Regulation of chromatin states and gene expression during HSN neuronal maturation is mediated by EOR-1/PLZF, MAU-2/cohesin loader, and SWI/SNF complex

Newborn neurons mature by distinct and sequential steps through the timely induction of specific gene expression programs in concert with epigenetic changes. However, it has been difficult to investigate the relationship between gene expression and epigenetic changes at a single-cell resolution during neuronal maturation. In this study, we investigated the maturation of hermaphrodite-specific neurons (HSNs) in C. elegans, which provided the link between chromatin dynamics, gene expression, and the degree of neuronal maturation at a single-cell resolution. Our results demonstrated that chromatin composition in the promoter region of several genes acting for neuronal terminal maturation was modulated at an early developmental stage, and is dependent on the function of the transcription factor EOR-1/PLZF and the cohesin loader MAU-2/MAU2. Components of the SWI/SNF chromatin remodeling complex were also required for the proper expression of terminal maturation genes. Epistasis analyses suggested that eor-1 functions with mau-2 and swsn-1 in the same genetic pathway to regulate the maturation of HSNs. Collectively, our study provides a novel approach to analyze neuronal maturation and proposes that predefined epigenetic modifications, mediated by EOR-1, MAU-2, and the SWI/SNF complex, are important for the preparation of future gene expression programs in neuronal terminal maturation.

accessibility changes have already occurred in immature newborn neurons and they remain static until the terminal maturation step 6,8 , raising the question of whether such pre-defined chromatin changes affect gene expression during neuronal terminal maturation. However, it has been difficult to define the relationship between gene expression and the underlying chromatin alterations within a heterogeneous cellular population including various developmental stages of neurons.
To avoid the difficulties caused by heterogeneity in a cellular population, we used hermaphrodite-specific neurons (HSNs) in the nematode Caenorhabditis elegans. In these hermaphrodite worms, HSNs are born from neuroblast cells at the tail and migrate to the mid-body region during embryogenesis, and the migration is completed by the L1 larval stage. Axon outgrowth initiates at the L3 larval stage. Finally, HSNs change their membrane and transmission properties by expressing genes required for functional maturation from the L4 larval stage onward, and to regulate egg-laying behavior at the adult stage 9 . This dynamic but slow maturation of HSNs and the transparent body of C. elegans allow easy access to gene expression activity and the chromatin state of each developmental stage by using extra-chromosomal reporter arrays 10,11 . These features provide a powerful means for analyzing the interplay between transcriptional activities and chromatin modifications during neuronal maturation at the single-neuron level.
To modify the chromatin states, chromatin remodeling complexes can be targeted to specific genomic loci by interacting with sequence-specific transcription factors [12][13][14] . Various transcription factors involved in the development of HSNs have been identified, including EOR-1, the C. elegans homolog of the Promyelocytic Leukemia Zinc Finger (PLZF) 15 . A recent study revealed that EOR-1 may regulate chromatin accessibility at transcriptional regulatory regions during the worm development 16 . Here, we show that EOR-1 affects chromatin dynamics in immature HSNs at the L1 larval stage and cell-autonomously enhances the later expression of genes required for HSN terminal maturation in adult hermaphrodite worms. We also showed that the core component of the SWI/ SNF chromatin remodeling complex SWSN-1 and the cohesin loader subunit MAU-2 cell-autonomously regulate the expression of reporters for several genes acting for HSN maturation. Moreover, genetic analyses revealed that eor-1 functions with mau-2 and swsn-1 in the same genetic pathway, suggesting previously unknown interactions between PLZF and the SWI/SNF and cohesin loader complexes during neuronal development. Our results provide a unique approach to analyze the relationship between chromatin alterations and gene expression during neuronal development and insight into the neurological mechanisms of neurodevelopmental disorders.

eor-1 regulates the expression of genes required for the HSN terminal maturation.
