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In Saccharomyces cerevisiae, the forkhead (Fkh) transcription factor Fkh1 (forkhead homolog) enhances the activity of many DNA replication origins that act in early S-phase (early origins). Current models posit that Fkh1 acts directly to promote these origins' activity by binding to origin-adjacent Fkh1 binding sites (FKH sites). However, the post-DNA binding functions that Fkh1 uses to promote early origin activity are poorly understood. Fkh1 contains a conserved FHA (forkhead associated) domain, a protein-binding module with specificity for phosphothreonine (pT)-containing partner proteins. At a small subset of yeast origins, the Fkh1-FHA domain enhances the ORC (origin recognition complex)-origin binding step, the G1-phase event that initiates the origin cycle. However, the importance of the Fkh1-FHA domain to either chromosomal replication or ORC-origin interactions at genome scale is unclear. Here, S-phase SortSeq experiments were used to compare genome replication in proliferating FKH1 and fkh1-R80A mutant cells. The Fkh1-FHA domain promoted the activity of ≈ 100 origins that act in early to mid- S-phase, including the majority of centromere-associated origins, while simultaneously inhibiting ≈ 100 late origins. Thus, in the absence of a functional Fkh1-FHA domain, the temporal landscape of the yeast genome was flattened. Origins are associated with a positioned nucleosome array that frames a nucleosome depleted region (NDR) over the origin, and ORC-origin binding is necessary but not sufficient for this chromatin organization. To ask whether the Fkh1-FHA domain had an impact on this chromatin architecture at origins, ORC ChIPSeq data generated from proliferating cells and MNaseSeq data generated from G1-arrested and proliferating cell populations were assessed. Origin groups that were differentially regulated by the Fkh1-FHA domain were characterized by distinct effects of this domain on ORC-origin binding and G1-phase chromatin. Thus, the Fkh1-FHA domain controlled the distinct chromatin architecture at early origins in G1-phase and regulated origin activity in S-phase., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Hoggard et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The piRNA pathway is a conserved germline-specific small RNA pathway that ensures genomic integrity and continued fertility. In C. elegans and other nematodes, Type-I piRNAs are expressed from >10,000 independently transcribed genes clustered within two discrete domains of 1.5 and 3.5 MB on Chromosome IV. Clustering of piRNA genes contributes to their germline-specific expression, but the underlying mechanisms are unclear. We analyze isolated germ nuclei to demonstrate that the piRNA genomic domains are located in a heterochromatin-like environment. USTC (Upstream Sequence Transcription Complex) promotes strong association of nucleosomes throughout piRNA clusters, yet organizes the local nucleosome environment to direct the exposure of individual piRNA genes. Localization of USTC to the piRNA domains depends upon the ATPase chromatin remodeler ISW-1, which maintains high nucleosome density across piRNA clusters and ongoing production of piRNA precursors. Overall, this work provides insight into how chromatin states coordinate transcriptional regulation over large genomic domains, with implications for global genome organization., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Paniagua et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

In eukaryotic cells, the one-dimensional DNA molecules need to be tightly packaged into the spatially constraining nucleus. Folding is achieved on its lowest level by wrapping the DNA around nucleosomes. Their arrangement regulates other nuclear processes, such as transcription and DNA repair. Despite strong efforts to study nucleosome positioning using Next Generation Sequencing (NGS) data, the mechanism of their collective arrangement along the gene body remains poorly understood. Here, we classify nucleosome distributions of protein-coding genes in Saccharomyces cerevisiae according to their profile similarity and analyse their differences using functional Principal Component Analysis. By decomposing the NGS signals into their main descriptive functions, we compared wild type and chromatin remodeler-deficient strains, keeping position-specific details preserved whilst considering the nucleosome arrangement as a whole. A correlation analysis with other genomic properties, such as gene size and length of the upstream Nucleosome Depleted Region (NDR), identified key factors that influence the nucleosome distribution. We reveal that the RSC chromatin remodeler-which is responsible for NDR maintenance-is indispensable for decoupling nucleosome arrangement within the gene from positioning outside, which interfere in rsc8-depleted conditions. Moreover, nucleosome profiles in chd1Δ strains displayed a clear correlation with RNA polymerase II presence, whereas wild type cells did not indicate a noticeable interdependence. We propose that RSC is pivotal for global nucleosome organisation, whilst Chd1 plays a key role for maintaining local arrangement., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Zeitler et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

In Trypanosoma cruzi DNA is packaged into chromatin by octamers of histone proteins that form nucleosomes. Transcription of protein coding genes in trypanosomes is constitutive producing polycistronic units and gene expression is primarily regulated post-transcriptionally. However, chromatin organization influences DNA dependent processes. Hence, determining nucleosome position is of uppermost importance to understand the peculiarities found in trypanosomes. To map nucleosomes genome-wide in several organisms, digestion of chromatin with micrococcal nuclease followed by deep sequencing has been applied. Nonetheless, the special requirements for cell manipulation and the uniqueness of the chromatin organization in trypanosomes entails a customized analytical approach. In this work, we adjusted this broadly used method to the hybrid reference strain, CL Brener. Particularly, we implemented an exhaustive and thorough computational workflow to overcome the difficulties imposed by this complex genome. We tested the performance of two aligners, Bowtie2 and HISAT2, and discuss their advantages and caveats. Specifically, we highlight the relevance of using the whole genome as a reference instead of the commonly used Esmeraldo-like haplotype to avoid spurious alignments. Additionally, we show that using the whole genome refines the average nucleosome representation, but also the quality of mapping for every region represented. Moreover, we show that the average nucleosome organization around trans-splicing acceptor site described before, is not just an average since the same chromatin pattern is detected for most of the represented regions. In addition, we extended the study to a non-hybrid strain applying the experimental and analytical approach to Sylvio-X10 strain. Furthermore, we provide a source code for the construction of 2D plots and heatmaps which are easy to adapt to any T. cruzi strain., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Beati et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

High-throughput sequencing technology is central to our current understanding of the human methylome. The vast majority of studies use chemical conversion to analyse bulk-level patterns of DNA methylation across the genome from a population of cells. While this technology has been used to probe single-molecule methylation patterns, such analyses are limited to short reads of a few hundred basepairs. DNA methylation can also be directly detected using Nanopore sequencing which can generate reads measuring megabases in length. However, thus far these analyses have largely focused on bulk-level assessment of DNA methylation. Here, we analyse DNA methylation in single Nanopore reads from human lymphoblastoid cells, to show that bulk-level metrics underestimate large-scale heterogeneity in the methylome. We use the correlation in methylation state between neighbouring sites to quantify single-molecule heterogeneity and find that heterogeneity varies significantly across the human genome, with some regions having heterogeneous methylation patterns at the single-molecule level and others possessing more homogeneous methylation patterns. By comparing the genomic distribution of the correlation to epigenomic annotations, we find that the greatest heterogeneity in single-molecule patterns is observed within heterochromatic partially methylated domains (PMDs). In contrast, reads originating from euchromatic regions and gene bodies have more ordered DNA methylation patterns. By analysing the patterns of single molecules in more detail, we show the existence of a nucleosome-scale periodicity in DNA methylation that accounts for some of the heterogeneity we uncover in long single-molecule DNA methylation patterns. We find that this periodic structure is partially masked in bulk data and correlates with DNA accessibility as measured by nanoNOMe-seq, suggesting that it could be generated by nucleosomes. Our findings demonstrate the power of single-molecule analysis of long-read data to understand the structure of the human methylome., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Kerr et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

