Chromatin transactions are typically studied in vivo, or in vitro using artificial chromatin lacking the epigenetic complexity of the natural material. Attempting to bridge the gap between these approaches, we established a system for isolating the yeast genome as a library of mononucleosomes harboring the natural epigenetic signature, suitable for biochemical manipulation. Combined with deep sequencing, this library was used to investigate the stability of individual nucleosomes and, as proof of principle, the nucleosome preference of the chromatin remodeling complex, RSC. This approach uncovered a distinct preference of RSC for nucleosomes derived from regions with a high density of histone variant H2AZ, and this preference is indeed markedly diminished using nucleosomes from cells lacking H2AZ. The preference for H2AZ remodeling/nucleosome ejection can also be reconstituted with recombinant nucleosome arrays. Together, our data indicate that, despite being separated from their genomic context, individual nucleosomes can retain their original identity as promoter- or transcription start site (TSS)-nucleosomes. Besides shedding new light on substrate preference of the chromatin remodeler RSC, the simple experimental system outlined here should be generally applicable to the study of chromatin transactions. The eukaryotic genome is stored as a polymer of nucleosomes in which ∼147 base pairs (bp) of DNA are wrapped around a core of eight histone proteins separated by linkers of variable length . BecauseA long-standing challenge in the field of chromatin research has been the lack of a reconstituted genome-wide system for studying the role of epigenetic modifications in the context of their natural sequences. Current approaches typically involve either assembling chromatin in vitro from naked DNA and free histones, or studying it inside the cell, in vivo. Even though both approaches have substantially advanced our understanding of chromatin function in general, they also suffer some limitations: Reconstituted chromatin has virtually none of the complexity of natural chromatin, being devoid of the natural genomic sequences and/or the combinatorial assortment of histone marks. Conversely, chromatin studied in vivo obviously retains its natural composition but is trapped in a context in which a process of interest is continuously subjected to the direct or indirect influence of the numerous biochemical reactions taking place inside the cell. Protocols in which chromatin is isolated directly from the host organism in a manner suitable for biochemical reconstitution have been described, but they are generally limited to the study of one or a few loci at a time . As suchPHO5 gene rings (RSC (Remodels the Structure of Chromatin) is a member of the Swi2/Snf2 family of ATP-dependent chromatin remodelers and the most abundant chromatin remodeling factor in yeast . It is nne rings . It alsone rings . We chosne rings , 2015. THTZ1 gene in budding yeast) is a variant of histone H2A that shares 60% sequence identity with its canonical H2A counterpart . The majority of the genome was recovered in the purification: 94% was thus covered by at least five reads. As might be expected, areas with less, or no, reads were mainly found toward the gene-poor ends of the chromosomes . This agrees with the previous finding that yeast chromatin is mostly open and active . Over limiting time, RSC remodels only a subset of the input nucleosomes . Although we here confined our analysis to mononucleosomes, the reaction also worked on dinucleosomes . The extracted DNA was sequenced after paired-end adapter ligation, enabling us to map each nucleosome to the reference yeast genome. Fragments from all four bands had the length expected for nucleosomal DNA containing a short linker . Indeed, the mean length of nucleosome fragments was similar between the four different categories  and across the different stability groups defined below . Even after experimentation and gel-extraction of the underlying DNA . These patterns are highly similar to those previously reported by A number of assays for measuring chromatin remodeling in vitro have been developed. We chose a simple disassembly assay, which involves incubating the nucleosome library with ATP and the histone chaperone Nap1, with or without RSC . In thisying DNA A,B, the Nucleosome position calling from MNase-derived sequencing data is very dependent on the specific software, so we chose a window-based approach as a robust method for quantifying nucleosome occupancy. For this purpose, we divided the yeast genome into 167-bp sliding windows with a step size of 25 bp. As previously reported for MNase-seq for nucleosomal DNA , we obse2[DNA/NUC]) measures the relative stability of nucleosomes across the genome, with high scores suggesting the presence of unstable nucleosomes. Similarly, we quantified the degree of RSC-dependent nucleosome disassembly by calculating the log-ratio of reads from the DNAR and NUCR samples, before subtracting the Instability Score to account for instability. The resulting Remodeling Score (RS = log2[DNAR/NUCR] – IS) measures the effect of RSC-dependent remodeling, with higher scores reflecting a more strongly remodeled nucleosome. We divided the sets in eight percentiles . Finally, overlapping or adjacent windows in the same IS/RS-percentile were merged to obtain continuous unstable or remodeled regions. For simplicity, we will hereafter often refer simply to ‘nucleosomes,’ although these are strictly speaking merged, overlapping windows in the same IS/RS-percentile. We hereby obtained sets of 450,257 windows  in which nucleosome instability could be measured and 437,761 windows  to study RSC remodeling.In order to quantify the stability of each nucleosome, we calculated the normalized log-ratio of reads between the DNA and NUC samples per window using both replicates. The resulting Instability Score . Unlike protein-coding genes, which are occupied by multiple nucleosomes, the short tRNA genes are typically covered by only a single nucleosome, which indeed appeared to often be highly unstable . We also observed an enrichment of protein-coding gene promoters among nucleosomes in the higher percentiles compared with the lower percentiles . Plotting the position of the nucleosomes in the 99th percentile, a broad area of instability was uncovered, peaking in the promoter upstream of protein-coding genes , and whatever the underlying cause, instability may therefore be intrinsic to the structure of the nucleosome.The nucleosomes represented by the different instability percentiles were not randomly distributed across the genome. Instead, the most unstable nucleosomes were enriched in protein-coding genes, tRNA genes, and promoters compared with nucleosomes in the lower percentiles , with no statistically significant difference detected (two-sided Wilcoxon rank-sum test). Similarly, there were no differences in the lengths of DNA reads across the different stability groups, nor between DNA and NUC reads . We also compared the nucleosomes in the different IS percentiles with a recently published nucleosome data set measured by chemical cleavage . Inspection of nucleosome traces indicated that many of the relevant areas were often relatively nucleosome-free in the input sample, although the same regions were detected among the gel-purified mononucleosomes and enriched in the free DNA sample after incubation , and given that we were not aiming at studying ‘nucleosome stability,’ the data sets obtained on ‘unstable nucleosomes’ were hereafter merely used as controls for experiments with RSC, to ensure that any nucleosome detected as remodeled was not merely unstable. We note, however, that one distinguishing feature of the nucleosomes classed as the most ‘unstable’ was that they were relatively enriched for ‘hot’ nucleosomes . In contrast to the Instability Scores, no marked difference between protein-coding and tRNA genes was observed for Remodeling Scores across this area , so for simplicity, the analysis below is focused on mRNA and tRNA genes together (‘genes’). Analysis of the RS within 1 kb of the TSS thus showed a marked increase of the score just upstream of, and on, the TSS  compared with nucleosomes in lower percentiles . The lack of significant size differences between the different percentiles indicated that the highly remodeled nucleosomes do not carry additional DNA, such as adjacent promoter DNA that might have served to recruit RSC, and that the recruitment signal(s) must thus be contained within the nucleosome itself.When we aligned the most strongly remodeled nucleosomes  around the TSSs of genes, we found a broad peak upstream of the TSS, covering both the  the NFR C. Of the the NFR C, lower,We conclude that RSC prefers to remodel mononucleosomes derived from promoter and TSS regions and particularly from the so-called nucleosome-free regions.Supplemental Fig. S4C). Poly(dAdT) tracts were generally modestly enriched in remodeled nucleosomes , in apparent agreement with the previous finding that RSC shows a preference for reconstituted nucleosomes bearing poly(dAdT) tracts . We failed to find a general correlation between RS and the transcription levels of the corresponding genes across the genome, as measured by RNA-seq . This suggests that the characteristics of chromatin at these genes are preserved to some degree in isolated mononucleosomes and, vice versa, that recognition of individual nucleosomes by RSC in vivo may indeed have significant consequences for transcription of the adjoining gene.RSC harbors eight bromodomains, at least one of which has been shown to bind acetylated histone tails . We fail RNA-seq . However RNA-seq . When th RNA-seq D, with mSupplemental Fig. S5A