Cell type-specific functions of the PBAF chromatin-remodeling complex in neuronal diversification

Domain: biology
Published: 2025-12-14
Editorial outcome: major_revision

Abstract
Mutations in two highly related chromatin-remodeling complexes, BRG1/BRM-associated factor (BAF) and Polybromo-associated BAF (PBAF), cause various neurodevelopmental conditions. Although BAF activity is required at multiple steps of nervous system development, the specific neural functions of PBAF remain largely unexplored. Through an unbiased genetic screen in C. elegans, we identify here critical roles for PBAF in postmitotic neuronal identity. In nerve cord motor neurons, PBAF represses gene expression by antagonizing the terminal selector-type transcription factor UNC-3 (EBF1-4). In contrast, PBAF activates gene expression in caudal motor neurons. This dichotomy in PBAF function generates distinct motor neuron identities. PBAF disruption leads to locomotion defects. Genetic, genomic, and biochemical data suggest that the cell type-specific functions of PBAF in different motor neuron groups arise via its recruitment to specific gene loci by conserved transcription factors, such as MAB-9/TBX20. Altogether, our findings provide a conceptual framework to understand specific neuronal defects that arise in neurological conditions caused by mutations in broadly expressed chromatin-remodeling complexes.

Introduction
Nervous systems are strikingly complex structures composed of numerous types of neurons and glia. Neuronal types have been traditionally defined based on anatomical and electrophysiological criteria1,2. Single-cell RNA sequencing (scRNA-Seq) has recently revolutionized neuronal classification efforts by generating molecular profiles of individual neurons3–7. Studies in model organisms (e.g., nematodes, fruit flies, zebrafish, and mice) have revealed a pervasive organizational principle: the existence of distinct neuronal types that share a number of traits (e.g., neurotransmitter usage) but can be further subdivided into subtypes based on anatomical, functional, or molecular criteria. In C. elegans, anatomically defined neuronal types can be molecularly subdivided into distinct subtypes5,8, and this is the case in every systematically studied component of animal nervous systems, such as the D. melanogaster optic lobe9,10, the mouse cortex11,12 and the mouse retina13. Given the ubiquity of neuronal subtype diversity, probing its underlying mechanisms is a key step in understanding neuronal development, function, and evolution.

To date, known molecular mechanisms underlying neuronal subtype diversification primarily involve transcription factors (TFs). At the level of neural progenitor cells, studies in the fly ventral nerve cord and optic lobe uncovered the concept of temporal TFs, whose sequential activation and cross-repressive interactions diversify neural progenitor cells, and thereby their postmitotic neuronal progeny14,15. TF-based mechanisms also operate in the vertebrate neural tube, where morphogen gradients determine TF expression in neural progenitors, ultimately generating distinct neuronal types and subtypes16–19. While studies like these enrich our understanding of diversification at the level of neural progenitors, much less is known about the transcriptional mechanisms that operate in post-mitotic neurons to diversify neuronal fate.

Terminal selectors are TFs that act in post-mitotic neurons20,21. They control the identity of individual neuron types and subtypes (e.g., sensory, inter-, and motor neurons) by directly activating the transcription of key effector genes, termed “terminal identity genes”. These encode proteins essential for neuronal function, such as neurotransmitter biosynthesis proteins, ion channels, and neuropeptides. To date, terminal selectors have been described in nematodes (C. elegans), fruit flies (Drosophila), cnidarians (Nematostella vectensis), marine chordates (Ciona robusta), and mice (Mus musculus), indicating a deeply conserved role for these critical regulators of neuronal identity20,21. For example, in the mouse brainstem, the terminal selector Pet-1 controls the identity of serotonergic neurons by activating scores of terminal identity genes22,23. Similarly, in the mouse midbrain, the terminal selector Nurr1 controls the identity of dopaminergic neurons24. As with most neuron types, serotonergic and dopaminergic neurons are heterogeneous populations of cells that are further subdivided into distinct subtypes based on anatomical, functional, and molecular criteria25,26. However, the precise mechanisms through which Pet-1 and Nurr1, respectively, control the identity of individual subtypes of serotonergic and dopaminergic neurons remain unclear.

These two examples highlight a conceptual problem broadly applicable to most neuron types: a common TF (e.g., terminal selector) specifies a group of neurons with shared features, but additional gene regulatory mechanisms must exist to diversify those neurons into subtypes (Fig. 1a). Understanding these mechanisms is biomedically relevant because mutations and variants in dozens of human terminal selector orthologs (e.g., EBF3, OTX2, FEV, and NURR1) cause neurodevelopmental and neurodegenerative disorders27–32.

The inherent complexity of vertebrate nervous systems poses a significant challenge in efforts to uncover molecular mechanisms that control neuronal diversification. On the other hand, unbiased genetic screens in more tractable organisms, such as fruit flies and nematodes, have revealed a broadly applicable gene regulatory principle: TFs with either activator or repressor activities are expressed in specific neuronal subtypes to either collaborate with or counteract a common TF (terminal selector) shared by all subtypes of a given neuronal class, thereby generating unique subtype identities (Fig. 1a). For example, the terminal selector CHE-1 controls the identity of a bilateral pair of chemosensory neurons (ASEL/R) in C. elegans33,34. CHE-1 directly activates scores of terminal identity genes expressed in both ASE neurons. However, ASE diversification into ASEL and ASER subtypes occurs via the activity of distinct, subtype-specific TFs that can act as activators or repressors of subtype-specific terminal identity genes35. Similar TFs that either counteract or collaborate with a terminal selector have been identified in C. elegans touch receptors and motor neurons36–38, as well as in D. melanogaster photoreceptors39,40. Altogether, our current knowledge of neuronal diversification principles that operate in post-mitotic neurons is primarily based on two types of TFs: (a) terminal selectors that act in all subtypes of a given neuron type, and (b) additional TFs that act either as activators or repressors in select subtypes. However, the roles of epigenetic mechanisms, such as DNA methylation, histone modifications, and chromatin remodeling, in generating neuronal subtype diversity remain poorly understood.

In this study, we investigate the mechanisms of neuronal diversification in C. elegans cholinergic motor neurons (MNs) that control locomotion. We recently showed that these MNs are a heterogeneous population of cells subdivided molecularly into at least 22 subtypes, referred to as “subclasses” herein (Fig. 1b–d)8. These MNs also share several features, such as connectivity to muscle and use of the same neurotransmitter (acetylcholine, ACh) (Fig. 1b). They also share the same terminal selector, UNC-3, the sole C. elegans ortholog of the Collier/Olf/Ebf (COE) family of TFs41,42. Mutations in EBF3, the human ortholog of UNC-3, cause a neurodevelopmental syndrome characterized by intellectual disability and motor developmental delay30–32,43. It is known that UNC-3 acts directly to activate expression of terminal identity genes that are shared among all MN subclasses (e.g., those encoding ACh biosynthesis proteins), as well as terminal identity genes with subclass-specific expression (e.g., encoding ion channels and neuropeptides) (Fig. 1c)44. However, UNC-3 does not operate alone. At the level of post-mitotic MNs, additional conserved TFs have been identified to enable cholinergic MN diversification. These fall into two categories: (a) TFs that collaborate with UNC-3, such as the HOX proteins LIN-39, MAB-5, and EGL-5, to co-activate subclass-specific terminal identity genes (Fig. 1d)44, and (b) repressor TFs, such as the T-box ortholog MAB-9 and ARID3 ortholog CFI-1, that antagonize the ability of UNC-3 to broadly activate terminal identity genes in all MN subclasses (Fig. 1d)37,38. Altogether, the terminal selector UNC-3 and additional TFs (activators and repressors) act directly by recognizing their cognate DNA binding sites at the cis-regulatory regions of specific genes, and thereby generate distinct MN subclass identities (Fig. 1a, d).

Using an unbiased genetic screen in C. elegans, we identified here a crucial role for the polybromo-associated BRG1-associated factor (PBAF) chromatin-remodeling complex in MN diversification. PBAF belongs to the highly conserved SWI/SNF family of multi-subunit chromatin-remodeling complexes, mutations in which cause cancer and neurodevelopmental disorders such as Coffin–Siris syndrome, Nicolaides-Baraitser syndrome, Kleefstra syndrome, and autism spectrum disorders45,46. Despite its broad expression across tissues, PBAF exerts specific and strikingly different effects on post-mitotic MNs depending on cell body position. In nerve cord MNs, PBAF antagonizes the terminal selector UNC-3 by repressing UNC-3-dependent terminal identity genes. In posteriorly located (caudal) MNs, PBAF collaborates with UNC-3 to activate gene expression and thereby diversify these cells from their nerve cord counterparts. Mechanistically, we propose that the different effects of PBAF are due to the activity of distinct TFs in nerve cord (MAB-9/TBX20) and caudal (EGL-5/HOX) MNs. Altogether, our findings illuminate the epigenetic mechanisms that drive neuronal diversification by uncovering cell type-specific roles for an ancient and broadly expressed chromatin-remodeling complex.

Results
Defining MN diversity in the C. elegans ventral nerve cord
Based on distinct axodendritic morphologies, the MNs that populate the C. elegans ventral nerve cord (VNC) and its flanking ganglia (retrovesicular ganglion [RVG], preanal ganglion [PAG]) are divided into nine cardinal classes (SAB, DA, DB, DD, VA, VB, VC, VD, and AS) in hermaphrodite animals47. The number of neurons for each class ranges from 3 (SAB) to 13 (VD), totaling 78 cells (SAB=3 neurons, DA=9, DB=7, DD=6, VA=12, VB=11, VC=6, VD=13, AS=11) (Fig. 1b). These classes differ in cell body position and muscle innervation: (a) SAB neurons innervate head muscle, (b) VC neurons innervate vulva muscle essential for egg laying, and (c) MNs of the remaining seven classes (AS, DA, DB, DD, VA, VB, and VD) innervate body wall muscles essential for sinusoidal locomotion. Except for the SAB and VC neurons, cell bodies of the remaining seven classes (DA, DB, DD, VA, VB, VD, and AS) intermingle along the VNC and its flanking ganglia (RVG, PAG) (Fig. 1b).

Based on neurotransmitter usage, seven MN classes are cholinergic (SAB, DA, DB, VA, VB, VC, and AS) and two are GABAergic (DD and VD)47. These seven cholinergic classes share a number of molecular features, such as expression of acetylcholine (ACh) biosynthesis genes (unc-17/VAChT and cho-1/ChT), neurotransmitter receptors (e.g., acr-2/CHRNA3/6), and ion channels (e.g., slo-2) (Fig. 1c). These features are not shared by the two GABAergic classes (DD, VD), which also express distinct sets of genes (e.g., unc-25/GAD and unc-47/VGAT)48. Through single-cell RNA-Sequencing (scRNA-Seq), we recently uncovered a striking degree of molecular diversity within cardinal MN classes in the adult8. For example, the five cholinergic MN classes (DA, DB, VA, VB, and AS), which control locomotion and express the terminal selector unc-3/EBF, can be further subdivided into 22 subclasses based on gene expression (DA subclasses: DA1, DA2-5, DA6-8, and DA9; DB subclasses: DB1, DB2, and DB3-7; VA subclasses: VA1, VA2, VA3-8, VA9-10, VA11, and VA12; VB subclasses: VB1, VB2, VB3, VB4-9, and VB10-11; AS subclasses: AS2-3, AS4-8, AS9-10, and AS11) (Fig. 1c). For the current study, we re-analyzed our scRNA-Seq dataset and found that the head muscle-innervating SAB neurons, which also require unc-3/EBF for their terminal identity, display a distinct molecular signature, albeit their limited representation in the dataset prevented possible identification of SAB subclasses (Fig. 1c, Supplementary Fig. 1). Altogether, both anatomical and molecular studies have firmly established that C. elegans MNs are remarkably diverse, offering a powerful platform to uncover molecular mechanisms of neuronal subclass diversification.

A genetic screen for regulators of MN diversity
The terminal selector UNC-3/EBF is expressed in six classes (SAB, DA, DB, VA, VB, and AS) of cholinergic MNs (Fig. 1d). Mechanistically, UNC-3 acts directly to activate expression of shared (e.g., unc-17/VAChT and cho-1/ChT) and subclass-specific terminal identity genes42,49. Our previous work showed that additional TFs act either as activators (e.g., LIN-39/Hox and MAB-5/Hox) or repressors (e.g., CFI-1/ARID3 and MAB-9/TBX20), thereby generating distinct signatures of terminal identity gene expression in individual MN subclasses37,50 (Fig. 1b–d). However, it remains unknown whether and how epigenetic mechanisms (e.g., DNA or histone modifications, chromatin remodeling) influence the activity of UNC-3, thereby dictating MN diversity.

To address this knowledge gap, we leveraged an available genetic tool that specifically labels a subset of unc-3-expressing MNs41. A transgenic red fluorescent reporter for glr-4 (glr-4prom::tagRFP), ortholog of human GRIK4 (glutamate receptor, ionotropic, and kainite 4) is expressed in SAB neurons (RVG ganglion) and the DA9 subclass (PAG ganglion) in an unc-3-dependent manner (Fig. 2a)41, partially recapitulating endogenous glr-4 expression (Fig. 1c)38. Our scRNA-Seq data show that glr-4 transcripts are detected at high levels in MNs located outside the ventral nerve cord (e.g., SAB and DA9) (Fig. 1c). Within the ventral nerve cord, glr-4 transcripts are normally expressed at low (basal) levels, or are undetectable in MNs. This pattern of expression is consistent with our recent study, where we analyzed the expression of the endogenous mScarlet::glr-4 reporter with single-cell resolution38. Nevertheless, the restricted expression pattern of the glr-4prom::tagRFP reporter offers an opportunity to conduct a genetic screen for mutants that display ectopic glr-4prom::tagRFP expression in MN subclasses located in the VNC, and thereby identify—in an unbiased manner—regulatory factors that prevent UNC-3 from activating glr-4 in VNC subtypes (Fig. 2a)38.

Following random ethylmethanesulfonate [EMS] mutagenesis of the P0 generation, we screened F2 progeny (~1500 haploid genomes) for homozygous recessive mutants displaying altered glr-4prom::tagRFP expression in MNs at larval stage 4 (L4) (Fig. 2a). We retrieved 3 mutant alleles: two alleles (ot785 and ot786), which affect two TFs (CFI-1, LIN-13), have been previously described elsewhere37,38. This study focuses on the third allele, ot792. In homozygous ot792 mutant animals, we observed ectopic glr-4prom::tagRFP expression in MNs of the nerve cord, but no effect on SAB and DA9 neurons (Fig. 2a–d). Of the 58 MNs that normally reside in the nerve cord, 11.6±2.2 cells express glr-4prom::tagRFP in ot792 mutant animals compared to 0.6±0.9 cells in control animals (Fig. 2b). We obtained similar results with an endogenous mScarlet::glr-4 reporter; both the number of MNs expressing mScarlet::glr-4 and its fluorescent intensity are increased in ot792 mutants (Supplementary Fig. 2a–c)38. Hence, ot792 mutants display increased glr-4 expression in a subset of the 58 nerve cord MNs. Next, we used a previously published strain that does not carry the ot792 mutation but selectively overexpresses glr-4 in nerve cord MNs due to mutated binding sites for CFI-1, a TF that normally represses glr-438. We found defects in thrashing behavior of this strain (Supplementary Fig. 3a), indicating that increased glr-4 levels in MNs are detrimental to C. elegans motility. Therefore, the ot792 mutants offer an opportunity to identify a mechanism that tunes down (limits) glr-4 expression in nerve cord MNs.

