC2 allelic genes.
(g) Live-cell imaging revealed that EGFP-MORC2FL exhibits dynamic subcellular localization throughout mitosis, without forming detectable condensates. Scaler bar: 10 μm.
The C-terminal domain (CTD) of MORC2 mediates nuclear condensate formation.
(a) EGFP-tagged MORC2 CTD forms nuclear condensates in HeLa cells, whereas the NTD does not. Scaler bar: 10 μm.
(b) Truncation analysis of EGFP-tagged CTD reveals that both the CC3-IBD and IDR-NLS regions are important for condensate formation. Scaler bar: 10 μm.
(c) EGFP-tagged CTD constructs lacking the IDR-NLS region but fused to an exogenous N-terminal nuclear localization signal (“PKKKRKV”) successfully localize to the nucleus but fail to form condensates, indicating that the IDR is essential for phase separation. Scaler bar: 10 μm.
(d) Targeted expression of IDR-NLS and CC3-IBD fragments in the nucleus failed to induce condensate formation, indicating that these domains alone are not sufficient to drive phase separation. Scaler bar: 10 μm.
(e) The statistical data for droplet area in (Fig. 3a) demonstrate that the deletion of IDR or CC3 is imperative for condensate formation. The data are expressed as the mean ± standard error of the mean (SEM). n ≥ 15 fields; one-way ANOVA with Tukey’s post hoc test. ****P < 0.0001.
The biophysical behavior of CTD and CTDΔCW in vitro.
(a) SDS-PAGE analysis of Cy3-labeled CTD showing expected size and degradative behavior.
(b) Fluorescence microscopy images of Cy3-labeledCTD at increasing protein concentrations (1.25LμM, 2.5LμM, 5LμM, and 10LμM) in standard phase separation buffer (50LmM Tris-Cl, pH 8.0, 150LmM NaCl). Despite increasing concentration, CW-CTD formed only small, dense puncta that did not exhibit robust size expansion or morphological changes. Scaler bar: 10 μm.
(c) Representative bright-field and Cy3 fluorescence microscopy images of labeled CTDΔCW protein droplets, showing intact condensate morphology under standard phase separation conditions. Scaler bar: 5 μm.
(d) SDS-PAGE analysis comparing Cy3-labeled and unlabeled CTDΔCW, indicating no degradation or mobility shift.
(e) Static light scattering (SLS) profiles of Cy3-labeled and unlabeled CTDΔCW proteins reveal similar molecular weight distributions and hydrodynamic behavior.
(f) Time-lapse imaging of Cy3-labeled CTDΔCW over 180 s reveals dynamic droplet behavior in vitro. Scaler bar: 2 μm.
Improved visualization of MORC2 nuclear condensates in HEK293T cells.
(a) Representative single-cell images of EGFP-MORC2-expressing HEK293T cells, reformatted from original Fig. 4i to enhance resolution and comparability with Fig. 4j. Nuclear puncta are clearly visible at this scale. Scaler bar: 10 μm.
(b) Quantification of the percentage of cells showing visible nuclear condensates in each construct. Data are presented as mean ± SEM; n ≥ 11 replicates. Statistical significance was assessed using unpaired Student’s t-test. ****p < 0.0001.
MORC2 phase separation regulates gene transcription.
(a) Abundance trajectories of all 240 regulated genes were normalized to their respective mean values.
(b) GO-based cellular component analysis of the regulated genes mapped in DAVID (v2025_1) knowledgebase. A substantial number of MORC2-regulated genes are primarily localized to the nucleus, chromatin, and nucleoplasm.
(c) GO-based biological process analysis. MORC2-regulated genes are predominantly involved in the biological process of transcriptional regulation.
(d) GO-based molecular function analysis. MORC2 primarily regulates genes encoding transcription factors with transcriptional regulatory activity. Additionally, MORC2 modulates the transcription of several cytokine genes.
(e) A selected panel of 12 genes that were regulated by MORC2 in this sequencing context but lost responsiveness to the LLPS-deficient mutant was shown, including 8 downregulated and 4 upregulated genes.
Representative SDS-PAGE gels for all purified protein constructs.
(a) MORC2 fragments used for conformational analysis by FPLC-MALS (related to Fig. 1).
(b) MORC2 CC3 domain fragments used for dimerization analysis by FPLC-MALS (related to Fig. S1).
(c) MORC2 fragments expressed in E. coli for EMSA-based DNA-binding assays (related to Fig. 5).
(d) MORC2 fragments expressed in HEK293F cells for ATPase activity measurements with or without 601 DNA (related to Fig. 5).
(e) Disease-associated MORC2 mutants expressed in HEK293F cells for ATPase and DNA-binding assays,while E236G mutant exhibited poor biochemical properties, precluding further analysis in vitro (related to Fig. 7).
