A Conserved Mycobacterial Nucleomodulin Hijacks the Host COMPASS Complex to Reprogram Pro-Inflammatory Transcription and Promote Intracellular Survival

Reviewer #2 (Public review):

Summary:

The manuscript by Chen et al addresses an important aspect of pathogenesis for mycobacterial pathogens, seeking to understand how bacterial effector proteins disrupt the host immune response. To address this question, the authors sought to identify bacterial effectors from M. tuberculosis (Mtb) that localize to the host nucleus and disrupt host gene expression as a means of impairing host immune function.

Strengths:

The researchers conducted a rigorous bioinformatic analysis to identify secreted effectors containing mammalian nuclear localization signal (NLS) sequences, which formed the basis of quantitative microscopy analysis to identify bacterial proteins that had nuclear targeting within human cells. The study used two complementary methods to detect protein-protein interaction: yeast two-hybrid assays and reciprocal immunoprecipitation (IP). The combined use of these techniques provides strong evidence of interactions between MgdE and SET1 components and suggests that the interactions are, in fact, direct. The authors also carried out a rigorous analysis of changes in gene expression in macrophages infected with the mgdE mutant BCG. They found strong and consistent effects on key cytokines such as IL6 and CSF1/2, suggesting that nuclear-localized MgdE does, in fact, alter gene expression during infection of macrophages.

Weaknesses:

There are some drawbacks in this study that limit the application of the findings to M. tuberculosis (Mtb) pathogenesis. The first concern is that much of the study relies on ectopic overexpression of proteins either in transfected non-immune cells (HEK293T) or in yeast, using 2-hybrid approaches. Some of their data in 293T cells is hard to interpret, and it is unclear if the protein-protein interactions they identify occur during natural infection with mycobacteria. The second major concern is that pathogenesis is studied using the BCG vaccine strain rather than virulent Mtb. However, overall, the key findings of the paper - that MgdE interacts with SET1 and alters gene expression are well-supported.

Reviewer #3 (Public review):

In this study, Chen L et al. systematically analyzed the mycobacterial nucleomodulins and identified MgdE as a key nucleomodulin in pathogenesis. They found that MgdE enters into host cell nucleus through two nuclear localization signals, KRIR108-111 and RLRRPR300-305, and then interacts with COMPASS complex subunits ASH2L and WDR5 to suppress H3K4 methylation-mediated transcription of pro-inflammatory cytokines, thereby promoting mycobacterial survival. This study is potentially interesting, but there are several critical issues that need to be addressed to support the conclusions of the manuscript.

(1) Figure 2: The study identified MgdE as a nucleomodulin in mycobacteria and demonstrated its nuclear translocation via dual NLS motifs. The authors examined MgdE nuclear translocation through ectopic expression in HEK293T cells, which may not reflect physiological conditions. Nuclear-cytoplasmic fractionation experiments under mycobacterial infection should be performed to determine MgdE localization.

(2) Figure 2F: The authors detected MgdE-EGFP using an anti-GFP antibody, but EGFP as a control was not detected in its lane. The authors should address this technical issue.

(3) Figure 3C-3H: The data showing that the expression of all detected genes in 24 h is comparable to that in 4 h (but not 0 h) during WT BCG infection is beyond comprehension. The issue is also present in Figure 7C, Figure 7D, and Figure S7. Moreover, since Il6, Il1β (pro-inflammatory), and Il10 (anti-inflammatory) were all upregulated upon MgdE deletion, how do the authors explain the phenomenon that MgdE deletion simultaneously enhanced these gene expressions?

(4) Figure 5: The authors confirmed the interactions between MgdE and WDR5/ASH2L. How does the interaction between MgdE and WDR5 inhibit COMPASS-dependent methyltransferase activity? Additionally, the precise MgdE-ASH2L binding interface and its functional impact on COMPASS assembly or activity require clarification.