and Supplemental Fig. S5B), in contrast to the IS  and compared the resulting preference with that observed for wild-type nucleosomes. Initially, we selected four individual nucleosomes that we had found to be strongly remodeled and which carry H2AZ . Using qPCR to detect remodeling in these regions, we found that RSC indeed showed a preference for H2AZ-containing nucleosomes at the TSS when presented with nucleosomes from wild-type cells but not with nucleosomes from htz1Δ cells, even though htz1Δ nucleosomes were, of course, still remodeled due to the general ‘background’ activity of RSC .In order to experimentally address a putative causative role for H2AZ in the preference of RSC for TSS nucleosomes, we performed remodeling assays using nucleosomes prepared from rry H2AZ . These whtz1Δ nucleosomes for deep sequencing and analyzed the resulting data in the same manner as before, computing Instability and Remodeling Scores genome-wide. Parameters such as genome coverage , nucleosome positioning, nucleosome fragment lengths, and the distribution of raw DNA and NUC reads to windows in the different IS percentiles were similar to those observed in wild type, indicating that the data sets were suitable for comparison . The nucleosome occupancy metaprofiles in the different RS percentiles were similar between WT and htz1Δ nucleosomes around the TSS of genes, with some notable exceptions: The large peak of the 99th percentile in WT was much reduced with htz1Δ nucleosomes and the 95th percentile peak was largely absent  in WT were also detected in the 90th percentile RS with htz1Δ nucleosomes, and a subgroup from the 99th percentile in WT of four times the size (16%) moved to the bottom 10th percentile of the htz1Δ RS. This indicates an underlying reordering of the preferred nucleosome remodeling positions in htz1Δ cells.We also prepared DNA libraries from experiments with y absent D. This whtz1Δ (upper panel on the right): The WT RS thus increased on the TSS, while the opposite was observed with the htz1Δ RS, which reached a minimum in the same area. The ISs for the same regions were similar and so cannot account for this difference  and computed the average RS. We then divided all bins into H2AZ+   and H2AZTo finally investigate nucleosome remodeling by RSC using an independent, complementary system, we employed nucleosome arrays that were assembled on circular DNA plasmids using recombinant histone proteins in the presence of DNA topoisomerase I . NucleosThese results strongly support the finding that RSC prefers nucleosomes containing H2AZ.The challenge of understanding the processes that take place in natural, eukaryotic chromatin, particularly at the molecular or biochemical level, is substantial. Reconstituting such chromatin with purified components in vitro is extremely challenging, and the study of biochemical mechanisms in vivo is very difficult as well. Here, we present a new molecular tool which we believe can help fill the gap between these approaches: a ‘library’ of mononucleosomes, which can be used to study different biochemical characteristics of natural chromatin as well as the substrate preference of chromatin binding factors, ATP-dependent remodelers, and chromatin-modifying enzymes. The validity of the approach is suggested by the results themselves: The finding that RSC-remodeled nucleosomes have specific and highly sensible characteristics  indicates that the methodology works. One might have feared, for example, that random nucleosome regions had resulted from these experiments. Instead, the nucleosome-selecting factor tested—the chromatin remodeler RSC—does indeed make meaningful choices from this library, which helps further the understanding of the biology of both nucleosomes, chromatin, and RSC.What specifies a particular genomic location—whether it is a promoter, a TSS, a telomere, or a centromere? In many cases, there are conserved DNA sequence motifs, as is the case with telomeres and recognition-sites for DNA-binding transcription factors, for example. But what about when there are no conserved sequence elements, as is the case for many promoters and TSSs? One obvious possibility is that it is exclusively through the combined presence of nearby protein-binding sites that a genomic locus comes to acquire its functional identity. According to that model, the spatial context in which a region exists is crucial for that area to ‘know’ its function. Some evidence for this idea has grown with the advent of techniques for mapping long-range interactions on chromatin . HoweverPHO5 chromatin circle  the entire genome can be interrogated simultaneously, (2) the ‘indirect’ influences from processes occurring on chromatin in vivo have been removed, and (3) nucleosomes harbor most, if not all, the epigenetic features as they are found inside the cell. While the experimental system was established based on a yeast nucleosome library and using RSC as an example of the ‘nucleosome selectivity factor,’ it should be possible to similarly apply it to nucleosomes and chromatin-associated factors from other cell types.htz1Δ. Nuclei were prepared largely as described in Nucleosomes were prepared from strain W303 (wild type) or isogenic The assay was adapted from the protocol described in Adapters were ligated to mononucleosomal DNA using the TruSeq ChIP-seq Sample Prep (Illumina), and sequenced on Illumina HiSeq 2500.YEF3 and SSP120 were designed according to Relative recoveries of individual sequences in the four bands  were determined by standard qPCR using the DNA obtained from a nucleosome disassembly assay. Native chromatin immunoprecipitation was performed using Protein A Dynabeads (Life Technologies); DNA was purified using a commercial PCR purification kit (Life Technologies GeneJET). Antibodies used were all from Abcam: #8580 (H3K4me3), #9050 (H3K36me3), and #1791 (H3). Primers for Escherichia coli according to TAP-tagged RSC was purified from Rsc2-TAP cells according to Complementary paired-end reads were merged using FLASH  and thenSupplemental Materials.Further details on all the methods briefly outlined above can be found in www.ebi.ac.uk/arrayexpress) under accession number E-MTAB-7926.All raw and processed sequencing data from this study have been submitted to the ArrayExpress database at EMBL-EBI (


Three-dimensional (3D)-chromatin organization is critical for proper enhancer-promoter communication and, therefore, for a precise execution of the transcriptional programs governing cellular processes. The emergence of Chromosome Conformation Capture (3C) methods, in particular Hi-C, has allowed the investigation of chromatin interactions on a genome-wide scale, revealing the existence of overlapping molecular mechanisms that we are just starting to decipher. Therefore, disentangling Hi-C signal into these individual components is essential to provide meaningful biological data interpretation. Here, we discuss emerging views on the molecular forces shaping the genome in 3D, with a focus on their respective contributions and interdependence. We discuss Hi-C data at both population and single-cell levels, thus providing criteria to interpret genomic function in the 3D-nuclear space. The three-dimensional 3D) spatial organization of chromatin within the nucleus represents a crucial step that bridges the linear information encoded in genomes with its capacity to execute complex but precise transcriptional programs. Therefore, chromatin organization underlies the molecular basis of a wide range of biological processes such as cell cycle control and DNA replication, repair, or maintenance . By physD spatialFor decades, the scientific community has been intrigued by the nature of the physical interactions between regulatory elements and genes, prompting the development of novel analytical and experimental strategies for their investigation. While microscopy-based approaches, such as Fluorescent In-Situ Hybridization (FISH), successfully settled the mechanistic basis of these functional contacts , it was Almost 10 years after the release of the first Hi-C maps, the field has expanded rapidly, in part due to the continuous improvement and decreasing costs of Next Generation Sequencing technologies. These advances led to the identification of chromatin interactions at an unprecedented resolution, yielding a massive amount of data, on a wide range of cells, tissues, and organisms. Yet, translating the signal of Hi-C maps into a meaningful biological output remains one of the biggest challenges of the field. It has become increasingly evident that Hi-C maps are the results of multiple, overlapping mechanisms, with a highly dynamic nature, that converge in a temporal “snapshot” of nuclear interactions. The individual dissection of these mechanisms is central to understand the underlying biology of such datasets.In this review, we discuss current knowledge about the biology of chromatin interactions, to provide a framework that enables the interpretation of Hi-C maps. We evaluate fundamental mechanisms under the prism of the population-based nature of the technology, but contextualizing them with recent single-cell and super-resolution microscopy advances. The appropriate interpretation of chromatin interaction datasets will allow the navigation through a 3D-nuclear space, thus unlocking the true potential of these approaches to facilitate the interpretation and prediction of genomic function in biological processes.As highlighted previously, 3C-based technologies and, in particular, Hi-C represented a novel approach to interrogate chromatin interactions in a high-throughput manner. With this technology, large genome-wide matrices of interaction could be reconstructed and visualized, with squares displaying interaction