Dual genomic localizations and gene regulatory functions of MBD-2 with and without NuRD in Caenorhabditis elegans which lacks DNA methylation

Domain: biology
Published: 2026-01-20
Editorial outcome: major_revision

Abstract
5-methylcytosine (5mC) DNA methylation is an ancient epigenetic mechanism. Methyl-CpG-binding domain protein 2 or 3 (MBD2/3) is a component of the Nucleosome Remodeling and Deacetylase (NuRD) complex, essential for gene regulation in mammals. The MBD2/3 ortholog is surprisingly preserved even in 5mC-free invertebrates, such as nematodes and flies. We found that Caenorhabditis elegans MBD-2 (Cel-MBD-2), despite without the MBD domain, is a conserved component of the NuRD complex. Cel-MBD-2 null mutation or deletion of its NuRD-interacting coiled-coil domain causes morphological defects and infertility. While a minority of Cel-MBD-2 colocalizes with NuRD at active chromatin, the majority of Cel-MBD-2 localizes to repressive chromatin environments enriched with H3K27me3 and H3K9me2/3, not overlapping with NuRD. The mechanism by which Cel-MBD-2 localizes to the repressive chromatin is still unknown, but is independent of NuRD or MBD-5mC interaction. The potential compensation of DNA methylation by histone modifications highlights the adaptability of epigenetic regulation during evolution.

Introduction
5-methylcytosine (5mC) DNA methylation is indispensable in vertebrate developmental processes, including imprinting, X-inactivation, and silencing of transposable elements. 5mC is recognized by methyl-CpG binding domain (MBD)-containing proteins, including MBD2, MBD3 and MeCP2. Vertebrate paralogs MBD2 and MBD3 are believed to have derived from duplication of the single ancestral invertebrate MBD2/31. Mammalian MBD2 isoforms all contain a functional MBD domain2. MBD3 isoforms possess either a mutation on a critical residue in MBD or a truncated MBD domain (Fig. 1A, B). Consequently, mammalian MBD3 can only weakly bind to 5mC3. Therefore, MBD3 primarily associates with hypomethylated CG-rich regions in the genome, whereas mammalian MBD2 predominantly binds to 5mC-dense CG-rich regions3,4.

In mammals, MBD2 and MBD3 mutants show different phenotypes. Mammalian MBD2 knockout leads to behavioral phenotypes, including defects in maternal nurturing behavior and downregulation of autism-related genes in mice5,6. In contrast, knockout of MBD3 leads to preimplantation lethality in mice owing to its essential role in stem cell differentiation7. Interestingly, the longest isoform MBD2a (containing N-terminal MBD and GR-rich domain, both of which confer 5mC affinity) with the highest affinity for 5mC could only partially rescue the differentiation block in mbd3 knockout2. On the other hand, overexpression of MBD2 isoform b or mutants with a lower 5mC affinity (mutation at MBD or deletion of GR-rich domain) could fully rescue the differentiation defect of mbd3 knockout2. This suggests that MBD2 has retained the conserved MBD domain that confers 5mC affinity, allowing it to diversify its targets compared to MBD3. Without these domains, MBD2 is functionally redundant with MBD3. In addition, depletion of Nucleosome Remodeling and Deacetylase (NuRD) components leads to a similar differentiation block as seen in MBD3 mutant8. Therefore, these essential functions in stem cell differentiation and survival likely arise from the genes regulated by the MBD2/3-NuRD interaction. On the other hand, MBD2-specific gene targets mediated by 5mC-binding may confer cognitive ability, which is non-essential but may provide survival advantages5,6. In invertebrates with 5mC, for example in sponges, knockdown of the single MBD2/3 induces a highly penetrant developmental defect9.

On the other hand, the function of MBD2/3 in invertebrates that have lost 5mC methylation is less studied. For example, in the nematode P. pacificus, knockout of Ppa-mbd-2 leads to a fully penetrant egg-laying defect (Egl) related to a neurotransmitter defect and downregulation of eud-1, the master regulator of mouth form plasticity, but the mbd-2 mutants are viable10,11. Knockdown of C. briggsae Cbr-mbd-2 leads to strong uncoordinated movement (Unc) in > 50% of the F1 progenies and larval arrest in 35%12. In C. elegans, Cel-mbd-2 transcript has been detected at all larval stages and in adults12. Knockdown of Cel-mbd-2 led to 18% animals with mild Unc phenotype without paralysis, 11% with exploding vulva, and <1% with multivulva (Muv)12. The mild, low penetrance of the RNA interference (RNAi) knockdown phenotypes was surprising, but no mutant analysis has been performed before current study.

Regardless of their different affinities to 5mC, mammalian MBD2 and MBD3 are mutually exclusive scaffolding components of the highly conserved Nucleosome Remodeling and Deacetylase (NuRD) complex. The NuRD complex is highly conserved across metazoans. The mammalian NuRD complex can be functionally split into two subcomplexes: (1) a histone deacetylase core consisting of MTA1/2/3, HDAC1/2, and RBBP4/7, and (2) a nucleosome remodeling core consisting of CHD3/4/5 (Mi-2) and GATAD2A/B (Table 1). MBD2/3 acts as a scaffolding component connecting the two subcomplexes. This holds true for the most ancestral metazoan sponges (which possess 5mC in their genomic DNA)9, Drosophila (in which the presence of genomic 5mC is disputed)13, planarians (which lack 5mC14), Xenopus15, mice16, and humans17.

In mice, MBD3 is confined to the nucleus and has been proposed to facilitate localization of the NuRD complex to chromatin. In mouse embryonic stem cells (mESCs), sequestering MBD3 in the cytoplasm produced transcriptomic changes similar to mbd3 knockout mutants, resulting in a loss of the NuRD component CHD4 binding to certain loci18. Mammalian MBD3’s binding sites are largely encompassed by the NuRD complex component binding sites, featuring transcriptional mark P300 and RNA polymerase II, and histone modifications of open chromatin, such as H3K27ac, H3K4me1, and H3K4me33,7,18,19. The general affinity of the NuRD complex to open chromatin is likely conserved from mammals18 to C. elegans20 based on ChIP-seq data of some NuRD components.

A conventional model posits that mammalian MBD2 recruits the NuRD complex to methylated promoters, silencing transcription through histone deacetylation. Supporting this view, histone deacetylase inhibitor trichostatin A leads to de-repression of certain MBD2-NuRD target genes21,22. However, recent evidence shows that only a small subset of vertebrate MBD2 binds to hypomethylated regions marked by active histone modifications4,23, overlapping with those of MBD3 and NuRD complex4,7,18. Consistently, MBD2t, a MBD2 isoform lacking the NuRD-interacting coiled-coil domain, does not bind to these MBD2-NuRD-overlapping sites4.

Surprisingly, MBD2 can also function independently of NuRD. In both normal and cancer cells, human MBD2 mainly binds to silent promoters enriched with methylated CpG3,24. Recent studies suggest that MBD2 binds to a large subset of methylated promoter regions without recruiting NuRD4. These 5mC-dense promoters bound by MBD2 are CG-rich, silent, and without correlation with any histone marks3,4,23,24. The above information leads to a new model of dual binding patterns of mammalian MBD2a: (1) a major subset of NuRD-independent binding to silent 5mC-dense regions, and (2) a minor subset of NuRD-overlapping binding to open chromatin, hypomethylated regions, similar to MBD3.

This ancient 5mC DNA methylation and its associated DNA methyltransferases (DNMT1/3) are lost in diverse invertebrate lineages, including C. elegans, Drosophila25, and planarians14. C. elegans is a well-validated 5mC-free model organism, as confirmed by various approaches, including high-performance liquid chromatography26,27, anti-5mC dot blotting of genomic DNA and immunofluorescence staining of germline28. The loss of genomic 5mC and the respective DNA methyltransferases (DNMT1 and DNMT3) in C. elegans has been proposed as a mechanism to evade DNMT-induced alkylation damage29. While basal members of marine nematodes29 and most insects30 retain functional 5mC methylation, the loss of 5mC has occurred independently in derived nematodes and insects, representing a striking case of convergent evolution. The convergent evolution of 5mC loss presents a critical question: how is the gene silencing function by DNA methylation upheld by compensatory epigenetic mechanisms? Alternative epigenetic tools such as repressive histone modifications (e.g., H3K27me3, H3K9me2) and noncoding RNAs may facilitate transcriptional silencing. However, the detailed mechanisms remain unresolved.

Intriguingly, despite the loss of 5mC, Methyl-CpG Binding Domain protein 2 (MBD2), a canonical 5mC reader historically linked to DNA methylation, is present in all these lineages that lack 5mC. For example, C. elegans MBD-2 (Cel-MBD-2) lacks the methyl-CpG binding domain. Despite limited information from previous RNA interference (RNAi) knockdown phenotypes, the function of Cel-MBD-2 in development remains largely unknown12, and whether it is part of the NuRD complex in C. elegans is unclear. Here, we have constructed Cel-mbd-2 null mutants and characterized their phenotypes and transcriptomes. By tagging Cel-MBD-2 with GFP, we observed its expression and localization during development and performed immunoprecipitation followed by mass spectrometry (IP-MS), which confirmed that Cel-MBD-2 indeed interacts with NuRD components. We also performed ChIP-seq on Cel-MBD-2, and compared the results with existing data on NuRD components and various histone modifications to determine its binding patterns. The results reveal dual binding patterns of Cel-MBD-2, resembling those of mammalian MBD2, in which MBD2 either binds its target genes together with or independently of the NuRD complex to modulate gene regulation.

Results
Cel-MBD-2 lacks the methyl-binding domain (MBD) and is expressed ubiquitously in the nucleus
Cel-MBD-2 does not possess the methyl-binding domain (MBD), but retains the coiled-coil (CC) domain and intrinsically disordered region (IDR) implicated in NuRD complex interactions (Fig. 1A, B). To determine the cellular localization and dynamics of Cel-MBD-2, we tagged it with GFP::3xFLAG at its N-terminus to avoid interference with the C-terminal coiled-coil domain essential for interacting with NuRD components. MBD-2 is highly expressed in the nuclei of embryos, and its localization is cell cycle-dependent (Fig. 1C). Specifically, during interphase, MBD-2 is localized in the nucleoplasm. In prometaphase to anaphase, MBD-2’s nuclear signal gradually decreases and is excluded from chromosomes. During cytokinesis, MBD-2’s signal gradually accumulates back into the nucleoplasm. MBD-2 is also enriched in the nucleoplasm ubiquitously in larvae and adult worms, including the germline and intestine (Fig. 1D, Supplementary Fig. 1). Such nuclear localization resembles some of the other NuRD components, including CHD-3, LET-418, and HDA-1 (Supplementary Fig. 2A–D). Expression of Cel-MBD-2 in the nuclei in all life stages is consistent with its potential role in gene regulation throughout development.

Cel-MBD-2 is essential for fertility and vulval development
To investigate Cel-MBD-2’s functions beyond the RNAi knockdown approach12 (Supplementary Fig. 3B, C), we constructed a mbd-2 null mutant. mbd-2 is located in an intron of pigo-1, which is on the opposite strand of mbd-2 (Fig. 2A). To generate the null mutation of MBD-2, we inserted a 43-nucleotide-long knock-in cassette (STOP-IN)31, including five premature stop codons, in the first exon (Fig. 2A). Such an approach ensures premature termination of translation at all three reading frames31. This approach also alleviates the need to delete any DNA sequence in pigo-1’s intron, and thus should cause less effects on the expression of the overlapping gene pigo-1. Since homozygous mbd-2-/- mutant is sterile (Fig. 2B), the strain is maintained as heterozygous with a balancer chromosome, hT2, containing a mbd-2 wildtype copy (see Materials and methods). To verify that the observed phenotypes, including sterility, are due to mbd-2 loss-of-function mutation, but not related to the overlapping gene pigo-1, we expressed gfp::3xflag::mbd-2 single-copy transgene (tg) driven by mbd-2 endogenous promoter at the oxTi365 locus. The tg fully rescues the infertility and morphology defects of mbd-2-/- mutant (Fig. 2B).

Phenotypically, all of the P0 mbd-2-/- worms are small and relatively immobile adults with a fully penetrant protruding vulva (Pvl) phenotype (Fig. 2C). The P0 mbd-2-/- worms are able to produce on average 10 progenies (Fig. 2B). Almost all of the P0 mbd-2-/- worms died on day 3-4 of adult stage, likely from bagging, in which embryos that cannot be laid hatch inside the uterus and cause lethality to the mothers (Fig. 2D). Interestingly, the P0 mbd-2-/- mutants also frequently avoid the bacterial lawn.

The F1 mbd-2-/- worms, which also lack maternal mbd-2 transcripts, are even smaller and uncoordinated. The F1 mbd-2-/- worms have fully penetrant protruding vulva (Pvl) and sterility. Thirty-two percent (32.4%) of the F1 mbd-2-/- living adults have two vulvas (Fig. 2E). Thirty-seven percent (37.3%) of F1 mbd-2-/- mutants arrest in various developmental stages (L3-L4 larval stages), and 12.2% also show a deformed anal region (Dar) phenotype (Fig. 2F).

The severity of the phenotypes, including sterility and morphological defects, increases when both maternal and zygotic mbd-2 expression is depleted in F1 mbd-2-/-, as compared to P0 mbd-2-/-, suggesting that both maternal and zygotic mbd-2 contribute to the development and fertility of the offspring. On the other hand, heterozygous mbd-2+/- worms produced by mating P0 mbd-2-/- hermaphrodites with N2 wildtype males are fertile and morphologically indistinguishable from wildtype (Supplementary Fig. 3A), indicating that zygotic expression of mbd-2 is sufficient for its reproduction function. Thus, mbd-2 is not strictly maternal-effect sterile (mes), as is the case in PRC2 complex mutants32. The cause of sterility in mbd-2-/- is unclear, but it is unlikely to be caused solely by the protruding vulva (Pvl), as RNAi of mbd-2 resulted in a reduced brood size in F1 worms even without a protruding vulva (Supplementary Fig. 3B). Taken together, we have identified the role of Cel-MBD-2 in vulva development and fertility.

Drastic dysregulation of the transcriptome in C. elegans mbd-2 null mutants
To investigate the potential roles of MBD-2 in regulating genes involved in development and reproduction, we analyzed the transcriptomic changes in mbd-2-/- null mutants. We performed RNA-sequencing (RNA-seq) on young adult worms from N2 wildtype, P0, and F1 mbd-2-/- strains, with two biological replicates for each group. Using an adjusted p-value < 0.05 and Fold Change (FC) ≥ 2, we identified 5356 protein-coding genes that exhibited differential expression in P0 mbd-2-/- when compared to wildtype N2 worms (Supplementary Data 3). This represents more than a quarter of all protein-coding genes in C. elegans, indicating the importance of MBD-2 in master gene regulation. Specifically, 4206 and 1150 genes were significantly upregulated and downregulated, respectively, in the P0 mbd-2-/- worms (Supplementary Fig. 5A, Supplementary Data 3). The dysregulation is further enhanced in F1 mbd-2-/- worms with 5099 and 1547 genes significantly upregulated and downregulated, respectively (Supplementary Fig. 5B, Supplementary Data 4). The higher proportion of upregulated genes as compared to downregulated genes suggests that Cel-MBD-2 functions primarily as a gene repressor, similar to its mammalian ortholog MBD233.

As expected, the upregulated genes overlap extensively between the P0 and F1 mbd-2-/- mutants, but to a lesser extent for the downregulated genes (Fig. 3A, B). In general, the gene ontology (GO) term analysis suggests that P0 and F1 mbd-2-/- consistently led to upregulation of genes involved in nervous system development, and downregulation of genes involved in metabolic pathways (Fig. 3C, D, Supplementary Fig. 6A–D).