Transcription factors have been reported to regulate the target specificity of epigenetic enzymes in various organisms 13,17,18 , and the development of HSNs in C. elegans 19,20 . To uncover the mechanisms that govern gene expression and the underlying chromatin dynamics in developing HSNs, we performed a candidate screen for transcription factors that affect gene expression of HSNs in the adult hermaphrodite worms. HSNs express genes required for their terminal maturation at the L4 stage onward, such as abts-1, kcc-2, and ida-1, which encodes a sodium-driven chloride bicarbonate exchanger, a sodium chloride co-transporter, and a protein tyrosine phosphatase-like receptor, respectively 9,21 (Fig. 1A). We found that eor-1(cs28) null mutants show decreased expression of GFP, which is under the control of the abts-1b promoter, in HSNs (Fig. 1B,C). On the other hand, eor-1(cs28) mutation did not appear to affect the expression of GFP, which is under the control of the same abts-1b promoter, in both head and tail neurons (Fig. 1B). Mutants in eor-2, which encodes a binding partner of EOR-1 22 , also showed decreased expression of GFP in HSNs (Fig. 1C). The phenotype observed in eor-1 and eor-2 mutants may be explained by the defect in the specific regulation of abts-1 gene expression or a defect in the maturation of HSNs. To distinguish between these possibilities, we analyzed the expression of kcc-2 and ida-1, which are also expressed from the L4 larval stage onward and required for the functional maturation of HSNs 21,23 . We found that eor-1(cs28) mutants show decreased expression of GFP in HSNs under the control of each promoter (Fig. 1C). Furthermore, eor-1(cs28) mutants were reported to show a defect in egg-laying, which is controlled by mature HSNs 24 . The defect of eor-1(cs28) mutants in the expression of Pabts-1b::gfp was rescued by the genomic eor-1 DNA fragment fused with mCherry, eor-1::mCherry (Fig. 1D). On the other hand, unlike other transcription factors regulating HSN maturation steps 19,20,25,26 , the eor-1 mutation did not affect migration or neurite elongation of HSNs (Fig. 1B, Supplementary Fig. S1). These results indicate that eor-1 regulates the terminal maturation of HSNs by modulating expression of multiple genes required for the maturation, rather than indirectly through earlier deficits in HSN development.

EOR-1 functions in HSNs.
Neurite elongation of HSNs has been shown to be regulated by FGF signaling derived from vulval epithelium cells 27 . This suggests that the maturation of HSNs may be affected by surrounding cells. However, mutations in egl-15, a gene encoding the sole C. elegans FGF receptor, did not affect the expression of Pabts-1b::gfp in HSN neurons ( Supplementary Fig. S2), raising the possibility that eor-1 plays a cell-autonomous role in the maturation of HSNs. To determine whether eor-1 cell-autonomously functions to regulate the maturation of HSNs, we generated transgenic eor-1(cs28) mutants expressing EOR-1 under the control of the unc-86 promoter, which drives gene expression in multiple neurons including HSNs from embryogenesis onward, but not in surrounding cells. The unc-86 promoter has previously been used to examine the cell-autonomous function of genes in HSNs 28,29 . The expression of EOR-1 under the control of unc-86 promoter recovered the phenotype of eor-1(cs28) mutants, and the transgenic eor-1(cs28) mutants showed almost the same expression of Pabts-1b::gfp in HSNs as wild-type animals (Fig. 1D). However, other phenotypes, such as uncoordinated movement and the larval lethality of eor-1(cs28) mutants, did not recover with the expression of EOR-1 under the control of the unc-86 promoter. These results suggested that the expression of EOR-1 in specific neurons is sufficient for the normal expression of Pabts-1b::gfp in HSNs.
