Genome-wide association studies identified almost 100 sequence variants linked to PD in SNCA. However, the consequences of this genetic variability are rather unclear. Herein, our analysis on selective single nucleotide polymorphisms (SNPs) which are highly associated with the PD susceptibility revealed that several SNP sites attribute to the nucleosomes and overlay with bivalent regions poised to adopt either active or repressed chromatin states. We also identified large number of transcription factor (TF) binding sites associated with these variants. In addition, we located two docking sites in the intron-1 methylation prone region of SNCA which are required for the putative interactions with DNMT1. Taken together, our analysis reflects an additional layer of epigenomic contribution for the regulation of the SNCA gene in PD.Parkinson disease (PD) is characterized by a pivotal progressive loss of substantia nigra dopaminergic neurons and aggregation of  SNCA gene (encoding α-SYN) has repeatedly been identified in several genome-wide association studies (GWAS)3. The importance of α-SYN in the pathogenesis of PD is highlighted by the fact that a multiplication of SNCA lead to severe Parkinsonism, which suggests that SNCA expression level, determines the severity of the PD pathology4.Parkinson’s disease (PD) is a heterogeneous age-associated incurable neurodegenerative syndrome, occurring in both sporadic and familial forms. Although phenotypically diverse, the defining pathological hallmark of PD is the loss of midbrain dopaminergic neurons in the substantia nigra pars compacta. Surviving dopaminergic neurons display characteristic extensive aggregate confirmations , which among other proteins contain alpha-synuclein (α-SYN). Although it is unclear why exactly neurons degenerate in PD, it is striking that the SNCA increased the expression of α-SYN5.In addition to genetic changes, understanding of epigenetic alterations is equally important to explain the dynamics of histone modifications and transcription factor binding sites that strongly regulate the gene expression. Among genetic regulators, microRNAs and transcription factors (TF) plays an essential role for regulating gene expression. Furthermore, the alterations induced by environmental factors also contribute to the disease pathology. In previous experiments our team demonstrated that decreasing methylation of intron-1 of SNCA gene on the epigenetic landscape . Furthermore, we provide structural insights into the methylation prone intron-1 region of SNCA as binding site of DNMT1 (DNA methyltransferase 1).Herein, we focused especially on a putative impact of the genetic variants (SNPs) distributed throughout the et al.6). The distribution of PD associated SNPs appeared to be non-random as most of them were found to be embedded in the intron 4 region. To address how the sequence variation is reflected in the occupancy profiles of nucleosomes, we categorized them on the basis of nucleosome occupancy i.e. SNPs located within nucleosomes and SNPs lacking nucleosomes in their vicinity. In order to control the processing for the sequenced datasets, we first checked the nucleosome occupancy profiles near transcription start sites (TSSs). The profiles showed a typical distribution pattern of nucleosome-depleted region (NDR) flanked by two well positioned nucleosomes, followed by equally spaced nucleosome array downstream of TSSs. The nucleosome occupancy around the selective SNPs was measured in three different cell types  , thus SNPs in the TF binding sites may exert effects upon binding affinity of TFsoid Fig. , which aSNCA also contains SNPs but they have not yet been linked to PD risk or phenotype. REP1 may however, play a role in the regulation of SNCA expression as its longest (263 bp) and intermediate length (261 bp) alleles are associated with an increased risk for PD13. Previously, the binding to transcription factor poly (ADP-ribose) transferase/polymerase-1 (PARP-1) with REP1 has been described14. Herein, using computationally predicted binding sites, we described additional transcription factor motifs enriched in this tandem repeats region , located in the promoter region (~10 kb upstream) of  longest 63 bp and17. In our analysis, we found SNP rs1372519 within a peak of H3K27ac (enhancer and promoter marks) and within the DNA binding motif sequence of NeuroD1 (NDF1), which had been involved in a specific neurogenic program, including differentiation and migration earlier18. Likewise, SNP rs2301134 is located within a peak of H3K9me3 histone modification mark (repressor and heterochromatin marks) and the DNA binding motif sequence of PRDM1 (BLIMP1), which has been shown previously to modulate the divergence of neural or germline fates through repression of SOX2 during human development19. SNP rs2619364 was located near a peak of H3K27me3 histone modification mark (repressor and heterochromatin marks) and a DNA binding motif sequence of RE1-Silencing Transcription factor (REST), also known as Neuron-Restrictive Silencer Factor (NRSF). NRSF is a zinc-finger transcription factor initially described as a nuclear negative regulator of differentiation20, now known to play a role in neuronal cells. Furthermore, we found that selective SNP sites lay within bivalent regions poised to adopt either active or repressed chromatin states. Overall, SNPs located in promotor region and intron 4 site were found to be enriched with histone modifications . To undermine the bivalency of histone marks, we further analyzed H3K4me1, H3K4me3, H3K36me3, H3K9me3, H3K27me3, H3K27ac and Input DNA in human monocyte, stomach, fetal muscle leg, H9 human embryonic stem cell (ESC), neural progenitor populations of neuroepithelial (NE), early radial glial (ERG) and mid radial glial (MRG). The bivalency of H3K4me3 and H3K27me3 around the transcriptional start site (TSS) of SNCA was observed in all tissue and cell types, while the co-existence of H3K27ac and H3K27me3 was limited to few sample types. Interestingly, the co-existence of H3K4me1 and H3K27me3 was also noticed in all the tissue and the cell types  was predicted21. But the CAGE analysis, enrichment of CpG sites and several putative transcription factor binding sites point towards an additional promotor region in the intron 1. Importantly, the methylation levels in the intron-1 of SNCA have been associated with expression of α-SYN in PD22. Therefore, we searched for potential docking sites required for putative interactions with DNMT1 in intron-1. Based on our previously published methodology, the molecular docking was conducted between the motifs identified from nucleotides and DNMT123.24. Docking of linear B-DNA (flexible) 8 bp from the identified motif to DNMT1 (stationary) identified the similar distinct DNA-binding sites in the 109 top-ranked conformations  by using our established methodons Fig. . The B-DBecause of the observed high binding affinity, we took the docked position as prime site and used it for further molecular dynamic simulations. We observed that the complementary interactions between DNMT1 and DNA fit well along with the basic patches of DNA. From the crystal structure 3SWR, it appears that the region near to DNA binding site might play a role to hold the DNA which we observed in different orientation and in different crystals. This region also shows very low electron density and highest B-factor, indicating its structural flexible.From the crystal structure 3SWR, it appears that the missing link near DNA binding site might play a role to hold the DNA as we observed in different orientation in different crystals. This region also shows very low electron density and highest B-factor, indicating its structural flexible.25. Our data showed that the residues K649, R650, R651 and R652 make important contributions in increasing the stability of DNA-protein complexes , according to our previously shown methodologyxes Fig. . It was xes Fig. , upper. xes Fig. . The intxes Fig. , middle.et al.5 might be the result of DNMT1 interactions.These results are supported by hydrogen bond (HB) occupancy analysis as well, in which we observed a similar trend of contribution from the residues in terms of their establishment of HBs Fig. , lower. SNCA gene, we checked the correlation of SNCA expression and DNA methylation around SNCA in iPSC-derived dopaminergic neurons using public available data (GSE51921)26. The analysis showed that during the differentiation there is a slight increase in DNA methylation at upstream of SNCA, however, the gene expression was also increased  , H3K9me3 was uniformly present in most SNP sites located at promoter region, while H3K27me3 correlates only to rs2583988. Similarly, histone marks for active chromatin  were also found to be overlapping with certain variants. In intron 4 specifically, we could define 2 regions , where most of PD related SNPs were located. How the interplay of active/repressive histone marks contributes towards SNCA regulation through these SNP sites is unclear. However, previously studies provided a hint that the genetic variants might engage in crosstalk with sequence-specific transcription factors and epigenetic patterns (including DNA methylation31) thereby impacting the transcription of genes locally or remotely33. Importantly, each histone modification is unique to regulate the