MORC2 Mediates Transcriptional Regulation Through Liquid-Liquid Phase Separation and DNA Binding

Reviewer #1 (Public review):

Summary:

This work demonstrates that MORC2 undergoes phase separation (PS) in cells to form nuclear condensates, and the authors demonstrate convincingly the interactions responsible for this phase separation. Specifically, the authors make good use of crystallography and NMR to identify multiple protein:protein interactions and use EMSA to confirm protein:DNA interactions. These interactions work together to promote in vitro and in cell phase separation and boost ATPase activity by the catalytic domain of MORC2.

However, the authors have very weak evidence supporting their potentially valuable claim that MORC2 PS is important for the appropriate gene regulatory role of MORC2 in cells. Exploring causal links between PS and function is an important need in the phase separation field, particularly as regards the role of condensates in gene regulation, and is a non-trivial matter. Any study with convincing data on this matter will be very important. For this reason, it is crucial to properly explore the alternative possibility that soluble complexes, existing in the same conditions as phase-separated condensates, are the functional species. It is also critical to keep in mind that, while a specific protein domain may be essential for PS, this does not mean its only important function pertains to PS.

In this study, the authors do not sufficiently explore the role that soluble MORC2 complexes may play alongside MORC2 condensates. Neither do they include enough data to solidly show that domain deletion leads to phenotypes via a loss of phase separation per se, rather than the loss of phase separation being a microscopically visible result, not cause, of an underlying shift in protein function. For these reasons, the authors' conclusions regarding the functional role of MORC2 condensates are based on incomplete data. This also dampens the utility of this work as a whole, since the very nice work detailing the mechanism of MORC2 PS is not paired with strong data showing the importance of this observation.

Strengths:

Static light scattering and crystallography are nicely used to demonstrate the dimerization of MORC2FL and to discover the structure of the CC3 domain dimer, presumably responsible for the dimerization of MORC2FL (Figure 1).

Extensive use of deletion mutants in multiple cell lines is used to identify regions of MORC2 that are important for forming condensates in the nucleus: the IBD, IDR, and CC3 domains are found to be essential for condensate formation, while the CW domain plays an unknown role in condensate morphology (Figure 3). The authors use NMR to further identify that the IBD domain seems to interact with the first third of the centrally located IDR, termed IDRa, but not with the latter two-thirds of the IDR domain (Figure 4). This leads them to propose that phase separation is the product of IDB:IDRa interaction, CC3 dimerization, and an unknown but important role for the CW domain.

Based on the observation that removal of the NLS resulted in diffuse cytoplasmic localization, they hypothesized that DNA may play an important role in MORC2 PS. EMSA was used to demonstrate interaction between DNA and several MORC2 domains: CC1, CC2, IDR, and TCD-CC3-IBD. Further in vitro microscopy with purified MORC2 showed that DNA addition significantly reduces MORC2 saturation concentration (Figure 5).

These assays convincingly demonstrate that MORC2 phase separates in cells, and identify the protein domains and interactions responsible for this phenomenon, with the notable caveat that the role of the CW domain here is left unexplored.

Weaknesses:

Although the authors demonstrated phase separation of MORC2FL, their evidence that this plays a functional role in the cell is incomplete.

Firstly, looking at differentially upregulated genes under MORC2FL overexpression, the authors acknowledge that only 10% are shared with differentially regulated genes identified in other MORC2FL overexpression studies (Figure 6c,d). No explanation is given for why this overlap is so low, making it difficult to trust conclusions from this data set.

Secondly, of the 21 genes shared in this study and in earlier studies, the authors note that the differential regulation is less pronounced when a phase-separation-deficient MORC2 mutant is overexpressed, rather than MORC2FL (Figure 6e). This is taken as evidence that phase separation is important for the proper function of MORC2. However, no consideration is made for the alternative possibility that the mutant, lacking the CC3 dimerization domain, may result in non-functional complexes involving MORC2, eliminating the need for a PS-centric conclusion. To take the overexpression data as solid evidence for a functional role of MORC2 PS, the authors would need to test the alternative, soluble complex hypothesis. Furthermore, there seems to be low replicate consistency for the MORC2 mutant condition (Figure S6a), with replicate 3 being markedly upregulated when compared to replicates 1 and 2.