ScIeNtIfIc REpORtS | (2018) 8:7942 | DOI:10.1038/s41598-018-26149-2 EOR-1 binds to abts-1 promoter regions. eor-1 encodes the zinc-finger transcription factor homologous to human PLZF. The binding consensus sequence of EOR-1 has been previously obtained from the analysis of ChIP-Seq data for EOR-1 30 . To test whether EOR-1 directly regulates the expression of Pabts-1b::gfp, we analyzed the existence of the binding consensus sequence of EOR-1 in the abts-1b promoter. We found a fully identical motif (motif 1: GAGACGCAGA) to the EOR-1 binding consensus sequence at about 200 bp upstream of the start codon of abts-1b. Furthermore, according to the JASPAR database, we found six additional motifs (motif 2−7) similar to the EOR-1 binding consensus sequence in the abts-1b promoter ( Fig. 2A). To characterize the importance of each EOR-1 binding motif, we compared the activity of the abts-1b promoter by mutating each motif. Simultaneous mutation of all seven motifs eliminated expression in HSNs, while expression in head neurons was not affected (Fig. 2B,C). These results are consistent with the expression pattern of Pabts-1b::gfp of eor-1(cs28) mutants. Next, we searched the motif sufficient for the expression in HSNs. When we mutated the fully identical motif (motif 1) to the EOR-1 binding consensus sequence at about 200 bp upstream of the start codon of abts-1b, expression in HSNs disappeared. In contrast, mutating the six additional motifs (motif 2-7) did not eliminate expression in HSNs, suggesting that the motif 1 at about 200 bp upstream of the start codon is required and sufficient for the expression of Pabts-1b::gfp in HSNs. As mentioned above, the expression of Pabts-1b::gfp in HSNs was eor-1-dependent (Fig. 1B,C). Moreover, analysis of ChIP-Seq data for EOR-1 from modENCODE indicated that EOR-1 was localized among the abts-1b promoter ( Supplementary Fig. S3). Therefore, these findings suggested that EOR-1 directly regulates the expression of abts-1 in HSNs by binding to the promoter region. Analysis of ChIP-Seq data for EOR-1 also indicated that EOR-1 was localized to the kcc-2 and ida-1 promoters, in addition to the abts-1 promoter ( Supplementary Fig. S3), suggesting the broad role of EOR-1 in regulating the expression of genes involved in the maturation of HSNs.
Expression of EOR-1 at an early developmental stage is sufficient for normal HSN maturation. Transcription of genes involved in the maturation of HSNs begins at the L4 larval stage. To determine when EOR-1 functions to regulate the expression of genes required for the maturation of HSNs, we utilized worms that express EOR-1 under the control of a heat shock promoter. We delivered a 2h-heat shock to the worms soon after egg-laying (0 h) or at 72 h after egg-laying, which corresponds to the gastrulation stage of embryogenesis and the developmental stage L4 to young adult, respectively. Surprisingly, the expression of EOR-1 at 72 h after egg-laying did not recover the expression of Pkcc-2c::gfp in eor-1(cs28) mutants, while the expression of EOR-1 soon after egg-laying did rescue the defect (Fig. 3A). Combined with the previous data showing that EOR-1 functions at ~470 min after first cell division during embryogenesis to induce male-specific apoptosis of HSNs 15,31 , these results motivated us to analyze the temporal expression pattern of eor-1. The eor-1::mCherry that contains eor-1-rescuing activity for the terminal maturation showed broad mCherry expression starting at embryogenesis ( Supplementary Fig. S4). We confirmed the nuclear localization of EOR-1::mCherry in HSNs, visualized by myristoyl-tagged GFP under the control of the unc-86 promoter, at the L1 larval stage of wild-type worms (Fig. 3B). These results suggested that EOR-1 may function to regulate the expression of genes required for the maturation of HSNs at an early developmental stage, when expression of the genes is not yet observed. However, it could be possible that the EOR-1 proteins expressed at L1 larval stage are stable during developmental stages and can affect the gene expression required for the maturation of HSNs at L4 larval stage. To rule out the possibility, we analyzed the time course of degradation of EOR-1::mCherry expressed soon after egg-laying in HSNs under the control of a heat shock promoter (Fig. 3C). The amount of EOR-1::mCherry protein was significantly abundant at 24 hr after heat shock, which corresponds to the L1 stage. However, the mCherry signal in HSNs of transgenic animals was reduced to the background levels observed in non-transgenic animals at 48 hr and 72 hr after heat shock, which correspond to the L2/L3 and L4/young adult stage, respectively. These results reinforced the significance of EOR-1 expressed at an early developmental stage for the later gene expression during HSN maturation.