predictive chromatin signature at promoters, enhancer and TSS, still they can also share overlapping chromatin34 signatures35. This is what we have observed in several SNP sites, where bivalent signature  poised to adopt either active or repressed chromatin states. It is noteworthy to mention that even though the bivalency of H3K4me3 and H3K27me3 around the transcriptional start site (TSS) of SNCA was observed in all tissue and cell types, the low SNCA expression was also observed in some samples . Thus, it can be argued that the observed bivalency at the given locus simply reflects the cellular heterogeneity . Another possibility might be the repression of SNCA antisense overlapping with SNCA (at their 5′ ends) by H3K27me3 histone mark. Therefore, in the future, the emerging single-cell epigenomic methods hold the potential to add deeper insights into SNCA gene regulation.The last few years have seen expanding knowledge of the genetic-epigenetic insights of PD pathology38 can also contribute to disease susceptibility by changing the gene transcription39. Brenner et al. identified and confirmed the bindings of two zinc finger proteins ZSCAN21 (intron-1) and GATA2 (intron-2) in SNCA, but did not observe any SNP or mutations within these binding sites40. Recently, two RNA-binding proteins  that target SNCA 3′UTRs and controlling its expression were also discussed41. Herein, we specifically investigated TF motifs overlapping the SNPs and found that 11 SNPs shared the affinity towards specific TFs. Interestingly, 2 of them were splice site SNPs and rs356186 among them emerged as the one with preferential sites for TFs as well as for various chromatin modifications. We could also show many of these TFs  have previously been implicated in PD. We extended our analysis for repetitive microsatellite D4S3481  located upstream of SNCA gene and found several associated TFs motifs in and around the repetitive sequences. Recently, Afek et al. claimed that two TF families (GATA and ELK) have consensus binding sites in a very close proximity to REP-11 region, which appears to be potentially relevant for SNCA transcriptional regulation42. Till date, apart from the expansion of repetitive sequence, none of the SNP inside this locus have been proven to be predictive for PD. Previously, It was shown that the alternative splicing also contribute towards the dysregulation of SNCA43, therefore, our findings of chromatin regulation at splice site SNPs provide useful insights towards the molecular characteristics of SNCA gene.Apart from histone medications, disease variants are also known to alter transcriptional levels. Specifically, the variants in coding regions may confer disease risk through altered protein sequences, however, variants in non-coding regions or regulatory variants SNCA gene, we investigated promoter specific CpG rich region overlapping these SNP sites. We identified a DNMT1 binding site specifically at CpG8-CpG9 region of intron-1 region of SNCA, where a high level of methylation was previously observed by Jowaed et al.5. The control of DNMTs in SNCA locus is also confirmed by one recent study in which authors by using CRISPR-deactivated Cas9 (dCas9) fused with the catalytic domain of DNA-methyltransferase 3A (DNMT3A) claimed to reduce the expression levels of SNCA in human-induced pluripotent stem cell (hiPSC)–derived dopaminergic neurons from a PD patient with SNCA triplication44. Therefore, we propose that the sequence specific recognition of DNMT1 we identified in this study may impose the intrinsic control of SNCA specific gene regulation. Apart from DNMT1, other unknown factor(s) may also have an additional role in SNCA regulation e.g. some risk variant in a non-coding distal enhancer element were predicted to regulate the expression of α-synuclein45. Therefore, we checked the correlation of SNCA expression and DNA methylation in iPSC-derived dopaminergic neurons and observed an increase in both DNA methylation and expression at this particular locus. Hence, the contribution of multiple factors towards the expression of SNCA cannot be excluded. On a broader view, the multiplication of SNCA gene, hypomethylation of SNCA intron1, the A53T mutation and mutation of miRNA binding sites in the 3′UTR of SNCA, at least in part, may participate in the regulatory mechanisms of SNCA expression.To evaluate further evidence for genetic-epigenetic regulation of SNCA gene. The blueprint  and NCBI for computational analysis. For predicting the splice acceptor or donor sites and exonic enhancer and silencer elements, we used Human Splicing Finder tool (http://www.umd.be/HSF3/HSF.shtml). The input sequence used was 50 bp up- and downstream of the SNP position related to the disease associated ten rsIDs. From the output, only predictions with score more than 70 percent were considered in results. Transcription factor enrichment analysis was performed by using SNP2TFBS  tool which selects and visualizes user defined variants that affect single or multiple transcription factors. Apart from SNCA gene, Rep1 , the polymorphic microsatellite repeat located approximately 10 kb upstream of the SNCA gene, was also checked for 9 SNP sites and TF binding sites.We investigated the panel of 34 SNPs previously found in GWAS studies and from the literature in association to https://www.encodeproject.org). FASTQ files of ChIP-seq data of H3K27ac, H3K27me3, H3K4me1, H3K4me3 and Input DNA in H9 human embryonic stem cell (ESC) and neural progenitor populations of neuroepithelial (NE), early radial glial (ERG) and mid radial glial (MRG) were downloaded from SRA database referred from GEO database (GSE62193). Reads in FASTQ files of ChIP-seq data were aligned to the hg19 version of the human reference genome using BWA mem with default parameter46. BAM files produced by BWA were converted to SAM files, sorted and indexed by Samtools47. Peak calling of ChIP-seq data was performed by SICER with optional parameters ‘hg19 1 200 150 0.74 600 0.01’48. WIG files of ChIP-seq data were produced from the BAM files by bam2wig.pl with optional parameters ‘–extend–rpm–bin 200’ in Biotoolbox (https://github.com/tjparnell/biotoolbox), and were displayed using custom truck in UCSC Genome browser (http://genome.ucsc.edu/). The overlaps between SNPs and peaks of histone modification marks were investigated using intersectBed of Bed tools with default parameter49. In context to check the overlap between SNPs and open chromatin regions, we obtained the Bed file of peaks of DNase-DGF data in fetal brain from NIH Epigenome roadmap website (http://www.roadmapepigenomics.org and GEO database (GSM723021), and investigated the overlaps between SNPs and peaks of open chromatin regions using intersect Bed.To examine the overlap between SNPs and histone modification marks, FASTQ files of ChIP-seq data of H3K27ac, H3K4me1 and Input DNA in neuron were download from Sequence Read Archive (SRA) database referred from Gene Expression Omnibus (GEO) database (GSE71278), FASTQ files of ChIP-seq data of H3K4me3, H3K36me3, H3K9me3, H3K27me3 and Input DNA in fetal brain  were downloaded respectively. BAM files of ChIP-seq data of H3K4me1, H3K4me3, H3K36me3, H3K9me3, H3K27me3, H3K27ac and Input DNA in human monocyte, stomach, fetal muscle leg  were downloaded respectively from ENCODE project database (http://www.repeatmasker.org) and RepBase RepeatMasker Edition (http://www.girinst.org). The overlap of repeat DNA sequences and SNPs was examined using intersectBed.Repeat DNA sequences were searched for hg19 version of the human reference genome using RepeatMasker (http://genome.ucsc.edu) in human genome assemble hg19 (GRCh7.p13). For nucleosome occupancy profile, the raw reads was mapped to human genome (hg19) using Bowtie50. Only the uniquely mapped reads were used to further analysis. Nucleosome occupancy refers to reads count at each genomic locus which means the count of the reads covering the locus. Each read was extended to 73 bp in the 3′ direction and shifted by 36 bp towards the 3′ direction. The nucleosome occupancy profiles near special sites  were represented with the average nucleosome occupancy profile, which was calculated by summing the occupancy signal at each genomic site and then dividing the summed signal by the gene number. For the SNPs, the raw reads count data was normalized by dividing by the maximal reads count of whole genome and then multiplying by 100.To determine the nucleosome occupancy around SNPs, sequencing datasets of nucleosomes were retrieved from three cell types: lymphoblastoid (Gene expression omnibus (GEO) accession number: (GSM907783), keratinocytes (GSE65191), IMR90 (GSE44985) and neural progenitor cells (GSE117870). Reads in the datasets were determined with micrococcal nuclease digestion followed by next generation sequencing (MNase-Seq). Coordinates of single nucleotide polymorphisms (SNP) sites and transcription start sites (TSSs) were retrieved from UCSC using the Tables function . A SNP site with a P ≤ 0.05 was considered as being occupied by nucleosomes.(SNCA, previously evaluated for DNA methylation levels was used to find DNMT1 interacting sites5. The identification of motifs from nucleotide sequences, molecular docking and molecular dynamics simulation was performed according to the methods we have published previously25.The intron-1 region of Supplementary infoSupplementary Data