PBRM-1 (PBRM1/BAF180) represses glr-4/GRIK4 in MNs
Upon single nucleotide polymorphism (SNP) mapping and whole genome sequencing51 (Supplementary Fig. 4), we identified the molecular lesion in ot792 mutants: a C to T nucleotide substitution (at position +7115 bp from ATG) that generates a premature stop (TAG, Amber) codon in the 12th exon of pbrm-1, ortholog of human polybromo 1 (PBRM1/BAF180) (Fig. 2e). This point mutation of the ot792 allele affects all known pbrm-1 transcripts, C26C6.1a.1 and C26C6.1b.1-3 (Fig. 2e). We also characterized animals carrying an independent pbrm-1 deletion allele (tm415), which selectively affects a single transcript (C26C6.1a.1) (Fig. 2e). Again, we observed ectopic glr-4prom::tagRFP in MNs of the VNC, although the effect is weaker compared to pbrm-1(ot792) (Fig. 2b–d).

At the protein level, the C26C6.1a.1 transcript produces the long PBRM-1 isoform a, whereas transcripts C26C6.1b.1-3 produce the short PBRM-1 isoform b (Fig. 2e). Six bromodomains (BrD) and two bromo-adjacent homology (BAH) domains are present in the long PBRM-1 isoform but absent from the short one (Fig. 2e, f). Both short and long pbrm-1 isoforms contain the high mobility group (HMG) domain (Fig. 2e). All these domains are present in human PBRM1 (BAF180) (Fig. 2f). Next, we employed CRISPR/Cas9 mutagenesis to generate isoform-specific pbrm-1 mutant alleles. Mutation of the start ATG (Met) codon of the short isoform (C26C6.1b.1) into ATA (Ile) (pbrm-1(10332) allele) had no effect on glr-4 expression in nerve cord MNs (Fig. 2e, i). However, mutation of the start ATG (Met) codon of the long isoform (C26C6.1a.1) into ATA (Ile) (pbrm-1(10440) allele) led to ectopic glr-4 expression in MNs (Fig. 2e, i). Moreover, transgenic supply of the long pbrm-1 isoform driven by its endogenous promoter (pbrm-1prom::pbrm-1 cDNA) completely rescued the glr-4 phenotype in pbrm-1(ot792) mutants (Fig. 2g, h). Hence, the long isoform of PBRM-1 is specifically required for glr-4 repression.

Further, complementation testing showed that trans-heterozygote animals carrying both alleles (ot792 and tm415) also displayed ectopic expression of glr-4 in nerve cord MNs (7.7±1.2 cells, p<0.0001), corroborating that pbrm-1 gene activity is necessary to repress glr-4 (Fig. 2b). Altogether, our genetic screen identified pbrm-1 (PBRM1/BAF180) as a negative regulator of glr-4 expression in a subset of nerve cord MNs (Fig. 2i), implicating the long PBRM-1 isoform in the process of neuronal subclass diversification. As pbrm-1 is the signature subunit of the polybromo-associated BRG1/BRM-associated factor (PBAF) chromatin remodeling complex52, these results implicate PBAF-mediated epigenetic regulation in the process of MN diversification.

pbrm-1/PBRM1 acts in nerve cord MNs to repress glr-4/GRIK4
Due to its specific effect on glr-4/GRIK4 expression in a subset of nerve cord MNs, we wondered whether pbrm-1 expression is restricted to these cells. Analysis of available scRNA-Seq data of adult MNs8 revealed that pbrm-1 transcripts are present broadly in all MN subclasses (both cholinergic and GABAergic) (Fig. 3a). At the protein level, an endogenous C-terminal GFP reporter (PBRM-1::TY1::EGFP::3xFLAG, allele st12226) that captures expression of all PBRM-1 isoforms (Fig. 2e) is expressed broadly in most (if not all) C. elegans neurons and other cell types (Fig. 3b and Supplementary Fig. 5a), consistent with a recent study53.

This broad expression suggests that pbrm-1 could act either in post-mitotic MNs to control glr-4 or in other cells that indirectly influence glr-4 expression in MNs. To address this question, we conducted MN-specific RNAi against pbrm-154. We used two cis-regulatory elements, from lin-39 and cho-1, known to drive expression in post-mitotic nerve cord MNs42,50. RNAi against all isoforms, or just the long isoform resulted in ectopic glr-4 expression in nerve cord MNs (Figs. 3c and 2e). Hence, we conclude that pbrm-1 is necessary to repress glr-4 in post-mitotic MNs.

In addition to RNAi, we used ZIF-1-mediated protein degradation to selectively deplete PBRM-1 in post-mitotic MNs55 (Fig. 3d). We generated a transgenic line that expresses the GFP nanobody::ZIF-1 protein in nerve cord MNs. We crossed it to PBRM-1::TY1::GFP::3xFLAG animals (st12226 allele) and observed efficient PBRM-1::GFP depletion in nerve cord MNs, accompanied by ectopic glr-4 expression (Fig. 3d). Altogether, our MN-specific RNAi and protein degradation experiments demonstrate a cell-autonomous role for PBRM-1 in repressing glr-4 in post-mitotic MNs.

The ATPase activity of PBAF is needed for glr-4/GRIK4 repression
Through ATP hydrolysis, the PBAF complex facilitates ejection, sliding, and/or swapping of nucleosomes, thereby altering the chromatin state to affect gene transcription in a positive or negative manner60. We therefore asked whether SWSN-4, the sole SWI/SNF ATPase in C. elegans59, is necessary for glr-4 repression. Because complete elimination of swsn-4 gene activity leads to early lethality59, we used a hypomorphic, temperature-sensitive swsn-4(os13) allele and MN-specific RNAi. In both cases, we observed ectopic expression of glr-4 in nerve cord MNs at the restrictive temperature (20°C); the effect of RNAi was stronger than that of the swsn-4(os13) allele (Fig. 4e, Supplementary Fig. 6-7). To assess whether the effect on glr-4 is due to ATP-dependent chromatin remodeling, we used CRISPR/Cas9 gene editing to generate an ATPase-dead version of SWSN-4 by introducing a lysine (K)-to-arginine (R) mutation at a conserved residue (K564) essential for ATP hydrolysis61–64. Although homozygous swsn4K564R/K564R animals are embryonic lethal consistent with a previous study64, heterozygous swsn4K564R/+ animals are viable and fertile. Compared to controls, we found ectopic glr-4::tagRFP expression in MNs of swsn4K564R/+ animals (Fig. 4f, g). This finding not only indicates that ATP-dependent chromatin remodeling is required for glr-4 repression but also suggests that the PBAF effect on glr-4 is dosage-dependent.

Next, we conducted a similar analysis for four other shared subunits of BAF and PBAF (SWSN-1, SWSN-3, SWSN-5, SWSN-6) using available mutant alleles or RNAi (Supplementary Fig. 6). We observed ectopic glr-4 expression in nerve cord MNs of swsn-1(os22) and swsn-5(ok622) single mutants and in animals with MN-specific RNAi against swsn-6 (Fig. 4e and Supplementary Fig. 7). In shared subunit mutants, glr-4 is ectopically expressed in fewer MNs when compared to PBAF-specific mutants (Fig. 4c, e). Further, we observed no ectopic glr-4 expression in MNs of swsn-3 LOF mutants (Fig. 4e and Supplementary Fig. 7). These weaker or absent effects on glr-4 are likely due to the weak nature of LOF mutant alleles for these shared subunit-encoding genes, which do not affect organismal survival (Supplementary Fig. 6). Strong LOF alleles for genes encoding shared core BAF/PBAF subunits are lethal59,65, and thereby not examined herein. In conclusion, several shared subunits of PBAF and BAF, as well as the activity of the ATPase SWSN-4/BRG1, are needed for glr-4 repression in nerve cord MNs.

PBAF antagonizes the terminal selector UNC-3/EBF in MNs
The terminal selector UNC-3/EBF acts directly to activate multiple effector target genes (including glr-4) in subclasses of cholinergic MNs41,42. In prior work with the endogenous mScarlet::glr-4 reporter38, we showed that UNC-3 is required to activate basal/low levels of glr-4 in nerve cord MNs. Here, we show that expression of the transgenic glr-4prom::tagRFP reporter in SAB and DA9 neurons is completely lost in unc-3 (n3435) null mutant animals (Fig. 5a–d), consistent with the known role of UNC-3 as transcriptional activator41,44. We therefore hypothesized that, in nerve cord MNs, unc-3 gene activity is necessary for the increased glr-4 expression we observe in PBAF mutants (Fig. 4c). Through double mutant analysis, we found this to be the case. Expression of glr-4 in nerve cord MNs is present in single pbrm-1(-) or swsn-9(-) mutants, but completely lost in unc-3(-); pbrm-1(-) and unc-3(-); swsn-9(-) double mutants (Fig. 5a, c). Next, we over-expressed UNC-3 specifically in nerve cord MNs and witnessed a modest increase in glr-4prom::tagRFP expression. However, UNC-3 over-expression in a PBAF (swsn-9) mutant was able to substantially increase glr-4prom::tagRFP in MNs (Fig. 5e), suggesting that perturbation of the PBAF complex enables UNC-3 to robustly activate glr-4 transcription. Altogether, our genetic analysis suggests that the PBAF complex represses glr-4 in nerve cord MNs by antagonizing the transcriptional activator UNC-3/EBF, albeit the precise molecular mechanism remains elusive (Fig. 5f). Despite the broad expression of PBAF, this effect is remarkably specific to a subset of nerve cord MNs. Outside the nerve cord, no change was observed in glr-4 expression in SAB and DA9 neurons of pbrm-1 or swsn-9 mutants (Fig. 5a, b, d, f).

PBAF represses glr-4/GRIK4 in specific MN subclasses
Which MN subclasses show glr-4 expression in PBAF mutants? Our genetic analysis showed up to 14±1 (mean±STDV) nerve cord MNs ectopically express glr-4prom::tagRFP in PBAF mutants (Fig. 4c), indicating PBAF represses glr-4 in a subset of the 58 MNs that populate the VNC (Fig. 1a). Using a combination of criteria (stereotypic cell body position, birth order, and fluorescent MN reporters; see Methods) (Fig. 6a), we identified with single-cell resolution the specific MN subclasses that ectopically express glr-4 in three PBAF-specific mutants (pbrm-1, swsn-9, and phf-10). We found that PBAF represses glr-4prom::tagRFP primarily in subclasses of DA, VA, and AS neurons located specifically in the VNC (DA2-5, DA6-8, VA2, VA3-8, VA9-10, AS2-3, AS4-8, and AS9-10) (Fig. 6b, c). Compared to DA and VA, this effect is stronger in AS subclasses, where the percentage of animals ectopically expressing glr-4prom::tagRFP approaches 100% (Fig. 6b, c). We obtained similar results with MN-specific RNAi against swsn-7, which encodes the PBAF-specific subunit SWSN-7 (BAF200) (Fig. 6b, c). Because glr-4 transcripts are detected at low levels in AS and VA neurons (Fig. 1c), we propose that PBAF tunes down glr-4 to low levels in these neurons (Fig. 6d). On the other hand, glr-4 transcripts are undetectable in DA neurons (Fig. 1c) due to PBAF’s repressive activity (Fig. 6d).

Next, we wondered whether the molecular identities of the DA, VA, and AS subclasses are broadly affected in PBAF mutants. To test for this possibility, we evaluated expression of seven available terminal identity markers of DA, VA, and/or AS neurons (unc-17/VAChT, dbl-1/BMP7, del-1/SCNN1B/D/G, slo-2/KCNT1/2, acr-2/CHRNA3/6, unc-53/NAV1, and unc-129/GDF10). However, we found no changes in the number of MNs expressing each marker in multiple PBAF mutants (Supplementary Fig. 8). In addition, these findings show that the total number of MNs is not affected by lack of PBAF activity, indicating that PBAF does not affect MN generation in the C. elegans nerve cord. In summary, the PBAF chromatin remodeling complex selectively represses glr-4, and likely additional yet-to-be identified target genes, in DA, VA, and AS MN subclasses (Fig. 6d).

MAB-9/TBX20 represses glr-4/GRIK4 in AS subclasses of MNs
The specificity of the observed PBAF effect on glr-4 in distinct subclasses of DA, VA, and AS neurons is striking, given that PBAF subunit-encoding genes (pbrm-1, swsn-7, swsn-9, and phf-10) are broadly expressed in neurons and other C. elegans cell types (Fig. 7a). What is the molecular basis for this specificity? We hypothesized that in DA, VA, and AS subclasses, transcription factors with repressor activity recognize specific DNA sequences at the glr-4 locus and subsequently recruit the PBAF complex, resulting in glr-4 transcriptional repression. To test this idea, we focused on AS subclasses (AS2-3, AS4-8, and AS9-10) because the PBAF effect on glr-4 is stronger compared to DA and VA subclasses (Fig. 6b–d). The transcription factor MAB-9 (ortholog of human Tbx20) is expressed in AS neurons (Fig. 7a) and acts as a transcriptional repressor37,66. If our hypothesis is correct, we would expect mab-9(e2410) LOF mutants to exhibit a similar phenotype as PBAF mutant animals, expressing high levels of glr-4 in nerve cord AS subclasses. We found this to be the case using two transgenic reporters (glr-4prom::tagRFP, glr-4 4.9kbprom::GFP) and one endogenous (mScarlet::glr-4) reporter for glr-4 (Fig. 7b, c and Supplementary Fig. 9a, c–e). Further, we found the expression of a mab-9 reporter to be unaffected in MNs of various PBAF mutants (Supplementary Fig. 9f, g). We conclude that mab-9 is required to tune down glr-4 expression in AS subclasses of nerve cord MNs.

MAB-9/TBX20 and PBAF act directly to repress glr-4/GRIK4
If MAB-9 recruits PBAF to the glr-4 locus, which is normally activated by UNC-3 in various MNs, our hypothesis predicts that in WT animals, MAB-9, PBAF, and UNC-3 directly bind to cis-regulatory elements of the glr-4 locus. We therefore interrogated available chromatin immunoprecipitation followed by sequencing (ChIP-Seq) whole-animal data for MAB-9, two PBAF proteins (PBRM-1 and SWSN-7) and UNC-349,67, and found that all four proteins bind to the glr-4 locus (Fig. 7d). Interestingly, MAB-9 and PBAF (PBRM-1 and SWSN-7) binding coincides prominently within the glr-4 locus and modestly upstream of glr-4, supporting the possibility of MAB-9 recruiting PBAF (Fig. 7d). Further, binding of the glr-4 repressor (MAB-9) is mostly excluded from regions bound by the glr-4 activator (UNC-3) (Fig. 7d).

To test whether MAB-9 binding on the glr-4 locus is functional, we first followed a bioinformatic approach (see “Methods”) and identified 10 predicted MAB-9 binding sites upstream and within the glr-4 locus (Fig. 7d and Supplementary Table 1). Next, we individually deleted the three most proximal MAB-9 sites (#5 - #7) in the context of our transgenic glr-4 538bp prom::tagRFP animals (Fig. 7e, f). Deletion (Δ) of either site #5 or #7 yielded no effect on glr-4prom::tagRFP expression in nerve cord MNs (Fig. 7e, f). However, deletion of site #6 (Δ site #6) led to ectopic glr-4prom::tagRFP expression in AS subclasses in the VNC (Fig. 7e, f), suggesting that MAB-9 binds directly to site #6 to repress glr-4 transcription. Interestingly, MAB-9 site #6 lies next to UNC-3 site #1, which is necessary for driving basal levels of endogenous mScarlet::glr-4 expression in nerve cord MNs38.