Thirdly, the authors close by examining the in-cell PS capabilities and ATPase activity of several disease-associated mutants of MORC2 ( Figure 7). However, the relevance of these mutants to the past 6 figures is unclear. None of these mutations is in regions identified as important for PS. Two of the mutations result in a higher percentage of the cell population being condensate-positive, but this is not seemingly connected to ATPase activity, as only one of these two mutants has increased ATPase activity. Figure 7 does not add any support to the main hypotheses in the paper, and nowhere in the paper do the authors investigate the protein regions where the mutations in Figure 7 are found.

Reviewer #2 (Public review):

Summary:

The study by Zhang et al. focuses on how phase separation of a chromatin-associated protein MORC2, could regulate gene expression. Their study shows that MORC2 forms dynamic nuclear condensates in cells. In vitro, MORC2 phase separation is driven by dimerization and multivalent interactions involving the C-terminal domain. A key finding is that the intrinsically disordered region (IDR) of MORC2 exhibits strong DNA binding. They report that DNA binding enhances MORC2's phase separation and its ATPase activity, offering new insights into how MORC2 contributes to chromatin organization and gene regulation. The authors try to correlate MORC2's condensate-forming ability with its gene silencing function, but this warrants additional controls and validation. Moreover, they investigate the effect of disease-linked mutations in the N-terminal domain of MORC2 on its ability to form cellular condensates, ATPase activity, and DNA-binding, though the findings appear inconclusive in the manuscript's current form.

Strengths:

The authors determined a 3.1 Å resolution crystal structure of the dimeric coiled-coil 3 (CC3) domain of MORC2, revealing a hydrophobic interface that stabilizes dimer formation. They present extensive evidence that MORC2 undergoes liquid-liquid phase separation (LLPS) across multiple contexts, including in vitro, in cellulo, and in vivo. Through systematic cellular screening, they identified the C-terminal domain of MORC2 as a key driver of condensate formation. Biophysical and biochemical analyses further show that the IDR within the C-terminal domain interacts with the C-terminal end region (IBD) and also exhibits strong DNA-binding capacity, both of which promote MORC2 phase separation. Together, this study emphasizes that interactions mediated by multiple domains-CC3, IDR, and IBD- drives MORC2 phase separation. Finally, the authors quantified the effect of removing the CC3 on the upregulation and downregulation of target gene expression.

Weaknesses:

Though the findings appear compelling in isolation, the study lacks discussion on how its findings compare with previous studies. Particularly in the context of MORC2-DNA binding, there are previous studies extensively exploring MORC2-DNA binding (Tan, W., Park, J., Venugopal, H. et al. Nat Commun 2025), and its effect on ATPase activity (ref 22). The contradictory results in ref 22 about the impact of DNA-binding on ATPase activity, and ATPase activity on transcriptional repression, warrant proper discussion. The authors performed extensive in-cellulo screening for the investigation of domain contribution in MORC2 condensate formation, but the study does not consider/discuss the possibility of some indirect contributions from the complex cellular environment. Alternatively, the domain-specific contributions could be quantified in vitro by comparing phase diagrams for their variants. While the basis of this study is to investigate the mechanism of MORC2 condensate-mediated gene silencing, the findings in Figure 6 appear incomplete because the CC3 deletion not only affects phase separation of MORC2 but also dimerization. Furthermore, their investigation on disease-linked MORC2 mutations appears very preliminary and inconclusive because there are no obvious trends from the data. Overall, the discussion appears weak as it is missing references to previous studies and, most importantly, how their findings compare to others'.

Reviewer #3 (Public review):

Summary:

The manuscript by Zhang et al. demonstrates that MORC2 undergoes liquid-liquid phase separation (LLPS) to form nuclear condensates critical for transcriptional repression. Using a combination of in vitro LLPS assays, cellular studies, NMR spectroscopy, and crystallography, the authors show that a dimeric scaffold formed by CC3 drives phase separation, while multivalent interactions between an intrinsically disordered region (IDR) and a newly defined IDR-binding domain (IBD) further promote condensate formation. Notably, LLPS enhances MORC2 ATPase activity in a DNA-dependent manner and contributes to transcriptional regulation, establishing a functional link between phase separation, DNA binding, and transcriptional control. Overall, the manuscript is well-organized and logically structured, offering mechanistic insights into MORC2 function, and most conclusions are supported by the presented data. Nevertheless, some of the claims are not sufficiently supported by the current data and would benefit from additional evidence to strengthen the conclusions.