frequencies, depicted by color intensity, between pairs of loci on the linear genome. Early Hi-C maps profiling mammalian cells were generated at 1 Megabase (Mb) resolution, each bin containing interactions between fragments within a 1 Mb window . The anaThose early findings, based on FISH and chromosome painting techniques, also delineated a prominent separation between euchromatic and heterochromatic regions. This phenomenon is also observed in Hi-C maps, depicted by a plaid pattern of enriched or depleted interactions within and between chromosomes . These sTo date, it is not entirely clear how the segregation of these compartments occurs, but interaction data at high resolution revealed compartmental associations that correlate well with the existence of specialized sites within the nucleus, such as Polycomb-repressed, Lamina-Associated Domains (LADs), or Nucleolus-Associated Domains (NADs) . FurtherWith further improvements on Hi-C resolution , an additional level of chromatin folding was revealed in mammalian genomes, at the sub-megabase scale. These units were denominated Topologically Associated Domains (TADs), defined as genomic regions with increased contact frequency and largely insulated from other neighboring regions ,17 FiguA,B. CompTADs are closely related to another prominent feature of Hi-C maps: chromatin loops B. Loops The most prominent theory by which TADs and chromatin loops are formed is the loop-extrusion model ,31. In tThe identification of potential key players in 3D-chromatin organization, such as CTCF, cohesin or transcription, led to a plethora of genetic studies to elucidate their individual role. These experiments provided important information to understand the interdependence of the aforementioned mechanisms and how they are reflected in Hi-C maps.The acute depletion of CTCF in mouse cells, via induced-auxin degron, resulted in a loss of loop domains ,34. HoweStudies depleting the cohesin-releasing factor WAPL increase the persistence of the cohesin complex on chromatin, thus providing important insights on the nature of the loop extrusion process ,38,39. TDrosophila melanogaster, where initial low-resolution Hi-C maps also revealed the existence of TAD structures around the size of 100 kb [Drosophila is better explained by compartmentalization based in transcriptional status, with a minor influence of insulator proteins [Drosophila, TAD formation by loop extrusion does not plays a prominent role in chromatin organization [Caenorhabditis elegans display structures that are reminiscent of TADs and that have been associated with another extruding complex, condensin, as well as with the dosage compensation complex (DCC) [The extension of chromatin-interaction studies to other organisms also provided essential clues to understand the relation between compartment segregation and chromatin extrusion. Among vertebrates, loop extrusion, and the CTCF orientation code appear as conserved mechanisms of chromatin organization ,40,41, af 100 kb , with bof 100 kb . High-ref 100 kb ,45. Furtproteins . While iproteins , in flieproteins . Therefonization . Such finization ,49,50. Inization . It is aex (DCC) . These fDrosophila showed that at early phases of development, when transcription is limited, chromatin is largely unorganized [Finally, the role of transcription in establishing 3D-chromatin organization has been also thoroughly investigated. Experiments in rganized . Howeverrganized . In contrganized ,53. In mrganized ,55,56. Hrganized . OverallIn summary, two opposing mechanisms, a transcription-based that segregates chromatin into compartments according to epigenetics states, and a superimposed cohesin-dependent mechanism that generates TADs and loop-structures, act together to organize the genome of most vertebrate species. Outside this clade, transcriptional compartmentalization appears as the major contributor organizing chromatin in the 3D-nuclear space. Studies in additional species and further functional validations will help to infer the degree of evolutionary conservation of these two mechanisms and to better elucidate their functional relation, both important aspects to understand their individual contribution to Hi-C maps.Besides the prominent features described in the previous section, Hi-C maps also display additional structures that are linked to the explained mechanisms. In this section, we provide an overview of these features and the forces shaping them.in vitro. Meta-TADs reflect a nested hierarchy of TADs within A/B compartments, thus providing a link between the molecular mechanisms of both features. In a recent study, TAD cliques were identified, which represent associations between non-linear, B-compartment TADs that are prominently reinforced during differentiation according to changes in chromatin compaction and gene repression [In general, TADs represent a stable and largely invariant scaffold of genomic organization across different cells/tissues and during development. Nevertheless, additional levels of organization can be observed between and within TADs, in some cases displaying a marked cell-to-cell variability. One prominent example are meta-TADs, large spatial structures that connect groups of linearly-consecutive TADs  Figure A. These pression .Drosophila [Several studies have highlighted the further subdivision of TADs into smaller and nested structures such as sub-TADs, insulating neighborhoods or CTCF-contact domains (CCDs) ,59,60,61osophila ,45, highAnother important feature of Hi-C maps are stripes or tracks, which reflect large segments of interaction between a certain locus and a contiguous region of the genome B. A receA proper interpretation of a Hi-C signal relies on mapping the data against a genomic assembly that matches the profiled sample. When a discrepancy exists, specific signatures arise that, in most cases, can be identified by genome-wide analyses or even manual inspection. This property has been effectively exploited to expand the utilization of Hi-C methods beyond the study of chromatin interactions.One of these applications is the application of Hi-C as a tool to identify structural variations associated to human disease. This strategy takes advantage of the fact that genomic rearrangements generally alter the distance between genomic loci, causing signal discrepancies due to data alignment against a reference genome that does not contain the variation. Depending on the specific pattern observed in the Hi-C map, deletions, inversions, duplications or translocations can be identified upon visual inspection (reviewed in ). The idAnother prominent application of Hi-C relates to its utilization to scaffold highly-fragmented genomic assemblies into entire chromosomes   it was possible to visualize a 1.2 Mb region in mammalian cells with nanometer-scale precision . This me domains ,74.In summary, the application of these novel approaches, to profile 3D-chromatin organization, provides results at the individual cellular level that recapitulates observations obtained from bulk data. Therefore, TADs can adopt multiple configurations, with higher probabilities to match the structures observed in averaged cells, but where insulation and boundary positioning do not appear as absolute values.Hi-C datasets represent a powerful tool to understand how chromatin is organized within the nucleus to effectively exert its biological function. Notably, chromatin organization is reflected at multiple genomic scales: from specific chromosomal territories to functional loops that connect enhancers to their cognate promoters. Therefore, distinct molecular forces impose constrains at these levels, thus converging on the complex interaction patterns that are visualized in Hi-C maps.Great advances have been made towards the identification of the essential factors and mechanisms that organize the genome within the nuclear space. Among them, transcription and loop extrusion by CTCF/cohesin appear as prominent players, acting as opposing forces that lie in a delicate balance. However, interspecies comparisons, such as between flies and mouse or human, reveal an absence of universal rules of chromatin organization. While compartmentalization appears to be an ancient feature of 3D-chromatin organization, domain formation by loop extrusion seems to be a more recent evolutionary acquisition. This type of organization can impose over transcription-based compartmentalization, inducing additional spatial constrains in the form of boundary elements. Profiling chromatin interactions in additional species along the phylogenetic tree will not only help to understand the origin and functional implications of this innovation, but also to the potential identification of other factors with the capacity of shaping the genome in 3D.Novel methods, such as single-cell or super-resolution microscopy, are helping to fill the gap between average population and the events occurring at a cellular level. As a field in development, it is expected that these technologies will acquire better resolution and throughput capacity. This will enable profiling chromatin interactions on vast amounts of individual cells from tissues and organs, thus providing exciting insights on how average conformations are shaped to meet specific transcriptional requirements in time and space. Applying this knowledge to identify subtle changes in interaction maps and their underlying causes and effects represents the next frontier in the field. Such knowledge would have important implications for the identification of novel mechanisms underlying human disease or evolutionary traits, eventually leading to better predictive models that might improve the connection between visual patterns of 3D-chromatin interaction and biological processes.