Next, we used CRISPR/Cas9 to mutate site #6 by introducing nucleotide substitutions (WT: CCTTTTCTCCT, Mutated: CCAAAAAACT) in the context of the endogenous mScarlet::glr-4 reporter (Supplementary Table 1), but this manipulation had no effect on mScarlet::glr-4 expression in MNs (Fig. 7g). The discrepancy between the results obtained when MAB-9 site #6 was deleted in the transgenic glr-4 538bp prom::tagRFP versus the endogenous mScarlet::glr-4 context suggests redundancy among the 10 MAB-9 sites. To test this idea, we simultaneously mutated site #6 and two other sites (#8 and #9) (Supplementary Table 1), but observed no effect on endogenous mScarlet::glr-4 expression in MNs (Fig. 7g), raising the possibility that one or more of the remaining seven MAB-9 sites are involved in glr-4 repression. This is consistent with previous work where simultaneous mutation of 11 binding sites for the repressor TF CFI-1 led to increased glr-4 expression in C. elegans MNs38. Altogether, genetic and biochemical (ChIP-Seq) evidence suggests that MAB-9/Tbx20 and PBAF act directly to repress glr-4/GRIK4 transcription in AS neuron subclasses (AS2-3, AS4-8, and AS9-10) (Fig. 7h).

To expand the repertoire of PBAF target genes, we identified 1000 genes that—like glr-4—are expressed at low levels in AS neurons based on available sc-RNA-Seq data5. Specifically, we included 1000 genes with expression values (transcripts per million, TPM) centered around glr-4 (TPM: 120.5) (see Methods). Next, we found that—like glr-4—247 of these 1000 genes are co-bound by UNC-3, MAB-9 and SWSN-7 (Supplementary Fig. 9b and Supplementary Data 1). Although validation of these 247 genes is beyond the scope of this work, this analysis nevertheless offers a list of putative target genes that are repressed by MAB-9 and PBAF in AS neurons.

Prediction/validation of MAB-9/TBX20 and PBAF interactions
A plausible mechanism by which MAB-9 could provide specificity to PBAF to repress glr-4 in AS neurons is through direct PBAF recruitment via protein-protein interactions. This model is consistent with the prominent co-binding of MAB-9, SWSN-7 and PBRM-1 within the glr-4 locus (Fig. 7d). Because co-immunoprecipitation experiments on isolated C. elegans MNs are not feasible, we tested this model via an in silico interaction screen using the Local ColabFold implementation of AlphaFold2, a powerful tool for accurate prediction of protein-protein interactions68–71. We screened for pairwise interactions between MAB-9 and the PBAF-specific subunits (SWSN-7, SWSN-9, PBRM-1, and PHF-10), as well as other core/accessory subunits (SWSN-2.2, SWSN-4, SWSN-5, SWSN-3, SWSN-1, SWSN-6, and SWSN-2.1). Following stringent criteria established in a previous study71, we assessed prediction quality using an interface predicted template modeling (ipTM) score cut-off of 0.3 for the majority (at least 3 out of 5) of the predicted models for each pairwise interaction (Fig. 8a and Supplementary Fig. 10). We further assessed the proposed interaction using the predicted alignment error plots (Fig. 8b and Supplementary Fig. 10). With this analysis, we identified the PBAF-specific subunit SWSN-7 and the accessory subunit SWSN-6 as predicted interactors with MAB-9. Predicted contacts of the top-ranked structures for both dimers were assessed using PDBsum and visualized with PyMOL (Fig. 8c, d). The MAB-9-SWSN-7 interface had an area of 1947 Å2 and 2069 Å2 for each protein, respectively. The MAB-9-SWSN-7 interaction consisted of 3 salt bridges, 9 hydrogen bonds, and 486 non-bonded contacts. The MAB-9-SWSN-6 interface had an area of 2252 Å2 and 2720 Å2 for each protein, respectively. The MAB-9-SWSN-6 interaction consisted of 1 salt bridge, 18 hydrogen bonds, and 1820 non-bonded contacts. These predicted interactions support a model of PBAF recruitment via direct protein interactions with MAB-9.

To experimentally test this model, we conducted co-immunoprecipitation assays in HEK293 cells co-transfected with epitope-tagged versions of MAB-9 and SWSN-6, or MAB-9 and SWSN-7. Following pull-down of MAB-9::Myc with anti-c-Myc magnetic beads, we detected SWSN-6::HA (49.5 kDa) or SWSN-7:HA (140.2 kDa) with western blotting against the HA epitope (Fig. 8e). Conversely, following pull-down of either SWSN-6::3XFLAG or SWSN-7:3XFLAG with anti-FLAG M2 magnetic beads, we detected MAB-9::HA (40.5 kDa) with anti-HA western blotting (Fig. 8e). Together, these results validate the AlphaFold2 predictions, supporting a model where MAB-9/TBX20 physically interacts with both SWSN-6 and SWSN-7 to recruit PBAF and thereby influence gene expression in AS neurons.

UNC-3 and PBAF control caudal MN terminal identity
In the PAG ganglion immediately posterior to the VNC, there are five subclasses of caudal MN that are cholinergic (DA8, DA9, VA11, VA12, and AS11) (Fig. 9a); these co-express UNC-3 and PBAF components (Fig. 7a). UNC-3 activates glr-4 in DA9, but loss of PBAF has no effect on glr-4 expression in DA9 (Fig. 2d). We therefore asked whether other unc-3-dependent terminal identity markers for caudal MN subclasses are affected in PBAF mutant animals. Indeed, we found that PBAF gene activity (pbrm-1, swsn-9, phf-10) is required for the expression of itr-1/ITPR1 in DA9 (Fig. 9b, f), avr-15/GLRA1-3 in VA11 and AS11 (Fig. 9c, g), ser-2/HTR1A in DA8 (Fig. 9d, h), and glr-5/GRIK1/3 in DA8 and VA12 (Fig. 9e, i). Additional markers (flp-18, sra-36, nlp-7, and flp-7) for caudal MN subclasses are not affected by loss of PBAF gene activity (Supplementary Fig. 11). These findings point to a different role for PBAF in caudal MN subclasses: instead of a role in gene repression, which we observed in nerve cord MN subclasses, PBAF is required to activate expression of various terminal identity markers in caudal MN subclasses. Taken together, our results indicate a dichotomy in PBAF function in C. elegans cholinergic MNs. In nerve cord MN subclasses, PBAF antagonizes UNC-3, and thereby acts as a repressor of glr-4. In caudal MN subclasses, PBAF and UNC-3 are both required to activate expression of several terminal identity genes (e.g., itr-1, avr-15, ser-2, and glr-5).

Loss of pbrm-1 leads to locomotion defects
Because our molecular analyses revealed gene expression defects in MNs, we wondered whether PBAF mutant animals display locomotion defects. To test this, we performed high-resolution behavioral analysis of freely moving adult (day 1) pbrm-1 mutant animals using automated multi-worm tracking72,73. This analysis can quantify multiple features related to C. elegans locomotion (e.g., speed, crawling amplitude, curvature, pause, forward and backward locomotion) and each feature can be localized to a specific part of the nematode’s body (e.g., head, mid-body, tail). We assessed homozygous pbrm-1 mutants carrying either the tm415 or ot792 alleles (Fig. 2e). Hierarchical clustering analysis showed that pbrm-1(tm415) animals cluster more closely to control (N2) animals, whereas pbrm-1(ot792) animals displayed strong locomotion defects (Fig. 10a). These behavioral effects are consistent with the stronger effects we observed on glr-4 expression in pbrm-1(ot792) compared to pbrm-1(tm415) animals (Fig. 2b). Because the PBAF effect on glr-4 is stronger in AS neurons, which are known to control body wave propagation74, we asked whether this particular feature is disrupted in pbrm-1 mutants. Indeed, we found significant changes in the body wave, reflected in curvature features and the time derivative of curvature features (d_curvature) in pbrm-1(ot792) mutants (Fig. 10c–g). Further, median midbody speed is affected in pbrm-1(ot792) mutants (Fig. 10b). Altogether, this analysis shows that loss of pbrm-1 leads to significant defects in posture (curvature) and motion (speed), consistent with defective body wave propagation.

Last, we conducted thrashing assays on adult animals, in which we knocked-down PBRM-1 specifically in motor neurons with either cell-specific RNAi or ZIF-1 technology using the exact same worm strains shown in Fig. 3c, d. However, we were unable to identify any thrashing defects (Supplementary Fig. 12), likely due to partial decrease of PBMR-1 with RNAi or ZIF-1. In fact, we only achieve a modest decrease in the number of motor neurons expressing PBRM-1::GFP with ZIF-1-mediated degradation (Fig. 3d). We conclude that although our RNAi and ZIF-1 approaches lead to ectopic glr-4 expression in motor neurons (Fig. 3c, d), their partial knock-down effect on PBRM-1 is insufficient to lead to detectable behavioral effects. Alternatively, we cannot discard that the pbrm-1(ot792) locomotory defects are caused by non-cell autonomous effects in other neurons or non-neuronal cells.

Methods
Genetics
Strains were grown at 15°C, 20°C, or 25°C on nematodegrowth media (NGM) plates with E. coli (OP50)133. Mutant alleles used: pbrm-1(ot792) (point mutation, premature stop), pbrm-1(tm415) (427 nucleotide deletion, 5 nucleotide insertion), swsn-9(ok1354) (1,010 nucleotide deletion), phf-10(rd21) (4 nucleotide deletion), unc-3(n3435) (1,008 nucleotide deletion), mab-9(e2410) (single nucleotide substitution), swsn-1(os22) (missense temperature-sensitive allele), swsn-3(tm3647) (432 nucleotide deletion), swsn-4(os13) (missense temperature-sensitive allele), swsn-5(ok622) (2,026 nucleotide deletion), and dpff-1(tm4287) (318 nucleotide deletion). All C. elegans strains used in this study are listed in Supplementary Table 2. All primers used in this study are listed in Supplementary Table 3.

Forward genetic screen
EMS mutagenesis was performed on otIs476[glr-4prom::TagRFP] fluorescent reporter animals, before screening visually at a dissecting fluorescent microscope (Zeiss SteREO Discovery V12) for changes in reporter expression in VNC MNs. To identify the causal genes of the mutant alleles obtained, Hawaiian single nucleotide polymorphism (SNP) mapping and whole-genome sequencing were performed followed by data analysis using the CloudMap pipeline51,134.

Microscopy
To prepare slides for imaging, animals were anesthetized using 100 mM sodium azide (NaN) while being mounted on a 4% agarose pad on glass slides. Images were taken with an automated fluorescence microscope (Zeiss, Axio Imager Z2). Several z-stacks were taken with each image (0.50 μm intervals between stacks) with Zeiss Axiocam 503 mono using the ZEN software (Version 2.3.69.1000, Blue edition). Images and z-stacks were then transferred and processed in ImageJ135.

Generation of transgenic reporters
Reporter gene fusions were generated using PCR fusion136. Target gene promoter fragments were fused to the tagRFP coding sequence. Gene fusion products were then injected into wild-type young adult animals at a total DNA concentration of 100 ng/μl. The fusion product was accompanied by a myo-2::GFP or myo-2::mCherry co-injection marker at a concentration of 3-5 ng/μl. Individual lines were isolated from F1 progeny.

Generation of RNAi constructs and lines
DNA fragments spanning 2–4 exons were selected from target genes and fused in both sense and antisense orientation to enhance elements sufficient to drive MN-specific expression, lin-39 (intron 1) and cho-1 (280 bp distal element), by PCR fusion54. Each fused construct was injected into young adult animals at a concentration of 100 ng/μl, along with myo-2::GFP or myo-2::mCherry co-injection marker at a concentration of 3–5 ng/μl. Individual lines were isolated from F1 progeny.

Motor neuron cell identification
A combination of criteria was used for MN cell ID: stereotypic cell body position of MNs2, birth order, and previously characterized fluorescent reporters for specific MN classes137. Identity was determined based on co-localization with these previously characterized fluorescent reporters.

Fluorescence intensity quantification
Images of worms were taken with identical parameters, using z-stacks that span the entire cell bodies of nerve cord MNs. These stacks were processed in ImageJ, where background was subtracted to reduce background noise from the image (rolling ball radius= 50 pixels). The MN cell bodies were manually selected (by drawing an area over MN cell bodies to be measured), and the average fluorescence intensity was measured and recorded for each visible MN in the image.

ZIF-1 mediated protein degradation
A plasmid containing GFP nanobody fused to ZIF-1 (driven by MN-specific 280 bp cho-1 promoter fragment) was injected into wild-type young adult animals along with co-injection marker myo-2::mCherry. GFP nanobody binds to GFP-tagged proteins, while ZIF-1 recruits the E3 ubiquitin ligase complex to degrade tagged proteins55. Individual lines were selected from F1 progeny. One line obtained underwent spontaneous integration into the genome and was subsequently used for our studies. This line was crossed with pbrm-1::TY1::EGFP::3xFlag to degrade PBRM-1::TY1::EGFP::3xFLAG protein.

Single-molecule RNA (smRNA) FISH
Animals were grown until plates were full of gravid worms, and were subsequently collected in an egg prep to synchronize animals. Synchronized L1 animals were then incubated in 4% paraformaldehyde in 1× PBS for 45 min at room temperature, washed two times using 1× PBS, and then permeabilized with 70% ethanol at 4°C overnight. A glr-4 probe was designed using the Stellaris Probe Designer website (Biosearch Technologies). The probe was mixed in hybridization buffer (0.1 g/mL dextran sulfate [Sigma D8906-50G], 1 mg/mL E. coli tRNA [ROCHE 10109541001], 2 mM vanadyl ribonucleotide complex [New England Biolabs S1402s], 0.2 mg/mL RNase-free BSA [Ambion AM2618], 10% formamide) and added to the worms. The hybridization buffer was removed and worms were washed twice in wash buffer (DAPI was added during the second wash and incubated for 30 min in the dark for nuclear counterstaining). Worms were washed once in 2× SSC, incubated in GLOX buffer (0.4% glucose, 0.1 M Tris-HCl, 2× SSC) for 2 min for equilibration, and then resuspended in GLOX buffer with glucose oxidase and catalase added before imaging138.

Transcription factor motif analysis
The position weight matrix (PWM) of UNC-3 has been described49. The MAB-9 PWM was derived from MODENCODE ChIP-seq data139 of mab-9::TY1::EGFP::3xFLAG L1 larva (experiment file identifier: ENCFF396HF). De novo motif discovery for MAB-9 was conducted with Homer140 using findMotifsGenome.pl using a sequence size of size given. The PWM of C. elegans MAB-9 is similar to the PWM of M. musculus Tbx20141. To identify putative UNC-3 and MAB-9 sites in the glr-4 locus, we used FIMO (Find Individual Motif Occurrences)142, which is one of the motif-based sequence analysis tools of MEME (Multiple Expectation maximization for Motif Elicitation) bioinformatics suite (http://meme-suite.org/). The p-value threshold for the analysis of MAB-9 and UNC-3 site in the glr-4 locus was set at p<0.01.

Co-binding analysis of UNC-3, MAB-9, and SWSN-7 on 1000 genes
One thousand genes with expression values (transcripts per million, TPM) centered around glr-4 (TPM: 120.5) were selected from CenGEN (L4 nervous system-wide scRNA-seq) to define a list of candidates with likely similar regulatory inputs in AS MNs. The expression range for the 500 genes expressed lower than glr-4 in AS MNs was 75.9 to 120.4 TPM; the expression range for the 500 genes expressed higher than glr-4 in AS MNs was 120.5 to 282.8 TPM. A total of 5861 genes with non-zero TPM values were detected in AS MNs in this dataset, with 4414 genes expressed below (TPM range: 0.5-75.9) and 446 genes expressed above (TPM range: 283.0–216,684.1) the glr-4-centered range used for this analysis. Most known AS MN class markers were expressed well-above the glr-4-centered range, including unc-8 (351.7 TPM), elt-1 (444.1 TPM), unc-3 (499.8 TPM), mab-9 (581.2 TPM), and unc-53 (2898.7 TPM), whereas one AS-specific transcriptional repressor fell within the upper range (unc-55=152.4 TPM). Genes that were previously shown to not express in AS MNs typically had very low (within glr-4-centered range) or undetectable expression in this dataset (e.g., bnc-1=0 TPM, cfi-1=0 TPM, vab-7=0 TPM, dbl-1=107.8 TPM, slo-2=146.2 TPM). Next, we integrated ChIP-Seq data on these 1,000 genes. We found 247 genes to be co-bound by UNC-3, MAB-9 and SWSN-7 (Supplementary Data 1).