(5) Figure 6: The authors proposed that the MgdE-regulated COMPASS complex-H3K4me3 axis suppresses pro-inflammatory responses, but the presented data do not sufficiently support this claim. H3K4me3 inhibitor should be employed to verify cytokine production during infection.

(6) There appears to be a discrepancy between the results shown in Figure S7 and its accompanying legend. The data related to inflammatory responses seem to be missing, and the data on bacterial colonization are confusing (bacterial DNA expression or CFU assay?).

(7) Line 112-116: Please provide the original experimental data demonstrating nuclear localization of the 56 proteins harboring putative NLS motifs.

Reviewer #1 (Public review):

Summary:

This fundamental study identifies a new mechanism that involves a mycobacterial nucleomodulin manipulation of the host histone methyltransferase COMPASS complex to promote infection. Although other intracellular pathogens are known to manipulate histone methylation, this is the first report demonstrating the specific targeting of the COMPASS complex by a pathogen. The rigorous experimental design using state-of-the art bioinformatic analysis, protein modeling, molecular and cellular interaction, and functional approaches, culminating with in vivo infection modeling, provides convincing, unequivocal evidence that supports the authors' claims. This work will be of particular interest to cellular microbiologists working on microbial virulence mechanisms and effectors, specifically nucleomodulins, and cell/cancer biologists that examine COMPASS dysfunction in cancer biology.

Strengths:

(1) The strengths of this study include the rigorous and comprehensive experimental design that involved numerous state-of-the-art approaches to identify potential nucleomodulins, define molecular nucleomodulin-host interactions, cellular nucleomodulin localization, intracellular survival, and inflammatory gene transcriptional responses, and confirmation of the inflammatory and infection phenotype in a small animal model.

(2) The use of bioinformatic, cellular, and in vivo modeling that are consistent and support the overall conclusions is a strength of the study. In addition, the rigorous experimental design and data analysis, including the supplemental data provided, further strengthen the evidence supporting the conclusions.

Weaknesses:

(1) This work could be stronger if the MgdE-COMPASS subunit interactions that negatively impact COMPASS complex function were better defined. Since the COMPASS complex consists of many enzymes, examining the functional impact on each of the components would be interesting.

(2) Examining the impact of WDR5 inhibitors on histone methylation, gene transcription, and mycobacterial infection could provide additional rigor and provide useful information related to the mechanisms and specific role of WDR5 inhibition on mycobacterial infection.

(3) The interaction between MgdE and COMPASS complex subunit ASH2L is relatively undefined, and studies to understand the relationship between WDR5 and ASH2L in COMPASS complex function during infection could provide interesting molecular details that are undefined in this study.

(4) The AlphaFold prediction results for all the nuclear proteins examined could be useful. Since the interaction predictions with COMPASS subunits range from 0.77 for WDR5 and 0.47 for ASH2L, it is not clear how the focus on COMPASS complex over other nuclear proteins was determined.

Author response:

The following is the authors’ response to the original reviews.

Reviewer #1 (Public review):

Summary:

This fundamental study identifies a new mechanism that involves a mycobacterial nucleomodulin manipulation of the host histone methyltransferase COMPASS complex to promote infection. Although other intracellular pathogens are known to manipulate histone methylation, this is the first report demonstrating the specific targeting of the COMPASS complex by a pathogen. The rigorous experimental design using state-of-the art bioinformatic analysis, protein modeling, molecular and cellular interaction, and functional approaches, culminating with in vivo infection modeling, provides convincing, unequivocal evidence that supports the authors' claims. This work will be of particular interest to cellular microbiologists working on microbial virulence mechanisms and effectors, specifically nucleomodulins, and cell/cancer biologists that examine COMPASS dysfunction in cancer biology.

Strengths:

(1) The strengths of this study include the rigorous and comprehensive experimental design that involved numerous state-of-the-art approaches to identify potential nucleomodulins, define molecular nucleomodulin-host interactions, cellular nucleomodulin localization, intracellular survival, and inflammatory gene transcriptional responses, and confirmation of the inflammatory and infection phenotype in a small animal model.