Conflicts between replication and transcription can have life-threatening consequences. RNA polymerase (RNAP) is the major impediment to replication progression, and its efficient removal from DNA should mitigate the consequences of collisions with replication. Cells have various proteins that can resolve conflicts by removing stalled (or actively translocating) RNAP from DNA. It would therefore seem logical that RNAP-associated factors, such as the bacterial DNA translocase Mfd, would minimize the effects of conflicts. Despite seemingly conclusive statements in most textbooks, the role of Mfd in conflicts remains an enigma. In this review, we will discuss the different physical states of RNAP during transcription, and how each distinct state can influence conflict severity and potentially trigger the involvement of Mfd. We propose models to explain the contradictory conclusions from published studies on the potential role of Mfd in resolving conflicts. Conflicts can lead to various detrimental outcomes, including replication fork stalling, double-stranded DNA breaks (DSBs), and mutations. In general, these events are more severe in the head-on orientation. For comprehensive insight into the consequences of such conflicts in both prokaryotes and eukaryotes, see the following reviews: [DNA replication and transcription must occur in a timely and accurate fashion in all organisms. Individually, each one of these tasks is a remarkable undertaking. It is even more striking to consider that replication and transcription utilize the same DNA template, yet, the machineries responsible for these respective processes must not impede each other’s function. While cells are remarkably efficient at coordinating replication and transcription, there are unavoidable encounters between the replication machinery (the replisome) and RNAP polymerase (RNAP). The encounters between these machineries, termed replication-transcription reviews: .Cells have a complement of factors to deal with replication-transcription conflicts and their consequences. Research has largely focused on proteins that help replication progression at these encounters. These include proteins implicated in replication restart , replicaAs with most conflicts, there are two sides to the story; in the conflict between replication and transcription, it is the role of the latter that is less well understood. Various RNAP associated factors help remove RNAP from the sites of protein roadblocks  or bulky2RNAP can encounter the replisome in various, distinct states. During unimpeded transcription, RNAP adopts a long-lived active complex,which can readily incorporate incoming nucleotides . Impedim3Most of our understanding about conflicts has focused upon transcription elongation. The process of transcription is dynamic, and includes three stages: initiation, elongation, and termination. Conflict severity in general, and gene orientation-specific effects, may largely depend upon the stage of transcription.Transcription initiation: At the initiation stage, the RNAP holoenzyme  and a sigma factor subunit in bacteria [bacteria ) recognibacteria . Subsequbacteria ,35. The bacteria ,36. Initbacteria . HoweverWhile it is plausible that the replisome encounters initiating RNAPs at some frequency during conflicts, studies using the bacterial transcription inhibitor rifampicin suggest that transcription elongation can directly impede replication. Rifampicin arrests RNAP at the transcription initiation phase ,38, rapiTranscription elongation: During elongation, RNAP forms a stable, processive complex on DNA, moving at a speed of approximately 10–100 nucleotides/second [s/second . Since rThe rate of elongation can be altered by various factors. Naturally occurring RNAP pausing decreases the rate of elongation and is perhaps the rate-limiting step in transcription . MoreoveTranslation can also enhance transcription elongation, as the coupling of these two processes in bacteria helps RNAP processivity by reducing backtracking . This suArrested transcription elongation is only one of multiple factors during transcription that can significantly alter the severity of replication-transcription conflicts. In particular, as previously discussed, gene orientation is a major determinant of conflict severity given that head-on replication-transcription conflicts are far more deleterious than co-direction conflicts. It is also thought that higher levels of transcription and longer transcripts exacerbate conflicts ,47. ThisTranscription termination: In bacteria, there are two mechanisms of termination: intrinsic termination and factor-dependent termination (Reviewed in [iewed in ). Duringiewed in , or by siewed in .Saccharomyces cerevisiae reveal that the transcription termination factor Sen1 helicase helps mitigate transcription-induced genomic instability [There is evidence that efficient termination is important in reducing replication-transcription conflicts, as inhibition of Rho promotes conflict induced genomic instability ,51. Studtability . Currenttability . A more 4Work in both prokaryotes and eukaryotes has highlighted the importance of R-loop formation in exacerbating the consequences of replication-transcription conflicts ,53,54. ROverall, these findings paint a complex, nuanced understanding of how RNAP can become a significant obstacle for the replisome. In particular, the state of RNAP and transcription are critically important for the severity and consequences of an encounter with replication. However, much of what we know regarding the state of RNAP and conflicts are from studies utilizing specific conditions such as UV damage as well as engineered reporter systems. Greater insight into how factors such as RNAP pausing, sequence context, and translation efficiency alters the severity and/or presence of conflicts at endogenous sites would further our understanding of conflicts.5An overview of Mfd: RNAP faces an array of obstacles during transcription and it is therefore not surprising that multiple proteins can help RNAP deal with such impediments. One of the well-studied factors that helps with stalled RNAP in bacteria is the highly conserved Mfd translocase protein. Mfd is classically described as a DNA repair protein that links nucleotide excision repair (NER) and transcription via the transcription-coupled repair (TCR) pathway , iewed in ,64. In tiewed in ,66. Thisiewed in . TCR is iewed in ,69. Intein vitro and in vivo studies show that Mfd utilizes its translocase activity to move extensively backtracked RNAP forward as well as to remove arrested RNAPs at a wide variety of obstacles, ranging from protein roadblocks to sites of nucleotide starvation [in vitro work shows that Mfd translocates autonomously (independent of RNAP) upon binding to DNA [in vivo in the absence of exogenous stressors [in vitro findings and providing further evidence that Mfd may function as a fundamental transcription processivity factor outside of a RNAP-blocking lesion, such as a photoproduct from UV irradiation. This is consistent with evidence that CSB promotes transcription elongation [More recent work has broadened the role of Mfd outside of its DNA repair function and has provided new mechanistic insights. Various arvation . Recent g to DNA . Consequg to DNA . Additiotressors , corroboongation ,78 in maGiven that the replication fork is a significant obstacle to RNAP processivity, it is logical to examine the role of Mfd in reducing the severity of replication-transcription conflicts. Intriguingly, the answer seems complex.A relationship between Mfd and conflicts? Trautinger et al. [rpo*35), which destabilizes RNAP elongation [r et al.  providedr et al. . This efongation . The autin vitro and loaded it head-on to an arrested RNAP complex on a linear double stranded DNA fragment. They found that replication across the linear fragment was inhibited by the presence of the head-on RNAP. The addition of Mfd into the system significantly reduced the replication stall generated by RNAP [Pomerantz and O’Donnell undertook the first mechanistic study directly looking at the role of Mfd in conflicts. The authors of this study reconstituted the bacterial replisome  by RNAP . The aut by RNAP .Fig. 1Moin vivo system to investigate genomic instability at replication-transcription conflict regions as well as the importance of various RNAP associated factors in mitigating DNA damage at conflict sites. Using a plasmid based system, the authors measured the presence of DSBs at head-on and co-directional conflict sites and found that they were dependent on RNAP backtracking [Shortly after these findings, Dutta, et al.  developetracking . While ctracking . These fin vivo, Mfd is unable to access or remove RNAP at severe head-on conflicts. Mfd is known to require access to at least 25 basepairs of DNA upstream of RNAP to bind to the protein [Why Mfd is unable to reduce DSBs at head-on conflicts remains unclear. Perhaps  protein . It is p protein  show tha6in vitro, evidence suggests that, in contrast to all other Replisome-RNAP conflict resolution factors, Mfd does not help cells survive the consequences of conflicts. Boubakri, et al. [E. coli require the activity of the DinG, Rep, and UvrD helicases as well as the RecBC double-stranded end processing enzymes for cell viability. However, deletion of Mfd, either alone or in conjunction with the aforementioned helicases, does not affect cell viability [Many Replisome-RNAP conflict resolution factors are either essential proteins or become essential upon induction of a head-on conflict. Although Mfd can reduce DSBs at co-directional conflicts and help replisome progression , et al.  and De S, et al.  show thaiability . Mfd is 7Bacillus subtilis, initial work by Paul et al. [dependent on Mfd, as deletion of Mfd reduced mutation rates of the head-on genes studied. Additionally, the authors found an epistatic relationship between Mfd and other factors of the TCR pathway, and proposed a model by which TCR activity at head-on conflicts promoted error-prone DNA repair [impedes or slows resolution of head-on conflicts to some degree. Mfd could increase mutagenesis by delaying an unknown step during conflict resolution. For example, previous work suggests that Mfd can impede the activity of helicases in the context of DNA repair [One of the various consequences of replication-transcription conflicts is DNA mutagenesis ,81,82. Ul et al.  revealedA repair , so it iInterestingly, additional work shows that Mfd promotes mutagenesis under various other conditions, including stationary-phase mutagenesis ,85, cellFurther insights into Mfd’s mechanism of mutagenesis will allow for a more comprehensive understanding of how Mfd’s specific activity at conflict sites (as well as other sites where RNAP is arrested) is linked to its ability to promote mutagenesis and