Interestingly, GO terms relevant to development, such as cell fate specification and Wnt signaling pathway, are enriched in genes that are exclusively downregulated in F1 mbd-2-/- (Supplementary Fig. 6F). Such differences may explain why completely penetrant sterility, additional morphological defects and growth arrest are only observed in F1 mbd-2-/-, but not in P0 mbd-2-/- which may still possess maternal mbd-2 transcripts in their early development.

On the other hand, 1691 of the P0 mbd-2-/- dysregulated genes are not significantly changed in F1 mbd-2-/-. Moreover, 198 genes are dysregulated in the opposite direction in F1 compared to P0 mbd-2-/-. For example, the biological process “DNA-binding transcription factor activity, RNA polymerase II-specific” (GO:0000981) is overrepresented in P0 mbd-2-/- upregulated and F1 mbd-2-/- downregulated GO terms (Supplementary Fig. 6A, D). The reason for such opposite-direction dysregulation is not clear, but could possibly be due to negative feedback loops or secondary effects, as the affected genes regulate transcription as well.

The coiled-coil domain contributes to major gene regulation function of C. elegans mbd-2
MBD2/3 is known to be a conserved component of the NuRD complex throughout metazoa9,34. The coiled-coil domain (CC) at C-terminus is highly conserved from C. elegans to human MBD2 and MBD3 (Fig. 1A)10, essential for interacting with the NuRD complex35–38. To further elucidate the function of its coiled-coil domain, we constructed a truncated MBD-2 without its C-terminal CC. To design the deletion of the CC of Cel-MBD-2, DeepCoil239 was used to detect the residues with a coiled-coil-forming probability of ≥ 20%, and identified 15 amino acids (residue 190-204) (Fig. 1A, B, gray label). These residues fall within the Cel-MBD-2 CC (residue 178-210) based on sequence alignment with CC of human MBD2 (residue 361–393)40 (Fig. 1A, gray bracket above green box). We reasoned that the deletion of these 15 amino acids within the CC domain would be sufficient to disrupt the domain’s structure and generated a strain of gfp::3xflag::mbd-2ΔCC/ΔCC mutant maintained with the same balancer as mbd-2-/- mutant. Interestingly, the truncated GFP::3xFLAG::MBD-2ΔCC is stably expressed in embryo nuclei (Fig. 3E), consistent with a human MBD-2 study, which showed preserved nuclear location after deletion of the CC41.

Remarkably, gfp::3xflag::mbd-2ΔCC/ΔCC phenocopies the null mutation, as characterized by the low brood size in P0 gfp::3xflag::mbd-2ΔCC/ΔCC worms and complete sterility observed in F1 gfp::3xflag::mbd-2ΔCC/ΔCC worms (Fig. 2B). In addition, 14.7% of F1 gfp::3xflag::mbd-2ΔCC/ΔCC worms possess two vulvas (n=170), and a fully penetrant protruding vulva phenotype is observed in both generations.

Next, we performed RNA-seq on young adult whole worms with three biological replicates for gfp::3xflag::mbd-2 (control) and P0 gfp::3xflag::mbd-2ΔCC/ΔCC mutants, and two biological replicates for F1 gfp::3xflag::mbd-2ΔCC/ΔCC. Interestingly, the transcriptomic changes in P0 and F1 gfp::3xflag::mbd-2ΔCC/ΔCC are highly similar to those in mbd-2-/- worms when compared to their respective controls (Fig. 3F, Supplementary Fig. 4G, 5A–F). The majority of upregulated (91%) and downregulated (52%) genes in P0 mbd-2-/- mutants are also dysregulated in the same direction in P0 gfp::3xflag::mbd-2ΔCC/ΔCC (Supplementary Fig. 5E).

To determine if MBD-2 and other NuRD components co-regulate the same genes, we have reanalyzed published RNA-seq data of the NuRD components hda-1 RNAi42 and dcp-66 RNAi43 in young adult worms together with our mbd-2-/- and gfp::3xflag::mbd-2ΔCC/ΔCC RNA-seq. The RNAi experiments42,43, likely resulting in partial protein depletion, yielded fewer differentially expressed genes (4972 for hda-1 RNAi and 1677 for dcp-66 RNAi) than our mutants (>11000 genes in P0 mbd-2-/- and P0 gfp::3xflag::mbd-2ΔCC/ΔCC), with a filtering criteria of adjusted p-value < 0.05. Among them, 668 differentially expressed genes overlap in all our P0 mbd-2-/-, P0 gfp::3xflag::mbd-2ΔCC/ΔCC, hda-1 RNAi, and dcp-66 RNAi, mostly showing expression change in the same direction (Fig. 3G). Specifically, 317 out of 668 genes are consistently upregulated in P0 mbd-2-/-, dcp-66 RNAi, and hda-1 RNAi. These genes, normally repressed by NuRD, are involved in nucleosome assembly, axon guidance and structural constituents of chromatin (Supplementary Fig. 6G). On the other hand, 171 out of 668 genes are consistently downregulated in the four conditions. These NuRD-activated genes are involved in the regulation of immune response and metabolic pathways (Supplementary Fig. 6H). Since most of the dysregulated genes are dysregulated in the same direction (488 out of 668) in mbd-2 null mutant, coiled-coil mutant, and NuRD complex component depletion, it suggests a functional association between MBD-2 and the NuRD complex.

MBD-2 is a scaffolding component of the NuRD complex through its coiled-coil (CC) domain
The NuRD complex can be functionally split into two subcomplexes, a histone deacetylase core and a nucleosome remodeling core9. Mammalian MBD2/3 serves as the scaffold of the NuRD complex. At one end, MBD2/3’s intrinsically disordered region (IDR) interacts with histone deacetylase core through the interface formed between MTA1/2/3 dimer-HDAC1/2 dimer35,37,40 (Fig. 4A). At the other end, MBD2/3’s C-terminal coiled-coil domain (CC) interacts with the coiled-coil domain of GATAD2A/B, which then recruits the chromatin remodeler CHD3/4/5 (Fig. 4A)35–37. The two subcomplexes that are connected via MBD-2’s IDR and CC are structurally conserved throughout metazoan. C. elegans possesses at least one ortholog for each component (Table 1 and Fig. 4A). Unexpectedly, a previous study could not detect any interaction between NuRD components and Cel-MBD-2, challenging such structural conservation of the NuRD complex in a methylation-free context44.

To test if Cel-MBD-2 is indeed in the NuRD complex, we performed native Immunoprecipitation-Mass Spectrometry (IP-MS) on tagged MBD-2 (GFP::3xFLAG::MBD-2) in young adult worms. Immunoprecipitation was carried out on 0.5 ml worms using an anti-FLAG antibody with two biological replicates. Parallel IP-MS experiments were performed using untagged wildtype (N2) lysates as negative controls for background subtraction. High-confidence interactors were defined as proteins exhibiting ≥ 2-fold enrichment in iBAQ (Intensity-based absolute quantitation of protein peptides) intensity over the negative control (two-tailed t-test, p-value < 0.05). Using these criteria, we identified 140 MBD-2-interacting proteins (Fig. 4B, Supplementary Data 7), including NuRD components HDA-1/HDAC1/2, LIN-53/RBBP4/7, DCP-66/GATAD2A/B, CHD-3/Mi-2, and LET-418/Mi-2. The only exception is LIN-40/MTA2/3, which was not detected in our IP-MS results in adults and embryos.

In addition, DVE-1/SATB1/2, which is a NuRD-interacting transcription factor (TF) conserved from humans45 to C. elegans42, is also identified as a MBD-2 interactor. Notably, CHD-3 and DVE-1 are among the top 3 interactors of MBD-2 (Supplementary Data 7).

We also performed co-immunoprecipitation (Co-IP) followed by Western blot to confirm Cel-MBD-2 interactions with the NuRD histone deacetylase core components HDA-1/HDAC1/2 and LIN-53/RBBP4/7 and a nucleosome remodeling core component LET-418/Mi-2 (Fig. 4D, Supplementary Fig. 7). The above results reveal that MBD-2 is a conserved component of NuRD complex in C. elegans.

Next, we compared our MBD-2 IP-MS data with published IP-MS data on HDA-146 and DVE-147 of young adult worms (Fig. 4C). The 4 interacting proteins overlapping among the MBD-2, HDA-1 and DVE-1 interactors are NuRD complex components. Interestingly, HDA-1 IP-MS shows 32 interactors that are shared by MBD-2, but not by DVE-1. On the other hand, DVE-1 shares only 5 interactors with MBD-2 alone. This trend of having more interactors shared between MBD-2 and HDA-1 (total 36) than between MBD-2 and DVE-1 (total 9) is consistent with that DVE-1 is only one of the NuRD-interacting TFs but not a core component of the NuRD complex.

The CC of MBD2/3 in metazoans has been demonstrated as an essential anchor point for the nucleosome remodeling core of NuRD via its interaction with CC of GATAD2A/B35–38. We predicted that the deletion of MBD-2 CC would lead to the dissociation of the nucleosome remodeling core from the remaining MBD-2-histone deacetylase core of NuRD. Over a thousand gfp::3xflag::mbd-2ΔCC/ΔCC synchronized young adult worms were manually picked and collected for IP-MS of MBD-2ΔCC using anti-FLAG antibody, and 2 biological replicates were prepared. As expected, 123 out of 141 of the confident MBD-2 interactors showed a decreased interaction in ΔCC (Fig. 4E, Supplementary Data 7). We further identified 35 “confidently lost interactors” (defined in Materials and Methods) that became completely undetectable in both replicates of ΔCC IP-MS (Fig. 4E, gray highlighted interactors in Supplementary Data 7). Intriguingly, CHD-3 and DVE-1 are among the “confidently lost interactors” in ΔCC, and DCP-66 shows reduced interaction with MBD-2 ΔCC (Supplementary Data 7), consistent with our hypothesis that MBD-2 ΔCC would lead to dissociation of the nucleosome remodeling core from the rest of the NuRD complex. However, LET-418, which also belongs to the nucleosome remodeling core, did not show a significant loss of interaction with MBD-2 ΔCC. This could also indicate that LET-418 may interact with MBD-2 independent of MBD-2 CC-DCP-66 CC interaction, possibly outside of the NuRD complex. Our verification of the Cel-MBD-2-NuRD interaction, specifically through its coiled-coil domain, has shed light on the structural and functional conservation despite the loss of the methyl-binding domain.

The majority of Cel-MBD-2 binding sites are non-NuRD-overlapping
Previous ChIP-seq data of some C. elegans NuRD components show general affinity for open chromatin20. We hypothesized that Cel-MBD-2 would bind mainly to open chromatin in association with the NuRD complex, similar to MBD3 in mammals3,4, as both do not have an intact MBD. To test this hypothesis, we performed ChIP-seq experiments on the strain expressing GFP::3xFLAG::MBD-2 at the young adult stage. The experiment included two biological replicates of 1.5ml worms each, with matched input DNA controls collected for background normalization. Immunoprecipitation was performed using anti-FLAG antibody. To compare the genome-wide binding profile of Cel-MBD-2 to other NuRD components, we also obtained the published ChIP-seq data of the C. elegans NuRD components (HDA-1/HDAC1/242, CHD-3/Mi-248 and LET-418/Mi-248) and its known interacting transcription factor (DVE-1/SATB1/242) in the same life stage (synchronized young adult worms) (Supplementary Data 10). We discovered 32869 peaks bound by Cel-MBD-2. The number of MBD-2 peaks was much more abundant than those of other NuRD components (3731 for CHD-3, 3551 for LET-418, 10959 for HDA-1 and 7839 for DVE-1) when analyzed with the same peak-calling pipeline (see Materials and methods). This could be due to our deeper sequencing depth (≥11 GB) compared to published datasets for other Cel-NuRD components (2-5 GB). Alternatively, this may imply that MBD-2 has many more binding sites than other NuRD components.

By extracting the MBD-2 peaks that overlap with both HDA-1 and CHD-3 from the histone deacetylase core and nucleosome remodeling core, respectively, we have defined these peaks as “NuRD-overlapping MBD-2” peaks (6.6% of all MBD-2 peaks) (Fig. 5A, Supplementary Data 11). For this classification, we did not consider the data from LET-418, the mutually exclusive paralog for CHD-3 in the Cel-NuRD complex, even though LET-418 and CHD-3 share extensive overlapping targets in embryos and show partial functional redundancy in embryo development20. Indeed, HDA-1 and LET-418 are present not only in the NuRD complex but also in another chromatin remodeling complex called the MEC complex, which does not contain CHD-344. Thus, overlapping with both HDA-1 and CHD-3 is a stringent and reasonable criterion for “NuRD-overlapping MBD-2” peaks. The remaining MBD-2 peaks that do not overlap with both HDA-1 and CHD-3 are defined as “non-NuRD-overlapping” MBD-2 peaks (93.4% of all MBD-2 peaks) (Fig. 5A, Supplementary Data 12). Our classified NuRD-overlapping MBD-2 peaks and non-NuRD-overlapping MBD-2 peaks are depicted in heatmaps, with enrichment and depletion of other NuRD complex components, respectively (Fig. 5B).

We are aware that the deep sequencing coverage may allow us to discover many more MBD-2 peaks as compared to previous studies on other NuRD components. Therefore, when we increased the stringency in analysis and narrowed down MBD-2 peaks to those with fold enrichment ≥ 4 rather than the default 1.2 of MACS2 peak calling setting (refer to Materials and methods), it resulted in only 5820 MBD-2 peaks. Yet, consistent with the less stringent but default parameter, only 6.9% of these 5820 peaks could be classified as NuRD-overlapping MBD-2 peaks (Supplementary Fig. 8B). In addition, we performed unsupervised k-means clustering of all 32869 MBD-2 peaks using HDA-1 and CHD-3 ChIP signal (Supplementary Fig. 8C). This results in 5009 peaks in Cluster 1 (15.24% of total MBD-2 peaks), with relatively high enrichment with HDA-1 and CHD-3 binding, and 27860 peaks in Cluster 2 (84.76% of total MBD-2 peaks) which is relatively deprived of NuRD components. These results confirm that Cel-MBD-2 possesses many NuRD-independent binding sites across the genome.

When we searched in C. elegans motif database based on all MBD-2 peaks, the most significantly enriched motifs are C-rich and CG-rich helix-loop-helix (HLH)-family motifs (Fig. 5C). When we searched in human motif database, Cel-MBD-2 is surprisingly still enriched at the CG-rich motif bound by human MBD2 despite the loss of the MBD domain in Cel-MBD-2 (Fig. 5C). On the other hand, as expected, the NuRD-overlapping MBD-2 peaks are enriched with the top NuRD component HDA-1’s enriched motif42 (a TC-rich motif, Fig. 5C).

Predominant binding of Cel-MBD-2 to genomic sites independently of the NuRD complex is interesting as Cel-MBD-2 does not possess a MBD domain, and the genome itself is unmethylated. We speculate that MBD-2 could bind to non-NuRD-overlapping target sites via interactions with transcription factors or histone mark readers, potentially through its protein-interacting CC domain or IDR.

Localization Cel-MBD-2 to repressive chromatin without strong overlap with other NuRD components and transcription factors
To identify potential DNA or chromatin recruiter for MBD2, we compared our Cel-MBD-2 IP-MS data with an existing C. elegans transcription factor (TF) database, WTF2.049. We have identified a few transcription factors interacting with Cel-MBD-2, including DPL-1/Dp and STA-1/STATs, in addition to the mentioned NuRD-interacting TF DVE-1 (Supplementary Data 7). DPor STATs are not known as interactors of MBD2/3 in humans. Intriguingly, DPL-1 is a known interactor of HDA-150 and LIN-5351 in C. elegans, likely in the context of the DRM (DP/Rb/MuvB) complex50. On the other hand, STA-1/STATs is involved in the regulation of immune response in C. elegans and humans50. STA-1 possesses a conserved coiled-coil domain52, which may interact with Cel-MBD-2’s CC domain. Indeed, STA-1 is identified as a “confidently lost” interactor of MBD-2 ΔCC in our IP-MS. This may implicate that STA-1 could be competing with the NuRD remodeling core (DCP-66/GATAD2A/B) for MBD-2’s interaction through its CC.