A model for DNA and nucleosomes is introduced with the goal of studying chromosomes from a single base level all the way to higher-order chromatin structures. This model, dubbed the Widely Editable Chromatin Model (WEChroM), reproduces the complex mechanics of the double helix including its bending persistence length and twisting persistence length, and the temperature dependence of the former. The WEChroM Hamiltonian is composed of chain connectivity, steric interactions, and associative memory terms representing all remaining interactions leading to the structure, dynamics, and mechanical characteristics of the B-DNA. Several applications of this model are discussed to demonstrate its applicability. WEChroM is used to investigate the behavior of circular DNA in the presence of positive and negative supercoiling. We show that it recapitulates the formation of plectonemes and of structural defects that relax mechanical stress. The model spontaneously manifests an asymmetric behavior with respect to positive or negative supercoiling, similar to what was previously observed in experiments. Additionally, we show that the associative memory Hamiltonian is also capable of reproducing the free energy of partial DNA unwrapping from nucleosomes. WEChroM is designed to emulate the continuously variable mechanical properties of the 10nm fiber and, by virtue of its simplicity, is ready to be scaled up to molecular systems large enough to investigate the structural ensembles of genes. WEChroM is implemented in the OpenMM simulation toolkits and is freely available for public use., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Lu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

While the biochemistry of gene transcription has been well studied, our understanding of how this process is organised in 3D within the intact nucleus is less well understood. Here we investigate the structure of actively transcribed chromatin and the architecture of its interaction with active RNA polymerase. For this analysis, we have used super-resolution microscopy to image the Drosophila melanogaster Y loops which represent huge, several megabases long, single transcription units. The Y loops provide a particularly amenable model system for transcriptionally active chromatin. We find that, although these transcribed loops are decondensed they are not organised as extended 10nm fibres, but rather they largely consist of chains of nucleosome clusters. The average width of each cluster is around 50nm. We find that foci of active RNA polymerase are generally located off the main fibre axis on the periphery of the nucleosome clusters. Foci of RNA polymerase and nascent transcripts are distributed around the Y loops rather than being clustered in individual transcription factories. However, as the RNA polymerase foci are considerably less prevalent than the nucleosome clusters, the organisation of this active chromatin into chains of nucleosome clusters is unlikely to be determined by the activity of the polymerases transcribing the Y loops. These results provide a foundation for understanding the topological relationship between chromatin and the process of gene transcription., Competing Interests: The authors declare that they have no conflict of interest., (Copyright: © 2023 Ball et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Telomere chromatin structure is pivotal for maintaining genome stability by regulating the binding of telomere-associated proteins and inhibiting the DNA damage response. In Saccharomyces cerevisiae, silent information regulator (Sir) proteins bind to terminal repeats and to subtelomeric X-elements, resulting in transcriptional silencing. Herein, we show that sir2 mutant strains display a specific loss of a nucleosome residing in the X-elements and that this deficiency is remarkably consistent between different telomeres. The X-elements contain several binding sites for the transcription factor Reb1 and we found that Sir2 and Reb1 compete for stabilizing/destabilizing this nucleosome, i.e. inactivation of Reb1 in a sir2 background reinstated the lost nucleosome. The telomeric-repeat-containing RNAs (TERRAs) originate from subtelomeric regions and extend into the terminal repeats. Both Sir2 and Reb1 repress TERRAs and in a sir2 reb1 double mutant, TERRA levels increased synergistically, showing that Sir2 and Reb1 act in different pathways for repressing TERRAs. We present evidence that Reb1 restricts TERRAs by terminating transcription. Mapping the 5'-ends of TERRAs from several telomeres revealed that the Sir2-stabilized nucleosome is the first nucleosome downstream from the transcriptional start site for TERRAs. Finally, moving an X-element to a euchromatic locus changed nucleosome occupancy and positioning, demonstrating that X-element nucleosome structure is dependent on the local telomere environment., Competing Interests: The authors have declared that no competing interests exist.

Although poorly positioned nucleosomes are ubiquitous in the eukaryotic genome, they are difficult to identify with existing nucleosome identification methods. Recently available enhanced high-throughput chromatin conformation capture techniques such as Micro-C, DNase Hi-C, and Hi-CO characterize nucleosome-level chromatin proximity, probing the positions of mono-nucleosomes and the spacing between nucleosome pairs at the same time, enabling nucleosome profiling in poorly positioned regions. Here we develop a novel computational approach, NucleoMap, to identify nucleosome positioning from ultra-high resolution chromatin contact maps. By integrating nucleosome read density, contact distances, and binding preferences, NucleoMap precisely locates nucleosomes in both prokaryotic and eukaryotic genomes and outperforms existing nucleosome identification methods in both precision and recall. We rigorously characterize genome-wide association in eukaryotes between the spatial organization of mono-nucleosomes and their corresponding histone modifications, protein binding activities, and higher-order chromatin functions. We also find evidence of two tetra-nucleosome folding structures in human embryonic stem cells and analyze their association with multiple structural and functional regions. Based on the identified nucleosomes, nucleosome contact maps are constructed, reflecting the inter-nucleosome distances and preserving the contact distance profiles in original contact maps., Competing Interests: The authors have declared that no competing interests exist.

The nucleosome remodeling complexes (CRs) SWI/SNF, RSC, and Ino80C cooperate in evicting or repositioning nucleosomes to produce nucleosome depleted regions (NDRs) at the promoters of many yeast genes induced by amino acid starvation. We analyzed mutants depleted of the catalytic subunits of these CRs for binding of transcriptional activator Gcn4 and recruitment of TATA-binding protein (TBP) during preinitiation complex (PIC) assembly. RSC and Ino80 were found to enhance Gcn4 binding to both UAS elements in NDRs upstream of promoters and to unconventional binding sites within nucleosome-occupied coding sequences; and SWI/SNF contributes to UAS binding when RSC is depleted. All three CRs are actively recruited by Gcn4 to most UAS elements and appear to enhance Gcn4 binding by reducing nucleosome occupancies at the binding motifs, indicating a positive regulatory loop. SWI/SNF acts unexpectedly in WT cells to prevent excessive Gcn4 binding at many UAS elements, indicating a dual mode of action that is modulated by the presence of RSC. RSC and SWI/SNF collaborate to enhance TBP recruitment at Gcn4 target genes, together with Ino80C, in a manner associated with nucleosome eviction at the TBP binding sites. Cooperation among the CRs in TBP recruitment is also evident at the highly transcribed ribosomal protein genes, while RSC and Ino80C act more broadly than SWI/SNF at the majority of other constitutively expressed genes to stimulate this step in PIC assembly. Our findings indicate a complex interplay among the CRs in evicting promoter nucleosomes to regulate activator binding and stimulate PIC assembly., Competing Interests: The authors have declared that no competing interests exist.

We have performed a comprehensive analysis of the involvement of histone H3 and H4 residues in the regulation of chronological lifespan in yeast and identify four structural groups in the nucleosome that influence lifespan. We also identify residues where substitution with an epigenetic mimic extends lifespan, providing evidence that a simple epigenetic switch, without possible additional background modifications, causes longevity. Residues where substitution result in the most pronounced lifespan extension are all on the exposed face of the nucleosome, with the exception of H3E50, which is present on the lateral surface, between two DNA gyres. Other residues that have a more modest effect on lifespan extension are concentrated at the extremities of the H3-H4 dimer, suggesting a role in stabilizing the dimer in its nucleosome frame. Residues that reduce lifespan are buried in the histone handshake motif, suggesting that these mutations destabilize the octamer structure. All residues exposed on the nucleosome disk face and that cause lifespan extension are known to interact with Sir3. We find that substitution of H4K16 and H4H18 cause Sir3 to redistribute from telomeres and silent mating loci to secondary positions, often enriched for Rap1, Abf1 or Reb1 binding sites, whereas H3E50 does not. The redistribution of Sir3 in the genome can be reproduced by an equilibrium model based on primary and secondary binding sites with different affinities for Sir3. The redistributed Sir3 cause transcriptional repression at most of the new loci, including of genes where null mutants were previously shown to extend chronological lifespan. The transcriptomic profiles of H4K16 and H4H18 mutant strains are very similar, and compatible with a DNA replication stress response. This is distinct from the transcriptomic profile of H3E50, which matches strong induction of oxidative phosphorylation. We propose that the different groups of residues are involved in binding to heterochromatin proteins, in destabilizing the association of the nucleosome DNA, disrupting binding of the H3-H4 dimer in the nucleosome, or disrupting the structural stability of the octamer, each category impacting on chronological lifespan by a different mechanism., Competing Interests: The authors have declared that no competing interests exist.