evolution.8in vitro, Mfd terminates transcription during RNAP arrest. Furthermore, in vivo evidence implicates Mfd in relieving transcriptional roadblocks and subsequently decreasing transcription at various sites in the B. subtilis genome. These findings suggest that Mfd helps regulate transcription when RNAP encounters a roadblock. Whether or not Mfd functions in a similar manner at conflict regions remains to be seen. Given that a critical function of Mfd is to deal with consequences of halted transcription elongation complexes, this is an interesting and critical avenue for further study.Replication-transcription conflicts have deleterious consequences on replication, but little is known about the consequences of conflicts on transcription. Lang, et al.  provided9While we have predominantly focused on reviewing the activity of Mfd at replication-transcription conflict regions, it is important to discuss other proteins that help rescue arrested RNAPs, as these proteins may also have fundamental roles as Replisome-RNAP conflict resolution factors.GreA and GreB are critical proteins that also rescue backtracked RNAPs . BiochemE. coli, the transcription factor DksA functions to alleviate conflicts between replication and RNAPs that are arrested due to amino acid starvation [B. subtilis and other Gram-positive species, highlighting that different species may use divergent strategies to reduce conflict severity.In arvation . DksA fuarvation  as well arvation . Deletioarvation . In thisThe transcription termination factor Rho also helps reduce conflict severity. Chemical inhibition of Rho with the antibiotic bicyclomycin increased the presence of DSBs at conflict regions , but acc10The field of replication-transcription conflicts has made significant advances in understanding both the consequences of conflicts as well as the role of many Replisome-RNAP conflict resolution factors in minimizing these damaging events. Such factors affect a diverse range of processes, ranging from R-loop resolution, to RNAP antibacktracking, to replication restart. But fundamentally, these proteins share one thing in common: they help mitigate and reduce the life-threatening consequences of conflicts. While we have made great progress in identifying the importance of these proteins, many critical and in particular mechanistic questions regarding how they function in conflicts remain unanswered.in vitro, in vivo, in some studies, Mfd mitigates some consequences of only co-directional and not head-on conflicts. Additionally, Mfd promotes mutagenesis at head-on genes, suggesting that it does indeed play a role in such conflicts in vivo, but this involvement might actually be counter-productive and potentially even detrimental for conflict resolution. Given that Mfd increases mutagenesis at conflict regions, it is indeed possible that Mfd promotes genomic instability at head-on conflict regions. Whether this effect is a trade-off for other functions of Mfd during these encounters, such as efficient transcription elongation, or an inhibition of other Replisome-RNAP conflict resolution factors, remains to be seen. As we continue to gain greater insights into differences between head-on and co-directional conflicts, as well as insights into the mechanism of Mfd’s activity, a clearer understanding of how and why Mfd functions at conflict regions will arise.One such question is how the enigmatic Mfd translocase helps deal with conflicts, if at all. While Mfd promotes replication progression at a head-on conflict Lastly, the undiscovered frontier in the field of conflicts is understanding how transcription is altered when it encounters the replisome. Studies on RNAP accessory factors, such as Mfd, in mitigating conflicts have exclusively looked at the role of these proteins in helping reduce replication stress. Given the biochemical activities of this class of proteins, it is quite likely that they alter transcription, but the consequences on gene expression and RNAP dynamics at conflict regions still requires further research. Such work will provide novel insight into the consequences of conflicts.


The nucleosome remodeling and deacetylase (NuRD) complex presents one of the major chromatin remodeling complexes in mammalian cells. Here, we discuss current evidence for NuRD’s role as an important epigenetic regulator of gene expression in neural stem cell (NSC) and neural progenitor cell (NPC) fate decisions in brain development. With the formation of the cerebellar and cerebral cortex, NuRD facilitates experience-dependent cerebellar plasticity and regulates additionally cerebral subtype specification and connectivity in postmitotic neurons. Consistent with these properties, genetic variation in NuRD’s subunits emerges as important risk factor in common polygenic forms of neurodevelopmental disorders (NDDs) and neurodevelopment-related psychiatric disorders such as schizophrenia (SCZ) and bipolar disorder (BD). Overall, these findings highlight the critical role of NuRD in chromatin regulation in brain development and in mental health and disease. Epigenetic mechanisms are key to establish stable yet malleable gene expression during (neuro-) development and beyond . Perturbvia specialized proteins, the so-called histone, to fit the limited space. While “open” (euchromatic) regions are accessible to nuclear factors, “closed” (heterochromatic) regions preclude in general transcriptional activation. However, heterochromatic regions can be quickly modified  rather than by the order of their appearance and summarize key points in a tabular format Table 1.In vivo RNAi and conditional Chd4 knockout mice experiments showed that NuRD depletion in cerebellar cortex strongly impaired the development of granule neuron parallel fibers and of Purkinje cell synapses in vivo. Intersection of genome-wide RNA-seq and ChIP-seq data revealed a network of <200 repressed genes and decommissioned promoters at which NuRD turned off histone modifications associated with transcriptional activation during cerebellar development. A targeted in vivo RNAi screen of this network identified a subset of genes that encoded negative regulators of presynaptic differentiation: Nhlh1 (nescient helix loop helix) is a TF repressing the ubiquitously expressed bHLH factor TCF3 , and Elavl2 is an RNA-binding protein with a possible role in mRNA splicing and stability. Given the broad impact of these factors, they may serve as hub for NuRD’s effect on presynaptic development (Despite early evidence for NuRD’s role in development (see above), a role of NuRD in neuronal maturation has remained largely unexplored until recently. The formation of neuronal circuits depends critically on the differentiation of synapses in brain development and beyond  and is selopment Figure 2Chd4 knockout mice. Combining RNA-seq with H2A.z ChIP-seq analyses revealed upregulation of >90% of the genes with reduced H2A.z occupancy, but little change in otherwise PTMs, in Chd4-null mice.In a subsequent study, in vivo at the promoters of a large group of neuronal-activity-dependent signaling genes in the cerebellar cortex and that deposition of H2A.z promotes gene deactivation  showed hyperresponsive granule neuron activity in vivo. Such hyperresponsivity associated with impaired procedural learning capacity as evidenced in behavioral tests . By contrast, motor coordination was barely affected in Chd4-null mice.Activity-dependent transcription is well known to modulate neuronal connectivity . What iselopment Figure 3Overall, these studies show NuRD-dependent chromatin remodeling in granule neuron synapse formation and connectivity during sensitive time windows. NuRD remodeling involves repression of a subgroup of inhibitory genes in synaptogenesis Figure 2Formation of the highly organized cerebral cortex requires contributions from different classes of NPCs in a tightly spatiotemporally controlled sequence . Key feaMbd3 knockout mice. Because deletion of both Mbd3 alleles is embryonic lethal, only heterozygous mice with unaffected viability were used. Macroscopically, heterozygous mice showed a reduction in cortical thickness that associated at the cellular level with an impaired specification of cortical projection neuron (PN) progenitors relative to wild-type mice: Pax6-positive APs were maintained in NuRD/Mbd3-mutant cortex but failed to respond to signals regulating symmetric versus asymmetric divisions and exited prematurely the cell cycle. Thereby, they produced insufficient amounts of Tbr2-positive BPs and neurons, and cortical plate neurons with deficits in terminal differentiation.Mbd3, specification of upper layers  was compromised, and both deep and upper cortical layer markers became coexpressed. Expression profiling of microdissected tissues further revealed that NuRD/Mbd3 was necessary to guide neurodevelopmental differentiation programs by terminating the expression of proneural genes.The mouse cortex develops between embryonic days 11 and 18 in a characteristic inside–out sequence: deep layers (designated 4–6) are formed first and upper, more superficial, layers (designated 2 and 3) are formed later. As a result, prospective upper layer neurons need to migrate through the deeper layer to attain their final destination . Mbd3 waBriefly, this study indicates that NuRD/Mbd3 coordinates cerebral cell lineage choice and differentiation programs by terminating proneurogenic transcription in both progenitor and neural progeny.in vitro and in vivo  was found to bind to Mbd3  and to iTaken together, both Smek and Mbd3 tilt the balance between self-renewing and neurogenic cell divisions in the same direction. Since both proteins colocalize in NSCs, in which Smek counteracts NuRD/Mbd3, this findings raises the question to what degree Smek- and NuRD/Mbd3-dependent corticogenesis differ from each other.Mbd2 may be required only in a small subpopulation of cells that do not manifest robust changes. In support of this hypothesis, proliferation and differentiation of olfactory receptor neurons were impaired in Mbd2-knockout mice  and an accumulation of undifferentiated progenitors. In an ESC model of neurogenesis, Chd5-depleted cells failed to upregulate genes involved in late stage neural differentiation including synapse development, neuron projection, and neurotransmitter transport. At the same time, a subgroup of Polycomb target genes  and finally of cortical thickness, a finding resembling Mbd3-deficient mice  are executed by excitatory PNs, presenting the largest portion of cortical neurons, and by inhibitory interneurons .Excitatory PNs are classified into numerous subtypes based on three major criteria: first, by the location within six cortical layers that are defined by histological measures; second, by their axon projections to different intracortical, subcortical, and subcerebral