ETV6 are associated with hematological malignancies. Notably, the t translocation leading to an ETV6-AML1 fusion gene is the most common genetic alteration found in childhood acute lymphoblastic leukemia. Moreover, most of these patients also lack ETV6 expression, suggesting a tumor suppressor function. To gain insights on ETV6 DNA-binding specificity and genome wide transcriptional regulation capacities, we performed chromatin immunoprecipitation experiments coupled to deep sequencing in a t-positive pre-B leukemic cell line. This strategy led to the identification of ETV6-bound regions that were further associated to gene expression. ETV6 binding is mostly cell type-specific as only few regions are shared with other blood cell subtypes. Peaks localization and motif enrichment analyses revealed that this unique binding profile could be associated with the ETV6-AML1 fusion protein specific to the t background. This study underscores the complexity of ETV6 binding and uncovers ETV6 transcriptional network in pre-B leukemia cells bearing the recurrent t translocation.Genetic alterations in the transcriptional repressor  ETV6 is a member of the ETS superfamily of transcription factors that are critical modulators of cellular homeostasis in several tissues. The normal function of ETS factors is mandatory for appropriate cell fate as dysregulation or deleterious events affecting these factors are frequently observed in a variety of cancers1. ETV6 is essential in the establishment and maintenance of hematopoiesis within the bone marrow compartment3. ETV6 translocations are frequently observed in various hematological disorders4 and germline mutations have been associated to predispositions for such diseases10.ETV6 aberration is the t translocation which fuses ETV6 to the AML1 gene (or RUNX1) and generates an in-frame ETV6-AML1 chimeric protein11. This is the most frequent chromosomal abnormality in childhood pre-B cell acute lymphoblastic leukemia (pre-B ALL), occurring in 20% of cases12. However, the ETV6-AML1 fusion protein seems insufficient to induce leukemia by itself15, suggesting that additional events are required to fully develop pre-B ALL16. Interestingly, the complete inactivation of ETV6 in t-positive pre-B ALL cases was underscored by several studies21 and indicates that ETV6 depletion could lead to pre-B ALL initiation.The most common ETV6 acts as a transcriptional repressor23. ETV6 has a N-terminal pointed (PNT) helix-loop-helix domain required for protein-protein interactions and homodimerization24. Its central repressive domain is also implicated in protein-protein interactions with members of the SMRT/N-CoR/mSin3A/HDAC co-repressor complexes27. The C-terminal part of ETV6 contains an ETS DNA-binding domain that recognizes a consensus ETS-binding site consisting of a core GGAA/T sequence with adjacent purine-rich sequences24. Interestingly, the ETV6-AML1 fusion protein combines both PNT and central repressive domains of ETV6 with Runt DNA binding and transactivation domains of AML1, thus converting AML1 from a transcriptional activator to a putative repressor28.Unlike the majority of ETS members, ETV6-AML1 have been studied31, the exact role of ETV6 remains poorly understood. To gain insights into ETV6 function in t-positive pre-B leukemia cells, we sought to identify ETV6 binding sites using chromatin immunoprecipitation coupled to high throughput sequencing (ChIP-seq). By including expression data32, we extensively described ETV6 binding properties and transcriptional activity in this particular context.Although the molecular functions of ETV6 was subcloned into pcDNA3.1 (pcDNA3.1 ETV6). The C-terminal HA-tagged ETV6 construct was generated by restriction enzyme digestion as described previously32 using an oligomer containing 3 tandem HA tag repeats. Both ETV6 and ETV6-HA were subcloned into pCCL lentiviral vector (kindly provided by Dr. Christian Beauséjour) through enzymatic digestion and ligation.The complete wild-type coding sequence of 2 incubator at 37 °C.Reh (ATCC ® CRL-8286™), a t-positive pre-B ALL cell line, was maintained in RPMI 1640 (Wisent) 10% Fetal Bovine Serum  in a 5% CO7 HEK293T cells were seeded into 15 cm petri dishes in DMEM (Wisent) 10% FBS. The next day, cells were transfected with 9 μg pCCL plasmids together with 6 μg pRSV-Rev, 7.8 μg pMD2.VSVG and 15 μg pMDL third generation encapsidation plasmids (kindly provided by Dr. Christian Beauséjour) in fresh RPMI 1640 10% FBS medium using polyethylenimine (Polysciences) at a final concentration of 6.5 μg/mL. Media was removed 16 h post-transfection and replaced by fresh DMEM 10% FBS. After 30 h, viral particles were retrieved from media by ultracentrifugation (50 000 g) and quantified by p24 antigen ELISA (Advanced Bioscience Laboratories).1.5 × 106 Reh cells and two different Reh clones (generated in methylcellulose media) were seeded in 2 mL of RPMI 1640 10% FBS medium. 200 ng of concentrated virus were added to cells with polybrene (Sigma) to a final concentration of 8 μg/mL. 24 h post-infection, medium was changed with fresh RPMI 1640 10% FBS. These cells were maintained 2 weeks in culture before carrying out further experiments.2 × 10ETV6  or GAPDH  and HRP-coupled secondary antibodies anti- mouse  and anti-goat  IgG, respectively. Membranes were then assayed by enhanced chemiluminescence detection with Western Lightning Plus-ECL (PerkinElmer) according to the manufacturer protocol.20 μg of nuclear protein extracts were diluted in Laemmli buffer and migrated on SDS-denaturating 10% polyacrylamide gels. Transfer on polyvinylidene difluoride membranes was performed at 4 °C overnight. Membranes were blocked in Blotto A solution  prior to immunoblotting using the primary antibodies against 32. Briefly, cross-linked chromatin isolated from 1.0 × 107 transduced Reh cells was used for immunoprecipitation with anti-HA magnetic beads (Thermo Fisher Scientific). DNA-protein complexes were eluted from the beads by competition with HA peptides prior to reverse-crosslinking and standard purification using phenol/chloroform/isoamyl alcohol (Sigma). The purified ChIP DNA was processed through TruSeq ChIP Sample Preparation Kit (Illumina) according to the manufacturer protocol. As positive control, a fraction of the amplified ChIP material was used to assess the ETV6 binding enrichment at the CLIC5A promoter32 by quantitative PCR (qPCR) using primers listed in Supplementary Table Chromatin immunoprecipitation (ChIP) has been performed as previously described33 and filtered using a mapping quality threshold of 20. Reads were merged for both ETV6 and ETV6-HA populations (n = 3). Peaks were called in the ETV6-HA condition over the background obtained in the ETV6 condition (negative IP control) using MACS v2.1.1.2016030934 with a q-value threshold of 0.1. Peaks overlapping blacklisted regions  or having a fold enrichment above 50 were discarded. Peaks were associated to ensembl genes using the closest gene transcription start site (TSS) annotations from HOMER and genomic coordinates from bedops’ closest-features v2.4.1938. To define the minimal peak fold enrichment threshold (≥4.5), a Fisher’s exact test p-value was calculated for the overlaps between ETV6 peaks and ETV6-modulated genes32 against the proportion of ETV6 peaks in the complete ensembl genes repertoire. Genomic annotations of peaks and motif enrichment analyses were performed using HOMER v4.839.Raw reads were aligned on the Hg19 reference genome using bowtie v2.2.3ETV6 ChIP-seq data in GM12878 and K562 cells are publically available as part of the ENCODE project.https://www.encodeproject.org/files/ENCFF272DJU/@@download/ENCFF272DJU.bed.gzGM12878:https://www.encodeproject.org/files/ENCFF514SLV/@@download/ENCFF514SLV.bed.gzK562:ETV6 peaks in GM12878 and K562 cells were thus obtained directly from the ENCODE platform as conservative IDR bed files (narrowPeak). The intersection of ETV6 peaks between cell types was obtained using pybedtools v.0.7.7 venn_mpl with default parameters40.41.Chromatin state data of GM12878 cells were obtained from the NIH Roadmap Epigenomics Project Portal ETV6, we expressed ETV6 and HA-tagged ETV6 in 3 biological replicates of Reh pre-B leukemic cells  translocation and a 12p13 locus deletion. These cells were then used for ChIP experiments using the HA epitope as a bait. We confirmed ETV6 binding to the CLIC5A promoter region, a validated ETV6 target gene32, in the 3 replicates, both by qRT-PCR  obtained by RNA-seq32. This strategy was performed through the entire peak fold enrichment range  to those of normal lymphoblastoid cells (GM12878) and myelogenous leukemia cells (K562) obtained from the ENCODE project . The strongest overlap was however observed between K562 and GM12878 cells . Of note, the same antibody and immunoprecipitation protocol were used to generate these ENCODE datasets and might favor this overlap. Only 67 regions were bound by ETV6 in all 3 cell lines (3.47% of Reh peaks). Peaks shared across Reh and GM12878 cells were however not more associated to ETV6-modulated genes in Reh32  was observed within gene  were in the flanking region (+/− 10 kb) of a TSS. The highest number of peaks within this range is observed in the first 1,000 bp upstream of the TSS  was assessed Fig. . Of the 41 compared to Reh. Considering the