However, published ChIP-seq data of these transcription factors show that their peaks have little overlap with non-NuRD-overlapping MBD-2 peaks. Particularly, DVE-1 is known to be a NuRD-interacting TF thus enriched at NuRD-overlapping MBD-2 peaks (Fig. 5D). In addition, DPL-1, which is known to interact with two of the NuRD components50,51, is also enriched mainly at NuRD-overlapping MBD-2 peaks (Fig. 5D, E, upper panel). Therefore, DPL-1 and DVE-1 may represent NuRD-interacting TFs that interact with MBD-2. STA-1 only shows weak enrichment at MBD-2 peaks (Fig. 5D). STA-1 peaks overlap slightly more with NuRD-overlapping MBD-2 peaks than with non-NuRD-overlapping MBD-2 peaks (Fig. 5E, lower panel). STA-1 recruitment may only contribute partially to non-NuRD-overlapping MBD-2 peaks. Therefore, it is unlikely that these TFs are the major recruiter for non-NuRD-overlapping MBD-2 targets.

In addition, the genome-wide binding pattern of MBD-2 shows characteristic enrichment at chromosome arms (Fig. 6A), which is a signature pattern of repressive histone marks, particularly H3K9me2, H3K9me3 and H3K27me348). In addition, MBD-2 shows a relatively more uniform distribution across chromosome X, similar to H3K27me3 (Fig. 6A). MBD-2 is more depleted in the central regions of chromosomes, which are enriched with H3K4me3, H3K9ac, and H3K27ac. Such enrichment at chromosome arms is unlikely to be observed in transcription factor-mediated recruitment, such as DVE-1, DPL-1, and STA-1, which show punctuated enrichment at transcribed gene promoters across the chromosome (Fig. 6A). Such genome-wide binding pattern suggests that MBD-2 localization could be transcription factor-independent, but related to local histone modification.

Non-NuRD-overlapping MBD-2 peaks are enriched with silent histone modifications, particularly H3K27me3 and H3K9me2 (Fig. 6B–D, Supplementary Fig. 8B, D). Forty-six percent (45.7%) and forty-one percent (41.1%) of the non-NuRD-overlapping MBD-2 peaks overlap with H3K27me3 and H3K9me2, respectively (Fig. 6C and Supplementary Data 13). Examples of non-NuRD-overlapping MBD-2 peaks and H3K27me3 peaks colocalization are also observed in genome browser (Fig. 6D, Supplementary Fig. 8D). Interestingly, H3K27me3 enrichment is exclusively at non-NuRD-overlapping MBD-2 peaks, and the binding patterns of them are similar (Fig. 6B–D, Supplementary Fig. 8D). On the other hand, H3K9me2 and H3K9me3 peaks are low in intensity and evenly distributed between non-NuRD and NuRD-overlapping regions (Fig. 6B–D, Supplementary Fig. 8D).

In contrast, NuRD-overlapping MBD-2 peaks are enriched with active histone modifications, including H3K4me3, H3K9ac and H3K27ac (Fig. 6B–D, Supplementary Fig. 8D). Our MBD-2 ChIP-seq data have confirmed the conservation of the enrichment of NuRD complex on active histone marks, consistent with the evidence by CHD-3 and LET-418 ChIP-seq data20. About eighty-seven percent (86.6%) of NuRD-overlapping MBD-2 peaks overlap with H3K27ac peaks (Fig. 6C), and NuRD-overlapping MBD-2 peaks frequently coincide with the “valleys” at H3K27ac peak centers (Figs. 6B, D, 8A, Supplementary Fig. 8D), consistent with NuRD complex enrichment at the nucleosome-depleted region of active promoters near TSS20.

When looking at the k-means clustered MBD-2 peaks based on HDA-1 and CHD-3 signals, Cluster 1 (15.24% of all MBD-2 peaks) is relatively enriched with NuRD components and active histone marks (H3K4me3 and H3K27ac), and Cluster 2 (84.76% of all MBD-2 peaks) is relatively deprived of NuRD components and enriched with H3K27me3 (Supplementary Fig. 8C). Consistently, we classified MBD-2 peaks based on whether they overlap with H3K27me3. Non-H3K27me3-overlapping MBD-2 peaks are enriched with NuRD components, while H3K27me3-overlapping MBD-2 peaks are not (Supplementary Fig. 9A).

Reciprocally, MBD-2 is enriched at most H3K27me3 and H3K9me2 peaks, and partially on some H3K9me3 peak centers (Supplementary Fig. 9B–D). These features indicate the divergent epigenetic properties of MBD-2 non-NuRD-overlapping and NuRD-overlapping peaks.

NuRD-overlapping or MBD-2-activated gene targets are more highly expressed, and MBD-2 shows different binding patterns for gene repression and activation
In view of the divergent epigenetic properties of non-NuRD-overlapping and NuRD-overlapping targets, we perform gene-wise analysis of expression levels. We focused on the protein-coding genes identified in our RNA-seq analysis (10,779 genes with a non-zero read count in at least one sample (N2, P0 or F1 mbd-2-/-) that contain these MBD-2 peaks within 2kb upstream or downstream of their transcription start sites (TSS), which usually covers the gene body. MBD-2 binds to 9262 protein-coding genes, almost half of the C. elegans genes (Supplementary Data 9). Interestingly, only around half of the differentially expressed genes in P0 and F1 mbd-2-/- RNA-seq are bound by MBD-2 at their transcription start site (TSS) ± 2kb based on our MBD-2 ChIP-seq (Supplementary Fig. 10A, B). Surprisingly, the differentially expressed genes in P0 (1.03-fold; hypergeometric p-value = 0.00076) and F1 (1.01-fold; hypergeometric p-value = 0.23) are not enriched for MBD-2 binding, compared to expectation. This may suggest that MBD-2 binding at genes has unknown functions that cannot be revealed by gene dysregulation. Moreover, the remaining half of the differentially expressed genes unbound by MBD-2 could be its indirect or secondary targets.

We classified MBD-2 target genes based on the presence of non-NuRD-overlapping or NuRD-overlapping MBD-2 ChIP-seq peaks (peaks classified as in Fig. 5A), resulting in three categories (Fig. 7A), including: (1) 646 genes that have both NuRD-overlapping and non-NuRD-overlapping peaks near the TSS, (2) 9756 genes that exclusively only have non-NuRD-overlapping MBD-2 peaks near the TSS, and (3) 377 genes that exclusively only have NuRD-overlapping MBD-2 peaks near the TSS. We focus on the (2) exclusive non-NuRD-overlapping and (3) exclusive NuRD-overlapping MBD-2 gene targets for the following expression analysis.

When looking at the expression levels of MBD-2 gene targets in wildtype (N2) worms, the median native expression level of the exclusive NuRD-overlapping MBD-2 gene targets is higher than that of the exclusive non-NuRD-overlapping MBD-2 targets and that of all protein-coding genes (Fig. 7B). We further subdivided the gene targets into upregulated and downregulated genes, and genes with unchanged expression, based on P0 mbd-2-/- RNA-seq (Fig. 7A). Surprisingly, only 23-31% of MBD-2-bound gene targets had significantly altered expression levels in both the null mutant and coiled-coil domain deletion mutant, no matter in the exclusive NuRD-overlapping MBD-2 gene targets or the exclusive non-NuRD-overlapping MBD-2 gene targets (Fig. 7A, Supplementary Fig. 10C, D). Nevertheless, exclusive non-NuRD-overlapping MBD-2 gene targets seem to show a higher degree of dysregulation than exclusive NuRD-overlapping ones, as observed in both P0 mbd-2-/- and gfp::3xflag::mbd-2ΔCC/ΔCC RNA-seq (Supplementary Fig. 10E, F). These results suggest that for the majority of the MBD-2 target genes, binding of MBD-2 to them does not significantly affect the expression levels in the adult worm analyzed. Yet, other developmental stages or stressed conditions have not been tested.

Interestingly, P0 mbd-2-/--downregulated genes, which are normally MBD-2-activated genes, have a higher median expression level than the no change or upregulated genes, both in the exclusive NuRD-overlapping and exclusive non-NuRD-overlapping MBD-2 gene targets (Fig. 7C). On the other hand, MBD-2-repressed genes have a lower median expression level. This indicates that MBD-2 reinforces the activation of its highly expressed targets or repression of its lowly expressed target genes.

Next, we investigated the binding pattern of MBD-2 on its gene targets. MBD-2 generally shows sharp enrichment at the TSS of its target genes. The binding is also extended to gene bodies with lower average signal compared to the TSS (Supplementary Fig. 11A). This holds true for both the exclusive non-NuRD-overlapping gene targets and the exclusive NuRD-overlapping gene targets (Fig. 7D). The binding upstream of the TSS is slightly higher in the exclusive NuRD-overlapping gene targets (~1.35 fold enrichment over input) compared to exclusive non-NuRD-overlapping gene targets (~1.21 fold). However, the overall gene binding patterns of MBD-2 show no dependency on its interaction with NuRD.

On the P0 mbd-2-/--upregulated genes, which are normally MBD-2-repressing genes, the average MBD-2 binding peak is at the TSS (Fig. 7E). In contrast, on the P0 mbd-2-/--downregulated genes, which are normally MBD-2-activated genes, the average MBD-2 binding peak is more downstream of the TSS. It appears that the localization of MBD-2 at the gene promoter and TSS may be associated with repression of target genes, whereas MBD-2 at the gene body may promote gene activation. On the other hand, MBD-2 binding pattern on F1 mbd-2-/--downregulated genes is less distinct, and may represent secondary effects (Supplementary Fig. 11B). These analyses show that MBD-2 has different binding patterns at its repressing and activating targets.

H3K27me3 correlates specifically with non-NuRD-overlapping MBD-2 gene targets, but not with NuRD-overlapping MBD-2 gene targets
Consistently, active histone marks, including H3K4me3, H3K9ac, H3K27ac and H3K36me3, are more enriched at the gene body of exclusive NuRD-overlapping MBD-2 gene targets compared to exclusive non-NuRD-overlapping MBD-2 gene targets (Fig. 8A). This is consistent with NuRD’s tendency to bind to open chromatin regions marked with active histone marks, which likely include active genes. In contrast, repressive histone marks, including H3K9me2, H3K9me3, and H3K27me3, are more enriched at exclusive non-NuRD-overlapping genes (Fig. 8A).

Combining with the P0 mbd-2-/- RNA-seq data, we found that in wildtype, H3K9ac and H3K4me3 are more enriched at exclusive NuRD-overlapping MBD-2 gene targets that are downregulated, whereas H3K27ac and H3K36me3 are more enriched at MBD-2 gene targets with unchanged gene expression (Supplementary Fig. 12).

In agreement with our results that non-NuRD-overlapping MBD-2 peaks are enriched with H3K27me3 (Fig. 6B), H3K27me3 is enriched in the exclusive non-NuRD-overlapping MBD-2 gene targets, regardless of being upregulated or downregulated in P0 mbd-2-/- (Supplementary Fig. 12). However, H3K27me3 is relatively low in the exclusive non-NuRD-overlapping MBD-2 gene targets that have unchanged expression in P0 mbd-2-/- and exclusive NuRD-overlapping MBD-2 gene targets (Supplementary Fig. 12). H3K27me3 may cooperate with MBD-2 in the transcriptional regulation of non-NuRD-overlapping gene targets.

Surprisingly, H3K36me3, which usually localizes at gene bodies, is enriched at MBD-2 gene targets with unchanged expression in P0 mbd-2-/- (Supplementary Fig. 12). Such an enrichment pattern of H3K36me3 seems to be opposite to that of H3K27me3 at MBD-2-regulated gene targets.

Consistent with the arm enrichment (Fig. 6A), MBD-2 exhibits a general positive correlation with repressive histone marks H3K9me2 and H3K9me3, irrespective of whether the target genes are non-NuRD- or NuRD-overlapping. In contrast, H3K27me3 is enriched at all non-NuRD MBD-2 peaks, except at locations where H3K36me3 is enriched (Fig. 6B). Indeed, H3K27me3 and H3K36me3 show a negative correlation (-0.67) at MBD-2 exclusive non-NuRD-overlapping gene targets (Supplementary Fig. 13A). When excluding the 3,913 non-NuRD-overlapping MBD-2 gene targets that have overlapping peaks with H3K36me3 (Supplementary Fig. 13B, C), we found that H3K27me3 reaches a correlation of 0.56 with MBD-2 at 5843 exclusive non-NuRD-overlapping MBD-2 gene targets (Fig. 8B). In contrast, H3K27me3 shows no significant correlation (0.01) with MBD-2 at NuRD-overlapping target genes (Fig. 8C, Supplementary Fig. 13C). This finding aligns with the observation in Fig. 6B, where H3K27me3 is exclusively enriched at non-NuRD-overlapping MBD-2 peaks, but not at NuRD-overlapping MBD-2 peaks. Conversely, when excluding non-NuRD-overlapping MBD-2 gene targets that overlap with H3K27me3, the correlation of H3K36me3 with MBD-2 increases from 0.03 to 0.31 (Supplementary Fig. 13D). Therefore, at non-NuRD-overlapping MBD-2 gene targets, the mutually exclusive enrichment of H3K27me3 or H3K36me3 could be related to its different gene regulatory functions (Supplementary Fig. 12).

These results suggest that while MBD-2 generally colocalizes with repressive histone marks, H3K27me3 is particularly distinctive in differentiating between its non-NuRD-overlapping and NuRD-overlapping gene targets. Further studies are needed to identify MBD-2’s recruiter(s) to repressed regions, particularly to elucidate the causal relationship between the enrichment of H3K27me3 and MBD-2 at its non-NuRD-overlapping gene targets.

Methods
Worm maintenance
List of worm strains used in this study is available in Supplementary Data 1. All the newly constructed strains were outcrossed with N2 or hT2 balancer-containing males for at least 3 times. All experiments were then performed on young adult worms (3 days post-hatching) obtained by bleach synchronization. Briefly, bleach was used on adult worms to kill the worms and preserve the embryos. Upon hatching in M9 buffer, L1 larvae were fed with OP50 E. coli and grown on standard conditions at 20°C unless specified.

Worm strain construction
Cas9-mediated tagging of GFP on MBD-2. The tagging of Cel-MBD-2 with GFP::[TEV]::3xFLAG N-terminal tag (strain identifier: WYY49; see Supplementary Data 1) was performed according to a published protocol81. Briefly, the plasmids PDD282 (with the tag and homology arms flanking MBD-2 transcription start site) and PDD162 (containing the Cas9 and sgRNA for MBD-2) were injected along with co-injection markers into the gonads of wildtype (N2) young adults. Successful integration of the tag was verified with live imaging of GFP expression, single worm PCR, Sanger sequencing, and Western blot. Sequences of primers (for amplifying the homology arms from genomic DNA) and sgRNA are provided in Supplementary Data 2.

Cas9-mediated mbd-2 loss-of-function, null mutation with premature stop codons. Insertion of stop codon cassette was performed as described31. Specifically, the cassette inserted contains stop codons in all three reading frames, and the cassette was inserted in the first exon of mbd-2. Using the co-CRISPR strategy, dpy-10 allele was used as the CRISPR positive control. The repair oligos for dpy-10 and mbd-2 (containing mbd-2 homology arms flanking the stop codon cassette), crRNA (specific for the mbd-2 and dpy-10) (sequences available in Supplementary Data 2), and universal tracrRNA were injected to wildtype (N2) young adult gonads. Successful null mutants were isolated based on the dumpy phenotype caused by the dpy-10 mutation, and knock-in of the stop codon cassette (STOP-IN) at mbd-2 was confirmed by single-worm PCR.