The following suggestions may help strengthen the manuscript:

Major comments:

(1) The central model proposes that multivalent interactions between the IDR and IBD promote MORC2 LLPS. However, the characterization of these interactions is currently limited. It is recommended that the authors perform more systematic analyses to investigate the contribution of these interactions to LLPS, for example, by in vitro assays assessing how the IDR or IBD individually influence MORC2 phase separation.

(2) The authors mention that DNA binding can promote MORC2 LLPS. It is recommended that they generate a phase diagram to systematically assess how DNA influences phase separation.

(3) The authors use the N39A mutant as a negative control to study the effect of DNA binding on ATP hydrolysis. Given that N39A is defective in DNA binding, it could also be employed to directly test whether DNA binding influences MORC2 phase separation.

(4) Many of the cellular and in vitro LLPS experiments employ EGFP fusions. The authors should evaluate whether the EGFP tag influences MORC2 phase separation behavior.

Author response:

Reviewer #1 (Public review):

For summary:

Thank you for your insightful and rigorous review. We fully agree with your core concern: establishing a causal link between MORC2 phase separation (PS) and its gene regulatory function is not only a key need in the phase separation field but also essential to elevating the overall utility of our work. To resolve the current gap in causal evidence, we will design experiments that explicitly distinguish the role of phase-separated condensates from soluble MORC2 complexes: We will generate a phase-separation-deficient but dimerization-competent MORC2 mutant by mutating key hydrophobic residues in the IDRa region (critical for IDR-IBD multivalent interactions driving phase separation) without disrupting the CC3 domain’s dimerization interface. In addition, we plan to investigate whether introducing a KS sequence[1] at the C-terminus can effectively attenuate the phase separation propensity of MORC2. These mutants will allow us to decouple “phase separation capacity” from “protein dimerization” (a prerequisite for both soluble complex formation and condensates).

For strengths:

We appreciate the reviewer’s recognition of our characterization of MORC2 phase separation and its structural basis. Our understanding of the CW domain’s function remains preliminary. Although we observed that the CW domain can influence condensate size, the IDR, IBD, and CC3 domains constitute the core structural elements driving phase separation. Consequently, the CW domain was not a primary focus of the current study. Nonetheless, investigating its functional contributions represents an interesting avenue for future work.

For weaknesses:

(1) We appreciate the reviewer’s rigorous concern. Our RNA-seq data were generated from fully independent transfections performed in triplicate across different time points and cell culture batches, aiming to maximize sample independence. However, for sensitive sequencing experiments, we observed that variability in transfection efficiency and cell culture across batches can introduce experimental differences, resulting in variable regulation of differentially expressed genes across samples. During differential gene analysis, p-value filtering excluded an additional 40 overlapping genes. In total, 61 genes overlapped with those reported in reference 22[2] (ZNF91, ZNF721, ZNF66, ZNF493, ZNF462, ZNF221, ZNF121, VGLL3, TUFT1, TLE4, TGFB2, SYS1-DBNDD2, STXBP6, SPRY2, SAMD9, ROR1, PTGES, PLK2, PLCXD2, PEA15, PDE2A, OLR1, NYAP2, NTN4, NRXN3, NEXN, MYLK, MPP7, MDGA1, MAMDC2, LBH, KRT80, ITGB8, IGFBP3, IGF2BP2, ICAM1, HIVEP3, GRB14, GPRC5A, GLCE, GJB3, GADD45B, GADD45A, FOXE1, FOSL1, FGF2, ETV5, ERBB3, DNAJC22, DIRAS1, DBNDD2, CXCL16, CRB2, COL9A3, CLDN1, BDNF, ATP8A1, AMOTL2, AHNAK2, ADAMTS16, ACSF2). To further enhance reproducibility, we will perform additional sequencing experiments.