number of shared ETV6 peaks between Reh and GM12878 cell lines -positive background may have retrieved putative ETV6-AML1-bound regions in addition to ETV6-bound regions.ETS transcription factors such as lls Fig. . Intereslls Fig. . RUNX molls Fig. , indicatETV6 peaks according to the motif groups  of these peaks is expected to be higher than RUNX motifs peaks which are presumably bound through ETV6-AML1. A striking difference is indeed observed when we compared the percentage of peaks containing only ETS (direct binding) or RUNX (indirect binding) motifs according to different peak fold enrichment  are therefore significantly stronger (i.e. higher fold enrichment) than RUNX-unique peaks and ETS/RUNX depleted peaks -positive pre-B leukemia cells. We further associated ETV6 binding to transcriptional regulation by integrating expression data32. Despite the clear correlation between ETV6 binding and gene expression in the Reh cell line, ETV6 binding profile in these cells was markedly different of those obtained in other hematopoietic cell types. By including epigenetic data, we were able to unveil the importance of the cell type-specific chromatin environment on ETV6 binding. In this regard, roughly half of Reh-specific ETV6 peaks maps in quiescent regions of the lymphoblastoid cell line GM12878. The cell type-specific transcriptional programs and related active chromatin states thus modulate ETV6 binding. Although ubiquitously expressed, ETV6 impact on expression profiles might be different across tissues and cell types as its binding depends on higher order chromatin conformations. It is noteworthy that ETV6 silencing or overexpression in purified CD34+ cells from cord blood led to differentially expressed genes with however no recurrence with genes obtained in Reh cells , further demonstrating the cell-specific role of ETV6 in transcriptional regulation.In this study, we provided the first in-depth mapping of ETV6 binding might as well rely on other proteins44 whose availability would lead to additional complexity and divergences across cell types. For instance, ETV6-AML1 seems to shape ETV6 binding profile in Reh cells. Although additional experiments could further strengthen our observations, the over-representation of RUNX motifs is likely a consequence of their interaction42 as these sequences are expectedly targeted by the Runt domain of the fusion protein29. The contrasting distribution across TSS of ETV6 peaks in Reh cells compared to other cell types may be attributed to this interaction as the dissection per motif groups revealed a tighter distribution of ETS-unique peaks. Furthermore, not only ETV6 is recruited to RUNX-containing sequences but also induced changes in gene expression at these loci. RUNX-unique motifs were found in similar proportions within ETV6 peaks and expression-correlated peaks. More importantly, ETV6 peaks containing both ETS and RUNX consensus binding sequences were even more associated with differentially expressed genes. These regions may be bound synergistically by the complex formed by ETV6-HA and endogenous ETV6-AML1 through their ETS and Runt domains, respectively. Interestingly, the expression of ETV632 or the knock-down of ETV6-AML131 in Reh cells lead to common differentially expressed genes , further suggesting that ETV6 and ETV6-AML1 cooperate in transcriptional regulation. Additionally, it remains possible that a fraction of RUNX-containing peaks could have been retrieved through the recruitment of ETV6 to AML1 itself as several reports described an interaction between AML1 and other ETS transcription factors48.ETV6 seems to be recruited to additional sites, indicating that ETV6 can act as a cofactor without direct DNA-binding. The most striking example is the high occurrence of RUNX motifs that could be explained by the ETV6-AML1 fusion protein in Reh cells. However this is unique to the specific t-positive background of these cells and might not reflect the general impact of ETV6 in normal cells. Nonetheless, a significant proportion of peaks contained neither ETS nor RUNX motifs but were still associated to a change in gene expression. These peaks were enriched for IRF binding sites in combination or not with ETS sites. This result is supported by the known interaction between ETV6 and IRF843 and further suggests that the interaction between ETV6  and IRF proteins is a rather common mechanism of transcriptional regulation. The function of ETV6 as a cofactor is however poorly understood and additional efforts are required to uncover putative partners and characterize their functions.In addition to its canonical function to directly bind ETS sequences, ETV6 binding is highly flexible and therefore ETV6 inactivation is expected to induce unique transcriptional modifications in a given cellular context. The wide spectrum of hematological diseases associated with ETV6 alterations10 could be explained, at least partially, by the distinct ETV6 regulatory network of the cell originally affected by the mutation. Accordingly, the complete disruption of ETV6 observed in most t-positive childhood pre-B ALL cases21 may induce specific transcriptional changes required for complete leukemic transformation. Although this study dissected ETV6 binding and clarified its transcriptional network in t-positive pre-B leukemia cells32, it remains challenging to associate ETV6 target genes to leukemia-related phenotypes as their functions are, for most of them, still unclear.All together, this report indicates that ETV6 function is mandatory to fully understand its role in leukemogenesis. Towards this goal we built the first genome wide map of ETV6 binding sites in pre-B leukemia cells bearing the recurrent t translocation. By including expression data, we obtained the detailed transcriptional network of ETV6 in these cells. This comprehensive analysis exposed the binding properties of ETV6 and suggests that ETV6 could also act as a cofactor to regulate gene expression. With the recent reports connecting germline ETV6 mutations to a panel of familial hematological diseases and given the complexity of ETV6-mediated transcription, further characterization of ETV6 remains valuable.Molecular characterization of Supplementary informationTable S3Table S4Table S6Table S8


Gh1) promoter, suggesting that methylation is required for SMCHD1 DNA binding. The goal of this study was to further understand DNA methylation directed role of SMCHD1 in regulating gene expression. Therefore, we profiled SMCHD1 genome wide occupancy in human neuroblastoma SH-SY5Y cells and evaluated if DNA methylation is required for SMCHD1 genomic binding by treating cells with the DNA demethylating reagent, 5-azacytidine (5-azaC).DNA methylation is an epigenetic modification that mainly repress expression of genes essential during embryogenesis and development. There are key ATPase-dependent enzymes that read or write DNA methylation to remodel chromatin and regulate gene expression. Structural maintenance of chromosome hinge domain containing 1 (SMCHD1) is an architectural protein that regulates expression of numerous genes, some of which are imprinted, that are sensitive to DNA methylation. In addition, SMCHD1 germline mutations lead to developmental diseases; facioscapulohumoral muscular dystrophy (FSHD), bosma arhinia and micropthalmia (BAMS). Current evidence suggests that SMCHD1 functions through maintenance or de novo DNA methylation required for chromatin compaction. However, it is unclear if DNA methylation is also essential for genomic recruitment of SMCHD1 and its role as an architectural protein. We previously isolated SMCHD1 using a methylated DNA region from mouse pituitary growth hormone (KCNQ1) gene that associates with central nervous system is a known SMCHD1 target. We showed SMCHD1 binding to an intronic region of KCNQ1 that is lost following 5-azaC treatment suggesting DNA methylation facilitated binding of SMCHD1. Indeed, deletion of SMCHD1 by CRISPR- Cas9 increases KCNQ1 gene expression confirming its role in regulating KCNQ1 gene expression.Our data suggest that the majority of SMCHD1 binding occurs in intron and intergenic regions. Gene ontology analysis of genes associated with SMCHD1 genomic occupancy that is sensitive to 5-azaC treatment suggests SMCHD1 involvement in central nervous system development. The potassium voltage-gated channel subfamily Q member1 (These findings provide novel insights on DNA methylation directed function of SMCHD1 in regulating expression of genes associated with central nervous system development that impact future drug development strategies. Structural maintenance of chromosome hinge domain containing 1 (SMCHD1) is a chromatin regulator that adopts a homodimeric arrangement driven by its hinge domain to modify gene expression on the X-chromosome as well as autosomal genes . OriginaKCNQ1 gene that has a role in central nervous system development. Our study associates the role of an epigenetic regulator with DNA methylation and characterizes its molecular function and downstream action that will have implications for drug development.SMCHD1 is a non-canonical member of the SMC protein family . SMCHD1 Gh1) gene promoter. In addition, we demonstrated that in SH-SY5Y neuroblastoma cells, SMCHD1 regulates expression of imprinted genes associated with two imprinting