During cell devision, maintaining the epigenetic information encoded in histone modification patterns is crucial for survival and identity of cells. The faithful inheritance of the histone marks from the parental to the daughter strands is a puzzle, given that each strand gets only half of the parental nucleosomes. Mapping DNA replication and reconstruction of modifications to equivalent problems in communication of information, we ask how well enzymes can recover the parental modifications, if they were ideal computing machines. Studying a parameter regime where realistic enzymes can function, our analysis predicts that enzymes may implement a critical threshold filling algorithm which fills unmodified regions of length at most k. This algorithm, motivated from communication theory, is derived from the maximum à posteriori probability (MAP) decoding which identifies the most probable modification sequence based on available observations. Simulations using our method produce modification patterns similar to what has been observed in recent experiments. We also show that our results can be naturally extended to explain inheritance of spatially distinct antagonistic modifications., Competing Interests: The authors have declared that no competing interests exist.

Chromatin structure and underlying DNA accessibility is modulated by the incorporation of histone variants. H2A.Z, a variant of the H2A core histone family, plays a distinct and essential role in a diverse set of biological functions including gene regulation and maintenance of heterochromatin-euchromatin boundaries. Although it is currently unclear how the replacement of H2A with H2A.Z can regulate gene expression, the variance in their amino acid sequence likely contributes to their functional differences. To tease apart regions of H2A.Z that confer its unique identity, a set of plasmids expressing H2A-H2A.Z hybrids from the native H2A.Z promoter were examined for their ability to recapitulate H2A.Z function. First, we found that the H2A.Z M6 region was necessary and sufficient for interaction with the SWR1-C chromatin remodeler. Remarkably, the combination of only 9 amino acid changes, the H2A.Z M6 region, K79 and L81 (two amino acids in the α2-helix), were sufficient to fully rescue growth phenotypes of the htz1Δ mutant. Furthermore, combining three unique H2A.Z regions (K79 and L81, M6, C-terminal tail) was sufficient for expression of H2A.Z-dependent heterochromatin-proximal genes and GAL1 derepression. Surprisingly, hybrid constructs that restored the transcription of H2A.Z-dependent genes, did not fully recapitulate patterns of H2A.Z-specific enrichment at the tested loci. This suggested that H2A.Z function in transcription regulation may be at least partially independent of its specific localization in chromatin. Together, this work has identified three regions that can confer specific H2A.Z-identity to replicative H2A, furthering our understanding of what makes a histone variant a variant., Competing Interests: The authors have declared that no competing interests exist.

The most basic level of eukaryotic gene regulation is the presence or absence of nucleosomes on DNA regulatory elements. In an effort to elucidate in vivo nucleosome patterns, in vitro studies are frequently used. In vitro, short DNA fragments are more favorable for nucleosome formation, increasing the likelihood of nucleosome occupancy. This may in part result from the fact that nucleosomes prefer to form on the terminal ends of linear DNA. This phenomenon has the potential to bias in vitro reconstituted nucleosomes and skew results. If the ends of DNA fragments are known, the reads falling close to the ends are typically discarded. In this study we confirm the phenomenon of end bias of in vitro nucleosomes. We describe a method in which nearly identical libraries, with different known ends, are used to recover nucleosomes which form towards the terminal ends of fragmented DNA. Finally, we illustrate that although nucleosomes prefer to form on DNA ends, it does not appear to skew results or the interpretation thereof., Competing Interests: Two authors, DAB and CEB, are brothers. CEB owns and operates the software consulting company, Qubit Software LLC. The contributions of Qubit Software LLC were strictly pro bono, and the custom codes written are publicly available. This does not alter our adherence to PLOS ONE policies on sharing data and materials. Thus the authors declare no conflict of interest.

The organization of nucleosomes along the Eukaryotic genome is maintained over time despite disruptive events such as replication. During this complex process, histones and DNA can form a variety of non-canonical nucleosome conformations, but their precise molecular details and roles during nucleosome assembly remain unclear. In this study, employing coarse-grained molecular dynamics simulations and Markov state modeling, we characterized the complete kinetics of nucleosome assembly. On the nucleosome-positioning 601 DNA sequence, we observe a rich transition network among various canonical and non-canonical tetrasome, hexasome, and nucleosome conformations. A low salt environment makes nucleosomes stable, but the kinetic landscape becomes more rugged, so that the system is more likely to be trapped in off-pathway partially assembled intermediates. Finally, we find that the co-operativity between DNA bending and histone association enables positioning sequence motifs to direct the assembly process, with potential implications for the dynamic organization of nucleosomes on real genomic sequences., Competing Interests: The authors have declared that no competing interests exist.

Perception of microbes by plants leads to dynamic reprogramming of the transcriptome, which is essential for plant health. The appropriate amplitude of this transcriptional response can be regulated at multiple levels, including chromatin. However, the mechanisms underlying the interplay between chromatin remodeling and transcription dynamics upon activation of plant immunity remain poorly understood. Here, we present evidence that activation of plant immunity by bacteria leads to nucleosome repositioning, which correlates with altered transcription. Nucleosome remodeling follows distinct patterns of nucleosome repositioning at different loci. Using a reverse genetic screen, we identify multiple chromatin remodeling ATPases with previously undescribed roles in immunity, including EMBRYO SAC DEVELOPMENT ARREST 16, EDA16. Functional characterization of the immune-inducible chromatin remodeling ATPase EDA16 revealed a mechanism to negatively regulate immunity activation and limit changes in redox homeostasis. Our transcriptomic data combined with MNase-seq data for EDA16 functional knock-out and over-expressor mutants show that EDA16 selectively regulates a defined subset of genes involved in redox signaling through nucleosome repositioning. Thus, collectively, chromatin remodeling ATPases fine-tune immune responses and provide a previously uncharacterized mechanism of immune regulation., Competing Interests: The authors have declared that no competing interests exist.

In mammals, cellular identity is defined through strict regulation of chromatin modifications and DNA methylation that control gene expression. Methylation of cytosines at CpG sites in the genome is mainly associated with suppression; however, the reason for enhancer-specific methylation is not fully understood. We used sequential ChIP-bisulfite-sequencing for H3K4me1 and H3K27ac histone marks. By collecting data from the same genomic region, we identified enhancers differentially methylated between these two marks. We observed a global gain of CpG methylation primarily in H3K4me1-marked nucleosomes during mouse embryonic stem cell differentiation. This gain occurred largely in enhancer regions that regulate genes critical for differentiation. The higher levels of DNA methylation in H3K4me1- versus H3K27ac-marked enhancers, despite it being the same genomic region, indicates cellular heterogeneity of enhancer states. Analysis of single-cell RNA-seq profiles demonstrated that this heterogeneity correlates with gene expression during differentiation. Furthermore, heterogeneity of enhancer methylation correlates with transcription start site methylation. Our results provide insights into enhancer-based functional variation in complex biological systems., Competing Interests: The authors have declared that no competing interests exist.

Trypanosoma cruzi alternates between replicative and nonreplicative life forms, accompanied by a shift in global transcription levels and by changes in the nuclear architecture, the chromatin proteome and histone posttranslational modifications. To gain further insights into the epigenetic regulation that accompanies life form changes, we performed genome-wide high-resolution nucleosome mapping using two T. cruzi life forms (epimastigotes and cellular trypomastigotes). By combining a powerful pipeline that allowed us to faithfully compare nucleosome positioning and occupancy, more than 125 thousand nucleosomes were mapped, and approximately 20% of them differed between replicative and nonreplicative forms. The nonreplicative forms have less dynamic nucleosomes, possibly reflecting their lower global transcription levels and DNA replication arrest. However, dynamic nucleosomes are enriched at nonreplicative regulatory transcription initiation regions and at multigenic family members, which are associated with infective-stage and virulence factors. Strikingly, dynamic nucleosome regions are associated with GO terms related to nuclear division, translation, gene regulation and metabolism and, notably, associated with transcripts with different expression levels among life forms. Finally, the nucleosome landscape reflects the steady-state transcription expression: more abundant genes have a more deeply nucleosome-depleted region at putative 5' splice sites, likely associated with trans-splicing efficiency. Taken together, our results indicate that chromatin architecture, defined primarily by nucleosome positioning and occupancy, reflects the phenotypic differences found among T. cruzi life forms despite the lack of a canonical transcriptional control context., Competing Interests: The authors have declared that no competing interests exist.