swsn-1 is required for the maturation of HSNs.
Our results suggest that the effect of EOR-1 expression at an early developmental stage lasts until the adult stage. Combined with a recent report showing that eor-1 may regulate chromatin accessibility dynamics at transcriptional regulatory regions during development 16 , this fact implies that EOR-1 regulates gene expression by modulating the epigenetic state of the genes expressed during the maturation step of HSNs. The SWI/SNF chromatin remodeling complex has been reported to regulate the maturation process of HSNs 23 , and serves to alter the structure of nucleosomes in order to regulate transcriptional activity 32 . To test whether the SWI/SNF chromatin remodeling complex affects the expression of genes required for the maturation of HSNs, we analyzed the expression of Pabts-1b::gfp in HSNs of swsn-1(os22) mutants and ham-3(n1654) mutants, both of which have been reported to show egg-laying defects 23,33 . swsn-1 and ham-3 encode the C. elegans homolog of human SMARCC1 and human SMARCD, respectively. The encoded proteins are subunits of the SWI/SNF chromatin remodeling complex. swsn-1 has been shown to be ubiquitously expressed and localized to nuclei 33 . swsn-1(os22) and ham-3(n1654) mutants showed decreased expression of Pabts-1b::gfp in HSNs (Fig. 3D). To determine whether swsn-1 functions to regulate the expression of Pabts-1b::gfp cell-autonomously, we expressed SWSN-1 in HSNs under the control of the unc-86 promoter. swsn-1(os22) mutants expressing SWSN-1 in HSNs showed nearly the same expression of Pabts-1b::gfp as wild-type (Fig. 3E). Therefore, swsn-1 may regulate Pabts-1b::gfp expression in HSNs cell-autonomously. These data imply that chromatin remodeling complexes directly or indirectly regulate the expression of Pabts-1b::gfp in HSNs.
H2A.Z is localized to the abts-1b promoter at the L1 to L2 early larval stage. The SWI/SNF chromatin remodeling complex affects the composition of histone H2A.Z in nucleosomes at the target loci 35,36 . The accumulation of H2A.Z in promoter regions correlates with the activity of gene expression and may be a landmark for nucleosome-free regions in active promoters 37,38 . Therefore, we hypothesized that H2A.Z may be deposited in the abts-1b promoter region during HSN maturation. To examine the localization of H2A.Z in the abts-1b promoter region, we visualized HTZ-1, the C. elegans homolog of H2A.Z, and extra-chromosomal arrays containing abts-1b promoter sequences by using Nuclear spot assays 39 . We generated transgenic worms expressing both YFP::HTZ-1 and mTurquoise2::LacI, which bind to Lac operator sequences (LacO). Then, the extra-chromosomal array (reporter array) that contains multiple copies of LacO repeats and abts-1b promoter sequences was introduced into the transgenic worms. The reporter array in nuclei can be visualized by the binding of mTurquoise2::LacI to LacO, indicating the localization of exogenous abts-1b promoter sequences. To analyze whether HTZ-1 was deposited to the abts-1b promoter, we examined the co-localization of mTurquoise2::LacI and YFP::HTZ-1 in the nucleus of HSNs. We observed frequent co-localization of mTurquoise2::LacI and YFP::HTZ-1 in HSNs of L1 to L2 larval stage worms with the reporter array carrying exogenous abts-1b promoter sequences (Fig. 4A). The frequency was significantly higher than that of worms with reporter arrays that contained LacO repeats but not the abts-1b promoter (Fig. 4B). These results suggested that HTZ-1 was deposited to the abts-1b promoter in HSNs at the L1 to L2 larval stage, supporting the possibility that the abts-1b promoter directly receives chromatin remodeling at an early developmental stage in order to induce later gene expression starting from the L4 stage. Such frequent co-localization of HTZ-1 and the abts-1b promoter was attenuated at  Fig. S6A,B). These results are consistent with the widely reported concept that H2A.Z dissociates from chromatin once transcription is initiated [40][41][42][43] . Furthermore, the reporter array with abts-1b promoter sequences was localized to the nuclear periphery at the L1 larval stage and apparently changed the sub-nuclear localization from periphery to center during development ( Supplementary Fig. S6A). In general, heterochromatin regions and euchromatin regions are localized to the nuclear periphery and the center of nuclei, respectively. Therefore, the reporter array likely recapitulated proper chromatin modifications in vivo.