Given the homozygous sterility of mbd-2-/- null mutants, they were maintained as hT2 [bli-4(e937) let-?(q782) qIs48]-balanced heterozygous stock (WYY74, with GFP signal at pharynx). Four rounds of outcross with hT2 males were performed to remove any background mutations and the dpy-10 mutation. We picked homozygous mbd-2-/- worms (with no pharyngal GFP) from the heterozygous stock. Then, we verified the genotype by single-worm PCR using primers flanking the insertion region, followed by Sanger sequencing. Primer sequences are available in Supplementary Data 2.

Single-copy transgenic expression of GFP::3xFLAG::MBD-2, at the oxTi365 locus. A single copy of mbd-2p::gfp::3xflag::mbd-2 transgene (tg) driven by the endogenous promoter (1408 bp upstream of its start codon) was inserted ectopically into the oxTi365 locus present in the EG8082 strain using Mos1-mediated Single Copy Insertion (MosSCI)82. Specifically, gfp::3xflag::mbd-2 containing its 3’UTR and endogenous promoter was amplified from the strain with endogenous gfp::3xflag::mbd-2 (WYY49) using single-worm PCR and cloned into the pCFJ150 vector (containing flanking sequence for oxTi365 locus to allow insertion via homologous recombination) using Gibson Assembly. The cloned vector was sequenced with M13F and M13R to verify correct insertion. The verified vector was then injected into the gonads of EG8082 worms. Selection for successful insertion was performed according to the protocol82. GFP::3XFLAG::MBD-2 expression was visualized by DeltaVision microscope. The strain was further crossed with mbd-2-/- null mutants at the endogenous mbd-2 locus carrying hT2 balancer (WYY74). Double homozygous mbd-2-/-; tg/tg worms were selected by nuclear GFP::3XFLAG::MBD-2 expression and the presence of a PCR band corresponding to the null mutant (one of the primers is overlapping with the stop codon cassette). At least 10 worms were screened for 3 generations.

Cas9-mediated deletion of the coiled-coil domain of mbd-2. CRISPR/Cas9 was performed for the construction of the mbd-2 null mutant strain31. Fifteen (15) amino acid residues predicted to confer formation of coiled-coil domain by DeepCoil239 (MBD-2 residue 190-204) were deleted via exclusion of these sequences in the repair template for gfp::3xflag::mbd-2 (WYY49). Homozygous CC deletion was verified by single-worm PCR covering the deletion region, followed by Sanger sequencing. Primer sequences are available in Supplementary Data 2. The expression of GFP::3xFLAG::MBD-2ΔCC was verified by live imaging and Western blot.

Live imaging of GFP-tagged MBD-2
Preparation and mounting of GFP::3xFLAG::MBD-2 (WYY49) embryos were performed as described83. WYY49 was crossed with a strain containing mCherry-H2B (OD56), which enabled the visualization of chromatin in live-cell imaging. Time-lapse images were acquired using DeltaVision microscope at 22°C, with a 60× oil objective. Embryos were captured in z-stacks of 12 sections at 0.2 μm intervals. Images were taken at 20-s intervals at 475-nm green (10% intensity) and 575-nm red (10% intensity) excitation filter channels; 535-nm green and 625-nm red emission filter, with exposure time of 0.05 s. For whole-worm live images, the worms were mounted in 2% sodium azide on 3% agarose pads and covered with a coverslip.

Knockdown of mbd-2
Knockdown of mbd-2 was performed according to the published protocol84. Briefly, total RNA was purified from wildtype (N2) worms. ~1.2 μg of the purified RNA was used for reverse transcription using High-capacity cDNA reverse transcription kit (Applied Biosystems, Cat. 4368813). Primers containing the T7 promoter were used to amplify the cDNA of mbd-2 covering part of exon 1 to part of the last exon (502 bp). Primer sequences are available in Supplementary Data 2. The amplified cDNA was purified with PCR Purification Kit (QIAGEN, Cat. 28106). dsRNA was synthesized with MegaScript T7 kit (Ambion, AM1334) following the manufacturer’s instructions. L4 stage N2 worms were injected with the dsRNA and singled out to new plates to allow for egg laying and brood size counting. For brood size counting, the worms were allowed to lay eggs and transferred to new plates every 24 h for 3 days. Only the F1 worms laid by injected worms between 24–48 h post-injection would be used for phenotype screening and brood size counting, as it was the time point at which the loss of GFP::3xFLAG::MBD-2 from embryonic nuclei was verified by live-cell imaging when gfp::3xflag::mbd-2 was injected with dsRNA in parallel.

RNA-sequencing of mbd-2 null and coiled-coil domain deletion mutant worms
mbd-2-/- and gfp::3xflag::mbd-2ΔCC/ΔCC mutant worms were maintained as hT2 [bli-4(e937) let-?(q782) qIs48]-balanced heterozygous stock. At least 400 heterozygous worms were picked and bleach synchronized. Around 2500 hatched L1 larvae were then transferred onto 10 OP50 seeded plates to grow to adults. The selection of the homozygous P0 mbd-2-/- and gfp::3xflag::mbd-2ΔCC/ΔCC worms was achieved by first identifying the high penetrance phenotype of protruding vulva in small adults, and then verifying by the lack of the GFP in the pharynx, encoded by the balancer, under a fluorescent microscope. 100–150 P0 mbd-2-/- worms and 120-135 P0 gfp::3xflag::mbd-2ΔCC/ΔCC were used for RNA extraction. A portion of the P0 mbd-2-/- and P0 gfp::3xflag::mbd-2ΔCC/ΔCC worms were bleach synchronized to obtain homozygous F1 mbd-2-/- and F1 gfp::3xflag::mbd-2ΔCC/ΔCC embryos, respectively. Two biological replicates were prepared for each genotype. Only 65-90 F1 mbd-2-/- and 100-102 F1 gfp::3xflag::mbd-2ΔCC/ΔCC young adult worms were used due to the very low brood size of P0 mbd-2-/- and P0 gfp::3xflag::mbd-2ΔCC/ΔCC.

Total RNA was isolated using TRIzol-chloroform extraction, followed by isopropanol precipitation. To remove DNA, RNA was treated with TURBO DNA-free™ Kit (Ambion, Cat.#AM1907). The RNA was resuspended in 20 μl RNAse-free water. RNA quality was accessed with Bioanalyzer (Supplementary Fig. 4A, B) and concentration was measured by Qubit. A minimum amount of 196 ng of total RNA was obtained. The total RNA samples were then submitted to the Centre for PanorOmic Sciences (CPOS, HKU LKS Faculty of Medicine) for library preparation using KAPA mRNA HyperPrep Kit and Illumina pair-end sequencing using Illumina NovaSeq 6000.

RNA-sequencing analysis
Sequencing reads were filtered for adapter sequence, low quality sequence and retaining reads with read length ≥ 40 bp, using Cutadapt with parameter --max-n 0.05 and followed by fastp --unqualified_percent_limit 50 --qualified_quality_phred 12 --disable_adapter_trimming --n_base_limit 50 --length_required 40. Reads were mapped to the reference genome WBcel235 using STAR v2.7.9a (default parameters) with the following exception: --twopassMode Basic and gene and transcript counts were obtained with the built-in counting option --quantMode. Differential expression analysis was done using DESeq2 with the gene count output from STAR. Wald tests within DESeq2 were conducted to assess differential gene expression between groups. The two-tailed Wald test p-value, and the Benjamini-Hochberg adjusted Wald test p-value is displayed in Supplementary Data 3–6. Results from DESeq2 were then run through apeglm for LFC shrinkage based on effect size using the lfcShrink function from DEseq2 for volcano plot and MA plot visualization85. Differentially expressed protein-coding genes were filtered to possess Benjamini-Hochberg adjusted p-value < 0.05 and with log fold change (FC) shrinkage values ≥ 1.0 or ≤ -1.0. Genes with adjusted p-values of NA were excluded from downstream analysis. Gene ontology (GO) enrichment analysis was performed using DAVID. The Transcripts Per Million (TPM) value for each gene was calculated using RSEM v1.3.3 for plotting the heatmap (Supplementary Fig. 4E, F). MA plots and PCA plots are shown (Supplementary Fig. 4C, D, G).

Chromatin immunoprecipitation (ChIP)-sequencing of GFP::3xFLAG::MBD-2
ChIP-seq experiments were performed on 2 biological replicates. 3 rounds of 800 ml liquid culture was performed to obtain 1.5 ml of synchronized young adult worm pellet. The worm pellet was mixed with an equal volume of PBS supplemented with proteinase inhibitor, then snapped frozen as worm droplets by dripping into liquid nitrogen and stored at -80°C. The frozen worm droplets were then grinded in liquid nitrogen for 10 minutes with a mortar and pestle into fine powder. The worm powder was then thawed in 3 ml of fixing solution (1.1% formaldehyde in PBS with proteinase inhibitor) and dounced with pestle B for 10 strokes to break the cuticle of the adult worms and allow penetration of the fixative. The 3 ml dounced mix was then transferred to a falcon. 2 ml of the fixing solution was used to flush the remaining dounced mix to the Falcon tube and 38 ml of fixing solution was further added. Fixation was carried out at room temperature with gentle rocking until the total fixation time reached 15 minutes. Next, the fixation was quenched by adding glycine to a final concentration of 125 mM and the sample was further rocked for 5 min at room temperature. The fixed worms were pelleted at 4000 × g, 4°C for 5 min, and rinsed twice with ice-cold PBS supplemented with proteinase inhibitor, once with MNase buffer (NEB Cat# M0247S). The lysis and release of chromatin from worms were performed according to Tabuchi et al.86 with slight modifications. Firstly, the fixed worm pellet was partially lysed by resuspending in MNase buffer with lysis buffer (0.2% NP-40 and 0.5% sodium deoxycholate) for 5 min on ice with gentle tapping. The worm was re-pelleted at 4000 × g, 4°C for 5 min and washed once with 1.5 ml of MNase buffer. It was then resuspended with 5 volumes of MNase buffer and snap frozen. A pilot test for MNase digestion was carried out on a small aliquot according to the manufacturer's protocol (NEB Cat# M0247S). In our hands, 37°C for 20 min was enough to achieve mono- to di-nucleosome DNA fragment sizes.

The remaining worm lysate (700-800 μl) was thawed for a larger scale MNase digestion. Then chromatin was solubilized by adding EDTA to a final 10 mM concentration, one protease inhibitor tablet (MCE Cat# HY-K0011) and Triton X-100 to a final 1% concentration, nutated at 4°C for 2 hr. Two volumes of cold 150 mM NaCl FA buffer (1% Triton™ X-100 + 0.1% sodium deoxycholate + 0.1% sarkosyl + protease/phosphatase inhibitors) was added, followed by sonication with a Covaris M220 model (duty factor 20%, average power 8, cycles per burst 200, 60 s per cycle, total 5 cycles) to liberate chromatin. The lysate was centrifuged at 16,000 × g for 15 min at 4°C to retrieve solubilized chromatin in the supernatant. A total of 5.25 mg total protein containing the soluble chromatin was used to perform immunoprecipitation with anti-FLAG antibody-conjugated magnetic beads (MCE Cat# HY-K0207) in the ratio of 10 μl magnetic beads per 500 μl lysate as suggested by the manufacturer. The lysate was aliquoted into 1.5 ml LoBind tubes (700 μg per tube) and the tubes were rotated with the magnetic beads overnight at 4°C. 5% of the protein (262.5 μg) was stored as input. The washing of the beads was carried out under varying salt concentrations according to the protocol described by Askjaer et al.87. The antibody/protein/DNA complexes were then eluted by adding 125 μl fresh Elution buffer (1% SDS/0.1 M NaHCO3) and heated at 65°C in a heat block for 15 min, and vortexed for 5 sec every 3 min. The beads were then magnetized and the eluate was retrieved into a low-binding 1.5 ml tube. The elution was repeated with 50 μl elution buffer and the eluates were combined. 1x volume of 2x elution buffer was added to the input and treated in the same condition in parallel with the immunoprecipitation samples. A Western blot was performed to verify the successful pull-down of GFP::3xFLAG::MBD-2.

RNase A (Thermo Scientific Cat# EN0531) was added in a 1:4 ratio to the volume of the ChIP-ed DNA samples and the inputs followed by incubation at 37°C for 1 hr. 1 M Tris Cl (pH 8) and 0.5 M EDTA were added to achieve a final concentration of 40 mM and 20 mM, respectively. Proteinase K (Qiagen Cat# 19131) (2.5 μl to every 150 μl) was added to eliminate the RNase and protein. Reverse crosslink was performed in 0.2 M NaCl salt at 800 rpm 65°C, for 16 hours in the thermomixer. The DNA was purified with PCR Purification Kit (QIAGEN, Cat. 28106)87. Concentrations and quality for the input and ChIPed DNA were assessed with Qubit and the Agilent 2100 Bioanalyzer (Supplementary Fig. 8A). The samples were submitted to the Centre for PanorOmic Sciences (CPOS, HKU LKS Faculty of Medicine) for library preparation with KAPA HyperPrep Kit and 151 bp paired-end DNA sequencing using Illumina NovaSeq 6000. We generated 20.9 and 17.3 GB raw data for the input replicate 1 and 2, respectively, and 12.9 and 11.5 GB for ChIP replicate 1 and 2, respectively.

ChIP-sequencing analysis
The reads were first filtered for adapter sequence and low-quality reads, retaining only reads with read length ≥ 30 bp. Low Quality was defined as reads with more than 5% unknown bases (“N”) and/or reads having more than 50% of bases with quality value <= 11 were filtered out. Clean reads were mapped to the reference genome WBcel235 using BWA (Version 0.7.17.5) with default parameters. Samtools view was used to remove unmapped reads and retain only properly paired mapped reads (-f 2 -F 4) with a minimum mapping quality of 30. Samtools sort was utilized to sort the alignments by coordinates. When comparing with single-end ChIP-seq data generated by other studies, including CHD-348, LET-41848, STA-188 and DPL-1 (ModENCODE) ChIP-seq, only the forward reads of the paired-end data of MBD-2 in this study, HDA-142 and DVE-142 were used for analysis for fair comparison (Figs. 5A, B, D, E, 6, 7A–C, D, 8, Supplementary Fig. 8B-D, 9, 10C, D, 12, and 13). On the other hand, when comparing the RNA seq and ChIP-seq data generated in this study, both reads of paired-end ChIP-seq data were used (Fig. 7E Supplementary Fig. 10A, B, 11A, B). For the above-mentioned ChIP-seq data, MACS2 was used to call narrow peaks with default settings except (--nomodel, --extsize 200, --SPMR (Save signal per million reads for fragment pileup profiles) and q-value cut-off of 0.01. For ChIP-seq data of histone marks, MACS2 was used to call broad peaks with default settings except (--nomodel, --extsize 200, --SPMR (Save signal per million reads for fragment pileup profiles) and q-value cut-off of 0.05. A complete list of ChIP-seq source data is available in Supplementary Data 8. Peaks are filtered for blacklisted regions supplied by ENCODE89 using bedtools. List of annotated MBD-2 paired-end peaks and single-end (using forward strand) are available in Supplementary Data 9 and 10, respectively.

The normalized input and ChIP bedgraph file generated by MACS2 with --SPMR (signal per million reads) was converted to bigwig file using bedGraphToBigWig. BigWig compare was used to generate a BigWig file with Fold enrichment (-FE) of ChIP-seq signal over input for visualization on IGV (Fig. 6A, D and Supplementary Fig. 8D). For annotating the genes bound by MBD-2 peaks, ChIPseeker (V1.28.3) was used with default settings.