Prediction of protein–protein interactions
Protein-protein interaction was predicted using the LocalColabFold (https://github.com/YoshitakaMo/localcolabfold) implementation of Alphafold257,143, with multiple sequence alignments generated by MMseqs2144, and using 5 recycling steps. Quality of the prediction was assessed using outputted prediction confidence measures for the overall complex and for the relative subunit positions (pTM and ipTM, respectively) and PAE plots. An ipTM cut-off of 0.3 was used to identify structures warranting potential further investigation, as previously observed71. Top ranked models for structures of interest were visualized using PyMOL (https://pymol.org/).

Automated worm tracking
Worms were maintained as mixed stage populations by chunking on NGM plates with E. coli OP50 as the food source. The pbrm-1(tm415) and pbrm-1(ot792) mutants were 6× outcrossed to the wild N2 isolate. The day before tracking, 30-40 L4 larvae were transferred to a seeded NGM plate and incubated at 20°C for approximately 24 h. Five adults were picked from the incubated plates to each of the imaging plates (see below) and allowed to habituate for 30 min before recording for 15 min. Imaging plates are 35 mm plates with 3.5 mL of low-peptone (0.013% Difco Bacto) NGM agar (2% Bio/Agar, BioGene) to limit bacteria growth. Plates are stored at 4°C for at least 2 days before use. Imaging plates are seeded with 50 μl of a 1:10 dilution of OP50 in M9 the day before tracking and left to dry overnight with the lid on at room temperature.

Behavioral feature extraction and analysis
All videos were analyzed using Tierpsy Tracker145 to extract each worm’s position and posture over time. These postural data were then converted into a set of behavioral features72. For each strain comparison, we performed unpaired Welch’s t-tests independently for each feature. The false discovery rate was controlled at 5% across all strain and feature comparisons using the Benjamini–Yekutieli procedure (Kim and van de Wiel, 2008).

Thrashing assay
In preparation for thrashing assay, animals were picked at the L4 stage onto an agarose plate without food. Animals were then individually picked and placed into a drop of M9 buffer on a separate agarose plate. Animals were given 30 s to acclimate and then recorded over the next 30 s via phone camera mounted onto a benchtop microscope. Video recordings of animals were analyzed at 0.5x speed to accurately count the number of thrashes (body bends) in 30 s.

Plasmid generation
Plasmids were generated by Genscript FLASH Gene DNA synthesis service. Plasmids include: (1) swsn-6 cDNA ORF clone from C. elegans (OCa343956), tagged with HA, inserted into pcDNA3.1+146, swsn-6 cDNA ORF clone from C. elegans (OCa343956), tagged with 3XFLAG, inserted into pcDNA3.1+, (3) swsn-7 cDNA ORF clone from C. elegans (OCa339589), tagged with HA, inserted into pcDNA3.1+, (4) swsn-7 cDNA ORF clone from C. elegans (OCa339589), tagged with 3XFLAG, inserted into pcDNA3.1+, (5) mab-9 cDNA ORF clone from C. elegans (OCa339423), tagged with HA, inserted into pcDNA3.1+, and (6) mab-9 cDNA ORF clone from C. elegans (OCa339423), tagged with myc, inserted into pcDNA3.1+. Control: plasmid pcDNA3.1_3xFLAG::BAP147.

Cell culture
HEK293 cells were cultured in DMEM (Gibco Dulbecco’s Modified Eagle Medium 1× 11960-044) supplemented with 10% FBS (Gibco A56707-01) and 2 mM L-glutamine and grown at 37°C with 5% CO2. Cells were cultured in 10 cm dishes.

Transfection
For each 10 cm dish, 24 μg of DNA (12 μg of plasmid 1 and 12 μg of plasmid 2) was added to 1 mL of Opti-MEM I 1× (Gibco 11058-021) and 60 μl of Lipofectamine LTX (Thermo Fisher Scientific Invitrogen 15338100). Plasmid combinations transfected into each dish include: (1) mab-9::HA and swsn-6::3XFLAG146, (2) mab-9::HA and swsn-7::3XFLAG, (3) mab-9::myc and swsn-6::HA, (4) mab-9::myc and swsn-7::HA, (5) mab-9::HA and pcDNA3.1+ containing BAP::FLAG, (6) mab-9::myc and pcDNA3.1+ containing BAP::FLAG, and (7) pcDNA3.1+ containing BAP::FLAG. BAP refers to bacteria alkaline phosphatase. After incubation for 30 min, the sample was added dropwise to each plate. Cell media was replaced 24 h after co-transfection, and protein was harvested 24 h later.

Protein harvest and quantification
Cells were collected in 3 mL of ice-cold ion-free PBS, centrifuged to a pellet at 5000 xg for 5 min at 4°C, then lysed with 1 mL of: 50 mM Tris-HCl at pH 7.4 (Fisher Scientific BP152-500), 150 mM NaCl (Fisher Scientific S271-10), 1 mM EDTA, 1% Triton X-100, 1:100 phosphatase inhibitor cocktail 1 (Sigma Aldrich P2850-1ML), 1:100 phosphatase inhibitor cocktail 2 (Sigma Aldrich P5726-1ML), and 1:100 protease inhibitor (Sigma Aldrich P8340-1ML). Cells were incubated in lysis buffer for 20 min on ice, vortexed every 10 min. Lysed cells were centrifuged to pellet at 16,000 xg for 15 min at 4°C. Supernatant was kept for experiments. Protein concentrations were determined using BCA Protein Assay kit (Thermo Scientific Pierce 23227).

Co-immunoprecipitation with anti-FLAG M2 magnetic beads
Anti-FLAG M2 magnetic beads (Millipore Sigma Aldrich, M8823) were washed three times with TBS (50 mM Tris-HCl Fisher Scientific BP152-500, 150 mM NaCl Fisher Scientific S271-10, 1 mM EDTA). One milligram of protein was added to 1.75 mL of TBS with 1:100 phosphatase inhibitor cocktail 1 (Sigma Aldrich P2850-1ML), 1:100 phosphatase inhibitor cocktail 2 (Sigma Aldrich P5726-1ML), and 1:100 protease inhibitor (Sigma Aldrich P8340-1ML). Each sample was incubated with 50 μl of packed beads at constant rotation overnight at 4°C. Next day, beads were collected by magnetic stand and washed three times with 20 times the packed bead volume. Protein was eluted and denatured with Laemmli sample buffer (BioRad #1610747) with 10% 2-Mercaptoethanol (Sigma Aldrich M3148-25ML), boiled at 95°C for 10 min.

Co-immunoprecipitation with anti-c-Myc magnetic beads
Anti-c-Myc magnetic beads (Thermo Fisher Scientific, 88842) were washed twice with 1× TBS-T buffer (25 mM Tris Fisher Scientific BP152-500, 0.15 M NaCl Fisher Scientific S271-10, 0.05% Tween-20 detergent Sigma Aldrich P1379-500ML). One milligram of protein was added to 400 μL of 1X TBS-T with 1:100 phosphatase inhibitor cocktail 1 (Sigma Aldrich P2850-1ML), 1:100 phosphatase inhibitor cocktail 2 (Sigma Aldrich P5726-1ML), and 1:100 protease inhibitor (Sigma Aldrich P8340-1ML). Each sample was incubated with 50 μL of beads at constant rotation overnight at 4°C. Next day, beads were collected by magnetic stand and washed twice with 300 μL of 5X TBS-T buffer 125 mM Tris Fisher Scientific BP152-500, 0.75 M NaCl Fisher Scientific S271-10, 0.25% Tween-20 detergent (Sigma Aldrich P1379-500ML) and once with 300 μL of ultrapure water. Protein was eluted and denatured with Laemmli sample buffer (BioRad #1610747) with 10% 2-Mercaptoethanol (Sigma Aldrich M3148-25ML), boiled at 95°C for 10 min.

Western blot
Lysates (20 μg of lysate, 30 μl of co-IP sample) were subjected to electrophoresis on Mini-PROTEAN TGX Stain-Free Gels (Bio-Rad), and then transferred to Amersham Hybond P 0.45 μm PVDF membranes (GE Healthcare). The membrane was blocked with 5% non-fat skim milk (Difco Skim Milk 232100) in Tris-buffered saline (Fisher Scientific BP152-500) containing 0.05% Tween-20 (Sigma Aldrich P1379-500ML) for 1 h at room temperature, and then incubated overnight at 4°C with primary antibody against HA (1:1000, C29F4, Cell Signaling Technology). Following washing, the membrane was incubated for 1 h at room temperature with anti-rabbit IgG, horseradish peroxidase-linked whole antibody (1:5000, NA934-1ML, Cytiva). The signal was detected using SuperSignal West Dura Extended Duration Substrate (Thermo Fisher Scientific 34076) and analyzed using ChemiDoc MP Imaging System and Image Lab software (version 6.1.0, Bio-Rad). Membranes were stripped (Thermo Scientific) and reblotted with primary antibody against beta-Actin (1:1000, GeneTex GTX109639) and secondary anti-rabbit IgG, horseradish peroxidase-linked whole antibody (1:5000, NA934-1ML, Cytiva) following the same blotting, signal detection, and imaging protocol.