Necessity of eor-1 in the localization of H2A.Z in the abts-1b promoter. Next, we analyzed whether
eor-1 is required for the deposition of HTZ-1 to the abts-1b promoter. We generated transgenic lines that contained reporter arrays carrying the abts-1b promoter with a mutated EOR-1 binding motif (Δmotif1, Fig. 2B). The transgenic lines showed a significant decrease in the frequency of the co-localization of mTurquoise2::LacI and YFP::HTZ-1 in the nucleus of HSNs at the L1 to L2 larval stage, compared with worms containing reporter arrays of the wild-type abts-1b promoter (Fig. 4C,D). Furthermore, in an eor-1(cs28) mutant background, the frequency of the co-localization of mTurquoise2::LacI and YFP::HTZ-1 significantly decreased on the wild-type abts-1b promoter (Fig. 4E,F). These results revealed that eor-1 is required for the deposition of HTZ-1 to the abts-1b promoter region. The requirement of eor-1 for the deposition of HTZ-1 at the L1 to L2 larval stage suggested that EOR-1 functions to regulate the expression of genes required for HSN maturation by modifying the epigenetic state of the genes at an early developmental stage.

Cohesin loader MAU-2 affects the gene expression during HSN terminal maturation.
Our data suggested that epigenetic modifications occurred at an early developmental stage and lasted until later gene expression as an epigenetic memory. However, it is yet unclear how the epigenetic modifications could be established and be maintained during development. To get an insight into the molecular mechanism for the modification, we sought another mutant defective in the expression of genes required for HSN maturation. Among egg-laying defective mutants, we identified mau-2(qm160) null mutants, which showed common defects with eor-1(cs28) mutants. eor-1(cs28) and mau-2(qm160) mutants share multiple abnormalities such as egg-laying defects, uncoordinated movement, dye-filling defects, and rod-like lethality 24,44 . mau-2 encodes a homolog of yeast Scc4, which loads cohesin rings to the target genomic loci. The Scc2/Scc4 cohesin loader binds to nucleosome-free genomic loci with active transcription states 45 . In this study, mau-2(qm160) mutants showed decreased expression of Pabts-1b::gfp in HSNs (Fig. 5A). Defects in the expression of Pabts-1b::gfp were rescued by the expression of MAU-2 under the control of the unc-86 promoter. These results suggested that mau-2 functions to regulate the expression of genes required for the maturation of HSNs cell-autonomously.
To obtain mechanistic insight into how mau-2 regulates gene expression, we analyzed the localization of YFP::HTZ-1 in HSNs in mau-2(qm160) mutants. If MAU-2 acts only for the maintenance of the epigenetic modification rather than the establishment, YFP::HTZ-1 is assumed to properly localize in HSNs at an early developmental stage, and the localization should be disturbed as development proceeds. Unexpectedly, the mau-2(qm160) mutation significantly decreased the frequency of the co-localization of mTurquoise2::LacI and YFP::HTZ-1 in HSNs at the L1 to L2 larval stage (Fig. 5C,D). These results are consistent with the idea that mau-2 and eor-1 cooperate to regulate gene expression by establishing chromatin features in HSNs at an early developmental stage.