regions; and third, by the expression of genes signifying specific subtypes of neurons , subcerebral PNs (SCPNs), and corticothalamic PNs (CThPNs) is schematically summarized in Fezf2 and its upstream transactivator Sox11  was high in postmitotic cells of the developing cortical plate in superficial layers  in the Ctip2 locus , a transcriptional adaptor known to interact with several NuRD subunits, showed increasing peri- and postnatal expression in Ctip2/Satb2 coexpressing neurons. Overexpression and knockdown experiments demonstrated that Lmo4 acted in the specification of Ctip2/Satb2-double positive neurons primarily by modulating Ctip2 expression in layer 5: Lmo4 sequestered Hdac1 before its interaction with Satb2/NuRD and thus interfered progressively with Satb-2-dependent repression of Ctip2 locus and promotes Ctip2 expression by interfering with NuRD-mediated deacetylation.In short, regulators such as Ctip2 and Satb2 with opposite function during embryonic corticogenesis can colocalize postnatally and contribute to the generation of diverse PN subtypes. Thereby, transcriptional adaptor Lmo4 targets Satb2/NuRD complexes in the Overall, NuRD is a critical modulator of the molecular circuitry underlying specification of neocortical PNs in embryonic corticogenesis. Key regulators, such as Lhx2, Ctip2, and Satb2, increase the availability of NuRD at their target sites in a time- and area-specific manner to provide negative feedback control. Additionally, NuRD is a critical regulator of postnatal PN subtype specification through variation of molecular signatures that are shared with early corticogenesis. Similar connectivity or molecular code among neocortical areas has been hypothesized to originate, at least in part, from variations on a “common theme” rather than from the activity of many independent and region-specific genetic programs . Along tNDDs are complex conditions that result from anomalous brain development. Frequently, they present with impairments in multiple domains including cognition, language, social behavior and communication, and/or motor skills. ID, ASDs, attention deficit/hyperactivity disorder (ADHD), communication disorders, and SCZ fulfill the criteria of NDDs . Here, wde novo mutations) per individual  to gain or loss of large DNA regions comprising thousands of nucleotides .A newborn carries on an average between 50 and 100 genetic variants. This number corresponds to 0.86 new amino acid altering mutations  with a primary diagnosis of childhood apraxia of speech. This mutation has been predicted to disrupt CHD3’s helicase domain. Given this index case, the researches went on to apply a genotype-based strategy to identify additional unrelated individuals (N = 35) with de novo mutations in CHD3. All of them shared global developmental delay and/or ID. At 2 years age or older, most of them showed a delayed development of speech and language. About half of the carriers presented macrocephaly, about one-third autism or autism-like features, and further symptoms including widening of cerebrospinal fluid space, hypotonic, and distinct facial dysmorphisms. The majority of the mutations clustered within highly conserved residues of the ATPase/helicase domain and was predicted to disrupt motifs critical to substrate binding and interaction. Among six of the identified mutations, a subset showed impaired ATPase activity in vitro, and five were impaired in chromatin remodeling. Briefly, de novo mutations in CHD3 cause a syndrome featuring ID, impairments in speech and language, and macrocephaly. Well-fitting, cognitive capabilities depend on cortical functions that are under the control of NuRD/CHD3 during corticogenesis.Similarly to CHD4, the ATPase/helicase domain of CHD3 has been implicated in NDDs based on a candidate approach. de novo germline mutations that significant impact total NDD risk and thus present novel disease genes. Moreover, the same mutations were shared in a sizeable fraction of patients presenting the same or similar disorders. This suggests that these mutations increase in general risk for abnormal brain development, whereby the eventual phenotype reflects the interaction with each carrier’s genetic background and/or environmental exposures , more than 800 ID-related genes have been identified by now  for genome-wide significant association for NDD. Of further note, de novo mutations were also identified in NuRD-regulated genes driving cortical layer formation such as TBR1 and SATB2  that were enriched for missense and/or likely gene-disruptive mutation. About half of these genes (N = 124) reached exome-wide significance (P < 5 × 10e−7) including the NuRD core subunits CHD3/4 and the NuRD-regulated genes TBR1 and SATB2, arguing that these genes contribute significantly to disease risk.Most recently, de novo mutations including CHD3, CHD5, and TBR1 . Both SCZ and BD are hypothesized to arise, at least in part, from abnormal neurodevelopment. Here, we discuss recent evidence for a potential role of NuRD in these perturbations.SCZ is a highly heritable devastating mental disorder  with a lP < 5 × 10e−8) significant associations comprising 108 independent loci, of which 93 have been recently replicated (A landmark genome-wide association study (GWAS) meta-analysis  has idenplicated . These 1CTIP2, EP300, EPC2, GATAD2A, KDM3B, RERE, and SATB2). Furthermore, three of these genes  presented the only gene in the index region. Four variants  showed nominally significant association with one or several cognitive task, while the risk allele for GATAD2A associated also with lower “Full Scale IQ.”Collectively, this study indicates that genetic variation in the NuRD core subunit GATAD2A or in the NuRD-interacting regulators CTIP2 and SATB2 contributes to cognitive impairment in SCZ.CNTN4, GATAD2A, GPM6A, MMP16, PSMA4, and TCF4. Consistent with this finding, four of these top candidates, including GATAD2A, have been also previously identified as SCZ risk gene (CTIP2) were significantly higher expressed in neurons than in oligodendrocytes and microglia. This gene set also formed a densely interconnected protein–protein interaction network enriched in synaptic neurotransmission-related pathways. In postmortem hippocampus, the expression of GATAD2A and TCF4 was enhanced in cases with SCZ relative to controls. Functionally, shRNA-mediated knockdown of the six top candidate genes in vitro impaired proliferation of neuroblastoma cells.In a related approach, isk gene  through GATAD2A and in NuRD-regulated SATB2 and CTIP2 in SCZ/cognition.Taken together, GWAS and integrative pathway/biostatistics analyses indicate a role for genetic variation in NuRD subunit BD presents extreme mood swings with mood-congruent delusions . Sleep dGenetic variation in circadian genes, also known as clock genes, has been associated with different mental disorders, especially BD  and autiInterestingly, CHD4, MTA2, RBBP4, and HDAC2, all of which encode NuRD subunits. This cluster was also enriched for coexpressed genes regulating prenatal amygdala development. Collectively, this work indicates a critical function of the hippocampus and amygdala in brain development and associated BD risk and implicates NuRD in these processes.Apart from its role in the molecular clock, NuRD has been also implicated in early brain development in BD by systematic analysis of GWAS data. N = 20,129) and SCZ  together with clinically relevant data to identify 114 genome-wide significant loci that were shared between these disorders and were enriched in genes underpinning synaptic and neuronal pathways. An additional comparison between SCZ and BD identified four genomic regions that contributed to differences in their biology. Combined regional association and heritability estimates were used to assess the contribution of these genomic regions to each disorder. This analysis suggested a locus that contributed differentially to vulnerability to BD and SCZ: although the association peaks of both disorders overlapped at this locus, they originated from independent causal variants for each condition. Interestingly, the same gene, GATAD2A, was significantly regulated by these variants though in opposite directions. As noted before  and neurodevelopment-related psychiatric disorders . Owing to the nature of polygenic diseases, in which numerous variants are thought to contribute incrementally to risk, mutations in NuRD subunits will have only minute effects at the level of single variants. However, sufficiently empowered patient-specific iPSC studies  and/or assemble in other macromolecular complexes . Hence, NuRD’s bona fide involvement in gene regulation needs to be carefully assessed both in terms of the presence of critical core subunits and their actual assembly in vivo. Relatedly, single component knockouts and mutations might not only impact NuRD, for example by destabilizing other subunits, but may also have secondary effects due to a genuine role of the respective subunit outside of NuRD.As much as we know about NuRD in well-defined cellular models, the composition and function of this macromolecular complex elopment . Moreoveelopment . Thus, Nin vitro (Emerging evidence indicates that tissue- and cell-type-specific TFs interact with specific NuRD subunits in a spatiotemporal defined manner during neurodevelopment and beyond. Such diversity in the composition of NuRD subunits could serve to constrain NuRD function to distinct neurodevelopmental time windows as to fulfill distinct roles in cell lineage specification, neural differentiation, and neuronal maturation. All of these aspects are highly relevant to NDDs and neurodevelopment-related psychiatric disorders, for which genetic variations in several NuRD subunits have been identified in the past years. Moreover, the folding of the genomic DNA into higher-order assemblies is increasingly recognized to impact nuclear processes. In this regard, NuRD regulated genes have been recently shown to cluster in 3D space in mouse ESCs, indicating an additional layer in gene regulation beyond the linear chromatin template . In any in vitro .Interestingly, genetic variation in NuRD subunits and NuRD-regulated genes in NDDs and neurodevelopment-related psychiatric disorders such as SCZ and BD converge on pathways regulating neural development, neuronal maturation, synaptic connectivity and plasticity, and higher cognitive functions. The hypothesis that disturbances taking place in early brain development increase the risk for SCZ has become widely accepted as the “neurodevelopmental hypothesis of SCZ” . HoweverEpigenetic regulation of gene expression is reversible  and openAH and DS jointly wrote the manuscript. DS carried out the literature search. AH performed the artwork.Funding for this work was provided by Max Planck Society.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.