For visualization of ChIP-seq signal around transcription start sites (TSS) by plots or heatmaps, duplicated reads were marked and removed using Picard (V2.18.2) MarkDuplicates with “REMOVE_DUPLICATES=true”. Bigwig files with CPM (count per million) normalized read count was created with Deeptools bamCompare. Normalized ChIP read counts were divided by normalized input read counts to get the ratio of the number of reads. Deeptools ComputeMatrix was used to generate plots or heatmaps from the bamCompare output.

To compute correlations of MBD-2, NuRD components, and histone marks on exclusive non-NuRD- and NuRD-overlapping gene targets (as defined in Fig. 7A), we utilized DeepTools’ multiBigwig function. We calculated the Spearman’s correlation coefficient using previously generated BigWig files (SPMR-normalized ChIP signal fold enrichment over input) within the specified non-NuRD and NuRD-overlapping gene body.

Native co-immunoprecipitation (Co-IP) and immunoprecipitation-mass spectrometry (IP-MS) of GFP::3xFLAG::MBD-2
Ten 15-mm plates of mixed stage worms were bleached, followed by two rounds of 800 ml liquid culture were performed to obtain 0.5 ml of synchronized young adult worm pellet. The pellet was collected by washing three times with ice cold M9 and once with ice cold Co-IP lysis buffer (50 mM HEPES (pH 7.4), 1 mM EGTA, 1 mM MgCl2, 100 mM KCl, 10% glycerol, 0.05% NP-40, supplemented with proteinase inhibitor) according to a previous protocol90 and snapped frozen in liquid nitrogen. The lysate was thawed from -80°C on ice, and an equal volume of Co-IP lysis buffer was added and subjected to sonication for 10 cycles of 30 secon and 30 secoff using a Diagenode Bioruptor Pico. Highly purified total protein was obtained after two rounds of centrifugation: firstly at 20,000 × g for 10 min at 2°C, and secondly at 100,000 × g for 20 min at 2°C90.

For the IP-MS and Co-IP experiments of GFP::3xFLAG::MBD-2 worms, 0.8 mg of the total protein from whole worm lysate was incubated with 10 μl anti-FLAG antibody-conjugated magnetic beads (MCE Cat# HY-K0207) overnight at 4°C. The beads were washed 6 times with washing buffer (TBST: 50 mM Tris-HCl, 150 mM NaCl, 0.5% Tween-20, pH 7.4) prior to elution by boiling at 1x SDS page loading buffer. For Western blot, only half of the elution per 0.8 mg IP experiment was loaded for the IP wells, while 120 μg (30% of protein used for IP) was loaded as input in Fig. 4D. Full Western blots for Co-IP done on three biological replicates are displayed in Supplementary Fig. 7.

For IP-MS, the IP samples were run on a SDS page gel until the total immunoprecipitated proteins stacked together as a tight band at the boundary between the stacking and resolving gel. Subsequently, the gel was stained with Coomassie Blue. The total immunoprecipitated protein bands were then excised and submitted to LKS Faculty of Medicine, Proteomics and Metabolomics Core Facility (PMcore), Centre for PanorOmic Sciences (CPOS), the University of Hong Kong for liquid chromatography-mass spectrometry (LC-MS/MS) analysis, using Bruker timsTOF pro mass spectrometer. Log transformed intensity-based absolute quantitation of protein peptides (iBAQ) value was used to assess the absolute abundance of each protein based on their intensity (normalized to the expected number of tryptic peptides, which was affected by protein length)91. The relative iBAQ value (riBAQ) was calculated by subtracting the average log2 iBAQ of each protein in the negative control of wildtype (untagged MBD-2) samples from the average log2 iBAQ of each protein identified in GFP::3xFLAG::MBD-2 IP-MS92,93. The interactor threshold cutoff is defined by riBAQ >= 1 and a two-tailed t-test p-value < 0.05 (Supplementary Data 7).

For the IP-MS of GFP::3xFLAG::MBD-2ΔCC, ten 15-mm plates of mixed stage worms were bleached, followed by two rounds of 800 ml liquid culture. The worms were collected by centrifugation and transferred onto plates. 1000-2000 synchronized young adult worms were manually picked based on the protruding vulva and lack of balancer gfp into a LoBind tube containing 20 μl of cold M9. The selection of the homozygous ΔCC worms was achieved by identifying the high penetrance phenotype of protruding vulva in small adults, and verifying the lack of the GFP under a fluorescent microscope. After the worms had sunk to the bottom of the LoBind tube, the M9 was replaced with ice cold Co-IP lysis buffer and snapped frozen. Multiple rounds of worms picked in this approach were pooled together after thawing on ice, followed by measuring of protein concentration with a total of 280 and 490 μg for replicate 1 and 2, respectively. Co-IP lysis buffer was added to top up to 300 μl. Sonication and immunoprecipitation were performed with the same procedure as wildtype MBD-2. Same as the GFP::3xFLAG::MBD-2 IP-MS, riBAQ was calculated by subtracting the wildtype (untagged MBD-2) samples. The immunoprecipitation efficiency for the coiled-coil deletion mutated protein (MBD-2ΔCC) (log2 iBAQ = 12.80) was lower than for the full-length MBD-2 protein (log2 iBAQ = 17.32). The log2 iBAQ difference is 4.52 (see Supplementary Data 7). To prevent this difference from being misinterpreted as a loss of specific protein interactions, a normalization step was performed before plotting Fig. 4E. The negative control (on the X-axis) log2 iBAQ values of each interactor were subtracted by the immunoprecipitation efficiency difference (4.52). For defining “confidently lost interactors” (35 interactors highlighted in gray in Supplementary Data 7), we select the criteria of (1) being significant interactors in GFP::3xFLAG::MBD-2-expressing strain (defined as above), (2) with relative log2 iBAQ (log2 iBAQ in GFP::3xFLAG::MBD-2-expressing strain over that in the negative control) greater than the MBD-2 IP efficiency difference between GFP::3xFLAG::MBD-2 and GFP::3xFLAG::MBD-2ΔCC (4.52), and (3) being completely undetectable in both replicates of ΔCC IP-MS.

Statistics and reproducibility
The specific statistical tests used for data analysis are detailed in the corresponding figure legends. No statistical method was used to predetermine the sample size. Sequencing and IP-MS results are based on at least two biological replicates and associated significance values. For the Western blot, three biological replicates are included. For brood size counting and phenotype scoring, experiments were conducted with independent C. elegans animals with the indicated number of replicates (n).