Nucleosome positioning in the genome is essential for the regulation of many nuclear processes. We currently have limited capability to predict nucleosome positioning in vivo, especially the locations and sizes of nucleosome depleted regions (NDRs). Here, we present a thermodynamic model that incorporates the intrinsic affinity of histones, competitive binding of sequence-specific factors, and nucleosome remodeling to predict nucleosome positioning in budding yeast. The model shows that the intrinsic affinity of histones, at near-saturating histone concentration, is not sufficient in generating NDRs in the genome. However, the binding of a few factors, especially RSC towards GC-rich and poly(A/T) sequences, allows us to predict ~ 66% of genome-wide NDRs. The model also shows that nucleosome remodeling activity is required to predict the correct NDR sizes. The validity of the model was further supported by the agreement between the predicted and the measured nucleosome positioning upon factor deletion or on exogenous sequences introduced into yeast. Overall, our model quantitatively evaluated the impact of different genetic components on NDR formation and illustrated the vital roles of sequence-specific factors and nucleosome remodeling in this process., Competing Interests: The authors have declared that no competing interests exist.

Nucleosome positioning is crucial for the genome's function. Though the role of DNA sequence in positioning nucleosomes is well understood, a detailed mechanistic understanding on the impact of transcription remains lacking. Using numerical simulations, we investigated the dependence of nucleosome density profiles on transcription level across multiple species. We found that the low nucleosome affinity of yeast, but not mouse, promoters contributes to the formation of phased nucleosomes arrays for inactive genes. For the active genes, a heterogeneous distribution of +1 nucleosomes, caused by a tug-of-war between two types of remodeling enzymes, is essential for reproducing their density profiles. In particular, while positioning enzymes are known to remodel the +1 nucleosome and align it toward the transcription start site (TSS), spacer enzymes that use a pair of nucleosomes as their substrate can shift the nucleosome array away from the TSS. Competition between these enzymes results in two types of nucleosome density profiles with well- and ill-positioned +1 nucleosome. Finally, we showed that Pol II assisted histone exchange, if occurring at a fast speed, can abolish the impact of remodeling enzymes. By elucidating the role of individual factors, our study reconciles the seemingly conflicting results on the overall impact of transcription in positioning nucleosomes across species., Competing Interests: The authors have declared that no competing interests exist.

CpG-free pDNA was reported to facilitate sustained transgene expression with minimal inflammation in vivo as compared to CpG-containing pDNA. However, the expression potential and impact of CpG-free pDNA in in vitro model have never been described. Hence, in this study, we analyzed the transgene expression profiles of CpG-free pDNA in vitro to determine the influence of CpG depletion from the transgene. We found that in contrast to the published in vivo studies, CpG-free pDNA expressed a significantly lower level of luciferase than CpG-rich pDNA in several human cell lines. By comparing novel CpG-free pDNA carrying CpG-free GFP (pZGFP: 0 CpG) to CpG-rich GFP (pRGFP: 60 CpGs), we further showed that the discrepancy was not influenced by external factors such as gene transfer agent, cell species, cell type, and cytotoxicity. Moreover, pZGFP exhibited reduced expression despite having equal gene dosage as pRGFP. Analysis of mRNA distribution revealed that the mRNA export of pZGFP and pRGFP was similar; however, the steady state mRNA level of pZGFP was significantly lower. Upon further investigation, we found that the CpG-free transgene in non-integrating CpG-free pDNA backbone acquired increased nucleosome enrichment as compared with CpG-rich transgene, which may explain the observed reduced level of steady state mRNA. Our findings suggest that nucleosome enrichment could regulate non-integrating CpG-free pDNA expression and has implications on pDNA design., Competing Interests: The authors have declared that no competing interests exist.

Chromatin structure, including nucleosome positioning, has a fundamental role in transcriptional regulation through influencing protein-DNA interactions. DNA topology is known to influence chromatin structure, and in doing so, can also alter transcription. However, detailed mechanism(s) linking transcriptional regulation events to chromatin structure that is regulated by changes in DNA topology remain to be well defined. Here we demonstrate that nucleosome positioning and transcriptional output from the fission yeast fbp1 and prp3 genes are altered by excess topoisomerase activity. Given that lncRNAs (long noncoding RNAs) are transcribed from the fbp1 upstream region and are important for fbp1 gene expression, we hypothesized that local changes in DNA topological state caused by topoisomerase activity could alter lncRNA and fbp1 transcription. In support of this, we found that topoisomerase overexpression caused destabilization of positioned nucleosomes within the fbp1 promoter region, which was accompanied by aberrant fbp1 transcription. Similarly, the direct recruitment of topoisomerase, but not a catalytically inactive form, to the promoter region of fbp1 caused local changes in nucleosome positioning that was also accompanied by altered fbp1 transcription. These data indicate that changes in DNA topological state induced by topoisomerase activity could lead to altered fbp1 transcription through modulating nucleosome positioning., Competing Interests: The authors have declared that no competing interests exist.

A ~10-11 bp periodicity in dinucleotides imparting DNA bending, with shorter periods found in organisms with positively-supercoiled DNA and longer periods found in organisms with negatively-supercoiled DNA, was previously suggested to assist in DNA compaction. However, when measured with more robust methods, variation in the observed periods between organisms with different growth temperatures is not consistent with that hypothesis. We demonstrate that dinucleotide periodicity does not arise solely by mutational biases but is under selection. We found variation between genomes in both the period and the suite of dinucleotides that are periodic. Whereas organisms with similar growth temperatures have highly variable periods, differences in periods increase with phylogenetic distance between organisms. In addition, while the suites of dinucleotides under selection for periodicity become more dissimilar among more distantly-related organisms, there is a core set of dinucleotides that are strongly periodic among genomes in all domains of life. Notably, this core set of periodic motifs are not involved in DNA bending. These data indicate that dinucleotide periodicity is an ancient genomic architecture which may play a role in shaping the evolution of genes and genomes., Competing Interests: The authors have declared that no competing interests exist.

UltraPrep is an open-source, two-step method for purification of cell-free DNA that entails extraction of total DNA followed by size-selective enrichment of the smaller fragments that are characteristic of DNA originating from fragmentation between nucleosome. The advantages of the two related protocols that are described are that they can easily accommodate a wide range of sample input volumes, they rely on simple, magnetic bead-based technology, the yields of cfDNA are directly comparable to the most popular methods for cfDNA purification, and they dramatically reduce the cost of cfDNA isolation relative to currently available commercial methods. We provide a framework for physical and molecular quality analysis of purified cfDNA and demonstrate that the cfDNA generated by UltraPrep meets or exceeds the quality metrics of the most commonly used procedure. In addition, our method removes high molecular weight genomic DNA (hmwgDNA) that can interfere with downstream assay results, thereby addressing one of the primary concerns for preanalytical collection of blood samples., Competing Interests: All of the authors that contributed to this study are affiliated with either Ripple Biosolutions or PlasmaLab International as employees. In addition, the authors have an ownership stake in these entities. This affiliation does not alter our adherence to PLOS ONE policies on sharing data and materials. The open-source UltraPrep methods described in this manuscript are used to manufacture our commercial product, “UltraPure cfDNA”.