Dear Editor,Heterochromatin, the highly-condensed nucleosome arrays in chromosomes, plays an important role in regulating multiple cellular events including transcriptional silencing and chromosome segregation , telomeres and rRNA-encoding DNA (rDNA) region , consisting of two essential subunits, Spt16p and Pob3p (SSRP1 in humans), plays a critical role in transcription, replication, and other chromatin based processes in eukaryotes Formosa, . While bHMR locus (hmr::GFP) was used to monitor the degree of silencing by quantifying the percentage of yeast cells expressing GFP  and RTT106 was made as a query strain . Interestingly, spt16-m, a newly identified Spt16p mutant allele in our group, dramatically exacerbates the phenotype of cac1Δrtt106Δ (77.3%)  in its tandem pleckstrin homology (PH) domain and displays a subtle defect in transcription initiation but a substantial defect in DNA replication-coupled nucleosome assembly . GFP silencing appears to be normal in spt16-m cells (0.4%). However, spt16-m strongly exacerbates the silencing defect of sir1Δ (40.8%) . These results suggest that Spt16p cooperates with Sir1p, CAF-1, and Rtt106 in heterochromatin silencing at the HMR locus.In yeast, Sir1p is involved in the establishment of heterochromatin silencing at the HMR locus, we used reverse transcription quantitative PCR (RT-qPCR) to analyze the expression level of A1 gene that resides within HMR region  dependent histone deacetylase, which deacetylates nearby histones to facilitate binding of Sir3p and Sir4p. Subsequently, more Sir2p is recruited through direct interaction with Sir3p and Sir4p to spread Sir proteins Below is the link to the electronic supplementary material.


Sox30 promoter from the teleost fish Nile tilapia (Oreochromis niloticus) and predicted a putative cis-regulatory element (CRE) for doublesex and mab-3 related transcription factor 1 (Dmrt1), a male-specific transcription factor involved in male sex differentiation. Transcriptional profiling revealed that Sox30 and Dmrt1 similarly exhibited a high expression in tilapia testes from 90 days after hatching (dah) to 300 dah, and the transcription of the Sox30 gene was reduced about one-fold in the testes of male tilapia with Dmrt1 knockdown. Further dual-luciferase reporter assay confirmed that Dmrt1 overexpression significantly promoted transcriptional activity of the Sox30 promoter and this promotion was decreased following the mutation of putative CRE for Dmrt1 within the Sox30 promoter. Chromatin immunoprecipitation-based PCR (ChIP-PCR) and electrophoretic mobility shift assay (EMSA) demonstrated that Dmrt1 directly binds to putative CRE within the Sox30 promoter. These results together indicate that Dmrt1 positively regulates the transcription of the tilapia Sox30 gene by directly binding to specific CRE within the Sox30 promoter.The Sox family member Sox30 is highly expressed in the testis of several vertebrate species and has been shown to play key roles in spermiogenesis. However, its transcription regulation remains unclear. Here, we analyzed the  Homo sapiens), mouse (Mus musculus), chicken , Nile tilapia, the channel catfish , guppy (Poecilia reticulata), fathead minnow , little skate (Leucoraja erinacea), dogfish , lancelets (Branchiotoma belcheri), and elephant shark . Although the roles of the Sox30 protein in teleost are currently unclear, recent studies in mouse have demonstrated that Sox30 is involved in spermiogenesis and Sox30 knockout results in abnormality in germ cell development and a complete arrest of spermiogenesis [Sox transcription factors, containing a high mobility group (HMG)-box domain for DNA-binding, are common in metazoans and play important roles in various developmental processes, including sex determination, sexual differentiation, gonadal development, neural development . Sox30 iogenesis ,7,8.Sox30 gene exhibits a male-biased expression in teleost fish and mouse. In Nile tilapia, Sox30 is highly expressed in the testis at 90 days after hatching (dah) and 180 dah, especially in the sperms [Sox30 gene is transcriptionally regulated by retinoic acid and by the transcription factor MYBL1 [Sox30 gene in animals remains largely unknown.Increasing evidence revealed that the e sperms ,9. Simile sperms ,8. Previor MYBL1 . HoweverDmrt1 is dominantly expressed in the testis [Dmrt1 results in a lack of germ cells in males and sex reversal from male to female [Dmrt1 knockdown in Nile tilapia causes testis degradation, and Dmrt1 overexpression in XX fish results in sex reversal [Gsdf) gene critical for testicular differentiation [Cyp19a1a gene, a gene encoding aromatase responsible for the biosynthesis of estrogens from androgens [Sox30 and Dmrt1 raise a possibility that Dmrt1 may be also involved in regulating male-biased expression of the Sox30 gene in teleost and other animals.Doublesex and mab-3 related transcription factor 1 (Dmrt1) contains a zinc finger-like DM domain for DNA binding and is widely considered to be involved in male sex determination, differentiation and maintenance in vertebrates ,12,13. Ie testis ,15. The o female . Our prereversal ,18. In areversal . For exantiation  but alsondrogens . TherefoSox30 gene is highly expressed in Nile tilapia testis, here, we investigated the regulatory function of Dmrt1 on Sox30 transcription in Nile tilapia. We observed that both Sox30 and Dmrt1 are highly co-expressed in XY Nile tilapia gonads (testis) and that Sox30 is less transcribed in the testis of Nile tilapia with Dmrt1 knockdown (heterozygous mutant). We also predicted a putative cis-regulatory element (CRE) for Dmrt1 within the sequence of the Nile tilapia Sox30 promoter. Further experiments confirmed that Dmrt1 directly binds to that putative CRE within the Sox30 promoter to activate its transcriptional activity. Altogether, our results demonstrate that Dmrt1 directly and positively regulated the transcription of the male-biased Sox30 gene in Nile tilapia testis.The Nile tilapia as a vertebrate species is a good model for the study of sex determination and differentiation, due to the availability of genome sequences and transcriptome data ,23, the Sox30 is predominantly expressed in male gonads (testis) in teleost and mouse [Sox30 gene. Therefore, we cloned the proximal promoter containing 1982 bp sequence upstream of the translational start site of the tilapia Sox30 gene  from 90 dah to 300 dah  or CRISPR/Cas9 in tilapia resulted in testicular regression and an increase in the expression of its target gene Cyp19a1a in testis [Dmrt1 and Sox30 displayed a high expression in the XY gonad (testis) of Nile tilapia at 90 dah  analysis revealed that based on a PCR amplification with a specific primer pair covering the DNA region containing putative CRE for Dmrt1, a positive DNA fragment was detectable in the products precipitated with the antibody against Dmrt1 in Nile tilapia testes at 90 dah, as demonstrated by its size similar to the PCR products using input DNA as a template  to determine the direct binding of Dmrt1 to putative CRE within the Nile tilapia d probes B. Howeved probes C. These Dmrt1 gene leads to an abnormality in male sexual development, especially testicular regression in testis [Sohlh1, Gsdf, and Sox9b [Foxl2 and Cyp19a1a [Sox30 and Dmrt1 exhibit a co-expression in testis and Dmrt1 positively regulates the transcription of the Sox30 gene via a direct binding to a specific CRE within its promoter.The DM transcription factor Dmrt1 has been well-characterized as a master regulator in male sex determination, differentiation, and maintenance in vertebrates ,25,26,27n testis ,18. Incrnd Sox9b ,20,28, aCyp19a1a ,29. Our Sox30 gene is predominantly expressed in testis and Sox30 knockout results in an abnormal spermiogenesis [Sox30 gene is expressed in epithelial cells of the intratesticular efferent duct