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In this paper, the authors explore the intriguing phenomenon of C. elegans which are naturally deficient in DNA methylation.\nThey point out that the methyl-binding domain protein 2/3 (MBD2/3) ortholog, while losing the methyl-binding domain (MBD), is still\npresent, and they ask if this protein has any conserved functions in worms. They show that worm MBD2/3 retains its\nconserved role as a scaffolding component of NuRD, and demonstrates new functions in recognizing H3K27me3. Overall\nthe study is very interesting, well written, and the figures are well made. I do not have any suggestions for improvement, and\ncongratulate the authors on a very interesting and well designed work.", "reviewer_2": "The article “Dual genomic localizations and functions of MBD-2 with and without NuRD in DNA methylation-free C. elegans”\npresents an informative case study in the evolution of chromatin regulation. The authors tackle a clear outstanding question\n“what role does MBD-2 have if not through 5mc”? This question is particularly interesting given that 5mc is (parsimoniously)\nancestral, yet mbd-2/3 is maintained in several metazoan lineages that have lost 5mc. The authors perform a suite of genetic\nand biochemical experiments to address this question, and conclude that mbd-2 in C. elegans retains NuRD binding and\nfuncton(i.e., phenotype), however, it also has apparently gained NuRD independent chromatin binding (and possibly\nfunction). The two different categories can be distinguished by the presence/absence of repressive (non-NuRD) and\nactivating (NuRD +) histone modifications. They end by presenting a model in which H3K27me3 replaces 5mc as a\nmechanism of recruiting MBD-2 to genomic loci.\nOverall, this was an exciting paper that used mechanistic experiments to address a broadly interesting evolutionary\nquestion. The data quality, reproducibility and writing were all appropriate for the field. Their result of mbd-2 binding outside\nof NuRD raises interesting questions about what roles it evolved since the loss of 5mc - and how it is recruited in its\nabsence. I look forward to seeing this work in press, however, I have a few major-revision requests that if completed would\nsubstantially strengthen their manuscript and interpretations.\nMajor comments:\n1. The authors note that the CC-deletion phenocopies the mbd-2 knockout phenotype. This suggests that the interactions\nwith GATAD2A/B and CHD-3/4/5 are the dominant cause of infertility and vulva development. This is somewhat surprising\nin light of the large number of non-NURD peaks in the Chip-Seq data (93%), and the large number of genes which are\ndifferentially expressed but do not overlap with hda-1 and dcp-66 differentially expressed genes. If the CC-deletion\nphenocopies the null, what is the role of non-NURD mbd-2 binding? The authors note in the Discussion that coiled-coil\nmutants are too sick to do ChIP with, and I am receptive to that. However, an indirect but possible way to get at this question\nwould be RNA-seq of their coiled-coil mutant. The differentially expressed genes relative to wildtype, and comparison to\nmbd-2 knockout, should yield insight into the role of NuRD vs. non-NuRD mbd-2 binding. Given that the dual roles of mbd-2\nis a major feature of this paper, I would argue that this is a necessary (and again relatively easy) experiment for the authors\nto do.\n2. The authors emphasize that the non-NuRD MBD-2 ChIP-signal is enriched over H3K27me3. However, in Fig. 5D, the\ndata shows that 45.7% of peaks overlap with H3K27me3 peaks. Just slightly less enrichment (41.1%) can be seen with\nH3K9me2. Moreover, as the authors are likely aware, H3K27me3 covers almost half of the entire worm genome (Liu et al.,\n2011). Thus, overlap with H3K27me3 is not that different from overlap with all non-actively transcribing regions. When\ninspecting the genome tracks presented in Figure 5F, there does not appear to be any particular correspondence of MBD-2\npeaks with the broad domains of H3K27me3. When inspecting the chromosomal patterns in Fig. 7A, there is clearly a bias of\nMBD-2 to chromosome arms, but what is the evidence for specific enrichment with H3K27me3 compared to generally\nrepressed chromatin (also see Major Comment #3 below).\nLooking at Fig. 5C, it is true that the most distinguishing corollary of NuRD vs. non-NURD MBD-2 binding is the absence or\npresence of H3K27me3. While I agree that this is an interesting pattern, the inference is limited to the few histone\nmodifications that were assessed. Without more mechanistic evidence, or at the least, statistical support for enrichment, the\nauthors should omit this emphasis in their abstract, and probably minimize its emphasis in their Discussion.\n3. In general, the bioinformatic enrichment analyses lack statistical support. This is especially important for their\ninterpretations regarding overlap with histone modifications. For example, is the ChIP-seq peak overlap of MBD-2 with\nH3K27me3 significant relative to broad repressed domains? I would also appreciate more methodological information for\nRNA-seq, ChIP and IP-MS in the main text: sample size, p value and fold-change cut off, and replicate numbers. Most of\nthese are documented in the methods, but as they are critical for interpretation, in my opinion they should be incorporated\ninto their Results section as well.\nMinor comments:\n1. For clarity, could the authors label in Fig. 1C the cell cycles. Also for clarity, could the authors indicate in the legend that\nstrain OD56 has stronger mCherry-H2B expression in the germline.\n2. In Figure 2, could the authors please label by the relevant images whether the mutant worms were P0 or F1s?\n3. Could the authors please indicate how many (at least approximately) worms or full plates were used as input in the ChIP\nand IP-MS experiment? It was indeed impressive that they could obtain enough worms from the CC delta strain for IP-MS!\n4. It is indicated in their Methods section, but can the authors mention what the negative control was for the IP-MS\nexperiment in the Figure 4 legend?\n5. Line 516 references Fig. 4F; as far as I can see there is no figure 4F.\n6. Can the authors please define how Fig. 4C data is “normalized” in the legend?\n7. Figure 6 has some formatting issues (possibly just due to .pdf conversion).\n8. The phenotype of mbd-2 mutants is more dramatic than other maternal effect sterile mutants like mes-2. However, it does\nfollow a similar generational pattern. The hypothesis that MBD-2 may bind to H3K27me3 would be consistent with the\nmaternal effect sterile (or grandchildless) phenotype of PRC2 mutants. Do the authors agree that the mbd-2 phenotype is a\nmaternal effect sterile phenotype, and if so perhaps it’s worth acknowledging this so as to strengthen the link between these\ntwo repressive pathways?", "reviewer_3": "In their manuscript “Dual genomic localizations and functions of MBD-2 with and without NuRD in DNA methylation-free C.\nelegans”, the authors characterize C. elegans mbd-2, including detailed characterization of its expression pattern, mutant\nphenotypes, structure-function analysis (role of coiled-coil domain), protein interaction partners, and roles in gene regulation.\nDespite its name (Methyl - CpG - Binding - Domain - 2), mbd-2 lacks the methyl-cpg binding domain, but has conserved idr and\ncoiled-coil domains, which are important for interactions with the NuRD complex in other organisms. Whether mbd-2 is\npart of a C. elegans NuRD complex, and how mbd-2 is recruited to chromatin (in absence of CpG methylation) remained\nopen questions. The authors find that mbd-2 is broadly expressed and required for proper development and fertility, as is its\ncoiled coil domain. Through IP-MS, they find that mbd-2 is in a complex with most known NuRD components and with\nknown NuRD-interacting transcription factors. RNA-seq in mbd-2 mutants revealed many misregulated genes (~1/4 of\nprotein-coding genes), with a bias to up-regulation, Notably only a minority (~7%) of mbd-2 ChIP-seq peaks were co-\noccupied by other NuRD components. Rather, the majority of mbd-2-occupied sites are concentrated on chromosome arms\nand often co-localize with H3K27me3 (and H3K9me2), which leads the authors to conclude that mbd-2 has NuRD-\ndependent and NuRD-independent roles. The authors propose a model wherein H3K27me3 functionally substitutes for\nmethylated CpG in recruiting mbd-2 to chromatin.\nThis work provides new insight into a conserved and important chromatin modifying complex, which will make it of interest to\na broad readership. The authors make use of a broad toolkit of approaches (genome engineering, IP-MS, genomics). They\nconvincingly show that mbd-2 is in a complex with NuRD factors, but that the majority of mbd-2 does not colocalize along the\ngenome with NuRD factors. The work highlights the multi-tasking nature of many chromatin regulators, which can have\ndifferent effects depending on the genomic context and/or varying sets of cofactors. The authors speculate that repressive\nhistone modifications, and in particular H3K27me3, may provide an alternative mechanism for recruitment of mbd-2 in\nanimals without 5mC. While the study is overall well done, this last point is somewhat weak as it is based on correlation and\nthere is a lack of statistical analysis or additional experiments to support this conclusion. Overall, the rigor and statistical\nanalysis of the genome-wide analyses as well as the organization of the latter half of the manuscript/figures should be\nimproved prior to acceptance for publication.\nMajor Points:\n1. A more thorough analysis of the “non-NuRD-overlapping” mbd-2 peaks is needed. There is an emphasis on overlap with\nH3K27me3, but it appears that the overlap with H3K9me2 is similarly high (Fig. 5D), or even higher (Fig. S6D). In addition,\nthere does not seem to be much evidence for overlap with H3K9me3, in contrast to statements in the manuscript (e.g. Line\n556) and the model figure. The analysis of the overlap of the “non-NuRD-overlapping” mbd-2 peaks with H3K27me3 or\nH3K9me2/3 (or other histone modifications) should be more robust, and minimally include correlation of the signals, analysis\nof the ‘converse’ relationship (e.g. peaks of those histone marks overlapping mbd-2), and preferably a statistical analysis of\nthe overlaps.\n2. There is an implication that the overlap with H3K27me3 may be ‘specific’ to genomes without 5mC – it is difficult to\nevaluate this without an analysis of the relationship between H3K27me3 and mbd-2 (at “high 5mC site”) in mammals – has\nthis been analyzed? Is there data that could be used to investigate if mbd-2 also co-localizes with H3K27me3 in other\norganisms?\n3. The proposed model that H3K27me3 and/or H3K9me2/3 are involved in recruiting mbd-2 to genomic sites is based on the\nChIP-seq correlations. As noted by the authors (line 806), experiments to address causation (and the directionality of this\nrelationship) would strengthen the conclusions.\n4. Organization of the manuscript, errors, and figure presentation:\na. Ordering: From Fig. 5 onwards, please ensure that the order of the text and figures (and panels within figures) correspond\nto one another. Currently, the figure content is not discussed in sequence, and some panels are not discussed in the text at\nall.\nb. Errors in display panels: In Fig. 6D, it appears that the incorrect data have been plotted in either the left or right panel,\nsince the upper and lower clusters should have identical numbers of rows in the right and left panels. Of note, this panel\n(6D), is not discussed in the text and the trend is different than what is observed for the P0 data (Fig. 6C). In Fig 8D and Fig\nS8B, it appears that panel labels are swapped (the “up”/n= 2262 and “no change”/n=6763 labels on the clusters).\nc. There is unnecessary redundancy in the ChIP-seq heatmaps. For example, the left panels of Fig. 5B, 5C, and 7B (mbd-2\nChIP-seq in 2 clusters) are identical to one another, and the left panel of Fig. 5E is shows 2 clusters that are likely also very\nsimilar to these other 3 panels. These same data are also shown in Fig S6B/C as one cluster instead of 2 clusters. A more\nefficient grouping of heatmaps is warranted.\nd. Please simplify the labels on all multi-cluster heatmaps so that they are easier to understand and a legible font size can\nbe used.\ne. Fig. 7A the y-scale may need to be adjusted for the sta-1 whole genome tracks, as they are dominated by 3 “peak” sites.\nMinor Points:\n5. Title – consider rephrasing “in DNA methylation-free C. elegans” – as written, this could be misinterpreted to mean that C.\nelegans without DNA methylation were “made”, as opposed to that they naturally lack 5mC methylation\n6. Introduction Line 106 – “convergent evolution of 5mC loss” - Please clarify the meaning of and evidence for “convergent\nevolution” in this context.\n7. Line 397, Figure 2 – I think that “bagging” is C. elegans-specific jargon. Please explain or replace with a term that is more\nbroadly understandable.\n8. Expression pattern (Figure 1 and S1) – is described as “ubiquitous” in all stages. It would be helpful to include images of\nother larval stages (in addition to embryo and adults which are shown). There also appears to be minimal expression in the\ndistal germline. The Fig S1 caption also indicates young adults, but some of the animals have many embryos and do not\nappear to be “young”. The HDA-1::GFP in particular have abnormal looking oocytes/embryos as well.\n9. Role in fertility – the mbd-2 mutant animals have very few progeny, however it is unclear how much contribution the egg\nlaying defect has to this outcome, especially in the generation with maternal contribution. An analysis of the\nproliferation/health of the germline and presence of gametes (e.g. by DAPI staining or another method) in these animals\nwould more clearly show the essentiality in fertility.\n10. Line 446: ~half of differentially expressed genes are bound by mbd-2. The significance is not clear, since mbd-2 targets\n~50% of protein-coding genes. Does this overlap represent an enrichment over what is expected by chance?\n11. In the section discussing the RNA-seq results, e.g. Line 553 – “there is not a huge difference in …” , line 568 “seem to be\ndysregulated to a larger extent” – these descriptions would be improved by more formal language and quantitative analysis/\ncomparisons supported by statistical analysis.\n12. Line 528 – citations should be included providing evidence of the histone modification reader activity of the mentioned\ncandidate proteins.\n13. Line 693-4 – “transcribed” should be rephrased, as transcription is not being measured in these assays.\n14. Line 766 – Discussion - “This raises a possibility that Cel-MBD-2 binding to non-NuRD-overlapping targets could be\nnon-essential, as is the case for mammalian MBD2, but it provides conserved survival advantages.” – This sentence is\nsomewhat vague. Please provide a citation for the binding of mbd-2 to non-NuRD targets being non-essential in mammals,\nand please elaborate on what/how “conserved survival advantages” would be provided."}, "author_response": "Below are the 3 reviewer comments, and in blue are our responses:\nReviewer #1 (Remarks to the Author):\nIn this paper, the authors explore the intriguing phenomenon of C. elegans which are\nnaturally deficient in DNA methylation. They point out that the methyl-binding domain\nprotein 2/3 (MBD2/3) ortholog, while losing the methyl-binding domain (MBD), is still\npresent, and they ask if this protein has any conserved functions in worms. They\nshow that worm MBD2/3 retains its conserved role as a scaffolding component of\nNuRD, and demonstrates new functions in recognizing H3K27me3. Overall the study\nis very interesting, well written, and the figures are well made. I do not have any\nsuggestions for improvement, and congratulate the authors on a very interesting and\nwell designed work.\nThank you for your nice comment!\nReviewer #2 (Remarks to the Author):\nThe article “Dual genomic localizations and functions of MBD-2 with and without\nNuRD in DNA methylation-free C. elegans” presents an informative case study in the\nevolution of chromatin regulation. The authors tackle a clear outstanding question\n“what role does MBD-2 have if not through 5mc”? This question is particularly\ninteresting given that 5mc is (parsimoniously) ancestral, yet mbd-2/3 is maintained in\nseveral metazoan lineages that have lost 5mc. The authors perform a suite of\ngenetic and biochemical experiments to address this question, and conclude that\nmbd-2 in C. elegans retains NuRD binding and functon(i.e., phenotype), however, it\nalso has apparently gained NuRD independent chromatin binding (and possibly\nfunction). The two different categories can be distinguished by the presence/absence\nof repressive (non-NuRD) and activating (NuRD +) histone modifications. They end\nby presenting a model in which H3K27me3 replaces 5mc as a mechanism of\nrecruiting MBD-2 to genomic loci.\nOverall, this was an exciting paper that used mechanistic experiments to address a\nbroadly interesting evolutionary question. The data quality, reproducibility and writing\nwere all appropriate for the field. Their result of mbd-2 binding outside of NuRD\nraises interesting questions about what roles it evolved since the loss of 5mc - and\nhow it is recruited in its absence. I look forward to seeing this work in press,\nhowever, I have a few major-revision requests that if completed would substantially\nstrengthen their manuscript and interpretations.\nMajor comments:\n1. The authors note that the CC-deletion phenocopies the mbd-2 knockout\nphenotype. This suggests that the interactions with GATAD2A/B and CHD-3/4/5 are\nthe dominant cause of infertility and vulva development. This is somewhat surprising\nin light of the large number of non-NURD peaks in the Chip-Seq data (93%), and the\nlarge number of genes which are differentially expressed but do not overlap with\nhda-1 and dcp-66 differentially expressed genes. If the CC-deletion phenocopies the\nnull, what is the role of non-NURD mbd-2 binding? The authors note in the\nDiscussion that coiled-coil mutants are too sick to do ChIP with, and I am receptive\nto that. However, an indirect but possible way to get at this question would be RNA-\nseq of their coiled-coil mutant. The differentially expressed genes relative to wildtype,\nand comparison to mbd-2 knockout, should yield insight into the role of NuRD vs.\nnon-NuRD mbd-2 binding. Given that the dual roles of mbd-2 is a major feature of\nthis paper, I would argue that this is a necessary (and again relatively easy)\nexperiment for the authors to do.\nThanks for the great suggestion. We have performed RNAseq of the CC-deletion\nmutant as per reviewer advice (Result section title: The coiled-coil domain\ncontributes to major gene regulation function of C. elegans mbd-2).\nSurprisingly, the loss of coiled-coil domain in MBD-2 shows highly similar\ntranscriptomic profile (Figure 3F, G, Supplementary Figure 5) and PCA profile\n(Supplementary Figure 4G) as the null mutant, consistent with their comparable\nphenotypes (Figure 2B, 3E). In addition, we did not observe a higher number of\ndifferentially expressed genes when comparing the null mutant to CC mutant. This\nsuggest that the coiled-coil domain contributes to the major gene regulation function\nof MBD-2. The implication is discussed in discussion line 883-892 “Theoretically,\nanalysis of MBD-2∆CC may provide a glimpse of MBD-2 binding without NuRD\nremodeling core and potential separation of function. However, RNA-seq analysis of\nthe mbd-2∆CC/∆CC mutant found that deletion of the coiled-coil domain causes similar\ngene dysregulation as the mbd-2 null mutant (Figure 3F,G, Supplementary Figure\n4G, 5A-F). The number and fold change of dysregulated non-NuRD-overlapping and\nNuRD-overlapping genes are similar in mbd-2-/- and mbd-2∆CC/∆CC (Supplementary\nFigure 10C-F). This unexpected result suggests that the CC domain may not only\nbind to NuRD remodeling core, but also contribute to non-NuRD-overlapping MBD-2\nbinding and gene regulation. Alternatively, MBD-2 may bind to these non-NuRD-\noverlapping gene targets to fine-tune their expression. Thus, the minor\ntranscriptional changes in these genes among wildtype, null mutation or ∆CC may\nnot be detected in RNA-seq approach”\nIn addition, this RNA-seq result could also imply that the CC may confer both NuRD\nand non-NuRD target recruitment of MBD-2, which is discussed in line 936-945\n“Particularly, we could focus on those interactors that rely on MBD-2’s CC for\ninteraction in IP-MS for future studies on NuRD-independent recruiter of Cel-MBD-2.