Statistics and reproducibility
Individual data point dot plots and box and whisker plots were used to represent the quantification of the data. All data points are presented. The horizontal line in the box represents the mean value, while the box itself represents the interquartile range. Unpaired two-sided t-test with Welch’s correction was performed and p-values were annotated. Differences with p<0.05 were considered significant. Asterisks in figures indicate statistical significance as follows: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. For plots comparing percentage of animals, chi-square analysis was performed to determine statistical significance. Fluorescent microscopy images next to each dot plot show a representative animal. The number of animals (n) for each analysis was determined based on standards in the C. elegans field. No data were excluded from the analyses. The Investigators were not blinded to the allocation during experiments and outcome assessment.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The manuscript by Osuma and colleagues utilized a glr4-reporter line to label a subset of motor neurons (MNs) that are dependent on unc-3 expression for their genetic identity. Using this tool they performed a genetic screen to identify regulatory factors that alter ectopic glr-4 expression and presumably alter neuronal diversity in the ventral nerve cord. In this way, they identified Pbrm-1 (BAF180) as a negative regulator of glr-4 in a subset of nerve cord MNs. They perform a large number of very elegant and informative genetic experiments to demonstrate that it is the PBAF complex and not the BAF complex that is required for the repression. Mining TF expression and binding data they identify Mab-9 as the TF that likely recruits PBAF complex to unc-3 targets to repress activation, which they confirm a role using additional RNAi and genetic experiments. Lastly, they demonstrate that in other more caudal MN subtypes, Pbrm-1 cooperates with unc-3 to activate gene expression in these MNs. Overall, it is a very well-written manuscript showing the power of the model system and it highlights a mechanistic role for SWI/SNF chromatin remodeling during neuronal diversification of postmitotic neurons. The article is of interest given the role of PBAF subunits in neurodevelopmental disorders and it would be a good addition to the literature.\nSuggestions and questions:\n1. The authors do a nice job of generating mutants for other members of the BAF and PBAF complexes to clearly show that ectopic expression is dependent on PBAF and not BAF. Included in this analysis was the use of a temperature sensitive mutant of Brg1 (swsn-4) from which they conclude that ATP-dependent remodeling is required. In fact, this experiment is similar to the deletion of other subunits in that it just disrupts a functional complex, strengthening the idea that the complex is required. To demonstrate that ATPase activity is required they should repeat the experiment using an ATPase-dead mutant that would still incorporate into the PBAF complex but would not remodel, thereby demonstrating that remodeling is required to silence glr-4.\n2. It was challenging to decipher where the Mab-9 binding site within the 538bp promoter was located when examining the ChIP-seq tracks that were presented in Figure 7d. From the schematic it appears that it should be located upstream of the Unc-3 sites. The authors should add the location of both the TF binding sites into the schematic. In addition, using the endogenous gene reporter and CRISPR to inactivate the site in vivo would be a more thorough way to show the importance of this site over the many other Mab-9 binding sites, while providing some context on the complete regulatory regions of the glr-4 gene.\n3. Similar to the previous point, the publicly available ChIP-seq data from whole animals showed a large number of Mab-9 and Pbrm-1 binding sites along the glr-4 locus. The regions with the most prominent overlapping binding sites were in the gene body. Are these likely to be active/important in the MNs or are they active binding site in other tissues? It was also not clear what sites within the promoter region were important for mediating gene activity and where they were with respect to the two Unc-3 sites. Trying to repeat the ChIP-seq data using dissected ventral nerve cord from WT, Mab-9 and ot792 mutants would really add to the analysis by highlighting the important regulatory regions mediating repression.\n4. Co-IP experiments showing an interaction between Mab-9 and Pbrm-1 (or another member of the PBAF complex) would provide further support of a role for Mab-9 in recruiting the PBAF complex to the glr-4 locus.\n5. It would be helpful to offer some explanation in the Discussion on why the cell-type specific TFs (Mab-9, CFI-1) play a dominant role in PBAF recruitment when Unc-3 and PBAF are both present. For example, are there differences in Mab-9 vs Unc-3 expression levels which favor repression versus activation? Differences in binding site affinities at the glr-4 promoter? In caudal MN subtypes, Unc-3 and PBAF are required for activation, but when two antagonistic TFs that recruit PBAF are both present (Unc-1 and Mab-9) how does repression dominate in this instance? Does it come down to differences in PBAF subunit composition, similar to what is known during the change from proliferation to differentiation? Some additional insight/opinion seems warranted.", "reviewer_2": "In this study, Osuma and colleagues used a forward genetic screen to identify C. elegans PBAF as a regulator of neuronal identity. Specifically, they found that PBAF represses gene expression in ventral nerve cord motor neurons and activates gene expression in posterior motor neurons. This suggests that PBAF utilizes distinct mechanisms to control related, but different neuronal cohorts in C. elegans. The authors used forward genetics, RNAi, neuron-specific rescue experiments, single-molecule FISH and analysed ChIP-Seq data to posit the mechanistic basis of PBAF function. In particular, they suggest that PBAF represses glr-4 VNC MN expression by antagonizing UNC-3 - a terminal selector TF.\nThis is significant work that identifies the chromatin modifier PBAF as a regulator of specific neuron fates. The data justify the conclusions drawn. I do however have a few issues that should be addressed.\nMajor points\nIs PBAF continuously required to repress glr-4 expression in the VNC MNs or does PBAF set up a program early in development? Removing PBAF post-developmentally would provide an answer to this question.\nThe authors shown that expression of glr-4 (encoding a glutamate receptor) is derepressed when PBAF is depleted in the VNC motor neurons. What is the behavioral effect of inappropriate GLR-4 expression? If there a motility/body bend phenotype?\nThe authors state that “Hence, our genetic analysis suggests that the PBAF complex represses glr-4 in nerve cord MNs by antagonizing the transcriptional activator UNC-3/EBF”. However, no mechanism underlying this suggestion is posited.\nWhat evidence is there that the neurons ectopically expressing glr-4 in DEL_MAB-9 site animals (Fig 7F) are AS neurons?\nMinor points\nThe authors mention that the pbrm-1(tm415) allele has a ‘weaker’ glr-4 phenotype compared to the ot792 allele. However, no statistical test is shown to compare these alleles.\nPage 4 line 6 add ‘of’ between subtypes and serotonergic\nPage 7 line 10 add ‘for the’ between Except and SAB\nPage 13 5th line from bottom - capitalize MAB-9", "reviewer_3": "This work explores the role of PBAF chromatin remodeler stablishing the postmitotic neural identity of motoneurons in the ventral nerve cord and its flanking ganglia in the nematode model animal C. elegans. Authors state that PBAF complex has divergent roles repressing gene expression in motoneurons located in the VNC while activating gene expression in those located posteriorly, in the PAG. The work proposes an interesting framework of interplay between layers of regulation of gene expression (i.e., transcription factors activity, chromatin landscape) in the context of neural fate. However, the manuscript presents important concerns that significantly reduce its relevance and conclusions, thus not reaching the quality expected for a publication in Nature Communications.\nMajor concerns:\n• As read-out of their multiple genetic manipulations the ectopic expression of a multi-copy integrated transcriptional reporter (glr-4p::tagRFP) is used. This is an important concern since authors are looking at the effect of a chromatin remodeler, it cannot be assumed that effects in this synthetic chromatin context reflects glr-4 locus. Endogenous effect of PBAF mutants is only shown in the SFig2B, for phh-10 and pbrm-1 mutants, however this analysis is performed differently to the one with glr-4p::tagRFP, there is no quantification of number of positive neurons, but measure of intensity in each MN as a pool, with no identity. This is an important concern because, as shown in Fig1 scRNAseq data and with CRISPR tagged allele, glr-4 endogenous gene is expressed in the many of these neurons, in contrast to lack of expression of the reporter. Thus, it is unclear if PFAB restricts expression of glr-4 in MN or alternatively tunes down expression of a gene normally expressed in those neurons. Furthermore, the effect seen for pbrm-1 in CRISPR tagged glr-4 is very mild.\n• In addition to the lack of convincing evidence for the role of PBAF in endogenous glr-4, this gene is the only target described for the repressive actions of PBAF. To make the paper more robust, additional targets and locomotor behavioural phenotypes should be provided.\n• As second major concern is that authors speculate about the interplay between PBAF and TFs important for MN fate, however, results do not provide any mechanistic insight of how this interplay takes place. The only results provided are whole-worm ChIPseq data for MAB-9, UNC-3 and PBRM-1, which does not demonstrate, either co-binding in same MN or any particular mechanisms of action. Additionally, MAB-9, UNC-3 expression analysis in PBRM-1 mutants is not reported.\nOther concerns:\n• Authors conclude there is a role for both long and short pbrm-1 isoforms in the VNC motoneurons. Data does not support this conclusion, tm415 could be a hypomorphic allele, explaining the weaker phenotype, and RNAi response amplifies its triggers towards 5' of the gene, thus the greater effect of the 3' clone is expected. Indeed, rescue with long isoform only (F2h) supports the specific role of long isoform, contrary to authors conclusions.\n• For ChIPseq, no called peaks are shown nor the scale of coverage is stated\n• Same data is displayed in Fig3A, Fig7A and SFig4D.", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "RESPONSE TO REVIEWERS\nWe thank the reviewers for their time and valuable feedback. We have addressed all\nconcerns through additional experiments and analyses. The new data have significantly\nstrengthened the conclusions of our work.\nREVIEWER COMMENTS\nReviewer #1 (Remarks to the Author):\nThe manuscript by Osuma and colleagues utilized a glr4-reporter line to label a subset of\nmotor neurons (MNs) that are dependent on unc-3 expression for their genetic identity. Using\nthis tool they performed a genetic screen to identify regulatory factors that alter ectopic glr-4\nexpression and presumably alter neuronal diversity in the ventral nerve cord. In this way, they\nidentified Pbrm-1 (BAF180) as a negative regulator of glr-4 in a subset of nerve cord MNs.\nThey perform a large number of very elegant and informative genetic experiments to\ndemonstrate that it is the PBAF complex and not the BAF complex that is required for the\nrepression. Mining TF expression and binding data they identify Mab-9 as the TF that likely\nrecruits PBAF complex to unc-3 targets to repress activation, which they confirm a role using\nadditional RNAi and genetic experiments. Lastly, they demonstrate that in other more caudal\nMN subtypes, Pbrm-1 cooperates with unc-3 to activate gene expression in these MNs.\nOverall, it is a very well-written manuscript showing the power of the model system and it\nhighlights a mechanistic role for SWI/SNF chromatin remodeling during neuronal\ndiversification of postmitotic neurons. The article is of interest given the role of PBAF subunits\nin neurodevelopmental disorders and it would be a good addition to the literature.\nWe thank the reviewer for highlighting the strengths and significance of this work.\nSuggestions and questions:\n1. The authors do a nice job of generating mutants for other members of the BAF and PBAF\ncomplexes to clearly show that ectopic expression is dependent on PBAF and not BAF.\nIncluded in this analysis was the use of a temperature sensitive mutant of Brg1 (swsn-4) from\nwhich they conclude that ATP-dependent remodeling is required. In fact, this experiment is\nsimilar to the deletion of other subunits in that it just disrupts a functional complex,\nstrengthening the idea that the complex is required. To demonstrate that ATPase activity is\nrequired they should repeat the experiment using an ATPase-dead mutant that would still\nincorporate into the PBAF complex but would not remodel, thereby demonstrating that\nremodeling is required to silence glr-4.\nTo address this important point, we engineered a new C. elegans strain. Upon\nsurveying the literature, we found that mutating lysine 785 (K785) at the ATPase domain of\nhuman SMARCA4 (BRG1) specifically disrupts ATPase activity PMID: 8871545, 32494730,\n30858614, 23698369. The K785 residue in human SMARCA4 is highly conserved and\northologous to K564 in C. elegans SWSN-4. Using CRISPR/Cas9, we replaced lysine (K) 785\nwith arginine (R) - K564R- in SWSN-4 because two studies previously showed that a K785R\nmutation on human SMARCA4 result in a catalytically dead enzyme PMID: 30858614,\n23698369. We found that the swsn-4 ATPase-dead mutation K564R is homozygous lethal in\nembryos, consistent with a previous C. elegans study that showed that a similar mutation\n(K564A) also resulted in early lethality (PMID: 32494730). However, we were able to evaluate\nglr-4::tagRFP expression in MNs of heterozygous swsn4K564R/+ which are viable and fertile.\nCompared to controls, we found ectopic glr-4::tagRFP expression in MNs of swsn4K564R/+\nanimals (Fig. 4f-g), indicating that ATP-dependent chromatin remodeling is required for glr-4\nrepression.\nWe updated the Results: “To assess whether the effect on glr-4 is due to ATP-\ndependent chromatin remodeling, we used CRISPR/Cas9 gene editing to generate an\nATPase-dead version of SWSN-4 by introducing a lysine (K)-to-arginine (R) mutation at a\nconserved residue (K564) essential for ATP hydrolysis PMID: 8871545, 32494730,\n30858614, 23698369. Although homozygous swsn4K564R/K564R animals are embryonic lethal\nconsistent with a previous study (PMID: 32494730), heterozygous swsn4K564R/+ animals are\nviable and fertile. Compared to controls, we found ectopic glr-4::tagRFP expression in MNs of\nswsn4K564R/+ animals (Fig. 4f-g). This finding not only indicates that ATP-dependent\nchromatin remodeling is required for glr-4 repression, but also suggests that the PBAF effect\non glr-4 is dosage-dependent.”\n2. It was challenging to decipher where the Mab-9 binding site within the 538bp promoter was\nlocated when examining the ChIP-seq tracks that were presented in Figure 7d. From the\nschematic it appears that it should be located upstream of the Unc-3 sites. The authors\nshould add the location of both the TF binding sites into the schematic.\nWe regret the lack of clarity. We have significantly updated Figure 7. Using FIMO, we\nbioinformatically predicted 10 MAB-9 sites and 4 UNC-3 sites on the glr-4 locus. All sites are\nnumbered, and their location is shown in Fig. 7d and Suppl. File 1. We updated the Methods\nwith details on prediction of TF binding sites.\nIn addition, using the endogenous gene reporter and CRISPR to inactivate the site in vivo\nwould be a more thorough way to show the importance of this site over the many other Mab-9\nbinding sites, while providing some context on the complete regulatory regions of the\nglr-4 gene.\nIndeed, there are 10 bioinformatically predicted MAB-9 sites (Fig. 7d). We mutated 3 sites\n(#5, #6, #7) individually in the context of the glr-4::tagRFP transgenic reporter and observed\nectopic glr-4::tagRFP in AS neurons only when site #6 was mutated (new Fig. 7d-f).\nFollowing the reviewer’s suggestion, we used CRISPR/Cas9 to introduce nucleotide\nsubstitutions in the MAB-9 site #6 in the context of our endogenous mScarlet::glr-4 reporter\n(allele syb3680) (new Fig. 7d). However, we did not observe any effects neither on the\nnumber of MNs expressing mScarlet::glr-4 nor the fluorescence intensity (new Fig. 7g),\nsuggesting possible redundancy between site #6 and the remaining 9 MAB-9 sites. To\naddress this, we generated yet another strain with CRISPR/Cas9 where we simultaneously\nmutated sites #6, #8, and #9, but again found no effects on mScarlet::glr-4 in MNs,\nsuggesting redundancy between the MAB-9 sites. Importantly, we do observe effects on the\nnumber of MNs expressing mScarlet::glr-4 in mab-9 null mutants (Suppl. Fig. 9).