of the testis. The present finding, together with previous observation [Dmrt1 expression is co-located with Sox30 expression in testis efferent duct. These results suggested that Sox30 and Dmrt1 may be involved in regulating the development of testis efferent duct. As is well known, the efferent duct is responsible for the absorption of fluid from the lumen, spermatozoon maturation, spermiophagy, and secretion of steroid hormone [Cyp19a1b or mouse Gemc1 is primarily due to the defect of efferent duct development [Sox30 and Dmrt1 in the efferent duct of the Nile tilapia testis.Sox30 is a member of the Sox transcription factor family and exists in many animals, including teleosts and mammals ,9,30. Itogenesis ,8. In thervation , further hormone ,32,33. Relopment ,35. ThusSox30 gene in Nile tilapia. This conclusion is supported by the following evidences. First, the transcription of the Sox30 gene in Nile tilapia testis is obviously attenuated by Dmrt1 knockdown. Second, the overexpression of the Nile tilapia Dmrt1 gene enhanced transcriptional activities of the Sox30 promoter in which a specific CRE for the binding of Dmrt1 is included. Third, ChIP-PCR and EMSA assay demonstrated that Dmrt1 could directly bind to specific CRE within the Nile tilapia Sox30 promoter. This finding helps to better understand the regulatory network involving in male sex determination and differentiation, as well as male gland (testis) development.A striking finding in this study is that Dmrt1 positively regulates the transcription of the 2.Nile tilapia fishes were reared in recirculating freshwater tanks at 26 °C before use. Animal experiments were conducted in accordance with the regulations of the Guide for Care and approved by the Institutional Animal Care and Use Committee of Southwest University . Moreover, the human embryonic kidney-derived HEK293 cell line were maintained in Dulbecco’s Modified Eagle’s medium (DMEM) containing 10% heat inactivated fetal bovine serum, supplemented with penicillin and streptomycin at 37 °C in a humidified atmosphere containing 5% CODmrt1 mutant Nile tilapia fishes at 90 dah and subsequently was treated with DNase I to eliminate the genomic DNA contamination. The cDNA for RT-PCR examination was synthesized by using the PrimeScript RT Master Mix Perfect Real Time kit . Quantitative RT-PCR analysis of the Sox30 mRNA expression level in tilapia testis was conducted according to the instructions of the SYBR1 Premix Ex TaqTM II kit . The relative abundance was evaluated using the following formula as described previously [Ct−ΔΔ. The beta-actin gene was used as a reference gene. The experiments were independently repeated three times. Primers used for RT-PCR are listed in Total RNA (2.0 µg) was extracted from the testes of wild type and eviously : R = 2−ΔDmrt1 (including 301 bp coding sequence and 207 bp 3′ untranslated region) and Sox30 (including 287 bp coding sequence and 168 bp 3′ untranslated region) were amplified and cloned into pGEM-T Easy vector, respectively. The related primers are listed in Dmrt1 and pGEM-T Easy-Sox30 plasmids were used as the templates for probe preparation. The probes against Dmrt1 and Sox30 were labeled with digoxigenin (DIG) and fluorescein by using RNA labeling kits , respectively. In addition, the gonads from male tilapia at 90 dah were fixed in 4% paraformaldehyde in 0.1 mol/L phosphate buffer pH 7.4 at 4 °C overnight. The samples were embedded in paraffin and cross-cut into 5 µm sections. The sections were hybridized with DIG-labeled Dmrt1 and fluorescein-labeled Sox30 RNA probes at 60 °C overnight. The first signal was detected by horseradish peroxidase (HRP)-conjugated anti-DIG antibody  with the TSATM Plus Tetramethylrhodamine (TMR) system  according to the manufacturer’s instructions. In order to detect the second signal, the sections were incubated in 3% H2O2 for 60 min to deactivate the HRP from the first staining. HRP-conjugated anti-fluorescein antibody  was then added to the sections, followed by detection with the TSATM Plus Fluorescein system. Finally, the sections were stained with 4′,6′-diamidine-2-phenylindole- dihydrochloride (DAPI) . Confocal images were collected on a Zeiss LSM 880 Laser Scanning Microscope.Double-colored fluorescent in situ hybridization-based expression profiling was performed as described as previously . BrieflySox30 gene, from −1982 bp to −1 bp relative to the translational start site, was obtained by a PCR reaction with Nile tilapia genomic DNA as a template. Then, the PCR product was cloned into the pGL3-Basic Vector for sequencing. The related primers are listed in Sox30 promoter were predicted by using the programs of MatInspector (http://www.genomatix.de) or AnimalTFDB2.0 .The proximal promoter region of the Nile tilapia Sox30 promoter was determined based on dual-luciferase reporter assay as described previously [Dmrt1 overexpression vector for overexpressing the Nile tilapia Dmrt1 gene in HEK293 cells and a series of constructs containing a luciferase reporter gene under the control of either different 5′-truncations of the Sox30 promoter or the Sox30 promoter with a mutation of putative CRE for Dmrt1 were separately generated. Site-directed mutagenesis was performed by using the MutanBEST Kit . The related primers are listed in Dmrt1 vector to check Dmrt1 overexpression by western blotting analysis. The following antibodies were used, including rabbit anti-Dmrt1 serum , mouse anti-alpha tubulin , mouse anti-HRP , and rabbit anti-HRP . Subsequently, the pcDNA3.1-Dmrt1 overexpression vector was separately co-transfected into HEK293 cells with each of the constructs containing a luciferase reporter gene driven by different varieties of the Nile tilapia Sox30 promoter. At 48 h after transfection, we harvested the cells and measured the relative luciferase activity by normalizing the firefly luciferase level to the Renilla luciferase level according to the protocol for the dual luciferase assay system .Dmrt1 regulation on the activity of the Nile tilapia eviously . BrieflySox30 promoter, EMSA experiments were performed as described previously [Dmrt1 gene with NE-PER Nuclear and Cytoplasmic Extraction Reagents . Then, 3–5 μg nuclear protein was incubated with 500 nM biotin-labeled probes in 1× binding buffer for 10 min at room temperature. For the competition analysis, a 50-fold molar excess of the unlabeled or mutated probe was added to the nuclear extracts prior to the addition of the labeled probe. Protein-DNA complexes were separated on 4% polyacrylamide gels in 0.5× TBE via electrophoresis at 100 V for 90 min and transferred to nylon membranes. The biotin-labeled DNA on the membrane was detected by using the chemiluminescent EMSA kit.To test the binding of Dmrt1 to potential CRE in the Nile tilapia eviously . BrieflySox30 promoter according to the manufacturer’s instructions . Briefly, the testes from Nile tilapia at 90 dah were fixed with 37% formaldehyde to cross-link chromatin. The cross-linked chromatins were then sonicated to shear into fragments of 100–500 bp in length. Immunoprecipitation assays were performed using anti-Dmrt1 and nonspecific rabbit IgG antibodies. The purified DNA from the immunoprecipitated chromatin was used as a template for PCR amplification. The primers used to amplify the specific region covering putative CRE for Dmrt1 were listed in We further conducted ChIP assays to confirm the direct binding of Dmrt1 to its CRE within the Nile tilapia p < 0.05 was statistically significant; *p < 0.05, **p < 0.01, and ***p < 0.001.Data were expressed as the mean ± SE of three independent biological replicates. The statistical significance of differences between data means was determined using a two-tailed Student’s t-test or One-way ANOVA, followed by Turkey test for multiple comparisons performed using SPSS 22 software.