disorders, Beckwith-Wiedemann and Silver-Russell syndromes  [DUX4 gene was greatly affected by 5-azaC treatment and there was a significant reduction in overall SMCHD1 occupation over this region  . BWS is WS, SRS) . Thus, tWS, SRS) , 18. WhiWS, SRS) , therefoion Fig. a. To exaene Fig. b led to PCR Fig. c.Fig. 1To elucidate SMCHD1 binding across the genome, we plotted SMCHD1 ChIP-Seq data peaks relative to nearest TSSs. SMCHD1 peaks were mostly found in intergenic and intron regions. When cells were treated with 5-azaC there was a slight redistribution of SMCHD1 binding sites to intergenic regions (from 78.47 to 81.47%) at the expense of those located in introns (from 17.69 to 14.57%) Fig. a. The peNext, we sought to identify genes associated with SMCHD1 genomic binding. Using PAPST software, we assigned SMCHD1 binding peaks +/− 1750 Kb relative to transcriptional start site of genes. Using DAVID Bioinformatic Resources, we performed gene ontology analysis and identified biological processes associated with the selected genes. Out of 2345 genes associated with SMCHD1 binding in control cells, 1458 genes were sensitive to 5-azaC treatment that are significantly associated with central nervous system  that amplifies the severity of symptoms suggesting that SMCHD1 mutation can act as a modifier of the disease [This study provides a high-resolution mapping of SMCHD1 genomic occupancy sites that are sensitive to DNA demethylating reagent, 5-azaC. Consistent with a previous study in murine neuronal stem (NSC) cells which showed that SMCHD1 occupancy is not restricted to gene promoters , we founy SMCHD1 , our datent Fig. a. Here, ion Fig. c. There  disease . These fp-value). In our study, DNA motifs identified at high frequency in SMCHD1 ChIP-seq peaks include beta/beta/alpha-zinc fingers, helix-loop-helix families, hormone receptors and homeodomain proteins. The majority of DNA binding motifs were sensitive to 5-azaC treatment with only a limited number that were refractory to 5-azaC. These include beta/beta/alpha-zinc-finger proteins, nuclear hormone receptors, Rel Homology region proteins and the CENP-B box binding family. The beta/beta/alpha-zinc-finger motif was the most common zinc finger motif that acts as a DNA binding domain and is found in various transcription factors including GLI3 [GLI3 gene in a 5-azaC sensitive manner  family of nuclear receptors that mediate the effects of retinoic acid (RA), and NR2F6 acts as a transcriptional repressor [SMCHD1 binding sites overlap with specific transcription factor binding motifs. A previous study suggested that SMCHD1 action may oppose that of CTCF in murine NSCs  . Our Ching GLI3 . GLI3 acing GLI3 . The GLIepressor . The sigWe used gene ontology and functional annotation bioinformatics to determine biological processes that associate with SMCHD1 genomic binding and if they are affected by 5-azaC treatment. SMCHD1 genomic occupancy greatly influenced by 5-azaC treatment and the majority of binding sites were lost. However, there were binding sites that were not affected by 5-azaC treatment. GO analysis suggests that these genes are involved in microtubule-based movement. In addition, SMCHD1 occupied genomic regions unique to 5-azaC treated samples that are associated with mRNA processing suggesting that SMCHD1 might have gained new binding sites when DNA methylation level is reduced. These findings suggest that CpG demethylation may unmask SMCHD1 binding sites that regulate gene expression. However, we cannot discount the possibility that this may have arisen as a cellular response to 5-azaC toxicity or non-specific binding of SMCHD1 following loss of DNA methylation marks.DUX4 repeats [Previous data suggest that SMCHD1 might act to establish and/or maintain repressive chromatin marks. SMCHD1 mutation in FSHD2 individuals results in a dramatic loss of DNA methylation at several autosomal regions not limited to  repeats . In addi repeats . We prev repeats . Whether repeats . In addi repeats . Unlike  repeats . So far, repeats . NonetheKCNQ1 gene. We previously showed that SMCHD1 regulates expression of an imprinted gene cluster that overlaps with KCNQ1. KCNQ1 is a subunit of the voltage gated potassium channel, Iks, which mediates the slow delayed rectifying potassium current and crucial for cardiac action potential repolarization [+ current. Mutations in the KCNQ1 and KCNE1 interface cause long QT syndrome and atrial fibrillation which results in prolongation of the QT interval of heart repolarization [KCNQ1 gene which is lost upon 5-azaC treatment. In addition, loss of SMCHD1 results in upregulation of KCNQ1 at the mRNA and protein level. KCNQ1 gene has been shown to be imprinted and monoallelically expressed. However, Sanger sequencing suggests that KCNQ1 is not imprinted in SH-SY5Y cells and elevated levels of KCNQ1 gene expression is due to up-regulation of both alleles . 5-Azacytidine (5-azaC) was purchased from Sigma (A1287). Antibodies used in this study included, anti-β-tubulin , commercial anti-SMCHD1 antibody , anti-SMCHD1 antiserum (produced in house), anti-LSD1 antiserum (produced in house), anti-DNMT1 .SH-SY5Y cells were cultured in Dulbecco’s Modified Eagle’s Medium  containing 4.5 g/L Glucose and L-Glutamine  which was supplemented with 10% fetal bovine serum (FBS). The cells were maintained in a humidified atmosphere which contained 5% CO2 at 37 °C. For 5-azaC treatment, cells were treated with 10 μM 5-azaC every 24 h for a period of 72 h to induce global loss of DNA methylation.For preparation of whole cell lysate, cells were pelleted and washed once with PBS, then lysed in lysis buffer (PBS containing 1% triton X-100). Following resuspension of the pellet, the cells were sonicated briefly (Branson Sonifier 450. output 3.5 and constant duty cycle in pulses) and incubated on ice for 20 min, vortexed and then centrifuged for 5 min at 14,000 g. The supernatant was quantified, diluted and boiled in sample buffer for 5 min. Proteins were separated on 6% SDS-PAGE acrylamide gels using Tris-glycine buffering system . After tE. coli BL21(pLsyS) using the PET28a expression system (Novagen). The peptide antigen was isolated on a Nickel column using standard denaturing conditions . After elution, the denatured peptide was renatured using a stepwise dilution protocol until the final buffer contained PBS. Next the peptide was mixed with alum for inoculation into a guinea pig. Following a standard 90-day inoculation protocol with a number of boosts, blood was collected, and the serum tested for its efficiency in immunoprecipitation and used for ChIP seq.We designed immunogenic SMCHD1 peptides to generate anti-SMCHD1 antiserum from guinea pigs and used this antiserum for ChIP-seq. Nucleotides encoding amino acids 1620–1727 of human SMCHD1 were expressed in http://www.bcgsc.ca/platform/solexa/.We examined the ability of our ChIP-seq anti-SMCHD1 antiserum to immunoprecipitate SMCHD1 compared to a commercially available anti-SMCHD1 antibody. Chromatin immunoprecipitation with 5-azaC treated and control SH-SY5Y cells was performed as previously described . BrieflyP-value and q-value cutoffs were both set as 0.05. The raw sequencing files were submitted to NCBI (GEO number GSE99227). A total of 5051 peaks were identified for the control SH-SY5Y cells and 2100 peaks for the 5-azaC treated SH-SY5Y cells (BED file). For evaluating SMCHD1 peak positions relative to the transcriptional start sites (TSSs), the Genomic Regions of Enrichment Analysis Tool (GREAT) software and SeqMonk program [http://www.bioinformatics.babraham.ac.uk/projects/seqmonk/) were applied. For motif analysis the SMCHD1 peaks were submitted to the SeqPos motif tool available in Galaxy cistrome [To identify genomic sites bound by SMCHD1 with high resolution, we first mapped the reads to the human genome GRCh37, hg19) , then used MACS2 paired peak calling for identification of SMCHD1 peaks . The P-v, hg19  targeting SMCHD1 and cloned them into the CRISPR-Cas9 PX459 plasmid following the Zhang Lab protocol  . In brip-values.For RNA extraction, Trizol  was used according to the manufacturer’s protocol. About 200 ng of RNA was reverse-transcribed using Superscript II . StepOne Real Time PCR System (Life Technologies) and SYBR Advantage qPCR Premix  was used for quantification of cDNA. The oligonucleotides used in this work are listed in Additional file Genomic DNA of SH-SY5Y control and KO cells were prepared using the Qiagen Blood and Cell culture kit . The samples were treated with bisulfite using the Imprint DNA Modification Kit . DNA samples were amplified by PCR. PCR primers were designed using the PyroMark Assay Design software 2.0 from Qaigen. PCR products were bound to streptavidin Sepharose beads , 10 μL of samples were sequenced using PyroMark Q24 pyrosequencer. Percent DNA methylation was then measured for each CpG site.Additional file 1: Figure S1. Types of transcription factor (TF) family binding sites found associated with SMCHD1 binding.Additional file 2: Figure S2. KNCQ1 is expressed bi-allelically in SH-SY5Y cells.Additional file 3: Table S1. Genes associated with SMCHD1 genomic occupancy.Additional file 4: Table S2. Ontology analysis of genes associated with SMCHD1 genomic occupancy.Additional file 5: Table S3. Motif clusters associated with SMCHD1 genomic occupancy.Additional file 6: Table S4. List of Primers.