(2) The use of bioinformatic, cellular, and in vivo modeling that are consistent and support the overall conclusions is a strength of the study. In addition, the rigorous experimental design and data analysis, including the supplemental data provided, further strengthen the evidence supporting the conclusions.

Weaknesses:

(1) This work could be stronger if the MgdE-COMPASS subunit interactions that negatively impact COMPASS complex function were better defined. Since the COMPASS complex consists of many enzymes, examining the functional impact on each of the components would be interesting.

We thank the reviewer for this insightful comment. A biochemistry assays could be helpful to interpret the functional impact on each of the components by MgdE interaction. However, the purification of the COMPASS complex could be a hard task itself due to the complexity of the full COMPASS complex along with its dynamic structural properties and limited solubility.

(2) Examining the impact of WDR5 inhibitors on histone methylation, gene transcription, and mycobacterial infection could provide additional rigor and provide useful information related to the mechanisms and specific role of WDR5 inhibition on mycobacterial infection.

We thank the reviewer for the comment. A previous study showed that WIN-site inhibitors, such as compound C6, can displace WDR5 from chromatin, leading to a reduction in global H3K4me3 levels and suppression of immune-related gene expression (Hung et al., Nucleic Acids Res, 2018; Bryan et al., Nucleic Acids Res, 2020). These results closely mirror the functional effects we observed for MgdE, suggesting that MgdE may act as a functional mimic of WDR5 inhibition. This supports our proposed model in which MgdE disrupts COMPASS activity by targeting WDR5, thereby dampening host pro-inflammatory responses.

(3) The interaction between MgdE and COMPASS complex subunit ASH2L is relatively undefined, and studies to understand the relationship between WDR5 and ASH2L in COMPASS complex function during infection could provide interesting molecular details that are undefined in this study.

We thank the reviewer for the comment. In this study, we constructed single and multiple point mutants of MgdE at residues S⁸⁰, D²⁴⁴, and H²⁴⁷ to identify key amino acids involved in its interaction with ASH2L (Figure 5A and B; Figure S5). However these mutations did not interrupt the interaction with MgdE, suggesting that more residues are involved in the interaction.

ASH2L and WDR5 function cooperatively within the WRAD module to stabilize the SET domain and promote H3K4 methyltransferase activity with physiological conditions (Couture and Skiniotis, Epigenetics, 2013; Qu et al., Cell, 2018; Rahman et al., Proc Natl Acad Sci U S A, 2022). ASH2L interacts with RbBP5 via its SPRY domain, whereas WDR5 bridges MLL1 and RbBP5 through the WIN and WBM motifs (Chen at al., Cell Res, 2012; Park et al., Nat Commun, 2019). The interaction status between ASH2L and WDR5 during mycobacterial infection could not be determined in our current study.

(4) The AlphaFold prediction results for all the nuclear proteins examined could be useful. Since the interaction predictions with COMPASS subunits range from 0.77 for WDR5 and 0.47 for ASH2L, it is not clear how the focus on COMPASS complex over other nuclear proteins was determined.

We thank the reviewer for the comment. We employed AlphaFold to predict the interactions between MgdE and the major nuclear proteins. This screen identified several subunits of the SET1/COMPASS complex as high-confidence candidates for interaction with MgdE (Supplementary Figure 4A). This result is consistent with a proteomic study by Penn et al. which reported potential interactions between MgdE and components of the human SET1/COMPASS complex based on affinity purification-mass spectrometry analysis (Penn et al., Mol Cell, 2018).

Reviewer #2 (Public review):

Summary:

The manuscript by Chen et al addresses an important aspect of pathogenesis for mycobacterial pathogens, seeking to understand how bacterial effector proteins disrupt the host immune response. To address this question, the authors sought to identify bacterial effectors from M. tuberculosis (Mtb) that localize to the host nucleus and disrupt host gene expression as a means of impairing host immune function.