Nucleosome positioning DNA sequence patterns (NPS)-usually distributions of particular dinucleotides or other sequence elements in nucleosomal DNA-at least partially determine chromatin structure and arrangements of nucleosomes that in turn affect gene expression. Statistically, NPS are defined as oscillations of the dinucleotide periodicity of about 10 base pairs (bp) which reflects the double helix period. We compared the nucleosomal DNA patterns in mouse, human and yeast organisms and observed few distinctive patterns that can be termed as packing and regulatory referring to distinctive modes of chromatin function. For the first time the NPS patterns in nucleus accumbens cells (NAC) in mouse brain were characterized and compared to the patterns in human CD4+ and apoptotic lymphocyte cells and well studied patterns in yeast. The NPS patterns in human CD4+ cells and mouse brain cells had very high positive correlation. However, there was no correlation between them and patterns in human apoptotic lymphocyte cells and yeast, but the latter two were highly correlated with each other. By their dinucleotide arrangements the analyzed NPS patterns classified into stable canonical WW/SS (W = A or T and S = C or G dinucleotide) and less stable RR/YY (R = A or G and Y = C or T dinucleotide) patterns and anti-patterns. In the anti-patterns positioning of the dinucleotides is flipped compared to those in the regular patterns. Stable canonical WW/SS patterns and anti-patterns are ubiquitously observed in many organisms and they had high resemblance between yeast and human apoptotic cells. Less stable RR/YY patterns had higher positive correlation between mouse and normal human cells. Our analysis and evidence from scientific literature lead to idea that various distinct patterns in nucleosomal DNA can be related to the two roles of the chromatin: packing (WW/SS) and regulatory (RR/YY and "anti")., Competing Interests: The authors have declared that no competing interests exist.

Empirical evidence suggests that the malaria parasite Plasmodium falciparum employs a broad range of mechanisms to regulate gene transcription throughout the organism's complex life cycle. To better understand this regulatory machinery, we assembled a rich collection of genomic and epigenomic data sets, including information about transcription factor (TF) binding motifs, patterns of covalent histone modifications, nucleosome occupancy, GC content, and global 3D genome architecture. We used these data to train machine learning models to discriminate between high-expression and low-expression genes, focusing on three distinct stages of the red blood cell phase of the Plasmodium life cycle. Our results highlight the importance of histone modifications and 3D chromatin architecture in Plasmodium transcriptional regulation and suggest that AP2 transcription factors may play a limited regulatory role, perhaps operating in conjunction with epigenetic factors., Competing Interests: The authors have declared that no competing interests exist.

The SWR1 chromatin remodeling complex, which deposits the histone variant H2A.Z into nucleosomes, has been well characterized in yeast and animals, but its composition in plants has remained uncertain. We used the conserved SWR1 subunit ACTIN RELATED PROTEIN 6 (ARP6) as bait in tandem affinity purification experiments to isolate associated proteins from Arabidopsis thaliana. We identified all 11 subunits found in yeast SWR1 and the homologous mammalian SRCAP complexes, demonstrating that this complex is conserved in plants. We also identified several additional proteins not previously associated with SWR1, including Methyl-CpG-BINDING DOMAIN 9 (MBD9) and three members of the Alfin1-like protein family, all of which have been shown to bind modified histone tails. Since mbd9 mutant plants were phenotypically similar to arp6 mutants, we explored a potential role for MBD9 in H2A.Z deposition. We found that MBD9 is required for proper H2A.Z incorporation at thousands of discrete sites, which represent a subset of the genomic regions normally enriched with H2A.Z. We also discovered that MBD9 preferentially interacts with acetylated histone H4 peptides, as well as those carrying mono- or dimethylated H3 lysine 4, or dimethylated H3 arginine 2 or 8. Considering that MBD9-dependent H2A.Z sites show a distinct histone modification profile, we propose that MBD9 recognizes particular nucleosome modifications via its PHD- and Bromo-domains and thereby guides SWR1 to these sites for H2A.Z deposition. Our data establish the SWR1 complex as being conserved across eukaryotes and suggest that MBD9 may be involved in targeting the complex to specific genomic sites through nucleosomal interactions. The finding that MBD9 does not appear to be a core subunit of the Arabidopsis SWR1 complex, along with the synergistic phenotype of arp6;mbd9 double mutants, suggests that MBD9 also has important roles beyond H2A.Z deposition., Competing Interests: The authors have declared that no competing interests exist.

Pyruvate Kinase M2 (PKM2) mediates metabolic reshuffling and is ubiquitously upregulated in several cancer types. The non-metabolic function of PKM2 as key nuclear kinase and modulator of gene expression is instrumental in cancer progression and tumorigenesis. Here, we attempt to discern the non-canonical function of PKM2 as an epigenetic modulator and the underlying implication of this activity. Using 5'-FAM labelled reconstituted mononucleosome we have shown that PKM2 interacts with the complex through Histone H3 and possibly obstruct the access to DNA binding factors. Subsequently, the interaction negatively impacts the ATP dependent remodeling activity of Chromodomain Helicase DNA binding protein-7 (Chd7). Chd7 remodeling activity is required to ameliorate DNA damage and is crucial to genome stability. Our study shows that PKM2 blocks the Chd7 mediated sliding of nucleosome. It can be conjectured that stalling Chd7 may lead to impaired DNA damage and increased genomic instability. We propose a mechanism in which PKM2 negatively regulate nucleosome repositioning in chromatin and may exacerbate cancer by altering the nucleosome architecture. This research is imperative to our understanding of how altered cancer metabolism can potentially modulate the gene expression and sustain incessant proliferation by tweaking the chromatin topography., Competing Interests: The authors have declared that no competing interests exist.

Ultraviolet (UV) light-induced mutations are unevenly distributed across skin cancer genomes, but the molecular mechanisms responsible for this heterogeneity are not fully understood. Here, we assessed how nucleosome structure impacts the positions of UV-induced mutations in human melanomas. Analysis of mutation positions from cutaneous melanomas within strongly positioned nucleosomes revealed a striking ~10 base pair (bp) oscillation in mutation density with peaks occurring at dinucleotides facing away from the histone octamer. Additionally, higher mutation density at the nucleosome dyad generated an overarching "translational curvature" across the 147 bp of DNA that constitutes the nucleosome core particle. This periodicity and curvature cannot be explained by sequence biases in nucleosomal DNA. Instead, our genome-wide map of UV-induced cyclobutane pyrimidine dimers (CPDs) indicates that CPD formation is elevated at outward facing dinucleotides, mirroring the oscillation of mutation density within nucleosome-bound DNA. Nucleotide excision repair (NER) activity, as measured by XR-seq, inversely correlated with the curvature of mutation density associated with the translational setting of the nucleosome. While the 10 bp periodicity of mutations is maintained across nucleosomes regardless of chromatin state, histone modifications, and transcription levels, overall mutation density and curvature across the core particle increased with lower transcription levels. Our observations suggest structural conformations of DNA promote CPD formation at specific sites within nucleosomes, and steric hindrance progressively limits lesion repair towards the nucleosome dyad. Both mechanisms create a unique extended mutation signature within strongly positioned nucleosomes across the human genome., Competing Interests: The authors have declared that no competing interests exist.

Most active DNA replication origins are found within euchromatin, while origins within heterochromatin are often inactive or inhibited. In yeast, origin activity within heterochromatin is negatively controlled by the histone H4K16 deacetylase, Sir2, and at some heterochromatic loci also by the nucleosome binding protein, Sir3. The prevailing view has been that direct functions of Sir2 and Sir3 are confined to heterochromatin. However, growth defects in yeast mutants compromised for loading the MCM helicase, such as cdc6-4, are suppressed by deletion of either SIR2 or SIR3. While these and other observations indicate that SIR2,3 can have a negative impact on at least some euchromatic origins, the genomic scale of this effect was unknown. It was also unknown whether this suppression resulted from direct functions of Sir2,3 within euchromatin, or was an indirect effect of their previously established roles within heterochromatin. Using MCM ChIP-Seq, we show that a SIR2 deletion rescued MCM complex loading at ~80% of euchromatic origins in cdc6-4 cells. Therefore, Sir2 exhibited a pervasive effect at the majority of euchromatic origins. Using MNase-H4K16ac ChIP-Seq, we show that origin-adjacent nucleosomes were depleted for H4K16 acetylation in a SIR2-dependent manner in wild type (i.e. CDC6) cells. In addition, we present evidence that both Sir2 and Sir3 bound to nucleosomes adjacent to euchromatic origins. The relative levels of each of these molecular hallmarks of yeast heterochromatin-SIR2-dependent H4K16 hypoacetylation, Sir2, and Sir3 -correlated with how strongly a SIR2 deletion suppressed the MCM loading defect in cdc6-4 cells. Finally, a screen for histone H3 and H4 mutants that could suppress the cdc6-4 growth defect identified amino acids that map to a surface of the nucleosome important for Sir3 binding. We conclude that heterochromatin proteins directly modify the local chromatin environment of euchromatic DNA replication origins., Competing Interests: The authors have declared that no competing interests exist.