\nIn revised Results and Discussion, we bring up the notion of MAB-9 site redundancy,\ni.e., MAB-9 needs to recognize multiple of its cognate site to elicit repression on glr-4,\nconsistent with its ChIP-Seq binding pattern on the glr-4 locus. This is reminiscent of the\nmechanism of action of CFI-1/ARID3 in non-AS motor neurons. In a previous study, we found\nthat we had to eliminate 11 CFI-1 sites in the glr-4 promoter to observe an effect in non-AS\nmotor neurons of the nerve cord (PMID: 36897776). In the revised Results, we state:\n“To test whether MAB-9 binding on the glr-4 locus is functional, we first followed a\nbioinformatic approach (see Methods) and identified 10 predicted MAB-9 binding sites\nupstream and within the glr-4 locus (Fig. 7d, Supplementary File 1). Next, we individually\ndeleted the three most proximal MAB-9 sites (#5 - #7) in the context of our transgenic glr-\n4538bp prom::tagRFP (Fig. 7e-f). Deletion (D) of either site #5 or #7 yielded no effect on glr-\n4prom::tagRFP expression in nerve cord MNs (Fig. 7e-f). However, deletion of site #6 (D site\n#6) led to ectopic glr-4prom::tagRFP expression in AS subclasses in the VNC (Fig. 7e-f),\nsuggesting that MAB-9 binds directly to site #6 to repress glr-4 transcription. Interestingly,\nMAB-9 site #6 lies next to UNC-3 site #1, which is necessary for driving basal levels of\nendogenous mScarlet::glr-4 expression in nerve cord MNs PMID: 36897776).\nNext, we used CRISPR/Cas9 to mutate site #6 by introducing nucleotide substitutions\n(WT: CCTTTTCTCCT, Mutated: CCAAAAAACT) in the context of the endogenous\nmScarlet::glr-4 reporter (Supplementary File 1), but this manipulation had no effect on\nmScarlet::glr-4 expression in MNs (Fig. 7g). The discrepancy between the results obtained\nwhen MAB-9 site #6 was deleted in the transgenic glr-4538bp prom::tagRFP versus the\nendogenous mScarlet::glr-4 context suggests redundancy among the 10 MAB-9 sites. To test\nthis idea, we simultaneously mutated site #6 and two other sites (#8 and #9)(Supplementary\nFile 1), but observed no effect on endogenous mScarlet::glr-4 expression in MNs (Fig. 7g),\nraising the possibility that one or more of the remaining seven MAB-9 sites are involved in glr-\n4 repression. Altogether, genetic and biochemical (ChIP-Seq) evidence suggests that MAB-\n9/Tbx20 and PBAF act directly to repress glr-4/GRIK4 transcription in AS neuron subclasses\n(AS2-3, AS4-8, AS9-10) (Fig. 7h).”\n3. Similar to the previous point, the publicly available ChIP-seq data from whole animals\nshowed a large number of Mab-9 and Pbrm-1 binding sites along the glr-4 locus. The regions\nwith the most prominent overlapping binding sites were in the gene body. Are these likely to\nbe active/important in the MNs or are they active binding site in other tissues?\nThe reviewer is correct - the MAB-9 sites (#1 - #4) in the gene body are likely important for\nthree reasons:\na). Our mutagenesis analysis (discussed above) focused on five MAB-9 sites (#5 - #9) in the\nglr-4 promoter. Because simultaneous mutagenesis of three sites (#6, #8, #9) did not affect\nendogenous mScarlet::glr-4 expression in MNs (Fig. 7g), it is likely that the four MAB-9 sites\n(#1-#4) in the gene body are important for glr-4 repression.\nb). These four MAB-9 sites (#1 - #4) are located in a genomic region that shows prominent\nco-binding of MAB-9 and two PBAF-specific components (PBRM-1 and SWSN-7) based on\nChIP-Seq (Fig. 7d).\nc). As described in point #4 below, we have predicted physical interactions between MAB-9\nand SWSN-7.\nIt was also not clear what sites within the promoter region were important for mediating gene\nactivity and where they were with respect to the two Unc-3 sites.\nPlease see our response to point #2 above. We have now updated the figure and now\nclearly show all predicted MAB-9 and UNC-3 (COE) binding sites in the glr-4 locus (new Fig.\n7d). In a Supplementary File 1, we provide the location and nucleotide sequence for all\nsites. In a previous study (PMID: 36897776), we mutated the first UNC-3 site (COE1), which\nis located between MAB-9 sites #6 and #7, in the endogenous glr-4::mScarlet allele. We\nfound that COE1 is required for basal activation of glr-4 transcription in nerve cord MNs. As\ndetailed in point #2 above, deletion (D) of MAB-9 site #6 led to ectopic glr-4 expression in\nnerve cord MNs when the deletion occurred in the context of a transgenic glr-4::tagRFP\ncontext (Fig. 7e-f). However, mutation of MAB-9 site #6 by nucleotide substitutions in the\ncontext of the endogenous mScarlet::glr-4 reporter did not lead to any effects (Fig. 7d, g),\npossibly due to redundancy between site #6 and other MAB-9 sites within the gene body.\nTrying to repeat the ChIP-seq data using dissected ventral nerve cord from WT, Mab-9 and\not792 mutants would really add to the analysis by highlighting the important regulatory\nregions mediating repression.\nWe agree that FACS-sorting of AS neurons followed by ChIP-seq would be the ideal\napproach. However, MAB-9 is only expressed in a limited number of cells (~35 nerve cord\nmotor neurons) out of the ~3,000 cells (959 somatic cells + 2000 germ cells) of the entire\nworm. Hence, it is incredibly challenging to obtain enough material for the proposed ChIP-\nSeq experiments.\n4. Co-IP experiments showing an interaction between Mab-9 and Pbrm-1 (or another\nmember of the PBAF complex) would provide further support of a role for Mab-9 in recruiting\nthe PBAF complex to the glr-4 locus.\nBecause MAB-9 is only expressed in a limited number of cells (~35 nerve cord motor\nneurons) out of the ~3,000 cells (959 somatic cells + 2000 germ cells) of the entire worm, it is\nincredibly challenging to conduct co-IP experiments for MAB-9. Prompted by the reviewer’s\nsuggestion, however, we in silico predicted potential interactions between MAB-9 and several\nPBAF/BAF components using ColabFold, which offers accelerated prediction of protein\nstructures and complexes by combining the fast homology search of MMseqs2 with\nAlphaFold2 or RoseTTAFold (PMID: 35637307, 35273146, 30615063). Of note, AlphaFold2\ncorrectly predicted protein-protein interactions within the SNARE complex, which were\nsubsequently validated with crystal structure data (PMID: 38072052). Using stringent cut-off\ncriteria established in the SNARE study, we ran a ColabFold pulldown between MAB-9 and\nthe following proteins: four PBAF-specific components [SWSN-9, PBRM-1, SWSN-7, PHF-\n10] and six core/accessory components [SWSN-1, SWSN-4, SWSN-5, SWSN-2.1-2, SWSN-\n3, SWSN-6]. We found that ColabFold predicted two potential interactions: a) between MAB-\n9 and SWSN-7, and b) between MAB-9 and SWSN-6.\nThe predicted MAB-9-SWSN-7 interaction is particularly intriguing because there is\nsubstantial co-binding of MAB-9 and SWSN- along the glr-4 gene body based on ChIP-Seq\ndata (Fig. 7d), corroborating the possibility of MAB-9 recruiting PBAF (SWSN-7) to the locus\nto elicit repression.\nThe data from this analysis are included in new Figure 8 and new Suppl. Fig. 10. We\nupdated the Results as follows:\n“Predicted physical interactions between the MAB-9/Tbx20, the PBAF-specific subunit\nSWSN-7 and the accessory subunit SWSN-6\nA plausible mechanism by which MAB-9 could provide specificity to PBAF to repress glr-4 in\nAS neurons is through direct PBAF recruitment via protein-protein interactions. This model is\nconsistent with the prominent co-binding of MAB-9, SWSN-7 and PBRM-1 within the glr-4\nlocus (Fig. 7d). Because co-immunoprecipitation experiments on isolated C. elegans MNs\nare not feasible, we tested this model via an in silico interaction screen using the\nLocalColabFold implementation of AlphaFold2, a powerful tool for accurate prediction of\nprotein-protein interactions (PMID: 40233800, PMID: 36170365, PMID: 38072052, PMID:\n34762488). We screened for pairwise interactions between MAB-9 and the PBAF-specific\nsubunits (SWSN-7, SWSN-9, PBRM-1, PHF-10), as well as other core/accessory subunits\n(SWSN-2.2, SWSN-4, SWSN-5, SWSN-3, SWSN-1, SWSN-6, SWSN-2.1). Following\nstringent criteria established in a previous study PMID: 38072052, we assessed prediction\nquality using an interface predicted template modelling (ipTM) score cut-off of 0.3 for the\nmajority (at least 3 out of 5) of the predicted models for each pairwise-interaction (Fig 8a,\nSuppl. Fig. 10). We further assessed the proposed interaction using the predicted alignment\nerror (PAE) plots (Fig. 8b, Suppl. Fig. 10). With this analysis, we identified the PBAF-specific\nsubunit SWSN-7 and the accessory subunit SWSN-6 as predicted interactors with MAB-9.\nPredicted contacts of the top-ranked structures for both dimers were assessed using\nPDBsum and visualized with PyMOL (Fig 8c,d). The MAB-9-SWSN-7 interface had an area\nof 1947 Å2 and 2069 Å2 for each protein, respectively. The MAB-9-SWSN-7 interaction\nconsisted of 3 salt bridges, 9 hydrogen bonds, and 486 non-bonded contacts. The MAB-9-\nSWSN-6 interface had an area of 2252 Å2 and 2720 Å2 for each protein, respectively. The\nMAB-9-SWSN-6 interaction consisted of 1 salt bridge, 18 hydrogen bonds, and 1820 non-\nbonded contacts. These predicted interactions support a model of PBAF recruitment via\ndirect protein interactions with MAB-9, offering entry-points for future experimental validation.”\n5. It would be helpful to offer some explanation in the Discussion on why the cell-type specific\nTFs (Mab-9, CFI-1) play a dominant role in PBAF recruitment when Unc-3 and PBAF are\nboth present. For example, are there differences in Mab-9 vs Unc-3 expression levels which\nfavor repression versus activation? Differences in binding site affinities at the glr-4 promoter?\nIn caudal MN subtypes, Unc-3 and PBAF are required for activation, but when two\nantagonistic TFs that recruit PBAF are both present (Unc-1 and Mab-9) how does repression\ndominate in this instance? Does it come down to differences in PBAF subunit composition,\nsimilar to what is known during the change from proliferation to differentiation? Some\nadditional insight/opinion seems warranted.\nTo address this, we conducted new experiments and have significantly updated the Results\nand Discussion as follows:\nResults:\n“The PBAF complex antagonizes the terminal selector UNC-3/EBF in nerve cord MNs\nThe terminal selector UNC-3/EBF acts directly to activate multiple effector target genes\n(including glr-4) in subclasses of cholinergic MNs41,42. In prior work with the endogenous\nmScarlet::glr-4 reporter38, we showed that UNC-3 is required to activate basal/low levels of\nglr-4 in nerve cord MNs. Here, we show that expression of the transgenic glr-4prom::tagRFP\nreporter in SAB and DA9 neurons is completely lost in unc-3 (n3435) null mutant animals\n(Fig. 5a-d), consistent with the known role of UNC-3 as transcriptional activator41,44. We\ntherefore hypothesized that, in nerve cord MNs, unc-3 gene activity is necessary for the\nincreased glr-4 expression we observe in PBAF mutants (Fig. 4c). Through double mutant\nanalysis, we found this to be the case. Expression of glr-4 in nerve cord MNs is present in\nsingle pbrm-1(-) or swsn-9(-) mutants, but completely lost in unc-3(-); pbrm-1(-) and unc-3(-);\nswsn-9(-) double mutants (Fig. 5a, c). Next, we over-expressed UNC-3 specifically in nerve\ncord MNs and witnessed a modest increase in glr-4prom::tagRFP expression. However, UNC-\n3 over-expression in a PBAF (swsn-9) mutant was able to substantially increase glr-\n4prom::tagRFP in MNs (new Fig. 5e), suggesting that perturbation of the PBAF complex\nenables UNC-3 to robustly activate glr-4 transcription. Altogether, our genetic analysis\nsuggests that the PBAF complex represses glr-4 in nerve cord MNs by antagonizing the\ntranscriptional activator UNC-3/EBF, albeit the precise molecular mechanism remains elusive\n(Fig. 5f).”\nDiscussion:\n“In nerve cord MNs, however, we found that PBAF represses (tunes down) gene expression.\nIn these cells, our findings suggest that the conserved TF MAB-9/TBX20 may act as a PBAF\nrecruiter. This model is supported by several lines of evidence: First, MAB-9 is known to act\nas a transcriptional repressor in C. elegans nerve cord MNs37. In AS neurons, MAB-9 is\nexpressed at higher levels compared to the transcriptional activator UNC-3 (Fig. 1d),\nthereby favoring glr-4 repression. Second,…”\nReviewer #2 (Remarks to the Author):\nIn this study, Osuma and colleagues used a forward genetic screen to identify C. elegans\nPBAF as a regulator of neuronal identity. Specifically, they found that PBAF represses gene\nexpression in ventral nerve cord motor neurons and activates gene expression in posterior\nmotor neurons. This suggests that PBAF utilizes distinct mechanisms to control related, but\ndifferent neuronal cohorts in C. elegans. The authors used forward genetics, RNAi, neuron-\nspecific rescue experiments, single-molecule FISH and analysed ChIP-Seq data to posit the\nmechanistic basis of PBAF function. In particular, they suggest that PBAF represses glr-4\nVNC MN expression by antagonizing UNC-3 - a terminal selector TF.\nThis is significant work that identifies the chromatin modifier PBAF as a regulator of specific\nneuron fates. The data justify the conclusions drawn. I do however have a few issues that\nshould be addressed.\nWe thank the reviewer for highlighting the significance of this work.\nMajor points\nIs PBAF continuously required to repress glr-4 expression in the VNC MNs or does PBAF set\nup a program early in development? Removing PBAF post-developmentally would provide an\nanswer to this question.\nTo address the reviewer’s concern, we employed the auxin-inducible degron (AID) system.\nWe used CRISPR/Cas9 to introduce the AID degron together with a fluorescent reporter\n(mNeonGreen, mNG) at the C-terminus of PBRM-1. However, such tagging led to strong\nhypomorphic effects on glr-4 expression in MNs (Figure for reviewer 2), preventing us from\ntesting whether PBAF is required continuously in larval and adult stages. Because the glr-4\nhypomorphic effects were observed even in the absence of the TIR1 effector protein (Figure\nfor reviewer 2), we conclude that C-terminal tagging disrupted the normal function of PBRM-\n1.\nFigure legend: Quantification showing number of MNs expressing glr-\n4prom::tagRFP. Animals analyzed at L4 stage. WT, n=35; PBRM-\n1::mNG::AID; TIR1, n=19, PBRM-1::mNG::AID, n=15. Welch’s t-test,\np<0.0001 = ****.\nNevertheless, we conducted three sets of experiments that\ndemonstrate that PBAF represses glr-4 in post-mitotic MNs. These\nexperiments rely on two MN-specific promoter fragments (cho-1, lin-\n39), which we found to be active specifically in post-mitotic MNs at L2\nand later larval stages.\n1. Using the cho-1 promoter to drive a GFPnanobody fused to ZIF-1,\nwe degraded PBRM-1::GFP and observed glr-4 de-repression in MNs at L4 (Fig. 3d).\n2. Using the cho-1 and lin-39 promoters to knock-down pbrm-1 isoforms with RNAi, we\nagain observed glr-4 de-repression in MNs at L4 (Fig. 3c).\n3. Using the lin-39 promoter to drive swsn-9 cDNA in swsn-9 mutants, we observed\nrescue of glr-4 de-repression in MNs at L4 (Suppl. Fig. 7c).\nBecause the cho-1 and lin-39 promoter fragments are not active in MN progenitors, we\nconclude that PBAF acts in post-mitotic MNs (VA and AS) at larval stages to repress glr-4.\nThe authors shown that expression of glr-4 (encoding a glutamate receptor) is derepressed\nwhen PBAF is depleted in the VNC motor neurons. What is the behavioral effect of\ninappropriate GLR-4 expression? If there a motility/body bend phenotype?\nTo address this, we used a previously published allele, syb5348, that increases endogenous\nglr-4 expression specifically in ventral nerve cord MNs (PMID: 36897776). Although worm\nspeed and response to touch are unaffected (Suppl. Fig. 3), increased glr-4 expression led\nto a reduction of body bends in a thrashing assay compared to control animals (Suppl. Fig.\n3). This result indicates that high glr-4 expression in verve cord MNs affects C. elegans\nmotility.\nWe updated the Results as follows: “Using a previously published strain that does not\ncarry the ot792 mutation but selectively overexpresses glr-4 in nerve cord MNs38, we found\ndefects in thrashing behavior (Suppl. Fig. 3a), indicating that increased glr-4 levels in MNs\nare detrimental to C. elegans motility. Therefore, the ot792 mutants offer an opportunity to\nidentify a mechanism that tunes down (limits) glr-4 expression in nerve cord MNs.”\nThe authors state that “Hence, our genetic analysis suggests that the PBAF complex\nrepresses glr-4 in nerve cord MNs by antagonizing the transcriptional activator UNC-3/EBF”.\nHowever, no mechanism underlying this suggestion is posited.\nTo address this, we conducted new experiments and have significantly updated the Results\nand Discussion as follows:\n“The PBAF complex antagonizes the terminal selector UNC-3/EBF in nerve cord MNs\nThe terminal selector UNC-3/EBF acts directly to activate multiple effector target genes\n(including glr-4) in subclasses of cholinergic MNs41,42. In prior work with the endogenous\nmScarlet::glr-4 reporter38, we showed that UNC-3 is required to activate basal/low levels of\nglr-4 in nerve cord MNs. Here, we show that expression of the transgenic glr-4prom::tagRFP\nreporter in SAB and DA9 neurons is completely lost in unc-3 (n3435) null mutant animals\n(Fig. 5a-d), consistent with the known role of UNC-3 as transcriptional activator41,44. We\ntherefore hypothesized that, in nerve cord MNs, unc-3 gene activity is necessary for the\nincreased glr-4 expression we observe in PBAF mutants (Fig. 4c). Through double mutant\nanalysis, we found this to be the case. Expression of glr-4 in nerve cord MNs is present in\nsingle pbrm-1(-) or swsn-9(-) mutants, but completely lost in unc-3(-); pbrm-1(-) and unc-3(-);\nswsn-9(-) double mutants (Fig. 5a, c). Next, we over-expressed UNC-3 specifically in nerve\ncord MNs and witnessed a modest increase in glr-4prom::tagRFP expression. However, UNC-\n3 over-expression in a PBAF (swsn-9) mutant was able to substantially increase glr-\n4prom::tagRFP in MNs (new Fig. 5e), suggesting that perturbation of the PBAF complex\nenables UNC-3 to robustly activate glr-4 transcription. Altogether, our genetic analysis\nsuggests that the PBAF complex represses glr-4 in nerve cord MNs by antagonizing the\ntranscriptional activator UNC-3/EBF, albeit the precise molecular mechanism remains elusive\n(Fig. 5f).”