\nSince coiled-coil deletion phenocopies null mutation and drives similar transcriptomic\ndysregulation, MBD-2’s CC may contribute to non-NuRD-overlapping genes\nrecruitment. It is further supported by the fact that the proportion of dysregulated\ngenes in P0 mbd-2-/- overlapping with exclusive non-NuRD and NuRD-overlapping\ngenes are highly similar to that of P0 gfp::3xflag::mbd-2∆CC/∆CC (Supplementary\nFigure 10C,D). This suggests that the non-NuRD binding of MBD-2 could be\ncontributed by coiled-coil interaction with certain chromatin factors. If this is the case,\nsuch recruiter may compete with NuRD complex for coiled-coil interaction with MBD-\n2, thus recruiting MBD-2 independently of the NuRD-overlapping binding sites.”\nIn addition, it is worth noting that in the previous study that depleted hda-1 and dcp-\n66 by RNAi, the number of differentially expressed genes were much lower, and the\ndegree of dysregulation is less profound than our deletion/CC mutants of mbd-2.\nFor example, only 1677 genes could be considered as differentially expressed in dcp-66\nRNAi (adjusted p-value < 0.05, fold change >0), while 11058 genes were retained in\nP0 mbd-2-/- with the same filtering criteria (Please refer to the added lines 510-513 in\nResults that explain the larger number of differentially expressed genes compared to\ndcp-66 and hda-1 RNAi). We further focused on comparing the direction of\ndysregulation on these overlapping differentially expressed gene retained and found\nsimilar direction of gene regulation (genes upregulated in dcp-66 and hda-1 are\nupregulated in mbd-2-/- as well as mbd-2CC/CC).\n2. The authors emphasize that the non-NuRD MBD-2 ChIP-signal is enriched over\nH3K27me3. However, in Fig. 5D, the data shows that 45.7% of peaks overlap with\nH3K27me3 peaks. Just slightly less enrichment (41.1%) can be seen with\nH3K9me2. Moreover, as the authors are likely aware, H3K27me3 covers almost half of the\nentire worm genome (Liu et al., 2011). Thus, overlap with H3K27me3 is not that\ndifferent from overlap with all non-actively transcribing regions. When inspecting the\ngenome tracks presented in Figure 5F, there does not appear to be any particular\ncorrespondence of MBD-2 peaks with the broad domains of H3K27me3. When\ninspecting the chromosomal patterns in Fig. 7A, there is clearly a bias of MBD-2 to\nchromosome arms, but what is the evidence for specific enrichment with H3K27me3\ncompared to generally repressed chromatin (also see Major Comment #3 below).\nLooking at Fig. 5C, it is true that the most distinguishing corollary of NuRD vs. non-\nNURD MBD-2 binding is the absence or presence of H3K27me3. While I agree that\nthis is an interesting pattern, the inference is limited to the few histone modifications\nthat were assessed. Without more mechanistic evidence, or at the least, statistical\nsupport for enrichment, the authors should omit this emphasis in their abstract, and\nprobably minimize its emphasis in their Discussion.\nWe agree with the reviewer’s assessment. Accordingly, throughout the manuscript,\nwe have toned down to generally describe MBD-2 as localizing to repressive\nchromatin. We have added other repressive histone marks in genome browser view\n(Figure 6D, Supplementary Figure 8D).\nHowever, H3K27me3 remains the most distinguished histone mark on non-NuRD-\noverlapping peaks but not on NuRD-overlapping peaks (Figure 6B-D), whereas\nH3K9m2/3 enrichment on non-NuRD-overlapping and NuRD-overlapping peaks are\nsimilarly weak (with low fold enrichment over input). In addition, we added\nSupplementary Figure 9A to show that the H3K27me3-overlapping and non-\nH3K27me3-overlapping MBD-2 peaks are depleted and enriched for NuRD complex\ncomponent binding, respectively. This result support that MBD-2 is separately\nrecruited to NuRD- and H3K27me3-containing sites in the genome. Moreover, we\nalso added reciprocal enrichment heatmap of MBD-2 at all H3K27me3, H3K9me2\nand H3K9me3 peak centers (Supplementary Figure 9B-D), and included correlation\nanalysis of various histone marks on non-NuRD-overalapping versus NuRD-\noverlapping MBD-2 gene targets in the new Figure 8B,C and Supplementary Figure\n13. These analyses suggest that H3K9me2 and H3K9me3 have positive correlation\nwith MBD-2 at both non-NuRD-overlapping and NuRD-overlapping gene targets, but\nH3K27me3 is correlated with MBD-2 exclusively at non-NuRD-overlapping gene\ntargets except for instance when H3K36me3 is present (Figure 8B, Supplementary\nFigure 13B, C). Conversely, the non-NuRD-overlapping genes that do not overlap\nwith H3K27me3 is correlated with H3K36me3 (Supplementary Figure 13D).\n3. In general, the bioinformatic enrichment analyses lack statistical support. This is\nespecially important for their interpretations regarding overlap with histone\nmodifications. For example, is the ChIP-seq peak overlap of MBD-2 with H3K27me3\nsignificant relative to broad repressed domains? I would also appreciate more\nmethodological information for RNA-seq, ChIP and IP-MS in the main text: sample\nsize, p value and fold-change cut off, and replicate numbers. Most of these are\ndocumented in the methods, but as they are critical for interpretation, in my opinion\nthey should be incorporated into their Results section as well.\nAs suggested by the reviewer, we have added the sample size, replicate number and\nstatistical cut off for RNAseq (line 453-454, 501-503), IP-MS (line 540-544, 567-569)\nand ChIPseq (line 587-588) data. As mentioned in reply to comment 2, we have now\nincluded correlation analysis of various histone marks on non-NuRD-overlapping vs.\nNuRD-overlapping gene targets in the new Figure 8B,C, Supplementary Figure 13.\nWe also added reciprocal enrichment heatmap of MBD-2 at all H3K27me3,\nH3K9me2 and H3K9me3 peak centers (Supplementary Figure 9B-D)\nMinor comments:\n1. For clarity, could the authors label in Fig. 1C the cell cycles. Also for clarity, could\nthe authors indicate in the legend that strain OD56 has stronger mCherry-H2B\nexpression in the germline.\nThanks for the suggestion. Figure 1C embryo live images have been labelled with\nthe cell cycle stages accordingly and added the statement to Figure 1 legend (line\n1006-1008) “mCherry::H2B has strong germline expression, while\nGFP::3xFLAG::MBD-2 has minimal expression at the transition zone of germline” for\nclarity.\n2. In Figure 2, could the authors please label by the relevant images whether the\nmutant worms were P0 or F1s?\nThanks for the suggestion. We have now labelled both the legend and figures\nwhether the worms are P0 or F1 for enhanced readability.\n3. Could the authors please indicate how many (at least approximately) worms or full\nplates were used as input in the ChIP and IP-MS experiment? It was indeed\nimpressive that they could obtain enough worms from the CC delta strain for IP-MS!\nAccording to reviewer’s major comment 3, we have now moved sample size\ninformation for each experiment into main text. We have added the approximate\nnumber of plates/worms needed for RNA-seq (line 211-213), ChIP (line 247-248) and\nIP-MS (line 341-342, 370-374) in the Materials and Methods.\n4. It is indicated in their Methods section, but can the authors mention what the\nnegative control was for the IP-MS experiment in the Figure 4 legend?\nThe negative control is wildtype N2, with untagged MBD-2. We have included this\ninformation at line 1057-1058 Figure 4(B, E) legend.\n5. Line 516 references Fig. 4FC; as far as I can see there is no figure 4F.\nSorry for the mistake. It should be Fig. 4C only. We have corrected accordingly.\n6. Can the authors please define how Fig. 4C data is “normalized” in the legend?\nWe have now clarified the detailed normalization method in Materials and method\n(line 382-394) “The immunoprecipitation efficiency for the coiled-coil deletion\nmutated protein (MBD-2∆CC) (log iBAQ = 12.80) was lower than for the full-length\n2 2\nSupplementary table 7). To prevent this difference from being misinterpreted as a\nloss of specific protein interactions, a normalization step was performed before\nplotting Figure 4E. The negative control (on the X-axis) log iBAQ values of each\n2\ninteractor were subtracted by the immunoprecipitation efficiency difference (4.52).\nFor defining “confidently lost interactors” (35 interactors highlighted in pink in\nSupplementary Table 7) we select the criteria of (1) being significant interactors in\nGFP::3xFLAG::MBD-2-expressing strain (defined as above), (2) with relative log\n2\niBAQ (log iBAQ in GFP::3xFLAG::MBD-2-expressing strain over that in the negative\n2\ncontrol) greater than the MBD-2 IP efficiency difference between\nGFP::3xFLAG::MBD-2 and GFP::3xFLAG::MBD-2∆CC (4.52), and (3) being\ncompletely undetectable in both replicates of ∆CC IP-MS.”\nThis normalization method is also briefly described in Figure 4E legend (line 1071-\n1075) “The immunoprecipitation efficiency for ∆CC was lower than for the full-length\nMBD-2 protein (Supplementary table 7). To prevent this difference from being\nmisinterpreted as a loss of specific protein interactions, the log iBAQ values of each\n2\ninteractor in the negative control (on the X-axis) were normalized before plotting\n(refer to Materials and methods for the normalization).”\n7. Figure 6 has some formatting issues (possibly just due to .pdf conversion).\nWe have formatted the figure (new Figure 7D, E, Supplementary Figure 11A, B) to\nenhance readability.\n8. The phenotype of mbd-2 mutants is more dramatic than other maternal effect\nsterile mutants like mes-2. However, it does follow a similar generational pattern. The\nhypothesis that MBD-2 may bind to H3K27me3 would be consistent with the\nmaternal effect sterile (or grandchildless) phenotype of PRC2 mutants. Do the\nauthors agree that the mbd-2 phenotype is a maternal effect sterile phenotype, and\nif so perhaps it’s worth acknowledging this so as to strengthen the link between these\ntwo repressive pathways?\nPreviously, the Strome lab has shown that even mes-/+ hermaphrodites produced by\nmating mes-/- mutant mothers with wildtype males are sterile, revealing that maternal\nmes+ gene product is required for fertility1.\nWe have tested this in mbd-2 null mutant by mating the P0 mutant hermaphrodites\nwith wildtype N2 males and found that the heterozygous F1 (mbd-2+/-) are fertile\n(Supplementary Figure 3A), indicating the observed sterile F1/ grandchildless\nphenotype is not strictly “maternal”. Nevertheless, the fact that F1 mbd-2-/- animals\nhave stronger phenotype than P0 mbd-2-/- animals suggests that there may be a\nweak maternal rescue.\nReviewer #3 (Remarks to the Author):\nIn their manuscript “Dual genomic localizations and functions of MBD-2 with and\nwithout NuRD in DNA methylation-free C. elegans”, the authors characterize C.\nelegans mbd-2, including detailed characterization of its expression pattern, mutant\nphenotypes, structure-function analysis (role of coiled-coil domain), protein\ninteraction partners, and roles in gene regulation. Despite its name (Methyl - CpG -\nBinding - Domain - 2), mbd-2 lacks the methyl-cpg binding domain, but has\nconserved idr and coiled-coil domains, which are important for interactions with the\nNuRD complex in other organisms. Whether mbd-2 is part of a C. elegans NuRD\ncomplex, and how mbd-2 is recruited to chromatin (in absence of CpG methylation)\nremained open questions. The authors find that mbd-2 is broadly expressed and\nrequired for proper development and fertility, as is its coiled coil domain. Through IP-\nMS, they find that mbd-2 is in a complex with most known NuRD components and\nwith known NuRD-interacting transcription factors. RNA-seq in mbd-2 mutants\nrevealed many misregulated genes (~1/4 of protein-coding genes), with a bias to up-\nregulation, Notably only a minority (~7%) of mbd-2 ChIP-seq peaks were co-\noccupied by other NuRD components. Rather, the majority of mbd-2-occupied sites\nare concentrated on chromosome arms and often co-localize with H3K27me3 (and\nH3K9me2), which leads the authors to conclude that mbd-2 has NuRD-dependent\nand NuRD-independent roles. The authors propose a model wherein H3K27me3\nfunctionally substitutes for methylated CpG in recruiting mbd-2 to chromatin.\nThis work provides new insight into a conserved and important chromatin modifying\ncomplex, which will make it of interest to a broad readership. The authors make use\nof a broad toolkit of approaches (genome engineering, IP-MS, genomics). They\nconvincingly show that mbd-2 is in a complex with NuRD factors, but that the\nmajority of mbd-2 does not colocalize along the genome with NuRD factors. The\nwork highlights the multi-tasking nature of many chromatin regulators, which can\nhave different effects depending on the genomic context and/or varying sets of\ncofactors. The authors speculate that repressive histone modifications, and in\nparticular H3K27me3, may provide an alternative mechanism for recruitment of mbd-\n2 in animals without 5mC. While the study is overall well done, this last point is\nsomewhat weak as it is based on correlation and there is a lack of statistical analysis\nor additional experiments to support this conclusion. Overall, the rigor and statistical\nanalysis of the genome-wide analyses as well as the organization of the latter half of\nthe manuscript/figures should be improved prior to acceptance for publication.\nMajor Points:\n1. A more thorough analysis of the “non-NuRD-overlapping” mbd-2 peaks is needed.\nThere is an emphasis on overlap with H3K27me3, but it appears that the overlap\nwith H3K9me2 is similarly high (Fig. 5D), or even higher (Fig. S6D). In addition, there\ndoes not seem to be much evidence for overlap with H3K9me3, in contrast to\nstatements in the manuscript (e.g. Line 556) and the model figure. The analysis of\nthe overlap of the “non-NuRD-overlapping” mbd-2 peaks with H3K27me3 or\nH3K9me2/3 (or other histone modifications) should be more robust, and minimally\ninclude correlation of the signals, analysis of the ‘converse’ relationship (e.g. peaks\nof those histone marks overlapping mbd-2), and preferably a statistical analysis of\nthe overlaps.\nWe agree with the reviewer’s assessment. Please see our response to reviewer 2\nmajor comment 2:\nWe agree with the reviewer’s assessment. Accordingly, throughout the manuscript,\nwe have toned down to generally describe MBD-2 as localizing to repressive\nchromatin. We have added other repressive histone marks in genome browser view\n(Figure 6D, Supplementary Figure 8D).\nHowever, H3K27me3 remains the most distinguished histone mark on non-NuRD-\noverlapping peaks but not on NuRD-overlapping peaks (Figure 6B-D), whereas\nH3K9m2/3 enrichment on non-NuRD-overlapping and NuRD-overlapping peaks are\nsimilarly weak (with low fold enrichment over input). In addition, we added\nSupplementary Figure 9A to show that the H3K27me3-overlapping and non-\nH3K27me3-overlapping MBD-2 peaks are depleted and enriched for NuRD complex\ncomponent binding, respectively. This result support that MBD-2 is separately\nrecruited to NuRD- and H3K27me3-containing sites in the genome. Moreover, we\nalso added reciprocal enrichment heatmap of MBD-2 at all H3K27me3, H3K9me2\nand H3K9me3 peak centers (Supplementary Figure 9B-D), and included correlation\nanalysis of various histone marks on non-NuRD-overalapping versus NuRD-\noverlapping MBD-2 gene targets in the new Figure 8B,C and Supplementary Figure\n13. These analyses suggest that H3K9me2 and H3K9me3 have positive correlation\nwith MBD-2 at both non-NuRD-overlapping and NuRD-overlapping gene targets, but\nH3K27me3 is correlated with MBD-2 exclusively at non-NuRD-overlapping gene\ntargets except for instance when H3K36me3 is present (Figure 8B, Supplementary\nFigure 13B, C). Conversely, the non-NuRD-overlapping genes that do not overlap\nwith H3K27me3 is correlated with H3K36me3 (Supplementary Figure 13D).\n2. There is an implication that the overlap with H3K27me3 may be ‘specific’ to\ngenomes without 5mC – it is difficult to evaluate this without an analysis of the\nrelationship between H3K27me3 and mbd-2 (at “high 5mC site”) in mammals – has\nthis been analyzed? Is there data that could be used to investigate if mbd-2 also co-\nlocalizes with H3K27me3 in other organisms?\nThe relationship of MBD2 and histone marks appears to be complicated, since\ndifferent reports in the literature showed inconsistent results. We added the following\nparagraphs in the Discussion (lines 898-908).\n“The relationship of mammalian MBD2 with histone modifications is ambiguous2-5. A\nstudy found that the majority of regions bound by MBD2 are enriched with the\nrepressive histone mark H3K9me3 in mouse hippocampus2, but the same could not\nbe observed in MBD2 ChIP-seq studies in HeLa5 and MCF-7 (breast cancer) cells4.\nThere seems to be no consensus on whether repressive histone marks are enriched\nin mammalian MBD2 binding sites.\nMoreover, the relationship between 5mC DNA methylation and histone modifications,\nparticularly the repressive histone mark H3K27me3, in mammals remains\ncontroversial. For example, a previous study found that H3K27me3 colocalize\nextensively with 5mC outside of CpG islands in both human and mouse cells but are\nmutually exclusive in CpG islands6. However, a more recent studies report that\nH3K27me3 negatively regulates 5mC methylation via inhibiting H3 ubiquitination\nacross the genome7.”\n3. The proposed model that H3K27me3 and/or H3K9me2/3 are involved in recruiting\nmbd-2 to genomic sites is based on the ChIP-seq correlations. As noted by the\nauthors (line 806), experiments to address causation (and the directionality of this\nrelationship) would strengthen the conclusions.\nWe appreciate the reviewer's emphasis on the importance of the causational\nrelationship with MBD-2 localization. We have seriously considered these\nexperiments and agree they would be highly valuable. However, there are some\ntechnical challenges. Depletion of the writers for key repressive marks (e.g., MES-\n2/3/6 components of the PRC2 complex for H3K27me) often leads to germline\ndysfunction in C. elegans. These mutants are often maintained with balancer,\nmaking it difficult to obtain a large population of homozygous mutant animals for\nChIP-seq experiments. Reciprocally, it is also difficult to test whether MBD-2 affects\nH3K27me3, because MBD-2 itself is essential for fertility as well. It may be possible\nto use feeding RNAi or inhibitors to knockdown or inhibit histone methyltransferases,\nbut it is uncertain whether the remaining activities of the enzymes will mask the\neffects on MBD-2. Besides ChIP-seq, the CUT&RUN chromatin profiling may provide\nhigher sensitivity and thus require a smaller amount of materials. It will take\nconsiderable time and efforts to optimize those methods for the current study, which\nwill significantly delay the publication of the paper. So, we consider proving the\ncausal relationship between histone modification and MBD-2 recruitment beyond the\nscope of the study. However, we like to pursue this direction in the future and also\ntest the physical interactions between MBD-2 and candidates that may\nrecruit/interact with H3K27me3 and H3K9me2.