Streptomyces avermitilis is an important industrial producer of avermectins, which are widely utilized as effective anthelmintic and insecticidal drugs. We used gene deletion, complementation, and overexpression experiments to identify SAV4189, a MarR-family transcriptional regulator (MFR) in this species, as an activator of avermectin biosynthesis. SAV4189 indirectly stimulated avermectin production by altering expression of cluster-situated activator gene aveR, and directly repressed the transcription of its own gene (sav_4189) and adjacent cotranscribed gene sav_4190 (which encodes an unknown transmembrane efflux protein). A consensus 13-bp palindromic sequence, 5′-TTGCCYKHRSCAA-3′ , was found within the SAV4189-binding sites of its own promoter region, and shown to be essential for binding. The SAV4189 regulon was thus predicted based on bioinformatic analysis. Night new identified SAV4189 targets are involved in transcriptional regulation, primary metabolism, secondary metabolism, and stress response, reflecting a pleiotropic role of SAV4189. sav_4190, the important target gene of SAV4189, exerted a negative effect on avermectin production. sav_4189 overexpression and sav_4190 deletion in S. avermitilis wild-type and industrial strains significantly increased avermectin production. SAV4189 homologs are widespread in other Streptomyces species. sav_4189 overexpression in the model species S. coelicolor also enhanced antibiotic production. The strategy of increasing yield of important antibiotics by engineering of SAV4189 homologs and target gene may potentially be extended to other industrial Streptomyces species. In addition, SAV4189 bound and responded to exogenous antibiotics hygromycin B and thiostrepton to modulate its DNA-binding activity and transcription of target genes. SAV4189 is the first reported exogenous antibiotic receptor among Streptomyces MFRs.The bacterial species  Streptomyces are filamentous soil bacteria characterized by complex life cycle and production of numerous antibiotics . MarR-family transcriptional regulators (MFRs) comprise more than 19,000 members and are widespread in bacteria and archaea. MFRs control a variety of cellular processes, including multidrug resistance, pathogenicity, stress responses, metabolic pathways, and degradation of aromatic compounds (Streptomyces (average 50 per genome), but only a few members have been characterized in this genus, including Streptomyces coelicolor OhrR  directly represses several ave structural genes, rather than aveR  and thiostrepton (Thi), produced by other Streptomyces species, released SAV4189 from its target genes. Thus, SAV4189 appears to mediate interspecies communication in Streptomyces by responding to antibiotic signals.Forty-two MFRs are encoded by the  76–02-e . We obseTable 1, and primers are listed in Supplementary Table S. avermitilis and Escherichia coli were as described previously  strain ATCC31267 as template. A 511-bp 5′ flanking region (from positions -314 to +197 relative to the sav_4189 start codon) was amplified with primers ZX141 and ZX142, and a 522-bp 3′ flanking region (from positions +502 to +1023) was amplified with primers ZX143 and ZX144. The two fragments were fused by PCR using primers ZX141 and ZX144, and ligated into pKC1139 to generate sav_4189 deletion vector pD4189, which was transformed into WT protoplasts. sav_4189-deleted mutant D4189 was selected by the method described previously , and a 575-bp 3′ flanking region (from positions +2446 to +3020) were amplified with primer pairs ZX177/ZX178 and ZX179/ZX180, respectively. The two fragments were ligated into pKC1139 to obtain sav_4190 deletion vector pD4190, which was transformed into WT protoplasts. The resulting sav_4190-deleted mutant D4190 was confirmed by PCR using ZX239, ZX240, ZX241, and ZX236 as primers  was used as internal control for normalization of transcription levels. Gene expression was determined in triplicate and repeated for three independent samples.Total RNAs were isolated at various times from eviously  to detersav_4189 coding region (171 amino acids) was amplified by PCR with primers ZX205 and ZX206. The PCR fragment was digested with NdeI/EcoRI and inserted into expression vector pET-28a (+) to generate pET28-4189, which was verified by DNA sequencing. pET28-4189 was transformed into E. coli BL21 (DE3), and recombinant His6-SAV4189 protein overexpression was induced by 0.2 mM IPTG for 8 h at 16°C. Cells were collected, washed, and disrupted in lysis buffer  by sonication on ice. The preparation was centrifuged, and the supernatant containing soluble His6-SAV4189 was loaded onto a Ni2+-NTA column (Qiagen). After extensive washing with wash buffer , the His6-SAV4189 protein was specifically eluted from the resin with elution buffer . Concentrations of purified proteins were determined by Bradford assay, and proteins were stored at -80°C.The 516-bp 6-SAV4189 in binding reaction. To verify specificity of SAV4189-probe 4189p interaction, a ∼100-fold excess of unlabeled specific probe 4189p or non-specific probe hrdB was added to the reaction mixture.Electrophoretic mobility shift assays were performed as described previously . DNA proFor EMSAs with antibiotics, avermectin B1 (AveB1), oligomycin (Oli), and thiostrepton (Thi) were dissolved in DMSO, while hygromycin B (HygB), apramycin (Apr), and kanamycin (Kan) were dissolved in deionized water. Dissolved antibiotics were added at various final concentrations, and solvent control DMSO was added at 8 μl in the reaction mixture.sav_4189 TSS using a 5′/3′ RACE kit (Roche). Total RNA (2 μg) prepared from 84-h culture of WT grown on YMS was used for reverse transcription with 20 pmol of gene-specific primer 4189SP1. An oligo(dA) tail was added to the 3′ end of purified cDNA using terminal deoxynucleotidyltransferase, followed by PCR amplification of the tailed cDNA using specific nested primer 4189SP2 and oligo(dT) anchor primer. The resulting PCR product (diluted 1000-fold) was amplified in an additional round of PCR with nested primer 4189SP3 and an anchor primer, to yield a single specific band. The purified final PCR product was inserted into pMD18-T vector (TaKaRa) for sequencing. The first nucleotide following oligo(dA) sequence was determined as the complementary base of sav_4189 TSS.5′ rapid amplification of cDNA ends (5′ RACE) analysis was performed to identify the sav_4188-sav_4189 intergenic region was synthesized by PCR using primers FAM-89footS/89footAS, and purified. The probe (400 ng) and various concentrations of His6-SAV4189 were mixed in a 25-μl volume, and incubated for 30 min at 25°C. Following DNase I digestion, DNA samples were purified and subjected to capillary electrophoresis with 3730XL DNA analyzer (Applied Biosystems). Results were processed with GeneMarker v2.2.A non-radiochemical capillary electrophoresis technique  was usedsav_4189 gene contains 516 nucleotides (nt) and encodes a MFR of 171 amino acids (including a conserved wHTH DNA-binding motif homologous to MarR) whose function is unknown. Divergently transcribed gene sav_4188, located 165 nt upstream of sav_4189, encodes an unknown membrane protein. Convergently transcribed gene sav_4190, located downstream of sav_4189, encodes a putative transmembrane efflux protein . The sav_4189-sav_4190 intergenic region in only 22 nt long, suggesting that the two genes might be cotranscribed, and this was confirmed by RT-PCR   with that of WT. HPLC analysis showed that avermectin yield in WT strain was not observable until day 2, then increased gradually from day 2 onward . Relative to WT level, yields were lower for D4189, higher for O4189, and not significantly different for C4189. On the end of fermentation day 10, overexpression of sav_4189 (strain O4189) increased avermectin yield by ∼2.5-fold, and deletion of sav_4189 (strain D4189) led to a clear reduction (∼40%) in avermectin yield . These findings indicate that SAV4189 plays an activator role in avermectin production.To clarify the role of Figure 1C), indicating that SAV4189 is not involved in regulation of cell growth, and that the changed avermectin yields of D4189 and O4189 did not result from alteration of cell growth. D4189 and O4189 grew normally on YMS, SFM, RM14, and MM plates  of WT, D4189, and O4189 cultured in soluble FM-II. Cell weights of the three strains were similar . Thus, SAV4189 evidently exerts its regulatory function mainly during early fermentation stage.To elucidate the relationship between SAV4189 and avermectin production, we examined the aveR transcription levels of WT, D4189, and O4189 cultured in FM-I for 2 or 6 days. On both days, level relative to WT was reduced in D4189 and increased in O4189 , consistently with avermectin yields. Notably, aveR transcription level was particularly high for O4189 on day 2. These findings indicate that SAV4189 regulates avermectin production by stimulating cluster-situated activator gene aveR, primarily during early fermentation stage.To determine whether SAV4189 regulates avermectin production through CSR AveR, we performed qRT-PCR to examine sav_4189 and sav_4190 are cotranscribed. We therefore predicted that SAV4189 regulates expression of sav_4189-sav_4190 operon and adjacent divergently transcribed gene sav_4188. We measured transcription levels of these three genes using the same RNA samples as for examining aveR expression. No sav_4188 transcription was detectable in any of the three strains WT, D4189, and O4189, suggesting that this gene transcription was at very low level, or was not expressed under our fermentation conditions. We therefore did not investigate sav_4188 in subsequent experiments. In O4189, sav_4189 transcription level was higher than in WT, confirming sav_4189 overexpression . sav_4190 transcription was slightly reduced on day 2, and undetectable on day 6 . In D4189, transcription of both sav_4189 and sav_4190 was strongly upregulated on day 2. Transcription of sav_4189 was slightly increased, and that of sav_4190 was undetectable on day 6 . These findings indicate that SAV4189 functions as a repressor of its own operon sav_4189-sav_4190, mainly during early fermentation stage.A common regulatory mechanism of MFRs involves MarR protein regulating transcription of its own gene and a divergently oriented gene by binding to their intergenic region, which contains TSSs for both genes . sav_418aveR and sav_4189-sav_4190 operon, we performed EMSAs using soluble His6-SAV4189 protein expressed in E. coli. The aveR and sav_4189 promoter regions were labeled as probes aveRp and 4189p, respectively . Probe hrdB and BSA were used as negative probe and protein controls. His6-SAV4189 did not bind to probe aveRp or hrdB, but bound specifically to probe 4189p and generated several clearly retarded bands , suggesting that SAV4189 binds more than two sites on its own promoter region. Binding