Strengths:

The researchers conducted a rigorous bioinformatic analysis to identify secreted effectors containing mammalian nuclear localization signal (NLS) sequences, which formed the basis of quantitative microscopy analysis to identify bacterial proteins that had nuclear targeting within human cells. The study used two complementary methods to detect protein-protein interaction: yeast two-hybrid assays and reciprocal immunoprecipitation (IP). The combined use of these techniques provides strong evidence of interactions between MgdE and SET1 components and suggests that the interactions are, in fact, direct. The authors also carried out a rigorous analysis of changes in gene expression in macrophages infected with the mgdE mutant BCG. They found strong and consistent effects on key cytokines such as IL6 and CSF1/2, suggesting that nuclear-localized MgdE does, in fact, alter gene expression during infection of macrophages.

Weaknesses:

There are some drawbacks in this study that limit the application of the findings to M. tuberculosis (Mtb) pathogenesis. The first concern is that much of the study relies on ectopic overexpression of proteins either in transfected non-immune cells (HEK293T) or in yeast, using 2-hybrid approaches. Some of their data in 293T cells is hard to interpret, and it is unclear if the protein-protein interactions they identify occur during natural infection with mycobacteria. The second major concern is that pathogenesis is studied using the BCG vaccine strain rather than virulent Mtb. However, overall, the key findings of the paper - that MgdE interacts with SET1 and alters gene expression are well-supported.

We thank the reviewer for the comment. We agree that the ectopic overexpression could not completely reflect a natural status, although these approaches were adopted in many similar experiments (Drerup et al., Molecular plant, 2013; Chen et al., Cell host & microbe, 2018; Ge et al., Autophagy, 2021). Further, the MgdE localization experiment using Mtb infected macrophages will be performed to increase the evidence in the natural infection.

We agree with the reviewer that BCG strain could not fully recapitulate the pathogenicity or immunological complexity of M. tuberculosis infection. We employed BCG as a biosafe surrogate model since it was acceptable in many related studies (Wang et al., Nat Immunol, 2025; Wang et al., Nat Commun, 2017; Péan et al., Nat Commun, 2017; Li et al., J Biol Chem, 2020).

Reviewer #3 (Public review):

In this study, Chen L et al. systematically analyzed the mycobacterial nucleomodulins and identified MgdE as a key nucleomodulin in pathogenesis. They found that MgdE enters into host cell nucleus through two nuclear localization signals, KRIR^108-111 and RLRRPR^300-305, and then interacts with COMPASS complex subunits ASH2L and WDR5 to suppress H3K4 methylation-mediated transcription of pro-inflammatory cytokines, thereby promoting mycobacterial survival. This study is potentially interesting, but there are several critical issues that need to be addressed to support the conclusions of the manuscript.

(1) Figure 2: The study identified MgdE as a nucleomodulin in mycobacteria and demonstrated its nuclear translocation via dual NLS motifs. The authors examined MgdE nuclear translocation through ectopic expression in HEK293T cells, which may not reflect physiological conditions. Nuclear-cytoplasmic fractionation experiments under mycobacterial infection should be performed to determine MgdE localization.

We thank the reviewer for the comment. The MgdE localization experiment using Mtb infected macrophages will be performed.

(2) Figure 2F: The authors detected MgdE-EGFP using an anti-GFP antibody, but EGFP as a control was

We thank the reviewer for pointing this out. The new uncropped blots containing the EGFP band will be provided in Supplementary Information.

(3) Figure 3C-3H: The data showing that the expression of all detected genes in 24 h is comparable to that in 4 h (but not 0 h) during WT BCG infection is beyond comprehension. The issue is also present in Figure 7C, Figure 7D, and Figure S7. Moreover, since Il6, Il1β (proinflammatory), and Il10 (anti-inflammatory) were all upregulated upon MgdE deletion, how do the authors explain the phenomenon that MgdE deletion simultaneously enhanced these gene expressions?