In conclusion, eor-1 may act at an early developmental stage to regulate the expression of genes in later stages of HSN maturation, and this process is cooperative with chromatin remodeling complexes and cohesin loaders.

Discussion
The maturation of post-mitotic neurons consists of multiple processes that require temporally controlled gene expression associated with chromatin structure changes. To gain mechanistic insight into chromatin regulation during neuronal maturation, we studied gene expression during late stages of neuronal maturation at a single-neuron level by linking the degree of neuronal maturation with the chromatin state of the promoter region. Our study revealed that eor-1 regulates both the chromatin state and gene expression, which mediate the late stages of HSN maturation. Importantly, such chromatin regulation by eor-1 occurs at an early developmental stage, when the expression of genes that mediate the late maturation of HSN neurons is not observed. Our heat-shock rescuing experiment implies the critical period for the function of EOR-1, since the expression of EOR-1 at a later developmental stage could not recover the phenotype of eor-1(cs28) mutants. This observation is consistent with the potential model that EOR-1-dependent chromatin remodeling at the promoter region is a prerequisite for the later activation of gene expression (Supplementary Fig. S7). Although further studies such as the development of single-cell type ChIP-seq will be needed to compellingly support this model, our study demonstrates an alternative approach to analyze the relationship between gene expression and chromatin alterations during neuronal maturation.
eor-1 encodes a zinc finger transcription factor homologous to human PLZF. PLZF exerts various biological functions during the development of multiple tissues 46 . Although PLZF is expressed in a temporally dynamic pattern in the developing CNS, and functions to maintain neuronal progenitors in neurogenesis 47,48 , the role of PLZF in the terminal maturation of neurons is poorly understood. Our single-cell analysis indicated that eor-1 mutation did not eliminate the expression of genes for terminal maturation but rather decreased the expression. Furthermore, the extension of a neurite from HSNs was observed in eor-1 mutants. These observations suggest that eor-1 mutation did not affect the determination of HSN cell fate. Therefore, we propose that eor-1 functions to enhance lineage specific gene expression of HSNs by modulating the chromatin state of the specific genes. The function of EOR-1 in HSN maturation may be analogous to that of PLZF in the differentiation of mammalian stem cells.
In this study, we also showed that the components of the SWI/SNF chromatin remodeling complex such as SWSN-1/SMARCC1 and HAM-3/SMARCD are involved in the terminal maturation of HSNs. Furthermore, epistasis analysis suggested EOR-1 genetically interacted with SWSN-1. Interestingly, previous ChIP-seq analyses reported that two components of the SWI/SNF complex is highly enriched in a DNA motif nearly identical to the binding consensus sequence of EOR-1 in C. elegans 18,30 . Chromatin remodeling complexes may be recruited to target sites by interacting with pioneer transcription factors, which can bind to target sequences on condensed nucleosomal DNA 49 . Here, we showed that eor-1 is required for the chromatin remodeling on the abts-1b promoter region that contains the binding motif of EOR-1. Furthermore, recent ATAC-seq data showed that EOR-1 may regulate chromatin accessibility at transcriptional regulatory regions during development 16 . Although our genetic analysis has not shown the genetic hierarchy between eor-1 and swsn-1, it is highly possible that EOR-1 may function as a pioneer transcription factor for recruiting SWI/SNF chromatin remodeling complexes to target sites.
The cohesin complex also plays an important role in chromatin architecture and transcriptional regulation 50 . Our results implied a functional interaction between the cohesin loader complex and SWI/SNF chromatin remodeling complexes. Such functional similarity between the two complexes has also been observed in HSN neuronal migration 23,44 . Indeed, the yeast Scc2/Scc4 cohesin loader complex, recruited by the RSC chromatin remodeling complex, functions to maintain nucleosome-free regions, although it is unknown how the RSC complex is targeted to specific genomic sites 45 . Therefore, we propose a potential model where the interactions between EOR-1, SWSN-1, and MAU-2 alter chromatin structure and chromatin composition at EOR-1 binding sites in order to prepare for subsequent gene expression programs.