specificity was confirmed by competition experiments using ∼100-fold excesses of unlabeled specific probe 4189p (lane S), which reduced binding of SAV4189 to labeled probe 4189p, and of non-specific probe hrdB (lane N), which did not reduce the retarded signals. BSA did not retard probe 4189p, even at high protein concentration (750 nM). These findings indicate that the positive regulatory effect of SAV4189 on aveR expression is indirect, and that SAV4189 directly regulates sav_4189-sav_4190 operon through binding to the sav_4189 promoter region.To determine whether SAV4189 directly regulates sav_4189 TSS was localized by 5′ RACE to an A residue at position 22 nt upstream of the sav_4189 translational start codon , leading to the probable -10 and -35 promoter regions shown by boxes in Figure 4C.To identify precise binding sites and elucidate the regulatory mechanism of SAV4189 on its target promoter, we performed 5′ RACE and DNase I footprinting assays. The sav_4188-sav_4189 intergenic region revealed that SAV4189 protected two sites (termed A and B) on the sav_4189 coding strand . The two sites are 17 nt apart. Site A extends from positions -82 to -59, and site B extends from positions -41 to +5, relative to the sav_4189 TSS . Site A is close to, and site B overlaps the putative -35 and -10 regions of sav_4189 promoter, indicating that SAV4189 represses transcription of sav_4189-sav_4190 operon by hindering recruitment of RNA polymerase.DNase I footprinting assay with a 511-bp FAM-labeled probe covering the Figures 4B,C). The three palindromic sequences were all 13 bp (5-nt inverted repeats separated by 3 nt) and highly similar. To assess contributions of the three 13-bp sequences to SAV4189 binding, we performed EMSAs using 59-bp probes containing intact 13-bp sequences; i.e., both sides of sequences a, b1 and b2 were fused with non-specific DNA fragments from hrdB ORF by PCR to create probes A, B1 and B2, respectively . SAV4189 bound to each of these probes, indicating that all three 13-bp sequences are important for SAV4189 binding, and that SAV4189 binds at the three palindromic sequences on its own promoter region. These findings are consistent with that of several retarded bands in EMSAs for interaction of probe 4189p with purified His6-SAV4189, as described in the preceding section.MarR-family transcriptional regulators generally form homodimers and bind palindromic sequences within target promoter regions . DNAMAN 1 analysis of these three palindromic sequences generated a 13-bp consensus SAV4189-binding sequence 5′-TTGCCYKHRSCAA-3′  .WebLogo programFigure 5A). Further analysis using labeled probe B1  confirmed that affinity of SAV4189 to probe B1 was the lowest among the three probes. In experiments using 150-fold excesses, the dissociation of SAV4189 from labeled probe B2 caused by unlabeled probe B2 was more than that of the other two unlabeled probes , indicating the highest affinity of SAV4189 for probe B2, which was confirmed by comparison of intensities of SAV4189-probe B2 complex corresponding to 50-fold excess of unlabeled probes . The competitive ability of 150-fold excess of unlabeled probe A was higher than that of the same excess of unlabeled probe B1 . Therefore, we determined the affinity of SAV4189 to its target sequences in the order b2 > a > b1; however, the differences are slight.To compare affinity of SAV4189 for its three target sequences, we performed competitive EMSAs using labeled probes A, B1 and B2 that contained sequences a, b1 and b2, respectively, and ∼50-fold and 150-fold excesses of unlabeled specific competitor DNAs. In experiments using 50-fold excesses, unlabeled probe B1 dissociated less SAV4189 from labeled probe A than the other two unlabeled probes . The newly identified SAV4189 target genes were sav_2073 , olmRII , sav_5653 , pks1-3 , aspB1 , ectA , prpM3 , hmuO , and sav_1959 (encoding a putative secreted acyl esterase). SAV4189 did not interact with promoter regions of ccrA2 , add4 , savR1 , xylF , and ngcE .To test the accuracy of bioinformatic analysis, 14 putative target genes from various groups that have relatively high score or are well annotated were selected and confirmed experimentally by EMSAs. Among these, 9 targets were confirmed to bind directly to SAV4189 , indicating that sav_4190 plays a negative role in avermectin production. sav_4190 expression level on day 2 for D4189 grown in FM-I was ∼24-fold higher than that of WT , indicating that altered sav_4190 expression contributes to the reduced avermectin yield of D4189.The findings that SAV4189 positively regulates avermectin production, and that sav_4189 overexpression and by sav_4190 deletion, we experimentally engineered these two genes in industrial strain A-144 and evaluated effects on avermectin yield. sav_4189 overexpression vector pKC1139-4189 and sav_4190 deletion vector pD4189 were introduced into A-144 to construct mutants O4189/A-144 and D4190/A-144, respectively. Avermectin yield in shake-flask fermentation was ∼21% higher for O4189/A-144 and ∼15% higher for D4190/A-144 relative to parental A-144 . sav_4189 overexpression and sav_4190 deletion thus appear to be effective strategies for further enhancement of avermectin production in industrial strains.Because avermectin yield was strikingly increased by Streptomyces. To investigate possible regulation of antibiotic production by SAV4189 in other Streptomyces species, we transformed sav_4189 overexpression vector pKC1139-4189 into model strain S. coelicolor M145. Resulting transformant O4189/M145 was grown on YBP agar, and earlier, higher antibiotic production was visually observed . This finding suggests that SAV4189 (or its homolog) acts as an activator of antibiotic production in Streptomyces species other than S. avermitilis. Thus, a strategy for strain improvement based on engineering of SAV4189 and its target gene may be extended to other industrial Streptomyces strains having SAV4189 homologs, to promote antibiotic production.SAV4189 homologs are distributed widely in 4189p. SAV4189 DNA-binding activity was not inhibited by endogenous antibiotics Oli and AveB1, nor by exogenous antibiotics Kan and Apr, even at high concentration (20 mM) . In contrast, SAV4189-4189p complex was disrupted in dose-dependent manner by exogenous HygB and Thi , suggesting that SAV4189 may sense and respond to these antibiotics to reduce repression of its target genes. This concept was tested by measuring expression of SAV4189 targets sav_4189 and sav_4190 in WT in response to exogenous HygB and Thi. Cells were cultured in FM-II to stationary phase (96 h), added with these antibiotics at subinhibitory concentration, and total RNAs were isolated from fermentation culture after 3 h and analyzed by qRT-PCR. sav_4189 and sav_4190 transcription levels were notably increased by addition of HygB or Thi, but not of DMSO . HygB caused ∼143-fold increase of sav_4189 and ∼75-fold increase of sav_4190 expression. Thi caused ∼81-fold increase of sav_4189 and ∼71-fold increase of sav_4190 expression. These findings, taken together, indicate that SAV4189 recognizes various exogenous antibiotics such as HygB and Thi as ligands to modulate its DNA-binding activity and transcription of target genes.A defining characteristic of MFRs is their response to specific ligands, such as antibiotics and phenolic compounds, resulting in attenuated DNA-binding activity and derepressed gene expression . In viewS. avermitilis. Of the 42 MFRs of S. avermitilis, SAV4189 is the first to be characterized. The present findings enhance our understanding of control of secondary metabolism by MFRs in Streptomyces. Results of qRT-PCR and EMSAs indicate that SAV4189 plays an indirect role in stimulation of avermectin production by altering transcription of the CSR gene aveR, whose product is essential for activating all ave structural genes  may be extended to other industrially important antibiotics produced by Streptomyces.We identified two SAV4189 target genes: sav_4190, which encodes a transmembrane efflux protein belonging to the major facilitator superfamily (MFS), is repressed directly by SAV4189. sav_4190 expression level is low in WT strain; however, deletion of this gene results in increased avermectin yield in both WT and industrial strains, suggesting that sav_4190 plays an important negative role in avermectin production. In O4189, though sav_4190 transcription was undetectable on day 6, there is still a very low sav_4190 transcription level on day 2. Thus, deletion of sav_4190 in sav_4189 overexpression strain may further improve avermectin yield.S. coelicolor on the basis of their substrates. The putative substrates transported by SCO4031, a SAV4190 homolog in S. coelicolor, are multiple drugs. However, the substrates of SAV4190 could not be avermectins. If SAV4190 pumps out avermectins directly, deletion of sav_4190 will reduce avermectin yield. The fact is that SAV4190 has a negative effect on avermectin production. Thus, SAV4190 may be involved in expulsion from cells of intermediates of the avermectin biosynthetic pathway or some other precursors needed for avermectin biosynthesis when they accumulate to a certain level, thereby avoiding excessive avermectin production. We demonstrated recently that another MFS transporter in S. avermitilis, SAV7490 (AveM), also has a strong inhibitory effect on avermectin production, and that sav_7490 deletion significantly enhances avermectin yields in WT and industrial strains , which modulates morphological differentiation and endogenous undecylprodigiosin (Red) production , Kan, streptomycin (Strep), methicillin (Meth), penicillin G (PnG), and chloramphenicol (Chl), indicating the remarkable plasticity of the multidrug-binding pocket of this protein (Streptomyces species. These species may require differing types of receptors (such as TFRs and MFRs) to sense antibiotic signals produced by neighboring organisms, to elicit appropriate cellular responses. To our knowledge, SAV4189 is the first example of an exogenous antibiotic receptor among Streptomyces MFRs, and presumably plays an important role in interspecies communication. Our findings inform further investigations of ecologically relevant interactions between Streptomyces MFRs and exogenous antibiotic signals.SAV4189 did not bind to endogenous antibiotics AveB1 and Oli, but it did sense exogenous antibiotics HygB and Thi, which are produced by other  signals . Howeveroduction . We foun and Apr . AvaR2 a protein , 2013. Tsav_2073, olmRII, and sav_5653 are involved in transcriptional regulation, reflecting the complex cascades of SAV4189 regulatory network. olmRII encodes an oligomycin pathway-specific activator  associated with primary metabolism for evaluation and found that aspB1 involved in aspartate metabolism is SAV4189 target. ect genes encode enzymes for biosynthesis of compatible solute ectoine, which plays an important osmoprotective role (ectAp for ectA-ectB-ectC-ectD operon suggests that SAV4189 mediates protective responses to stress signals, such as osmotic stress. The other three identified SAV4189 targets are prpM3 involved in protein modification, hmuO involved in porphyrin metabolism, and sav_1959 with unknown function. Taken together, the present findings clearly demonstrate the pleiotropic effects of SAV4189 on cell physiological processes in S. avermitilis. Further studies in this important species will experimentally confirm the SAV4189 targets, clarify the broader regulatory roles of this protein, and eventually provide a comprehensive picture of cellular responses triggered by antibiotic and stress signals and mediated by SAV4189.The predicted SAV4189 regulon contains 250 genes, which have been extensively cataloged and are involved in numerous physiological processes. EMSA analysis led to identification of 9 new SAV4189 targets. Among these, ctivator . pks1-3 ive role . The finYW and XZ designed the research. JG, XZ, XL, and WL performed the experiments. ZC, LD, and JL contributed study materials. YW and JG wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