(2).Disease-associated mutants of MORC2

At the current stage, the results for disease-associated mutations are descriptive. While we observed that certain mutations clustered at the N-terminus can affect MORC2 condensate formation, ATPase activity, and DNA binding, we did not identify a mechanistic explanation for these correlations. Notably, the T424R mutation, previously reported to significantly enhance ATPase activity, also increased both intracellular condensate formation and in vitro DNA binding in our experiments. In contrast, other mutations did not show such consistent effects. Previous studies have established that MORC2’s ATP-binding and DNA-binding activities are independent[2]. Our results further suggest that MORC2’s phase separation behavior is also independent of both ATP and DNA binding, although existing evidence hints at potential cross-regulatory interactions among these three functions.

We are fully committed to implementing these revisions with strict rigor and plan to complete them within 8–10 weeks. We will submit a comprehensive response letter alongside the revised manuscript, explicitly mapping how each of your concerns has been addressed, and ensuring that our conclusions about MORC2 PS’s functional role are supported by solid, reproducible data. We believe these revisions will transform our study from a strong “mechanism-focused” work to a comprehensive one that bridges PS mechanisms and biological function—aligning with the high standards of the phase separation field. Thank you again for your invaluable guidance in improving our work.

Reviewer #2 (Public review):

For summary:

Thank you for your thorough and constructive review of our manuscript. We fully agree with the key concerns you raised and have developed a detailed revision plan to address each point comprehensively. We will perform additional control and validation experiments to directly link MORC2’s condensate-forming capacity with its gene silencing function. At the current stage, the results for disease-associated mutations are descriptive. While we observed that certain mutations clustered at the N-terminus can affect MORC2 condensate formation, ATPase activity, and DNA binding, we did not identify a mechanistic explanation for these correlations. Notably, the T424R mutation, previously reported to significantly enhance ATPase activity[3], also increased both intracellular condensate formation and in vitro DNA binding in our experiments. In contrast, other mutations did not show such consistent effects. Previous studies have established that MORC2’s ATP-binding and DNA-binding activities are independent[4]. Our results further suggest that MORC2’s phase separation behavior is also independent of both ATP and DNA binding, although existing evidence hints at potential cross-regulatory interactions among these three functions.

For strengths:

We thank the reviewer for their appreciation of the key findings presented in this manuscript.

For weaknesses:

We thank the reviewer for their careful assessment of MORC2’s DNA-binding properties and its relationship with ATPase and transcriptional activities. We would like to offer the following clarifications to address these concerns, which will also be incorporated into the Discussion section of the revised manuscript.

(1) Recent work by Tan et al.[4] similarly identified multiple DNA-binding sites in MORC2, consistent with our findings, though there are discrepancies in the precise binding regions. In particular, they reported that isolated CC1 and CC2 domains do not bind 60 bp dsDNA, which contrasts with our observations. We attribute this difference to the types of DNA used in the assays. In our study, we employed 601 DNA, a defined nucleosome-positioning sequence, which differs substantially from randomly designed short dsDNA. For instance, prior work by Christopher H. Douse et al.[3] also confirmed that MORC2’s CC1 domain can bind 601 DNA.

(2) In the study by Fendler et al.², DNA binding was reported to reduce MORC2’s ATPase activity—an observation that appears inconsistent with the results presented in our Fig. 5j. A critical distinction between the two studies lies in the experimental systems used: Fendler et al. employed a truncated MORC2 construct (residues 1–603) and 35 bp double-stranded DNA (dsDNA), whereas our experiments utilized full-length MORC2 and 601 bp DNA (a sequence with high nucleosome assembly potential). These differences—including the absence of potentially regulatory C-terminal regions in the truncated construct and the varying length/structural properties of the DNA substrates—introduce variables that substantially complicate direct comparative analysis of ATPase activity outcomes.

Separately, Douse et al.³ demonstrated that the efficiency of HUSH complex-dependent epigenetic silencing decreases as MORC2’s ATP hydrolysis rate increases, implying an inverse relationship between ATPase activity and silencing function. Notably, our current work has not established a direct mechanistic link between MORC2 phase separation and its ATPase activity. Thus, we refrain from inferring that the effect of MORC2 phase separation on transcriptional repression is mediated through modulation of its ATPase function—this remains an important question to address in future studies.