\nDiscussion:\n“In nerve cord MNs, however, we found that PBAF represses (tunes down) gene expression.\nIn these cells, our findings suggest that the conserved TF MAB-9/TBX20 may act as a PBAF\nrecruiter. This model is supported by several lines of evidence: First, MAB-9 is known to act\nas a transcriptional repressor in C. elegans nerve cord MNs37. In AS neurons, MAB-9 is\nexpressed at higher levels compared to the transcriptional activator UNC-3 (Fig. 1d), thereby\nfavoring glr-4 repression. Second,…”\nWhat evidence is there that the neurons ectopically expressing glr-4 in DEL_MAB-9 site\nanimals (Fig 7F) are AS neurons?\nWe regret the lack of clarity here. As shown in Fig. 1b, the AS neurons can be\nunambiguously identified based on their unique axonal morphology. All AS commissures to\nthe dorsal nerve cord are present on the right side of the body. Once the commissures reach\nthe dorsal nerve cord, they extend anteriorly and form synapses with dorsal body wall\nmuscles. Because we mutated the MAB-9 sites in the context of a glr-4::tagRFP fluorescent\nreporter, we counted the number of MNs that ectopically express tagRFP and also confirmed\nthat these cells belong to the AS class of MNs based on their commissural morphology. We\nupdated the legend of Fig. 7e-f accordingly.\nWe have also expanded our analysis of MAB-9 sites. We mutated additional sites\neither in the glr-4::tagRFP transgenic reporter or in the endogenous glr-4 locus (Fig. 7d-h).\nPlease, see revised Results, section “MAB-9/Tbx20 and PBAF act directly to repress glr-\n4/GRIK4 transcription”.\nMinor points\nThe authors mention that the pbrm-1(tm415) allele has a ‘weaker’ glr-4 phenotype compared\nto the ot792 allele. However, no statistical test is shown to compare these alleles.\nWe have now included the statistical comparison between the two genotypes. The pbrm-\n1(tm415) phenotype in terms of glr-4 expression is weaker than the pbrm-1 (ot792). P <\n0.0001 (Fig. 2b). We also extended our analysis at the behavioral level. Automated worm\ntracking revealed milder locomotion phenotypes for pbrm-1(tm415) animals compared to\npbrm-1(ot792) animals (new Fig. 10).\nPage 4 line 6 add ‘of’ between subtypes and serotonergic. Done. Thanks.\nPage 7 line 10 add ‘for the’ between Except and SAB. Done. Thanks.\nPage 13 5th line from bottom - capitalize MAB-9. Done. Thanks.\nReviewer #3 (Remarks to the Author):\nThis work explores the role of PBAF chromatin remodeler establishing the postmitotic neural\nidentity of motoneurons in the ventral nerve cord and its flanking ganglia in the nematode\nmodel animal C. elegans. Authors state that PBAF complex has divergent roles repressing\ngene expression in motoneurons located in the VNC while activating gene expression in\nthose located posteriorly, in the PAG. The work proposes an interesting framework of\ninterplay between layers of regulation of gene expression (i.e., transcription factors activity,\nchromatin landscape) in the context of neural fate. However, the manuscript presents\nimportant concerns that significantly reduce its relevance and conclusions, thus not reaching\nthe quality expected for a publication in Nature Communications.\nWe appreciate the reviewer’s critical evaluation. During the revision, we have added a\nsubstantial amount of new data that have significantly strengthened the manuscript’s major\nconclusions and proposed mechanism.\nMajor concerns:\n• As read-out of their multiple genetic manipulations the ectopic expression of a multi-copy\nintegrated transcriptional reporter (glr-4p::tagRFP) is used. This is an important concern since\nauthors are looking at the effect of a chromatin remodeler, it cannot be assumed that effects\nin this synthetic chromatin context reflects glr-4 locus. Endogenous effect of PBAF mutants is\nonly shown in the SFig2B, for phh-10 and pbrm-1 mutants, however this analysis is\nperformed differently to the one with glr-4p::tagRFP, there is no quantification of number of\npositive neurons, but measure of intensity in each MN as a pool, with no identity. This is an\nimportant concern because, as shown in Fig1 scRNAseq data and with CRISPR tagged\nallele, glr-4 endogenous gene is expressed in the many of these neurons, in contrast to lack\nof expression of the reporter. Thus, it is unclear if PFAB restricts expression of glr-4 in MN or\nalternatively tunes down expression of a gene normally expressed in those neurons. Furthermore,\nthe effect seen for pbrm-1 in CRISPR tagged glr-4 is very mild.\nWe regret the lack of clarity, which stems from the fact the glr-4::tagRFP transgenic\nreporter only partially recapitulates the endogenous glr-4 expression pattern – a very\ncommon phenomenon in gene regulation studies as transgenic reporters often lack the\nendogenous context.\nWe have addressed the reviewer’s concern in several ways.\n1. We clarify the differences between the glr-4::tagRFP transgenic reporter and the\nmScarlet::glr-4 endogenous reporter in a new panel (Fig. 7d) and in the Results\ntext.\n2. Our scRNA-Seq data show that glr-4 transcripts are detected at high levels in MNs\nlocated outside the ventral nerve cord (e.g., SAB, DA9) (Fig. 1c). Within the ventral\nnerve cord, glr-4 transcripts are normally expressed at very low (basal) levels, or\nare undetectable, in MNs. This pattern of expression is consistent with our recent\nstudy, where we analyzed the expression of the endogenous mScarlet::glr-4\nreporter with single-cell resolution (PMID: 36897776). Our transgenic glr-4::tagRFP\nreporter expresses strongly in the two MN classes outside the ventral nerve cord of\n(SAB and DA9). We clarified these differences in the revised Results: “Because\nglr-4 transcripts are detected at low levels in AS and VA neurons (Fig. 1c), we\npropose that PBAF tunes down glr-4 to low levels in these neurons (Fig. 6d). On\nthe other hand, glr-4 transcripts are undetectable in DA neurons (Fig. 1c) due to\nPBAF’s repressive activity (Fig. 6d).”\n3. In previous work (PMID: 36897776), we showed that the TF UNC-3 is required to\nactivate basal/low levels of glr-4 in nerve cord MNs, and this prior study has been\ncited in the current manuscript.\n4. Prompted by the reviewer’s comment, we analyzed the expression of the\nendogenous mScarlet::glr-4 reporter in two PBAF mutant strains. Compared to\ncontrols, we found an increased number of nerve cord MNs expressing\nmScarlet::glr-4 in phh-10 and pbrm-1 mutants (Suppl. Fig. 2c). Further, the\nfluorescent intensity of the mScarlet::glr-4 reporter is increased in nerve cord MNs\nof phh-10 and pbrm-1 mutants (Suppl. Fig. 2a).\n5. We show that glr-4 overexpression specifically in MNs affects C. elegans motility\n(Suppl. Fig. 3a).\nOverall, we conclude that PBAF limits (tunes down) glr-4 expression in AS and VA\nsubclasses, whereas it represses glr-4 in DA subclasses (Fig. 4d).\nRelevant to the reviewer’s concern are four additional points:\n1. We also evaluated endogenous glr-4 mRNA with sm RNA FISH in PBAF (phf-10)\nmutants and observed an increase in glr-4 mRNA levels in nerve cord MNs with\nsmRNA FISH (Suppl. Fig. 2d-e).\n2. Like the two PBAF mutants (phh-10 and pbrm-1), we found an increase in\nfluorescence intensity of the endogenous glr-4::mScarlet reporter in nerve cord\nMNs of mab-9 mutants (Suppl. Fig. 8a-c).\n3. To further corroborate our conclusions, we have used an additional transgenic glr-\n4::GFP reporter in which GFP is fused after the 14th exon under the control of 4.9kb\nof the glr-4 promoter. This reporter is normally expressed at very low levels in some\nnerve cord MNs, resembling the endogenous mScarlet::glr-4 reporter. Again, we\nobserved an increase in the number of MNs expressing this reporter in PBAF\nmutant (phf-10) and mab-9 mutant animals (Suppl. Fig. 8d).\n4. An ATPase-dead version of SWSN-4 (SMARCA4/BRG1) leads to increased glr-4\nexpression in MN (Fig. 4f-g).\nIn total, we have used four read-outs (endogenous reporter, two transgenic reporters,\nand sm RNA FISH) and have evaluated the expression of glr-4 in two PBAF mutants, as well\nas in mab-9 mutants. (Figure 4, Suppl. Figures 2, 8).\n• In addition to the lack of convincing evidence for the role of PBAF in endogenous glr-4,\nthis gene is the only target described for the repressive actions of PBAF. To make the paper\nmore robust, additional targets and locomotor behavioural phenotypes should be provided.\nBehavioral analysis: We evaluated locomotory behavior of pbrm-1 mutants using\nautomated worm tracking (see Methods). The pbrm-1 (tm415) and pbrm-1(ot792) mutants\nwere 6x outcrossed to the wild type (N2 strain). In revised Results, we report this data in a\nnew section:\n“Loss of pbrm-1 leads to locomotion defects\nBecause our molecular analyses revealed gene expression defects in MNs, we wondered\nwhether PBAF mutant animals display locomotion defects. To test this, we performed high-\nresolution behavioral analysis of freely moving adult (day 1) pbrm-1 mutant animals using\nautomated multi-worm tracking technology 73,74. This analysis can quantitate multiple features\nrelated to C. elegans locomotion (e.g., speed, crawling amplitude, curvature, pause, forward\nand backward locomotion) and, most importantly, each feature can be localized to a specific\npart of the nematode’s body (e. g., head, mid-body, tail). We assessed homozygous pbrm-1\nmutants carrying either the tm415 or ot792 alleles (Fig. 2e). Hierarchical clustering analysis\nshowed that pbrm-1(tm415) animals cluster more closely to control (N2) animals, whereas\npbrm-1(ot792) animals displayed strong locomotion defects (Fig. 10a). These behavioral\neffects are consistent with the stronger effects we observed on glr-4 expression for pbrm-\n1(ot792) compared to pbrm-1(tm415) animals (Fig. 2b). In Fig. 10b-g, we show examples of\nlocomotion features (e.g., curvature, speed, time paused) that are significantly affected in\npbrm-1(ot792) animals but mildly affected in pbrm-1(tm415) animals. We conclude that loss\nof pbrm-1 leads to locomotion defects.”\nMoreover, we assessed the effect of inappropriate glr-4 expression in locomotion.\nUsing a trashing assay, we found that animals that specifically overexpress glr-4 in nerve\ncord MNs display reduced motility. i.e., reduced number of body bends (Suppl. Fig. 3a).\nPBAF target analysis: Our work identified five new PBAF target genes: glr-4/GRIK4 in\nnerve cord MNs and itr-1/ITPR1, avr-15/GLRA1-3, ser-2/HTR1A, and glr-5/GRIK1/3 in caudal\nMNs. Because the roles of PBAF are poorly understood in nervous system development,\nespecially in the context of post-mitotic neurons, we feel that the identification of five PBAF\ntargets significantly contributes to the field.\nTo expand the repertoire of PBAF targets, we followed two approaches. First, we\nasked whether there are available transgenic reporter lines that – like glr-4 – are expressed\nboth in SAB and nerve cord MNs. We found several reporter lines that fall in this category\n(acr-2, del-1, dbl-1, slo-2, unc-53, etc.). However, when we crossed them into PBAF mutants\nwe found no effect in their expression in MNs (Suppl. Fig. 8).\nSecond, we identified 1,000 genes that – like glr-4 – are expressed at low levels in\nnerve cord MNs (AS neurons) based on sc-RNA-Seq data of larval MNs (CenGEN project,\nPMID: 34237253). Specifically, we included 1,000 genes with expression values (transcripts\nper million, TPM) centered around glr-4 (TPM: 120.5) (see updated Methods). Next, we found\nthat – like glr-4 – 247 of these 1,000 genes are co-bound by UNC-3, MAB-9 and SWSN-7\n(Suppl. Fig. 9b, Supplementary File 1). Although validation of these 247 genes is beyond\nthe scope of this manuscript, this analysis nevertheless offers a list of putative target genes\nthat are repressed by MAB-9 and PBAF in AS neurons.\n• As second major concern is that authors speculate about the interplay between PBAF and\nTFs important for MN fate, however, results do not provide any mechanistic insight of how\nthis interplay takes place. The only results provided are whole-worm ChIPseq data for MAB-\n9, UNC-3 and PBRM-1, which does not demonstrate, either co-binding in same MN or any\nparticular mechanisms of action. Additionally, MAB-9, UNC-3 expression analysis in PBRM-1\nmutants is not reported.\nPrompted by the reviewer’s comment, we conducted new experiments and analyses. Based\non the following data, we propose a model where MAB-9 acts as a PBAF recruiter in AS\nneurons to counteract the ability of UNC-3 to activate glr-4.\n- In previous work (PMID: 36897776), we showed that UNC-3 drives high levels of glr-4\nin head SAB neurons and basal/low levels of glr-4 in nerve cord MNs. As shown in\nFig. 7a, mab-9 is absent from SAB neurons, but expressed at higher levels than unc-3\nin AS neurons. We propose that these high levels of MAB-9 ultimately lead to\nrepression glr-4 expression in AS neurons.\n- Null mab-9 mutants and PBAF mutants show increased glr-4 expression in AS\nneurons (Fig. 7b-c, Suppl. Fig. 9).\n- In unc-3; PBAF double mutants, we observe the same phenotype as in unc-3 single\nmutants: no glr-4 expression in AS neurons, indicating that UNC-3 drives the increased\nlevels of glr-4 expression in AS when PBAF is disrupted (Fig. 5).\n- In a new experiment, we over-expressed UNC-3 in nerve cord MNs but this was\ninsufficient to increase glr-4 expression, presumably due to the high levels of MAB-9\nleading to PBAF recruitment. Interestingly, UNC-3 over-expression in a PBAF (swsn-9)\nmutant was able to increase glr-4 expression in MNs (Fig. 5e), suggesting that\nperturbation of the PBAF complex enables UNC-3 to activate glr-4 transcription.\n- ChIP-Seq shows extensive co-binding between MAB-9, SWSN-7, and PBRM-1 along\nthe glr-4 gene body, whereas UNC-3 binding is mostly confined upstream of the glr-4\nlocus (Fig. 7d).\n- Our in silico prediction analysis identified SWSN-7 as a putative physical interactor of\nMAB-9, supporting the model of PBAF recruitment by MAB-9 (Fig. 8). Please, see\nresponse #4 to Reviewer 1.\n- We also conducted extensive mutagenesis of MAB-9 binding sites either in the context\nof a transgenic glr-4::tagRFP reporter or in the endogenous mScarlet::glr-4 reporter\nusing CRISPR/ Cas9 (Fig. 7). This analysis uncovered possible redundancy between\nthe multiple MAB-9 sites in the glr-4 locus. between Please, see response #2 to\nReviewer 1.\n- We found the expression of a mab-9 reporter to be unaffected in MNs of various PBAF\nmutants (Suppl. Fig. 9f-g).\n- We used CRISPR/Cas9 to generate an ATPase-dead version of SWSN-4\n(SMARCA4/BRG1) by introducing a lysine-to-arginine mutation at a conserved residue\n(K564R) essential for ATP hydrolysis PMID: 8871545, 32494730, 30858614,\n23698369. Although homozygous swsn4K564R/K564R animals are embryonic lethal\nconsistent with a previous study (PMID: 32494730), heterozygous swsn4K564R/+\nanimals are viable and fertile. Compared to controls, we found ectopic glr-4::tagRFP\nexpression in MNs of swsn4K564R/+ animals (Fig. 4f-g), indicating that ATP-dependent\nchromatin remodeling is required for glr-4 repression.”\nAltogether, this data, described in Results and summarized in Discussion, offer\nmechanistic insights of how UNC-3, MAB-9 and PBAF control glr-4 gene expression in MNs.\nOther concerns:\n• Authors conclude there is a role for both long and short pbrm-1 isoforms in the VNC\nmotoneurons. Data does not support this conclusion, tm415 could be a hypomorphic allele,\nexplaining the weaker phenotype, and RNAi response amplifies its triggers towards 5' of the\ngene, thus the greater effect of the 3' clone is expected. Indeed, rescue with long isoform only\n(F2h) supports the specific role of long isoform, contrary to authors conclusions.\nThe reviewer is correct, and we have revised the text accordingly. Based on the\ntransgenic rescue experiments, the long isoform of PBRM-1 is necessary for glr-4 repression.\nTo corroborate this conclusion, we employed CRISPR/Cas9 mutagenesis to generate\nisoform-specific pbrm-1 alleles. Mutation of the start ATG (Met) codon of the short isoform\n(C26C6.1b.1) into ATA (Ile) (pbrm-1(10332) allele) had no effect on glr-4 expression in nerve\ncord MNs (Fig. 2e, i). However, mutation of the start ATG (Met) codon of the long isoform\n(C26C6.1a.1) into ATA (Ile) (pbrm-1(10440) allele) led to ectopic glr-4 expression in MNs\n(new Fig. 2e, i). Hence, the long isoform of PBRM-1 is necessary for glr-4 repression. This\nfinding has important mechanistic implications. For example, the BAH domains, which are\nonly present in the long isoform (Fig. 2f), may be implicated in glr-4 repression.\nIn Discussion, we mention: “… PBRM-1, the signature subunit of PBAF, contains two\nbromo-adjacent homology (BAH) domains (Fig. 2e), which are known protein-protein\ninteraction domains involved in transcriptional repression.”\n• For ChIPseq, no called peaks are shown nor the scale of coverage is stated.\nWe regret this oversight. We have significantly updated Figure 7. We reanalyzed the ChIP-\nSeq data for MAB-9, UNC-3, and SWSN-7. We now include tracks with called peaks\nunderneath the glr-4 locus, as well as the scale of coverage.\n• Same data is displayed in Fig3A, Fig7A and SFig4D.\nIn Fig 7a, we now show a partial version of the dot plot for unc-3, mab-9 and PBAF\nexpression because the remaining panels in the figure show additional data on these\nproteins. We have completely the data from Suppl. Fig. 4d to avoid redundancy.\nReviewer #4 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This\nis part of the Nature Communications initiative to facilitate training in peer review and to\nprovide appropriate recognition for Early Career Researchers who co-review manuscripts.