As the Drosophila embryo transitions from the use of maternal RNAs to zygotic transcription, domains of open chromatin, with relatively low nucleosome density and specific histone marks, are established at promoters and enhancers involved in patterned embryonic transcription. However it remains unclear how regions of activity are established during early embryogenesis, and if they are the product of spatially restricted or ubiquitous processes. To shed light on this question, we probed chromatin accessibility across the anterior-posterior axis (A-P) of early Drosophila melanogaster embryos by applying a transposon based assay for chromatin accessibility (ATAC-seq) to anterior and posterior halves of hand-dissected, cellular blastoderm embryos. We find that genome-wide chromatin accessibility is highly similar between the two halves, with regions that manifest significant accessibility in one half of the embryo almost always accessible in the other half, even for promoters that are active in exclusively one half of the embryo. These data support previous studies that show that chromatin accessibility is not a direct result of activity, and point to a role for ubiquitous factors or processes in establishing chromatin accessibility at promoters in the early embryo. However, in concordance with similar works, we find that at enhancers active exclusively in one half of the embryo, we observe a significant skew towards greater accessibility in the region of their activity, highlighting the role of patterning factors such as Bicoid in this process.

While nucleosome positioning on eukaryotic genome play important roles for genetic regulation, molecular mechanisms of nucleosome positioning and sliding along DNA are not well understood. Here we investigated thermally-activated spontaneous nucleosome sliding mechanisms developing and applying a coarse-grained molecular simulation method that incorporates both long-range electrostatic and short-range hydrogen-bond interactions between histone octamer and DNA. The simulations revealed two distinct sliding modes depending on the nucleosomal DNA sequence. A uniform DNA sequence showed frequent sliding with one base pair step in a rotation-coupled manner, akin to screw-like motions. On the contrary, a strong positioning sequence, the so-called 601 sequence, exhibits rare, abrupt transitions of five and ten base pair steps without rotation. Moreover, we evaluated the importance of hydrogen bond interactions on the sliding mode, finding that strong and weak bonds favor respectively the rotation-coupled and -uncoupled sliding movements.

Micrococcal nuclease (MNase) has been widely used for analyses of nucleosome locations in many organisms. However, due to its sequence preference, the interpretations of the positions and occupancies of nucleosomes using MNase have remained controversial. Next-generation sequencing (NGS) has also been utilized for analyses of MNase-digests, but some technical biases are commonly present in the NGS experiments. Here, we established a gel-based method to map nucleosome positions in Saccharomyces cerevisiae, using isolated nuclei as the substrate for the histone H4 S47C-site-directed chemical cleavage in parallel with MNase digestion. The parallel mapping allowed us to compare the chemically and enzymatically cleaved sites by indirect end-labeling and primer extension mapping, and thus we could determine the nucleosome positions and the sizes of the nucleosome-free regions (or nucleosome-depleted regions) more accurately, as compared to nucleosome mapping by MNase alone. The analysis also revealed that the structural features of the nucleosomes flanked by the nucleosome-free region were different from those within regularly arrayed nucleosomes, showing that the structures and dynamics of individual nucleosomes strongly depend on their locations. Moreover, we demonstrated that the parallel mapping results were generally consistent with the previous genome-wide chemical mapping and MNase-Seq results. Thus, the gel-based parallel mapping will be useful for the analysis of a specific locus under various conditions.

All eukaryotic genomes are packaged as chromatin, with DNA interlaced with both regularly patterned nucleosomes and sub-nucleosomal-sized protein structures such as mobile and labile transcription factors (TF) and initiation complexes, together forming a dynamic chromatin landscape. Whilst details of nucleosome position in Arabidopsis have been previously analysed, there is less understanding of their relationship to more dynamic sub-nucleosomal particles (subNSPs) defined as protected regions shorter than the ~150bp typical of nucleosomes. The genome-wide profile of these subNSPs has not been previously analysed in plants and this study investigates the relationship of dynamic bound particles with transcriptional control. Here we combine differential micrococcal nuclease (MNase) digestion and a modified paired-end sequencing protocol to reveal the chromatin structure landscape of Arabidopsis cells across a wide particle size range. Linking this data to RNAseq expression analysis provides detailed insight into the relationship of identified DNA-bound particles with transcriptional activity. The use of differential digestion reveals sensitive positions, including a labile -1 nucleosome positioned upstream of the transcription start site (TSS) of active genes. We investigated the response of the chromatin landscape to changes in environmental conditions using light and dark growth, given the large transcriptional changes resulting from this simple alteration. The resulting shifts in the suites of expressed and repressed genes show little correspondence to changes in nucleosome positioning, but led to significant alterations in the profile of subNSPs upstream of TSS both globally and locally. We examined previously mapped positions for the TFs PIF3, PIF4 and CCA1, which regulate light responses, and found that changes in subNSPs co-localized with these binding sites. This small particle structure is detected only under low levels of MNase digestion and is lost on more complete digestion of chromatin to nucleosomes. We conclude that wide-spectrum analysis of the Arabidopsis genome by differential MNase digestion allows detection of sensitive features hereto obscured, and the comparisons between genome-wide subNSP profiles reveals dynamic changes in their distribution, particularly at distinct genomic locations (i.e. 5'UTRs). The method here employed allows insight into the complex influence of genetic and extrinsic factors in modifying the sub-nucleosomal landscape in association with transcriptional changes.

Nucleosome organization affects the accessibility of cis-elements to trans-acting factors. Micrococcal nuclease digestion followed by high-throughput sequencing (MNase-seq) is the most popular technology used to profile nucleosome organization on a genome-wide scale. Evaluating the data quality of MNase-seq data remains challenging, especially in mammalian. There is a strong need for a convenient and comprehensive approach to obtain dedicated quality control (QC) for MNase-seq data analysis. Here we developed CAM, which is a comprehensive QC pipeline for MNase-seq data. The CAM pipeline provides multiple informative QC measurements and nucleosome organization profiles on different potentially functional regions for given MNase-seq data. CAM also includes 268 historical MNase-seq datasets from human and mouse as a reference atlas for unbiased assessment. CAM is freely available at: http://www.tongji.edu.cn/~zhanglab/CAM.

The fidelity of epigenetic inheritance or, the precision by which epigenetic information is passed along, is an essential parameter for measuring the effectiveness of the process. How the precision of the process is achieved or modulated, however, remains largely elusive. We have performed quantitative measurement of epigenetic fidelity, using position effect variegation (PEV) in Schizosaccharomyces pombe as readout, to explore whether replication perturbation affects nucleosome-mediated epigenetic inheritance. We show that replication stresses, due to either hydroxyurea treatment or various forms of genetic lesions of the replication machinery, reduce the inheritance accuracy of CENP-A/Cnp1 nucleosome positioning within centromere. Mechanistically, we demonstrate that excessive formation of single-stranded DNA, a common molecular abnormality under these conditions, might have correlation with the reduction in fidelity of centromeric chromatin duplication. Furthermore, we show that replication stress broadly changes chromatin structure at various loci in the genome, such as telomere heterochromatin expanding and mating type locus heterochromatin spreading out of the boundaries. Interestingly, the levels of inheritable expanding at sub-telomeric heterochromatin regions are highly variable among independent cell populations. Finally, we show that HU treatment of the multi-cellular organisms C. elegans and D. melanogaster affects epigenetically programmed development and PEV, illustrating the evolutionary conservation of the phenomenon. Replication stress, in addition to its demonstrated role in genetic instability, promotes variable epigenetic instability throughout the epigenome.