(3) Finally, we plan to perform additional experiments to rule out the potential effects of CC3 dimerization. We will generate a phase-separation-deficient but dimerization-competent MORC2 mutant by mutating key hydrophobic residues in the IDRa region (critical for IDR-IBD multivalent interactions driving phase separation) without disrupting the CC3 domain’s dimerization interface. In addition, we plan to investigate whether introducing a KS sequence[1] at the C-terminus can effectively attenuate the phase separation propensity of MORC2. These mutants will allow us to decouple “phase separation capacity” from “protein dimerization”.

We are committed to implementing these revisions with strict rigor and plan to complete them within 8–10 weeks. We will submit a detailed response letter alongside the revised manuscript, explicitly mapping how each of your concerns has been addressed, and ensuring the Discussion section is robust, context-rich, and fully integrates our work with the existing literature. We believe these improvements will significantly enhance the reliability, contextual relevance, and impact of our study, and we sincerely thank you for guiding us to elevate its quality.

Reviewer #3 (Public review):

For summary:

Thank you for your insightful review and constructive suggestions, which have been invaluable in refining our manuscript. We greatly appreciate your recognition of the study’s strengths, including its logical structure, integration of multi-disciplinary approaches (in vitro LLPS assays, cellular studies, NMR, and crystallography), and the establishment of a functional link between MORC2 phase separation, DNA binding, and transcriptional control. Your identification of areas needing stronger evidence has provided clear, actionable directions for improvement, and we are fully committed to addressing each point comprehensively.

For Major comments:

To strengthen the manuscript as per your recommendations:

(1) For the characterization of IDR-IBD interactions in PS: We will perform systematic in vitro assays, including PS turbidity measurements and confocal imaging of MORC2 variants lacking IDR or IBD (ΔIDR, ΔIBD) and truncated constructs (IDR alone, IBD alone). These experiments will quantify how each domain individually or synergistically contributes to phase separation propensity (e.g., critical concentration, condensate size/distribution).

(2) To assess DNA’s influence on PS: We will generate phase diagrams by testing a range of MORC2 concentrations (0.5–10 μM) or with 601 DNA (147bp) and concentrations (0–2 μM), using turbidity assays and microscopy to map phase boundaries. This will systematically clarify how DNA modulates MORC2 phase separation.

We plan to complete these experiments within 3–4 weeks, with rigorous quantification and statistical analysis to support our conclusions. The revised manuscript will include a detailed response letter mapping each of your suggestions to specific data additions, ensuring enhanced robustness and conviction. We believe these revisions will significantly strengthen the study’s conclusions, and we sincerely thank you for guiding us to improve its quality.

Reference:

[1] Mensah, M. A., Niskanen, H., Magalhaes, A. P., Basu, S., Kircher, M., Sczakiel, H. L., Reiter, A. M. V., Elsner, J., Meinecke, P., Biskup, S., et al. (2023). Aberrant phase separation and nucleolar dysfunction in rare genetic diseases. Nature 614, 564-571. https://doi.org/10.1038/s41586-022-05682-1.

[2] Fendler, N. L., Ly, J., Welp, L., Lu, D., Schulte, F., Urlaub, H., and Vos, S. M. (2024). Identification and characterization of a human MORC2 DNA binding region that is required for gene silencing. Nucleic Acids Res 53, gkae1273. https://doi.org/10.1093/nar/gkae1273.

[3] Douse, C. H., Bloor, S., Liu, Y. C., Shamin, M., Tchasovnikarova, I. A., Timms, R. T., Lehner, P. J., and Modis, Y. (2018). Neuropathic MORC2 mutations perturb GHKL ATPase dimerization dynamics and epigenetic silencing by multiple structural mechanisms. Nat Commun 9, 651. https://doi.org/10.1038/s41467-018-03045-x.

[4] Tan, W., Park, J., Venugopal, H., Lou, J. Q., Dias, P. S., Baldoni, P. L., Moon, K. W., Dite, T. A., Keenan, C. R., Gurzau, A. D., et al. (2025). MORC2 is a phosphorylation-dependent DNA compaction machine. Nat Commun 16, 5606. https://doi.org/10.1038/s41467-025-60751-z.