\nConsidering the above limitations, we have toned down the statements regarding the\npotential causational relationship of repressive marks with MBD-2 and simply\nreported the observed correlations in the revised manuscript. From this aspect, we\nhope the reviewer agrees that our current findings are still complete and compelling\nto provide a solid foundation for future investigations.\n4. Organization of the manuscript, errors, and figure presentation:\na. Ordering: From Fig. 5 onwards, please ensure that the order of the text and\nfigures (and panels within figures) correspond to one another. Currently, the figure\ncontent is not discussed in sequence, and some panels are not discussed in the text\nat all.\nThanks for the careful observation. We have performed major rearrangement on the\nfigures, supplementary tables and the main text to ensure that they are all discussed\nin corresponding order.\nb. Errors in display panels: In Fig. 6D, it appears that the incorrect data have been\nplotted in either the left or right panel, since the upper and lower clusters should\nhave identical numbers of rows in the right and left panels. Of note, this panel (6D),\nis not discussed in the text and the trend is different than what is observed for the P0\ndata (Fig. 6C).\nThanks for the detailed observation. The seemingly incorrectness was due to the fact\nthat during scaling of the gene body region, a significant number of genes are\nskipped due to gene length limit. We have now adjusted the parameter for this panel\nto no scaling, so the number of rows is equal (Supplementary Figure 11B).\nIn Fig 8D and Fig S8B, it appears that panel labels are swapped (the “up”/n= 2262\nand “no change”/n=6763 labels on the clusters).\nThanks for the correction. For simplicity, we now display only the line plot in the\npanels (Supplementary Figure 12) and removed the heatmap.\nc. There is unnecessary redundancy in the ChIP-seq heatmaps. For example, the\nleft panels of Fig. 5B, 5C, and 7B (mbd-2 ChIP-seq in 2 clusters) are identical to one\nanother, and the left panel of Fig. 5E is shows 2 clusters that are likely also very\nsimilar to these other 3 panels. These same data are also shown in Fig S6B/C as\none cluster instead of 2 clusters. A more efficient grouping of heatmaps is warranted.\nThank you for the detailed observation and suggestion. We also agree that there is\nsome redundancy. So, we have simplified by showing the NuRD-overlapping peaks\nare manually classified with the overlap with NuRD components (Figure 5B). To\nverify the manual clustering result, we showed the 2 cluster heatmap (by\nunsupervised clustering with NuRD components) (Supplementary Figure 8C). This\nverified the depletion of H3K27me3 and enrichment of active histone marks on\nNuRD-overlapping peaks. We have deleted the heatmap displaying all MBD-2\npeaks.\nd. Please simplify the labels on all multi-cluster heatmaps so that they are easier to\nunderstand and a legible font size can be used.\nThanks, we have increased the font size to at least 6 for all heatmaps. In addition,\nthe labels of the current Figure 8A and the related Figure S12 has now been\nsimplified.\ne. Fig. 7A the y-scale may need to be adjusted for the sta-1 whole genome tracks,\nas they are dominated by 3 “peak” sites.\nThanks for the advice. The 3 “peak” sites display was due to the fact that when we\ndisplayed all chromosome together in a condensed view, the autoscale function of all\nsix chromosomes together leads to the reduction of peaks displayed. To accurately\ndisplay the enrichment along each chromosome, we decided to show all\nchromosome one by one with their own autoscale for each track (new Figure 6A).\nMinor Points:\n5. Title – consider rephrasing “in DNA methylation-free C. elegans” – as written, this\ncould be misinterpreted to mean that C. elegans without DNA methylation were\n“made”, as opposed to that they naturally lack 5mC methylation\nThanks for pointing out the potential confusion, we have revised to “Caenorhabditis\nelegans which lacks DNA methylation”.\n6. Introduction Line 106 – “convergent evolution of 5mC loss” - Please clarify the\nmeaning of and evidence for “convergent evolution” in this context.\nWe have elaborated the statement by adding the following explanation to the\nintroduction line 108-110 “While basal members of marine nematodes8 and most\ninsects9 retain functional 5mC methylation, the loss of 5mC has occurred\nindependently in derived nematodes and insects, representing a striking case\nof convergent evolution.”\n7. Line 397, Figure 2 – I think that “bagging” is C. elegans-specific jargon. Please\nexplain or replace with a term that is more broadly understandable.\nThanks, we have now explained it in results “in which embryos that cannot be laid\nhatch inside the uterus and cause lethality to the mothers” (line 429-430) to show\nwhat the “bagging” phenotype means.\n8. Expression pattern (Figure 1 and S1) – is described as “ubiquitous” in all stages. It\nwould be helpful to include images of other larval stages (in addition to embryo and\nadults which are shown).\nThanks for the suggestion, we now added Supplementary Figure 1 to show MBD-2’s\nnuclear expression in L1 and L4.\nThere also appears to be minimal expression in the distal germline. The Fig S1\ncaption also indicates young adults, but some of the animals have many embryos\nand do not appear to be “young”. The HDA-1::GFP in particular have abnormal\nlooking oocytes/embryos as well.\nThanks for the careful observation, the HDA-1::GFP figure is replaced. The adults\nwere day 1 adults which were obtained by maturing L4 worms overnight at 20ºC.\n9. Role in fertility – the mbd-2 mutant animals have very few progeny, however it is\nunclear how much contribution the egg laying defect has to this outcome, especially\nin the generation with maternal contribution. An analysis of the proliferation/health of\nthe germline and presence of gametes (e.g. by DAPI staining or another method) in\nthese animals would more clearly show the essentiality in fertility.\nThe cause of sterility of mbd-2 mutants is still unclear, but not fully caused by egg-\nlaying defect or protruding vulva. We have observed complete sterility in F1 null\nmutant compared to P0, but bagging was not observed in F1 null mutant as they did\nnot even produce any embryos in their bodies. We observed germ cells in the small\nP0 mutants, yet the mutant body size and germline is smaller compared to wildtype,\nresulting in difficulty in dissection. In addition, RNAi of mbd-2 results in a highly\npenetrant reduction in brood size in the F1 generation (Supplementary Figure 3B),\nyet almost all of these F1 worms were morphologically wildtype. Therefore egg-\nlaying defect related to bagging or protruding vulva is unlikely the major cause for\nsterility. The fertility of P0 mothers injected with dsRNA at L4 stage was surprisingly\nunaffected. In contrast, F1 were not injected with dsRNA, but have reduced brood\nsize. Therefore, Cel-mbd-2 could be important for fertility before L4 stage.\n10. Line 446: ~half of differentially expressed genes are bound by mbd-2. The\nsignificance is not clear, since mbd-2 targets ~50% of protein-coding genes. Does\nthis overlap represent an enrichment over what is expected by chance?\nThe enrichment of MBD-2 binding genes in P0 differentially expressed genes is\nminimal, whereas the enrichment is not significant in F1. We have now added the\nstatistical analysis in line 708-710 “Surprisingly, the differentially expressed genes in\nP0 (1.03-fold; hypergeometric p-value = 0.00076) and F1 (1.01-fold; hypergeometric\np-value = 0.23) are not enriched for MBD-2 binding, compared to expectation”. This\nmay suggest that MBD-2 has additional roles beyond strong transcription regulation\nupon gene binding, for example fine-tuning of gene expression.\n11. In the section discussing the RNA-seq results, e.g. Line 553 – “there is not a\nhuge difference in …” , line 568 “seem to be dysregulated to a larger extent” – these\ndescriptions would be improved by more formal language and quantitative analysis/\ncomparisons supported by statistical analysis.\nThanks for the advice. Wordings have been revised to point to the quantitative\nanalysis and statistical analysis in line 744-746 “The binding upstream of the TSS is\nslightly higher in the exclusive NuRD-overlapping gene targets (~1.35 fold\nenrichment over input) compared to exclusive non-NuRD-overlapping gene targets\n(~1.21 fold).”\n12. Line 528 – citations should be included providing evidence of the histone\nmodification reader activity of the mentioned candidate proteins.\nWe have included the citations in the following sentence (line 959-968).\n13. Line 693-4 – “transcribed”) should be rephrased, as transcription is not being\nmeasured in these assays.\nThanks, the sentence is removed and the related sentences are all rephrased into\n“expression”.\n14. Line 766 – Discussion - “This raises a possibility that Cel-MBD-2 binding to non-\nNuRD-overlapping targets could be non-essential, as is the case for mammalian\nMBD2, but it provides conserved survival advantages.” – This sentence is somewhat\nvague. Please provide a citation for the binding of mbd-2 to non-NuRD targets being\nnon-essential in mammals, and please elaborate on what/how “conserved survival\nadvantages” would be provided.\nThe non-essential function of non-NuRD targets is studied in mouse indirectly\nthrough comparing MBD2 and MBD3 (which lacks the MBD domain). Specifically,\nMBD3 is essential for stem cell differentiation and survival of the organism, but\nMBD2 is non-essential. Interestingly, overexpressing MBD2 could rescue the\ndifferentiation defect in mbd3-/- mESCs10. This suggest that the essential function\ncomes from the NuRD interacting domains shared by these two paralogs but not the\nMBD domain. On the other hand, MBD2 contribute to mouse nurturing behavior and\nautism gene regulation, which may confer cognitive ability but are not essential to the\norganism2,10. This also matches the fact that higher organisms possess 5mC\nmethylation. We have added the discussion and citations to the introduction (line 42-\n56) and discussion (line 873-882).\nReferences:\n4 and mes-6, maternal-effect genes required for survival of the germline in\nCaenorhabditis elegans, is sensitive to chromosome dosage. Genetics 148,\n167-185 (1998).\nof autism-risk genes and cognition. Translational Psychiatry 13, 259 (2023).\n-Independent Genomic Targeting Principles of the MBD Protein Family. Cell\n153, 480-492 (2013).\nfor highly methylated loci. PLoS One 9, e99603 (2014).\nislands, active promoters and binding to exon sequences. Nucleic Acids Res\n41, 3010-3021 (2013).\ngenome-scale investigation of chromatin and DNA methylation cross-talk.\nGenome Res 22, 1128-1138 (2012).\nmediated H3 ubiquitination. Sci China Life Sci 65, 1685-1700 (2022).\na byproduct of cytosine DNA methyltransferase activity. Nat Genet 50, 452-\n459 (2018).\nMethylation across Insects. Mol Biol Evol 34, 654-665 (2017).\nregulating NuRD complex function during cellular differentiation. Nature\nCommunications 14, 3848 (2023)."}, {"round": 2, "reviewer_comments": {"reviewer_2": "The revised version of the manuscript addressed Major Comment #1: Does the cc-mutant exhibit a distinct transcriptional effect from the full deletion. The authors conducted the requested additional experiment and came to the conclusion that the transcriptional profiles are highly similar between the cc-mutant and the full mbd-2 knockout. These data put some cold water on the possibility of non-NuRD MBD-2 function, unless these functions are also mediated by the cc-domain. Both possibilities are addressed in the Discussion section. I found these data and analyses useful, and believe the field will appreciate it, even if they are somewhat confusing.\nRegarding Major Comment #2: The model of H3K27me3 and non-NuRD recruitment; the authors adequately toned down the discussion of this model given the lack of direct evidence for it. I would argue that it still gets too much coverage, but they appropriately indicate that it remains speculative at this point. I have one remaining sticky point here though, the title of the relevant Results section is \"Transcription factor-Independent recruitment of mbd-2 to repressive chromatin\". Logically however, all they have shown is an absence of evidence for transcription factor recruitment, which is not evidence of absence. Indeed, the authors conclude in the Discussion that other proteins like TFs are likely responsible for chromatin recruitment - I recommend that the authors modify the title of the Results section to reflect that.\nRegarding Major Comment #3: The authors did a great job addressing the presentation and description of bioinformatic analyses. The manuscript is much stronger as a result.\nIn summary, I am satisfied with the revised manuscript and think it will be a valuable addition to the field.", "reviewer_3": "In their substantially revised manuscript and response, the authors have addressed the concerns raised in the previous reviews very well. I do not have any major comments.\nMinor points:\n1. I think there may be a small nomenclature error for the mbd-2 allele expressing GFP-3xflag-tagged mbd-2. According to the methods, this is a CRISPR/Cas9-mediated knock-in. However, the designation in Table S1 contains “Si” (hkuSi10[mbd-2p::GFP::TEV::FLAG::mbd-2, I:6064091]), which would generally be used for a transgene. I think this allele should instead have a non-“Si” allele designation, in accordance with C. elegans nomenclature guidelines (https://wormbase.org/about/userguide/nomenclature#gi9m5c264k8be0afldh71j3--10 under Genome Engineering).\n2. Introduction line 50-51: “This suggests that MBD2 has evolved to acquire domains conferring 5mC affinity” – this statement sounds at odds with the earlier description of the evolution of mbd genes and their duplication. I think that the summary of previous work suggested that the ancestral gene had an MBD, which would mean that the non-MBD2 genes lost the MBD, rather than MBD2 gained an MBD?\n3. Line 637 subtitle “Transcription factor-independent recruitment of Cel-MBD-2 to repressive chromatin” – I suggest rephrasing this subtitle, as it is difficult to provide evidence for a negative statement such as this, and recruitment requirements have not been directly tested.\n4. Line 758 subtitle “H3K27me3 exclusively correlates with MBD-2 binding at non-NuRD-overlapping MBD-2 gene targets” – As written, this title seems to say that all H3K27me3 (in the genome) is at MBD-2 sites, whereas I believe the intended meaning is that H3K27me3 correlates with the non-NuRD-overlapping and not with the NuRD-overlapping MBD-2 sites.\n5. Line 810 Discussion “Surprisingly, not all MBD-2-bound gene targets are dysregulated.” – I think this statement can be removed as this seems typical and there is not a justification for why it is not expected."}, "author_response": "RESPONSES TO REVIEWERS' COMMENTS\nReviewer #2 (Remarks to the Author):\nThe revised version of the manuscript addressed Major Comment #1: Does the cc-\nmutant exhibit a distinct transcriptional effect from the full deletion. The authors\nconducted the requested additional experiment and came to the conclusion that the\ntranscriptional profiles are highly similar between the cc-mutant and the full mbd-2\nknockout. These data put some cold water on the possibility of non-NuRD MBD-2\nfunction, unless these functions are also mediated by the cc-domain. Both\npossibilities are addressed in the Discussion section. I found these data and\nanalyses useful, and believe the field will appreciate it, even if they are somewhat\nconfusing.\nThank you for the comments.\nRegarding Major Comment #2: The model of H3K27me3 and non-NuRD recruitment;\nthe authors adequately toned down the discussion of this model given the lack of\ndirect evidence for it. I would argue that it still gets too much coverage, but they\nappropriately indicate that it remains speculative at this point. I have one remaining\nsticky point here though, the title of the relevant Results section is \"Transcription\nfactor-Independent recruitment of mbd-2 to repressive chromatin\". Logically\nhowever, all they have shown is an absence of evidence for transcription factor\nrecruitment, which is not evidence of absence. Indeed, the authors conclude in the\nDiscussion that other proteins like TFs are likely responsible for chromatin\nrecruitment - I recommend that the authors modify the title of the Results section to\nreflect that.\nThe Results section subtitle has been toned down into “Localization Cel-MBD-2 to\nrepressive chromatin in the absence of other NuRD components and transcription\nfactors”.\nRegarding Major Comment #3: The authors did a great job addressing the\npresentation and description of bioinformatic analyses. The manuscript is much\nstronger as a result.\nThank you for the compliment.\nIn summary, I am satisfied with the revised manuscript and think it will be a valuable\naddition to the field.\nThank you for the positive feedback!\nReviewer #3 (Remarks to the Author):\nIn their substantially revised manuscript and response, the authors have addressed\nthe concerns raised in the previous reviews very well. I do not have any major\ncomments.\nMinor points:\n1. I think there may be a small nomenclature error for the mbd-2 allele expressing\nGFP-3xflag-tagged mbd-2. According to the methods, this is a CRISPR/Cas9-\nmediated knock-in. However, the designation in Table S1 contains “Si”\n(hkuSi10[mbd-2p::GFP::TEV::FLAG::mbd-2, I:6064091]), which would generally be\nused for a transgene. I think this allele should instead have a non-“Si” allele\ndesignation, in accordance with C. elegans nomenclature guidelines\n(https://wormbase.org/about/userguide/nomenclature#gi9m5c264k8be0afldh71j3--10\nunder Genome Engineering).\nThank you for pointing this out. CRISPR/Cas9-mediated knock-in or knock-out\ngenotypes are now changed to hku10[mbd-2p::GFP::TEV::FLAG::mbd-2, I:6064091],\nand hku11[C27A12.10::exon 1 STOP/ hT2[bli-4(e937) let-?(q782) qIs48] in Table S1.\n2. Introduction line 50-51: “This suggests that MBD2 has evolved to acquire domains\nconferring 5mC affinity” – this statement sounds at odds with the earlier description\nof the evolution of mbd genes and their duplication. I think that the summary of\nprevious work suggested that the ancestral gene had an MBD, which would mean\nthat the non-MBD2 genes lost the MBD, rather than MBD2 gained an MBD?\nThanks. We agree with the reviewer and has rewritten the statement “This suggests\nthat MBD2 has retained the conserved MBD domain that confer 5mC affinity”\n3. Line 637 subtitle “Transcription factor-independent recruitment of Cel-MBD-2 to\nrepressive chromatin” – I suggest rephrasing this subtitle, as it is difficult to provide\nevidence for a negative statement such as this, and recruitment requirements have\nnot been directly tested.\nThe Results section subtitle has been toned down into “Localization Cel-MBD-2 to\nrepressive chromatin in the absence of other NuRD components and transcription\nfactors”.\n4. Line 758 subtitle “H3K27me3 exclusively correlates with MBD-2 binding at non-\nNuRD-overlapping MBD-2 gene targets” – As written, this title seems to say that all\nH3K27me3 (in the genome) is at MBD-2 sites, whereas I believe the intended\nmeaning is that H3K27me3 correlates with the non-NuRD-overlapping and not with\nthe NuRD-overlapping MBD-2 sites.\nThanks for pointing this out. Now the title of the Results section subtitle has been\ncorrected to “H3K27me3 correlates specifically with non-NuRD-overlapping MBD-2\ngene targets, but not with NuRD-overlapping MBD-2 gene targets”.\n5. Line 810 Discussion “Surprisingly, not all MBD-2-bound gene targets are\ndysregulated.” – I think this statement can be removed as this seems typical and\nthere is not a justification for why it is not expected.\nThanks, the statement has been removed."}]

Decision letter