\nThanks.\n14"}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have adequately addressed my concerns.", "reviewer_2": "The authors have provided extensive new data and explanations that have fully addressed my initial concerns.", "reviewer_3": "We appreciate the significant amount of work and data the authors have incorporated into this revised version of the manuscript and commend them for their efforts. In particular, we acknowledge the value of the experiments generating isoform-specific pbrm-1 mutants, and more importantly, the characterization of the glr-4 endogenous reporter and FISH analyses in select PBAF mutants, which represent a key advance for the paper, as well as the modelling for MAB-9 PBAF interactions.\nHowever, some important concerns remain:\n1. Despite the new experiments, the role of the PBAF complex in MNs is still limited to regulation of glr-4 gene expression; no additional direct targets have been identified. Although the authors perform behavioral assays to potentially compensate for this limitation, the use of pbrm-1 mutants restricts the conclusions. pbrm-1 is panneuronally or even ubiquitously expressed, making it difficult to attribute locomotor defects specifically to MN gene expression, rather than to defects in other neurons or tissues. Given that the authors have also characterized other loss-of-function strains with more restricted patterns (e.g., ZIF-1-mediated degradation or MN-specific RNAi), behavioral assays should also be conducted using these models. Moreover, the analysis of locomotor phenotypes requires clearer explanation and connection to specific neuronal functions.\nFor instance, since AS motor neurons are implicated in body wave propagation, is this particular feature disrupted in the MN-\nspecific PBAF loss-of-function strains?\n2. The authors provide predictive modeling of a potential interaction between the PBAF complex and MAB-9, which is a\npromising direction. However, this remains unvalidated experimentally. While we recognize the technical challenges of\nperforming co-immunoprecipitation in C. elegans, alternative strategies—such as heterologous expression in cell lines or in\nvitro protein-protein interaction assays—should be considered.\n3. The experiments involving mutations in predicted MAB-9 binding sites are fragmentary. These were initially performed\nusing a glr-4 reporter construct covering a region without a MAB-9 ChIP-seq peak. Although this analysis shows an effect on\nreporter expression, similar mutations introduced into the endogenous locus do not yield the same result. To strengthen the\nproposed model, the site 6 mutation in the endogenous locus should be combined with mutations at other predicted MAB-9\nbinding sites that overlap with MAB-9 ChIP peaks.\nSpecific Comments:\n• Figure 10: In addition to employing alternative, more specific models for behaviour, the authors should improve the\ngraphical presentation. In particular, the titles of panels b–g are not very informative and should be revised for clarity.\n• Figure 7 and 9: The PBRM-1 ChIP-seq track is missing in Figure 7, and tracks for all relevant factors are missing in Figure\n9.\n• Supplementary Figure 3a: Please include a brief explanation in the main text of how the allele leads to glr-4\noverexpression (e.g., through cfi-1 mutation).\n• The manuscript states: “Interestingly, MAB-9 and PBAF (PBRM-1, SWSN-7) binding coincides at several regions upstream\nand within the glr-4 locus, supporting the possibility of MAB-9 recruiting PBAF (Fig. 7d).” However, MAB-9 peaks are\nobserved only within the gene body and not upstream. Please revise the text accordingly.\n• Please use italics for all gene names and alleles, which are still missing in some graphs and figure legends (Main and Sup\nfigures)", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "RESPONSE TO REVIEWERS – MANUSCRIPT NCOMMS-24-71325A\nReviewer #1 (Remarks to the Author):\nThe authors have adequately addressed my concerns.\nWe are glad that reviewer #1 is satisfied with the revised manuscript.\nReviewer #2 (Remarks to the Author):\nThe authors have provided extensive new data and explanations that have fully addressed my initial\nconcerns.\nWe are glad that reviewer #2 is acknowledging the addition of extensive new data.\nReviewer #3 (Remarks to the Author):\nWe appreciate the significant amount of work and data the authors have incorporated into this revised\nversion of the manuscript and commend them for their efforts. In particular, we acknowledge the value\nof the experiments generating isoform-specific pbrm-1 mutants, and more importantly, the\ncharacterization of the glr-4 endogenous reporter and FISH analyses in select PBAF mutants, which\nrepresent a key advance for the paper, as well as the modelling for MAB-9 PBAF interactions.\nWe are glad that reviewers #3 and #4 are acknowledging the significant amount of data added to the\nprevious revision. To address the remaining issues, we conducted additional experiments that\nstrengthen our original conclusions, as described below.\nHowever, some important concerns remain:\n1. Despite the new experiments, the role of the PBAF complex in MNs is still limited to regulation of\nglr-4 gene expression; no additional direct targets have been identified. Although the authors perform\nbehavioral assays to potentially compensate for this limitation, the use of pbrm-1 mutants restricts the\nconclusions. pbrm-1 is panneuronally or even ubiquitously expressed, making it difficult to attribute\nlocomotor defects specifically to MN gene expression, rather than to defects in other neurons or\ntissues. Given that the authors have also characterized other loss-of-function strains with more\nrestricted patterns (e.g., ZIF-1-mediated degradation or MN-specific RNAi), behavioral assays should\nalso be conducted using these models. For these experiments, we used the exact same RNAi lines against pbrm-1 as shown in\nFig. 3c and the exact same ZIF-1 line as shown in Fig 3d. However, we were unable to identify any\nthrashing defects (Suppl. Figure 12), likely due to the partial decrease of pbrm-1/PBRM-1 when RNAi\nor ZIF-1 technology are used. For example, we only achieve a modest decrease in the numbers of\nmotor neurons expressing PBRM-1::GFP when we employ ZIF-1-mediated degradation (Fig. 3d).\nAlthough this modest decrease of PBRM-1::GFP is sufficient to induce ectopic glr-4 expression in\nmotor neurons as we show in Fig. 3d, it does not lead to thrashing defects. For unknown reasons,\n1\nexpression of GFPnanobody::ZIF-1 transgene by itself affected thrashing behavior (Suppl. Figure\n12). We conclude that although our RNAi and ZIF-1 approaches lead to ectopic glr-4 expression in\nmotor neurons (Fig. 3c-d), their partial knock-down effect on pbrm-1 is insufficient to lead to\ndetectable behavioral effects.\nWe update the Results as follows:\n“Last, we conducted thrashing assays on adult animals, in which we knocked-down PBRM-1\nspecifically in motor neurons with either cell-specific RNAi or ZIF-1 technology using the exact same\nworm strains shown in Fig. 3c-d. However, we were unable to identify any thrashing defects (Suppl.\nFigure 12), likely due to partial decrease of PBMR-1 when RNAi or ZIF-1 technology. In fact, we only\nachieve a modest decrease in the number of motor neurons expressing PBRM-1::GFP when we\nemploy ZIF-1-mediated degradation (Fig. 3d). We conclude that although our RNAi and ZIF-1\napproaches lead to ectopic glr-4 expression in motor neurons (Fig. 3c-d), their partial knock-down\neffect on PBRM-1 is insufficient to lead to detectable behavioral effects.”\nFurther, we have conducted a bioinformatic analysis to identify additional targets of the PBAF\ncomplex in motor neurons based on what we already know about glr-4 (Suppl. Fig. 9b). This analysis\nis presented in Results as follows:\n“To expand the repertoire of PBAF target genes, we identified 1,000 genes that – like glr-4 –\nare expressed at low levels in AS neurons based on available sc-RNA-Seq data5. Specifically, we\nincluded 1,000 genes with expression values (transcripts per million, TPM) centered around glr-4\n(TPM: 120.5) (see Methods). Next, we found that – like glr-4 – 247 of these 1,000 genes are co-bound\nby UNC-3, MAB-9 and SWSN-7 (Suppl. Fig. 9b, Supplementary File 2). Although validation of these\n247 genes is beyond the scope of this work, this analysis nevertheless offers a list of putative target\ngenes that are repressed by MAB-9 and PBAF in AS neurons.”\nMoreover, the analysis of locomotor phenotypes requires clearer explanation and connection to\nspecific neuronal functions. For instance, since AS motor neurons are implicated in body wave\npropagation, is this particular feature disrupted in the MN-specific PBAF loss-of-function strains?\nThanks for this suggestion. We have updated the text, Figure 10 and its legend. In revised Results,\nwe state:\n“Because the PBAF effect on glr-4 is stronger in AS neurons, which are known to control body wave\npropagation PMID: 30204083, we asked whether this particular feature of locomotion is disrupted in\npbrm-1 mutants. Indeed, we found significant changes in the body wave, reflected in curvature\nfeatures and in the time derivative of curvature features (d_curvature) in pbrm-1(ot792) mutants (Fig.\n10c-g). Further, median midbody speed is affected in pbrm-1(ot792) mutants (Fig. 10b). Altogether,\nthis analysis shows that loss of pbrm-1 leads to significant defects in posture (curvature) and motion\n(speed), consistent with defective body wave propagation.”\n2\n2. The authors provide predictive modeling of a potential interaction between the PBAF complex and\nMAB-9, which is a promising direction. However, this remains unvalidated experimentally. While we\nrecognize the technical challenges of performing co-immunoprecipitation in C. elegans, alternative\nstrategies—such as heterologous expression in cell lines or in vitro protein-protein interaction\nassays—should be considered.\nWe thank the reviewers for this valuable suggestion. Indeed, we have conducted co-IP experiments\nusing heterologous expression in HEK293 cells and validated the predicted physical interactions\nbetween MAB-9 and SWSN-6, as well as between MAB-9 and SWSN-7. We have updated the\nResults, Methods and Figure 8.\nIn revised Results, we now state:\n“These predicted interactions support a model of PBAF recruitment via direct protein interactions with\nMAB-9.\nTo experimentally test this model, we conducted co-immunoprecipitation assays in HEK293\ncells co-transfected with epitope-tagged versions of MAB-9 and SWSN-6, or MAB-9 and SWSN-7.\nFollowing pull-down of MAB-9::Myc with anti-c-Myc magnetic beads, we detected SWSN-6::HA (49.5\nkDa) or SWSN-7:HA (140.2 kDa) with western blotting against the HA epitope (Fig 8e). Conversely,\nfollowing pull-down of either SWSN-6::3XFLAG or SWSN-7:3XFLAG with anti-FLAG M2 magnetic\nbeads, we detected MAB-9::HA (40.5 kDa) with anti-HA western blotting (Fig 8e). Together, these\nresults validate the AlphaFold2 predictions, supporting a model where MAB-9/Tbx20 physically\ninteracts with both SWSN-6 and SWSN-7 to recruit PBAF and thereby influence gene expression in\nAS neurons.”\n3. The experiments involving mutations in predicted MAB-9 binding sites are fragmentary. These were\ninitially performed using a glr-4 reporter construct covering a region without a MAB-9 ChIP-seq peak.\nAlthough this analysis shows an effect on reporter expression, similar mutations introduced into the\nendogenous locus do not yield the same result. To strengthen the proposed model, the site 6\nmutation in the endogenous locus should be combined with mutations at other predicted MAB-9\nbinding sites that overlap with MAB-9 ChIP peaks.\nWe agree. These experiments are very challenging. The multiplicity of TF binding sites requires the\ngeneration of strains carrying mutations for multiple sites.\nFollowing the reviewer’s suggestion, we used CRISPR/Cas9 gene editing to simultaneously mutate\nsite #6 and four intronic sites #1-4 that overlap with intronic MAB-9 ChIP-Seq peaks. However, the\nintroduction of the intronic mutations #1-4 unexpectedly led to reduced expression (assessed by\nfluorescent intensity quantification) in motor neurons of the endogenous 2xNLS::mScarlet::SL2::glr-4\nreporter (Figure for reviewer), which is a transcriptional reporter. We surmise that these intronic\nmutations may have affected glr-4 transcript stability/processing by, for example, altering the mRNA\nsecondary structure or preventing the binding of RNA-binding proteins. Because we are uncertain\nabout the effects of these intronic mutations on glr-4 transcript’s stability/processing, we provide this\ndata only as Figure for reviewer.\nLike MAB-9, we note that a previously described repressor TF, CFI-1, also acts through multiple\ncognate sites to repress glr-4 in C. elegans MNs (PMID: 36897776). In that study, we had to mutate\n3\n11 binding sites simultaneously to observe an effect on glr-4 expression. In that case, however, all 11\nCFI-1 sites were upstream of glr-4, not located in introns.\nWe have updated the Results to highlight that redundancy of TF bindings sites appears to be a\ncommon feature between these two repressor TFs (MAB-9, CFI-1) in C. elegans MNs.\n“…suggests redundancy among the 10 MAB-9 sites. To test this idea, we simultaneously mutated site\n#6 and two other sites (#8 and #9)(Supplementary File 1), but observed no effect on endogenous\nmScarlet::glr-4 expression in MNs (Fig. 7g), raising the possibility that one or more of the remaining\nseven MAB-9 sites are involved in glr-4 repression. This is consistent with previous work where\nsimultaneous mutation of 11 binding sites for the repressor TF CFI-1 led to increased glr-4 expression\nin C. elegans MNs (PMID: 36897776).\nSpecific Comments:\n• Figure 10: In addition to employing alternative, more specific models for behaviour, the authors\nshould improve the graphical presentation. In particular, the titles of panels b–g are not very\ninformative and should be revised for clarity.\nWe have now revised all title panels and legend of Figure 10 to improve clarity.\n4\n• Figure 7 and 9: The PBRM-1 ChIP-seq track is missing in Figure 7, and tracks for all relevant factors\nare missing in Figure 9.\nThe PBRM-1 ChIP-seq track is included in Figure 7.\nIn Figure 9, we provide ChIP-Seq data for the terminal selector UNC-3 and two PBAF components\n(PBRM-1 and SWSN-7). To our knowledge, there are no available ChIP-Seq data for other PBAF-\nspecific proteins.\n• Supplementary Figure 3a: Please include a brief explanation in the main text of how the allele leads\nto glr-4 overexpression (e.g., through cfi-1 mutation).\nWe added the following explanation in the main text, as requested:\n“Next, we used a previously published strain that does not carry the ot792 mutation but\nselectively overexpresses glr-4 in nerve cord MNs due to mutated binding sites for CFI-1, a\nTF that normally represses glr-4 38. We found defects in thrashing behavior of this strain\n(Suppl. Fig. 3a), indicating that increased glr-4 levels in MNs are detrimental to C. elegans\nmotility.”\n• The manuscript states: “Interestingly, MAB-9 and PBAF (PBRM-1, SWSN-7) binding coincides at several regions upstream\nand within the glr-4 locus, supporting the possibility of MAB-9 recruiting PBAF (Fig. 7d).” However, MAB-9 peaks are\nobserved only within the gene body and not upstream. Please revise the text accordingly.\nWe have revised the text as follows:\n“Interestingly, MAB-9 and PBAF (PBRM-1, SWSN-7) binding coincides prominently within the glr-4\nlocus and modestly upstream of glr-4, supporting the possibility of MAB-9 recruiting PBAF (Fig. 7d).”\n• Please use italics for all gene names and alleles, which are still missing in some graphs and figure legends (Main and Sup\nfigures)\nDone. Thanks.\nReviewer #4 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of\nthe Nature Communications initiative to facilitate training in peer review and to provide appropriate\nrecognition for Early Career Researchers who co-review manuscripts.\nThanks for the constructive feedback.\n5"}, {"round": 3, "reviewer_comments": {"reviewer_3": "We appreciate the work done by the authors to address all our concerns and suggestions.\nRegarding the lack of behavioural phenotype for MN specific pbrm-1 knock down, please add the following sentence, as a possible explanation in addition to the partial knock down:\n\"Alternatively, we cannot discard that pbrm-1(ot792) locomotory defects are caused by non-cell autonomous defects in other neurons or non-neuronal tissues.\"\nWe have no further comments.", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "REVIEWERS' COMMENTS\nReviewer #3 (Remarks to the Author):\nWe appreciate the work done by the authors to address all our concerns and suggestions.\nRegarding the lack of behavioural phenotype for MN specific pbrm-1 knock down, please add\nthe following sentence, as a possible explanation in addition to the partial knock down:\n\"Alternatively, we cannot discard that pbrm-1(ot792) locomotory defects are caused by non-\ncell autonomous defects in other neurons or non-neuronal tissues.\"\nWe have no further comments.\nWe added the sentence recommended by reviewer #3 in the last paragraph of Results:\n“Alternatively, we cannot discard that the pbrm-1(ot792) locomotory defects are caused by non-\ncell autonomous effects in other neurons or non-neuronal cells.\"\nReviewer #4 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is\npart of the Nature Communications initiative to facilitate training in peer review and to provide\nappropriate recognition for Early Career Researchers who co-review manuscripts.\nThanks for the feedback."}]

Decision letter