We thank the reviewer for the comment. A relative quantification method was used in our qPCR experiments to normalize the WT expression levels in Figure 3C–3H, Figure 7C, 7D, and Figure S7.

The concurrent induction of both types of cytokines likely represents a dynamic host strategy to fine-tune immune responses during infection. This interpretation is supported by previous studies (Podleśny-Drabiniok et al., Cell Rep, 2025; Cicchese et al., Immunological Reviews, 2018).

(4) Figure 5: The authors confirmed the interactions between MgdE and WDR5/ASH2L. How does the interaction between MgdE and WDR5 inhibit COMPASS-dependent methyltransferase activity? Additionally, the precise MgdE-ASH2L binding interface and its functional impact on COMPASS assembly or activity require clarification.

We thank the reviewer for this insightful comment. We cautiously speculate that the MgdE interaction inhibits COMPASS-dependent methyltransferase activity by interfering with the integrity and stability of the COMPASS complex. Accordingly, we have incorporated the following discussion into the revised manuscript (Lines 298-310):

“The COMPASS complex facilitates H3K4 methylation through a conserved assembly mechanism involving multiple core subunits. WDR5, a central scaffolding component, interacts with RbBP5 and ASH2L to promote complex assembly and enzymatic activity (Qu et al., 2018; Wysocka et al., 2005). It also recognizes the WIN motif of methyltransferases such as MLL1, thereby anchoring them to the complex and stabilizing the ASH2L-RbBP5 dimer (Hsu et al., Cell, 2018). ASH2L further contributes to COMPASS activation by interacting with both RbBP5 and DPY30 and by stabilizing the SET domain, which is essential for efficient substrate recognition and catalysis (Qu et al., Cell, 2018; Park et al., Nat Commun, 2019). Our work shows that MgdE binds both WDR5 and ASH2L and inhibits the methyltransferase activity of the COMPASS complex. Site-directed mutagenesis revealed that residues D²²⁴ and H²⁴⁷ of MgdE are critical for WDR5 binding, as the double mutant MgdE-D²²⁴A/H²⁴⁷ A fails to interact with WDR5 and shows diminished suppression of H3K4me3 levels (Figure 5D).”

Regarding the precise MgdE-ASH2L binding interface, we attempted to identify the key interaction site by introducing point mutations into ASH2L. However, these mutations did not disrupt the interaction (Figure 5A and B; Figure S5), suggesting that more residues are involved in the interaction.

(5) Figure 6: The authors proposed that the MgdE-regulated COMPASS complex-H3K4me3 axis suppresses pro-inflammatory responses, but the presented data do not sufficiently support this claim. H3K4me3 inhibitor should be employed to verify cytokine production during infection.

We thank the reviewer for the comment. We have now revised the description in lines 824825 “MgdE may suppresses COMPASS complex-mediated inflammatory responses by inhibiting H3K4 methylation” and in lines 219-220 "MgdE suppresses host inflammatory responses probably by inhibition of COMPASS complex-mediated H3K4 methylation."

(6) There appears to be a discrepancy between the results shown in Figure S7 and its accompanying legend. The data related to inflammatory responses seem to be missing, and the data on bacterial colonization are confusing (bacterial DNA expression or CFU assay?).

We thank the reviewer for the comment. Figure S7 specifically addresses the effect of MgdE on bacterial colonization in the spleens of infected mice, which was assessed by quantitative PCR rather than by CFU assay.

We have now revised the legend of Figure S7 as below (Lines 934-938):

“MgdE facilitates bacterial colonization in the spleens of infected mice. Bacterial colonization was assessed in splenic homogenates from infected mice (as described in Figure 7A) by quantifying bacterial DNA using quantitative PCR at 2, 14, 21, 28, and 56 days post-infection.”

(7) Line 112-116: Please provide the original experimental data demonstrating nuclear localization of the 56 proteins harboring putative NLS motifs.

We thank the reviewer for the comment. We will provide this data in the new Supplementary Table 2.