Agrobacterium tumefaciens mediated T-DNA integration is a common tool for plant genome manipulation. However, there is controversy regarding whether T-DNA integration is biased towards genes or randomly distributed throughout the genome. In order to address this question, we performed high-throughput mapping of T-DNA-genome junctions obtained in the absence of selection at several time points after infection. T-DNA-genome junctions were detected as early as 6 hours post-infection. T-DNA distribution was apparently uniform throughout the chromosomes, yet local biases toward AT-rich motifs and T-DNA border sequence micro-homology were detected. Analysis of the epigenetic landscape of previously isolated sites of T-DNA integration in Kanamycin-selected transgenic plants showed an association with extremely low methylation and nucleosome occupancy. Conversely, non-selected junctions from this study showed no correlation with methylation and had chromatin marks, such as high nucleosome occupancy and high H3K27me3, that correspond to three-dimensional-interacting heterochromatin islands embedded within euchromatin. Such structures may play a role in capturing and silencing invading T-DNA.

The breadth of diagnostic procedures that utilize cell free DNA (cfDNA) from human plasma has increased dramatically in recent years. Here, we confirm that tumor-derived cfDNA fragments are similar in size distribution to cfDNA derived from normal tissues. Therefore, collection procedures optimized with healthy donor specimens are likely to be applicable to the diagnosis and monitoring of many different cancer types. We verify that the distribution and DNA sequences of fragmentation sites in cfDNA from both normal-germline and tumor-derived cfDNA are non-random. A broad survey of cfDNA from healthy donors suggests that average individuals possess ~6 ng of cfDNA per mL of plasma. Importantly, the cfDNA present in plasma samples that were initially collected as whole blood in K2-EDTA tubes and subsequently processed by centrifugation is stable for several days at ambient temperatures. This observation has the potential to significantly reduce the cost and logistical complexity of shipping clinical samples from the site of collection to centers proficient in diagnostic analysis. Finally, plasma samples collected with high-volume plasma collection devices possess abundant quantities of cfDNA. Since the quantity of analyzed cfDNA is directly proportional to sensitivity of diagnostic assays, this method of plasma collection, where available, could enable highly sensitive post-treatment disease monitoring and early detection of cancer in at-risk individuals.

Cell-free DNA (cfDNA) is short, extracellular, fragmented double-stranded DNA found in plasma. Plasma of patients with solid tumor has been found to show significantly increased quantities of cfDNA. Although currently poorly understood, the mechanism of cfDNA generation is speculated to be a product of genomic DNA fragmentation during cellular apoptosis and necrosis. Sequencing of cfDNA with tumor origin has identified tumor biomarkers, elucidating molecular pathology and assisting in accurate diagnosis. In this study, we performed whole-genome sequencing ofcfDNA samples with matching tumor and whole blood samples from five patients diagnosed with stage IV gastric or lung cancer. We analyzed the coverage spectrum of the human genome in our cfDNA samples. cfDNA exhibited no large regions with significant under-coverage, although we observed unbalanced coverage depth in cfDNA at transcription start sites and exon boundaries as a consequence of biased fragmentation due to ordered nucleosome positioning. We also analyzed the copy number variant status based on the whole-genome sequencing results and found high similarity between copy number profile constructed from tumor samples and cfDNA samples. Overall, we conclude that cfDNA comprises a good representation of the tumor genome in late stage gastric and lung cancer., Competing Interests: The authors wish it to be known that our affiliation with Geneseeq Technology Inc. and Nanjing Shihe Jiyin Biotechnology Inc. does not alter our adherence to PLOS ONE policies on sharing data and materials.

The packaging and organization of genomic DNA into chromatin represents an additional regulatory layer of gene expression, with specific nucleosome positions that restrict the accessibility of regulatory DNA elements. The mechanisms that position nucleosomes in vivo are thought to depend on the biophysical properties of the histones, sequence patterns, like phased di-nucleotide repeats and the architecture of the histone octamer that folds DNA in 1.65 tight turns. Comparative studies of human and P. falciparum histones reveal that the latter have a strongly reduced ability to recognize internal sequence dependent nucleosome positioning signals. In contrast, the nucleosomes are positioned by AT-repeat sequences flanking nucleosomes in vivo and in vitro. Further, the strong sequence variations in the plasmodium histones, compared to other mammalian histones, do not present adaptations to its AT-rich genome. Human and parasite histones bind with higher affinity to GC-rich DNA and with lower affinity to AT-rich DNA. However, the plasmodium nucleosomes are overall less stable, with increased temperature induced mobility, decreased salt stability of the histones H2A and H2B and considerable reduced binding affinity to GC-rich DNA, as compared with the human nucleosomes. In addition, we show that plasmodium histone octamers form the shortest known nucleosome repeat length (155bp) in vitro and in vivo. Our data suggest that the biochemical properties of the parasite histones are distinct from the typical characteristics of other eukaryotic histones and these properties reflect the increased accessibility of the P. falciparum genome., Competing Interests: The authors have declared that no competing interests exist.

The mammalian genome is organized into submegabase-sized chromatin domains (CDs) including topologically associating domains, which have been identified using chromosome conformation capture-based methods. Single-nucleosome imaging in living mammalian cells has revealed subdiffusively dynamic nucleosome movement. It is unclear how single nucleosomes within CDs fluctuate and how the CD structure reflects the nucleosome movement. Here, we present a polymer model wherein CDs are characterized by fractal dimensions and the nucleosome fibers fluctuate in a viscoelastic medium with memory. We analytically show that the mean-squared displacement (MSD) of nucleosome fluctuations within CDs is subdiffusive. The diffusion coefficient and the subdiffusive exponent depend on the structural information of CDs. This analytical result enabled us to extract information from the single-nucleosome imaging data for HeLa cells. Our observation that the MSD is lower at the nuclear periphery region than the interior region indicates that CDs in the heterochromatin-rich nuclear periphery region are more compact than those in the euchromatin-rich interior region with respect to the fractal dimensions as well as the size. Finally, we evaluated that the average size of CDs is in the range of 100-500 nm and that the relaxation time of nucleosome movement within CDs is a few seconds. Our results provide physical and dynamic insights into the genome architecture in living cells., Competing Interests: The authors have declared that no competing interests exist.

The recruitment of RNA-Pol-II to the transcription start site (TSS) is an important step in gene regulation in all organisms. Core promoter elements (CPE) are conserved sequence motifs that guide Pol-II to the TSS by interacting with specific transcription factors (TFs). However, only a minority of animal promoters contains CPEs. It is still unknown how Pol-II selects the TSS in their absence. Here we present a comparative analysis of promoters' sequence composition and chromatin architecture in five eukaryotic model organisms, which shows the presence of common and unique DNA-encoded features used to organize chromatin. Analysis of Pol-II initiation patterns uncovers that, in the absence of certain CPEs, there is a strong correlation between the spread of initiation and the intensity of the 10 bp periodic signal in the nearest downstream nucleosome. Moreover, promoters' primary and secondary initiation sites show a characteristic 10 bp periodicity in the absence of CPEs. We also show that DNA natural variants in the region immediately downstream the TSS are able to affect both the nucleosome-DNA affinity and Pol-II initiation pattern. These findings support the notion that, in addition to CPEs mediated selection, sequence-induced nucleosome positioning could be a common and conserved mechanism of TSS selection in animals., Competing Interests: The authors have declared that no competing interests exist.

Defects in chromatin proteins frequently manifest in diseases. A striking case of a chromatin-centric disease is NUT-midline carcinoma (NMC), which is characterized by expression of NUT as a fusion partner most frequently with BRD4. ChIP-sequencing studies from NMC patients revealed that BRD4-NUT (B4N) covers large genomic regions and elevates transcription within these domains. To investigate how B4N modulates chromatin, we performed affinity purification of B4N when ectopically expressed in 293-TREx cells and quantified the associated histone posttranslational modifications (PTM) using proteomics. We observed significant enrichment of acetylation particularly on H3 K18 and of combinatorial patterns such as H3 K27 acetylation paired with K36 methylation. We postulate that B4N complexes override the preexisting histone code with new PTM patterns that reflect aberrant transcription and that epigenetically modulate the nucleosome environment toward the NMC state., Competing Interests: The authors have declared that no competing interests exist.