Chromatin regulation and gene expression can be temporally disassociated in principle, since these events are performed by distinct machineries 51 . However, it is yet unclear why the "pre-defined" nucleosome alterations are required for future gene expression during development. Poised RNA polymerase II (Pol II) preferentially occupies the promoter region of genes that are dynamically regulated during development 52,53 . This poised Pol II occupancy prepares genes for future expression by establishing a permissive state that enables rapid and synchronous gene induction 54,55 . Interestingly, poised Pol II targeted to promoter regions by a pioneer transcription factor also helps nucleosome depletion and contributes to the accessibility of the promoters 56,57 . Hence, it is conceivable that poised Pol II and chromatin remodeling complexes recruited by a pioneer transcription factor have a cooperative function to prepare future gene expression during development. A recent report indicated that developmentally regulated genes generally lack canonical post-translational histone modifications 58 . Thus, it could be possible that "pre-defined" nucleosome remodeling may be an alternative pathway to dynamically regulate genes during development, and may help developmentally coordinated neuronal maturation by rapid and synchronous gene induction.
In this study, we showed that HTZ-1, the C. elegans homolog of H2A.Z, was transiently accumulated on the promoter region of a gene required for the functional maturation of HSNs during development. Interestingly, recent reports have revealed that H2A.Z accumulates specifically at promoter regions, where the occupancy of H2A.Z correlates with that of poised Pol II during C. elegans development, implying the role of H2A.Z in pausing Pol II and preparing for rapid and synchronous gene induction 59,60 . Consistently, our results demonstrated that H2A.Z deposition on abts-1b promoter was removed once transcription was initiated at the L4 stage, raising the possibility that H2A.Z deposition mediated by EOR-1 and MAU-2 affects the transcriptional activity of genes required for HSN maturation. Hence, the interactions between EOR-1, SWSN-1, and MAU-2 might coordinate neuronal maturation and gene expression through H2A.Z incorporation into the genes required for HSN maturation.
In summary, we report the significance and the molecular basis of chromatin alterations for neuronal maturation in C. elegans. A similar mechanism may operate in higher organisms, since the maturation of HSN neurons consists of typical processes observed in mammalian neurons including cell migration, neurite elongation, synapse formation, and the functional maturation of membrane potential. Importantly, the functional link between MAU-2, SWSN-1, and EOR-1 may explain overlapping features observed among human disorders. Cornelia de Lange syndrome, caused by abnormalities of the human cohesin loader complex, is in some cases difficult to distinguish from Coffin-Siris syndrome, a SWI/SNF chromatin remodeling complex disorder [61][62][63] . Furthermore, loss-of-function mutations in human PLZF cause phenotypes commonly observed in both syndromes 64 . Thus, it is possible that neurological abnormalities in these disorders arise from dysfunctions in common molecular pathways that affect chromatin structures and transcriptional regulation. Further analyses may provide insight on the molecular basis of temporally regulated gene expression programs through chromatin dynamics, and facilitate the molecular understanding of these serious human disorders.

DNA constructs and Germline transformation.
For the mutation analysis of putative EOR-1 binding motifs in the abts-1b promoter, the abts-1b promoter (1.1 kb) and the abts-1 3′UTR (0.4 kb) were amplified by PCR from genomic DNA and were sub-cloned into the pPD95.75 vector to construct Pabts-1b::gfp::abts-1 3′UTR. The putative EOR-1 binding motif was mutated by substituting four nucleotides in the core sequence (WT: GAGAcgcaga» mutant: CCCCcgcaga). For the generation of the insertion line expressing Pkcc-2c::gfp::kcc-2 3′UTR, the kcc-2c promoter (7.9 kb) and kcc-2 3′UTR (1.3 kb) were amplified by PCR from genomic DNA and were sub-cloned into the pPD95.75 vector. For the construction of the genomic eor-1::mcherry fusion gene, the eor-1 genomic fragment (6.2 kb) without the stop codon was fused to mcherry. For the rescue experiment, eor-1a cDNA, swsn-1a cDNA, and mau-2a cDNA were amplified by PCR and sub-cloned into the pPD49.26 vector. The unc-86 and hsp-16.2 promoters were inserted upstream of the multi-cloning site of each plasmid. The constructs used for the nuclear spot assay, yfp::htz-1 and cfp::LacI, were gifts from S. Mango. cfp::LacI was substituted by mTurquoise2, amplified by PCR. The unc-86 and htz-1 promoters were inserted upstream of the multi-cloning site of the yfp::htz-1 and mTurquoise2::LacI constructs, respectively.