CHD1, CHD2, CHD4, CHD7 and CHD8 have been associated with a range of neurological phenotypes, including autism spectrum disorder (ASD), intellectual disability (ID) and epilepsy. Pathogenic variants in CHD2 are associated with developmental epileptic encephalopathy (DEE) in humans, however little is known about how these variants contribute to this disorder. Of the nine CHD family members, CHD2 is the only one that leads to a brain-restricted phenotype when disrupted in humans. This suggests that despite being expressed ubiquitously, CHD2 has a unique role in human brain development and function. In this review, we will discuss the phenotypic spectrum of patients with pathogenic variants in CHD2, current animal models of CHD2 deficiency, and the role of CHD2 in proliferation, neurogenesis, neuronal differentiation, chromatin remodeling and DNA-repair. We also consider how CHD2 depletion can affect each of these biological mechanisms and how these defects may underpin neurodevelopmental disorders including epilepsy.The chromodomain helicase DNA-binding (CHD) family of proteins are ATP-dependent chromatin remodelers that contribute to the reorganization of chromatin structure and deposition of histone variants necessary to regulate gene expression. CHD proteins play an important role in neurodevelopment, as pathogenic variants in  Gene regulation is a complex process that is tightly regulated by many factors across tissue types and at different points in development. Chromatin remodelers play an essential role as both activators and repressors of transcription by manipulating the structure of chromatin to allow access for gene regulation machinery or conversely, to obscure sites where gene expression must be repressed. The human nervous system is composed of multiple neuronal cell types, as well as supporting glial cells. These diverse cell types are all derived from neural progenitor cells (NPCs), which then differentiate and migrate to their proper locations during development. Epigenetic mechanisms play an important role in regulating this process and controlling cell fate , while yeast (Saccharomyces cerevisiae) only has one such protein . The CHD7 in patients with coloboma, heart defects, atresia choanae, growth retardation, genital abnormalities and ear abnormalities (CHARGE) syndrome , mild to moderate ID (CHD4) and mild to profound ID (CHD2/8). Additional neurological features include epilepsy (CHD2) or seizures (CHD1/8) and autistic spectrum disorders . Despite the ubiquitous gene expression profiles of all CHD family members implicated in human disease, only CHD2 pathogenic variants cause a brain-restricted phenotype, suggesting a unique role for this gene in neurodevelopment , chronic lymphocytic leukemia (CHD2), endometrial cancer (CHD4) and neuroblastoma  and there are a number reported in DECIPHER  has been observed, and one mutant allele is sufficient to cause disease. However, there is some evidence that rare variants in CHD2 could be a risk factor for more common types of photosensitive epilepsies without ID  has been conserved for over 476 million years, indicated by its presence in divergent species such as opossum, chick and zebrafish  antisense oligomers results in larvae displaying seizure-like behavior and photosensitivity, recapitulating the phenotype seen in humans with pathogenic CHD2 variants  protein, including the chromodomains, helicase domains and N-terminus of the DNA binding domain  of the dorsal telencephalon Figures . Here raChd2 is predominantly expressed in Pax6+ radial glia in the VZ/SVZ from E12–E18  at the same embryonic stage. shRNA knockdown of Chd2 by in utero electroporation during mouse embryogenesis leads to a reduction in the number of Pax6+ radial glia and increase in Tbr1+ IPs and Tuj1+ neurons. Overall these results suggest Chd2 deficiency suppresses the self-renewal capacity of the radial glia and instead promotes premature neuronal differentiation  to cortical interneurons by defined factors + neurons with shorter neurites, suggesting delayed or impaired differentiation. CHD2 knockout interneurons also exhibited electrophysiological defects that suggest hyper-excitability in these neurons  results in differentiation defects, with decreased expression of differentiation-dependent myogenic genes and a lack of myotube formation  may also play a role in cell-type and species-specific differences observed.Taken together, these studies have demonstrated differentiation defects in mouse ESCs, muscle and neuronal progenitor cells and human cortical interneurons. However, to date, only radial glia in mice have been shown to have defects in cellular proliferation Chd2 is depleted by miRNA knockdown in C2C12s. CHD2 also showed an interaction with MyoD, a master regulator of skeletal muscle differentiation, and together MyoD and CHD2 bind to myogenic gene promoters. Suppression of Chd2 expression in C2C12 cells decreased myogenic gene expression and halted myotube formation, indicating that Chd2 is required for skeletal muscle differentiation. Combined, these data suggest a model where CHD2 is guided to differentiation-dependent genes by other transcription factors  and that the chromodomain of CHD2 facilitates H3.3 incorporation into the nucleosome, poising genes necessary for differentiation for expression  project profiled numerous histone modifications, transcription factors and chromatin remodelers using ChIP-seq to classify the genome into functional elements of the genome  repair through non-homologous end-joining (NHEJ) requires the expansion of chromatin, which is mediated by poly (ADP-ribose) polymerase (PARP) enzymes. PARP1 is able to sense DNA-damage, and when activated, forms poly ADP-ribose (PAR) chains that are able to act as a docking platform for other DNA repair factors . When DNA DSBs were inflicted by lasers in U2OS cells, CHD2 accumulated at sites of damage in a PARP1-dependent manner, indicating that PARP1 is required for recruitment of CHD2 to sites of DNA damage  and that REST expression was decreased with Chd2 loss. REST is a transcriptional repressor that prevents the expression of neuronal genes in non-neuronal cells. These results suggest that CHD2 binding at REST prevents its expression; this may be particularly important in the maintenance of proliferative state of neuronal progenitor cells, and this association may contribute to the premature neuronal differentiation observed with Chd2 knockdown  that control GABAergic development, cause seizures in mice  from individuals with MECP2 duplications differentiated to cortical excitatory cells showed increased dendritic arborization and increased synchronized activity of the neuronal network  these approaches will be more widely used and standards of use developed.Although progress has been made deciphering the mechanism of CHD2 and its role in epilepsy in recent years, much is still unknown about the intricacies of chromatin remodeling in neuronal development and epilepsy. Improved animal models of in vitro (Birey et al., CHD2 has been found to regulate the chromatin architecture of developmentally important genes, poising these genes for expression during differentiation in various cell types. To date genome-wide CHD2 ChIP-seq and epigenomic profiling has not been performed in neuronal cell types to identify CHD2 target genes. In the future, these unbiased genome-wide experiments should be pursued using stem cell modeling in patient-derived and genome-edited cells, to identify CHD2 targets as well as alterations to the chromatin architecture. These studies may illuminate potential targets for therapeutic interventions. Moreover, to date CHD2 has been found to play a role in inhibitory neurons derived from hESCs and cortical excitatory neurons in mice. In the future, it will be key to study these two opposing neuronal systems at both a cell autonomous and network level. Typically, these experiments have been performed in knockout, or conditional knockout, mice. However, the use of patient-derived organoids is also an exciting area of burgeoning research, including the development or both ventral and dorsal organoids, and fusion of these two subtypes to study GABAergic migration CHD2 and its potential binding partners in different cell types. The role of CHD2 in DNA-repair likely is linked to its effects on proliferation, and the interplay between DNA-repair, cell cycle exit, and the balance between proliferation and differentiation during neurogenesis should be explored. Chd2 deficient mice have a reduction in their ability to repair DSBs, leading to increased cell death. Whether this defect leads to the decreased proliferation and increased differentiation seen in neuronal models remains to be seen.Additional research is required to decipher the effects of CHD2 on proliferation, and why only some CHD2-deficient cell types show proliferation defects. This could be due to distinct expression patterns of CHD2 haploinsufficiency is a rare cause of genetic epilepsy, many of the pathogenic mechanisms, particularly during neuronal development are likely to be overlapping with other genetic epilepsies. Indeed, preliminary transcriptome studies suggest CHD2 may be an upstream regulator of other genes implicated in epilepsy and that novel therapeutics may be applicable to broader range of patients with these disorders. In addition, there is extensive overlap between risk genes for autism and cancer, including CHD2 and other chromatin remodelers, CHD7, CHD8, ARID1B and ATRX (Crawley et al., Finally, while K-ML and GC contributed equally to the writing of this manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.