Antisense transcription is a prevalent feature at mammalian promoters. Previous studies have primarily focused on antisense transcription initiating upstream of genes. Here, we characterize promoter-proximal antisense transcription downstream of gene transcription starts sites in human breast cancer cells, investigating the genomic context of downstream antisense transcription. We find extensive correlations between antisense transcription and features associated with the chromatin environment at gene promoters. Antisense transcription downstream of promoters is widespread, with antisense transcription initiation observed within 2 kb of 28% of gene transcription start sites. Antisense transcription initiates between nucleosomes regularly positioned downstream of these promoters. The nucleosomes between gene and downstream antisense transcription start sites carry histone modifications associated with active promoters, such as H3K4me3 and H3K27ac. This region is bound by chromatin remodeling and histone modifying complexes including SWI/SNF subunits and HDACs, suggesting that antisense transcription or resulting RNA transcripts contribute to the creation and maintenance of a promoter-associated chromatin environment. Downstream antisense transcription overlays additional regulatory features, such as transcription factor binding, DNA accessibility, and the downstream edge of promoter-associated CpG islands. These features suggest an important role for antisense transcription in the regulation of gene expression and the maintenance of a promoter-associated chromatin environment.

FACT, in addition to its role in transcription, is likely implicated in both transcription-coupled nucleotide excision repair and DNA double strand break repair. Here, we present evidence that FACT could be directly involved in Base Excision Repair and elucidate the chromatin remodeling mechanisms of FACT during BER. We found that, upon oxidative stress, FACT is released from transcription related protein complexes to get associated with repair proteins and chromatin remodelers from the SWI/SNF family. We also showed the rapid recruitment of FACT to the site of damage, coincident with the glycosylase OGG1, upon the local generation of oxidized DNA. Interestingly, FACT facilitates uracil-DNA glycosylase in the removal of uracil from nucleosomal DNA thanks to an enhancement in the remodeling activity of RSC. This discloses a novel property of FACT wherein it has a co-remodeling activity and strongly enhances the remodeling capacity of the chromatin remodelers. Altogether, our data suggest that FACT may acts in concert with RSC to facilitate excision of DNA lesions during the initial step of BER.

In many mammals, including humans and mice, the zinc finger histone methyltransferase PRDM9 performs the first step in meiotic recombination by specifying the locations of hotspots, the sites of genetic recombination. PRDM9 binds to DNA at hotspots through its zinc finger domain and activates recombination by trimethylating histone H3K4 on adjacent nucleosomes through its PR/SET domain. Recently, the isolated PR/SET domain of PRDM9 was shown capable of also trimethylating H3K36 in vitro, raising the question of whether this reaction occurs in vivo during meiosis, and if so, what its function might be. Here, we show that full-length PRDM9 does trimethylate H3K36 in vivo in mouse spermatocytes. Levels of H3K4me3 and H3K36me3 are highly correlated at hotspots, but mutually exclusive elsewhere. In vitro, we find that although PRDM9 trimethylates H3K36 much more slowly than it does H3K4, PRDM9 is capable of placing both marks on the same histone molecules. In accord with these results, we also show that PRDM9 can trimethylate both K4 and K36 on the same nucleosomes in vivo, but the ratio of K4me3/K36me3 is much higher for the pair of nucleosomes adjacent to the PRDM9 binding site compared to the next pair further away. Importantly, H3K4me3/H3K36me3-double-positive nucleosomes occur only in regions of recombination: hotspots and the pseudoautosomal (PAR) region of the sex chromosomes. These double-positive nucleosomes are dramatically reduced when PRDM9 is absent, showing that this signature is PRDM9-dependent at hotspots; the residual double-positive nucleosomes most likely come from the PRDM9-independent PAR. These results, together with the fact that PRDM9 is the only known mammalian histone methyltransferase with both H3K4 and H3K36 trimethylation activity, suggest that trimethylation of H3K36 plays an important role in the recombination process. Given the known requirement of H3K36me3 for double strand break repair by homologous recombination in somatic cells, we suggest that it may play the same role in meiosis.

Eukaryotic DNA is strongly bent inside fundamental packaging units: the nucleosomes. It is known that their positions are strongly influenced by the mechanical properties of the underlying DNA sequence. Here we discuss the possibility that these mechanical properties and the concomitant nucleosome positions are not just a side product of the given DNA sequence, e.g. that of the genes, but that a mechanical evolution of DNA molecules might have taken place. We first demonstrate the possibility of multiplexing classical and mechanical genetic information using a computational nucleosome model. In a second step we give evidence for genome-wide multiplexing in Saccharomyces cerevisiae and Schizosacharomyces pombe. This suggests that the exact positions of nucleosomes play crucial roles in chromatin function.

Beyond its role in genomic organization and compaction, the nucleosome is believed to participate in the regulation of gene transcription. Here, we report a computational method to evaluate the nucleosome sensitivity for a transcription factor over a given stretch of the genome. Sensitive factors are predicted to be those with binding sites preferentially contained within nucleosome boundaries and lacking 10 bp periodicity. Based on these criteria, the Acute Myeloid Leukemia-1a (AML-1a) transcription factor, a regulator of immune gene expression, was identified as potentially sensitive to nucleosomal regulation within the mouse Ly49 gene family. This result was confirmed in RMA, a cell line with natural expression of Ly49, using MNase-Seq to generate a nucleosome map of chromosome 6, where the Ly49 gene family is located. Analysis of this map revealed a specific depletion of nucleosomes at AML-1a binding sites in the expressed Ly49A when compared to the other, silent Ly49 genes. Our data suggest that nucleosome-based regulation contributes to the expression of Ly49 genes, and we propose that this method of predicting nucleosome sensitivity could aid in dissecting the regulatory role of nucleosomes in general.

NURF is a conserved higher eukaryotic ISWI-containing chromatin remodeling complex that catalyzes ATP-dependent nucleosome sliding. By sliding nucleosomes, NURF is able to alter chromatin dynamics to control transcription and genome organization. Previous biochemical and genetic analysis of the specificity-subunit of Drosophila NURF (Nurf301/Enhancer of Bithorax (E(bx)) has defined NURF as a critical regulator of homeotic, heat-shock and steroid-responsive gene transcription. It has been speculated that NURF controls pathway specific transcription by co-operating with sequence-specific transcription factors to remodel chromatin at dedicated enhancers. However, conclusive in vivo demonstration of this is lacking and precise regulatory elements targeted by NURF are poorly defined. To address this, we have generated a comprehensive map of in vivo NURF activity, using MNase-sequencing to determine at base pair resolution NURF target nucleosomes, and ChIP-sequencing to define sites of NURF recruitment. Our data show that, besides anticipated roles at enhancers, NURF interacts physically and functionally with the TRF2/DREF basal transcription factor to organize nucleosomes downstream of active promoters. Moreover, we detect NURF remodeling and recruitment at distal insulator sites, where NURF functionally interacts with and co-localizes with DREF and insulator proteins including CP190 to establish nucleosome-depleted domains. This insulator function of NURF is most apparent at subclasses of insulators that mark the boundaries of chromatin domains, where multiple insulator proteins co-associate. By visualizing the complete repertoire of in vivo NURF chromatin targets, our data provide new insights into how chromatin remodeling can control genome organization and regulatory interactions.

Within the genomes of metazoans, nucleosomes are highly organised adjacent to the binding sites for a subset of transcription factors. Here we have sought to investigate which chromatin remodelling enzymes are responsible for this. We find that the ATP-dependent chromatin remodelling enzyme SNF2H plays a major role organising arrays of nucleosomes adjacent to the binding sites for the architectural transcription factor CTCF sites and acts to promote CTCF binding. At many other factor binding sites SNF2H and the related enzyme SNF2L contribute to nucleosome organisation. The action of SNF2H at CTCF sites is functionally important as depletion of CTCF or SNF2H affects transcription of a common group of genes. This suggests that chromatin remodelling ATPase's most closely related to the Drosophila ISWI protein contribute to the function of many human gene regulatory elements.