Transgenic strains were generated by microinjecting test DNA at a concentration of 10−50 ng/μL, along with a 2−20 ng/μL co-injection of the marker Pmyo-2::gfp, Pmyo-2::mCherry, Punc-122::gfp, or Punc-122::mCherry, and 0−70 ng/μL carrier DNA as described by Mello and Fire 66 . For nuclear spot assays, to avoid the contamination of LacO sites into the extrachromosomal array expressing mTurquoise2::LacI, the Punc-86::yfp::htz-1 PCR product, the Phtz-1::mTurquoise2::LacI PCR product, and the co-injection marker Punc-122::gfp PCR product were injected into wild-type worms. Transgenes harboring the abts-1b promoter and LacO repeats were generated by injecting the 1.1 kb abts-1b promoter sequence, pSV2-DHFR8.32, which contains 256 copies of the LacO sequence, as well as the co-injection marker Pmyo-2::gfp. Quantitative fluorescent microscopy measurements. Mid-late L4 worms, identified by vulval morphology, were transferred to new NGM plates with OP-50 and incubated for 16-20 h at 20 °C. Staged adult worms were put on agar pads and anesthetized with 5 mM sodium azide. Cell body images of HSNs were obtained using a Carl-Zeiss LSM5 confocal microscope at a fixed setting. The sum of fluorescent intensities within the cell body was quantified by LSM5 Pascal software.
All data points represent the means of more than 30 worms. Asterisks indicate statistically significant differences (**p < 0.01). Statistical significance was determined by the Mann-Whitney U test for the comparison between wild-type and mutants, and for the rescue experiments.
In each set of experiment, the mean fluorescent intensity of Pkcc-2c::gfp in HSNs of wild-type animals was assigned as 1.0, and each mean fluorescent intensity in eor-1 mutants or transgenic animals were shown as relative intensity by comparing to wild-type animal. All data points represent the means of more than 30 worms. Asterisks indicate statistically significant differences (p < 0.01). Statistical significance was determined by the Mann-Whitney U test for the comparison between wild-type and mutants, and for the rescue experiments.
The mean fluorescent intensity of EOR-1::mCherry in HSNs of wild-type animals without Ex[Phsp16.2::eor-1a::mCherry] was assigned as a relative intensity of 1.0. All data points represent the means of more than 28 worms. Asterisks indicate statistically significant differences (p < 0.01). Statistical significance was determined by the Mann-Whitney U test for the comparison between worms carrying the transgene (+Ex) and worms not carrying the transgene (−Ex).
Nuclear spot assays. Nuclear spot assays were performed as described 39 , with some modifications. In brief, images were obtained using an Olympus FV1000 confocal microscope. Developmental stages were discriminated by distal tip cell migration and body length. HSN cells were identified based on YFP::HTZ-1 expression driven by the unc-86 promoter and its relative location against gonadal primordium and coelomocytes visualized by the co-injection marker Punc-122::gfp. All nuclei of HSNs with mTurquoise2 spots were analyzed for the co-localization of YFP and mTurquiose2 dots.
All data points were collected from more than 30 worms. P values were calculated using the Fisher's exact test. Asterisks indicate statistically significant differences (*p < 0.05. **p < 0.01).
Data availability. Strains are available upon request.