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Technological advancements in cell-free DNA (cfDNA) liquid biopsy have triggered exponential growth in numerous clinical applications. While cfDNA-based liquid biopsy has made significant strides in personalizing cancer treatment, the exploration and translation of epigenetics in liquid biopsy to clinical practice is still nascent. This comprehensive review seeks to provide a broad yet in-depth narrative of the present status of epigenetics in cfDNA liquid biopsy and its associated challenges. It highlights the potential of epigenetics in cfDNA liquid biopsy technologies with the hopes of enhancing its clinical translation. The momentum of cfDNA liquid biopsy technologies in recent years has propelled epigenetics to the forefront of molecular biology. We have only begun to reveal the true potential of epigenetics in both our understanding of disease and leveraging epigenetics in the diagnostic and therapeutic domains. Recent clinical applications of epigenetics-based cfDNA liquid biopsy revolve around DNA methylation in screening and early cancer detection, leading to the development of multi-cancer early detection tests and the capability to pinpoint tissues of origin. The clinical application of epigenetics in cfDNA liquid biopsy in minimal residual disease, monitoring, and surveillance are at their initial stages. A notable advancement in fragmentation patterns analysis has created a new avenue for epigenetic biomarkers. However, the widespread application of cfDNA liquid biopsy has many challenges, including biomarker sensitivity, specificity, logistics including infrastructure and personnel, data processing, handling, results interpretation, accessibility, and cost effectiveness. Exploring and translating epigenetics in cfDNA liquid biopsy technology can transform our understanding and perception of cancer prevention and management. cfDNA liquid biopsy has great potential in precision oncology to revolutionize conventional ways of early cancer detection, monitoring residual disease, treatment response, surveillance, and drug development. Adapting the implementation of liquid biopsy workflow to the local policy worldwide and developing point-of-care testing holds great potential to overcome global cancer disparity and improve cancer outcomes.

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BACKGROUND: Nucleosome repositioning in cancer is believed to cause many changes in genome organisation and gene expression. Understanding these changes is important to elucidate fundamental aspects of cancer. It is also important for medical diagnostics based on cell-free DNA (cfDNA), which originates from genomic DNA regions protected from digestion by nucleosomes. RESULTS: We have generated high-resolution nucleosome maps in paired tumour and normal tissues from the same breast cancer patients using MNase-assisted histone H3 ChIP-seq and compared them with the corresponding cfDNA from blood plasma. This analysis has detected single-nucleosome repositioning at key regulatory regions in a patient-specific manner and common cancer-specific patterns across patients. The nucleosomes gained in tumour versus normal tissue were particularly informative of cancer pathways, with ~ 20-fold enrichment at CpG islands, a large fraction of which marked promoters of genes encoding DNA-binding proteins. The tumour tissues were characterised by a 5-10 bp decrease in the average distance between nucleosomes (nucleosome repeat length, NRL), which is qualitatively similar to the differences between pluripotent and differentiated cells. This effect was correlated with gene activity, differential DNA methylation and changes in local occupancy of linker histone variants H1.4 and H1X. CONCLUSIONS: Our study offers a novel resource of high-resolution nucleosome maps in breast cancer patients and reports for the first time the effect of systematic decrease of NRL in paired tumour versus normal breast tissues from the same patient. Our findings provide a new mechanistic understanding of nucleosome repositioning in tumour tissues that can be valuable for patient diagnostics, stratification and monitoring.

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[This corrects the article DOI: 10.3389/fgene.2023.1222112.].

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Aging induces systematic changes in the distribution of nucleosomes, which affect gene expression programs. Here we reconstructed nucleosome maps based on cell-free DNA (cfDNA) extracted from blood plasma using four cohorts of people of different ages. We show that nucleosomes tend to be separated by larger genomic distances in older people, and age correlates with the nucleosome repeat length (NRL). Furthermore, we developed the first aging clock based on cfDNA nucleosomics. Machine learning based on cfDNA distance distributions allowed predicting person's age with the median absolute error of 3-3.5 years.

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Nucleosomes not only serve as the basic building blocks for eukaryotic chromatin but also regulate many biological processes, such as DNA replication, repair, and recombination. To modulate gene expression in vivo, the histone variant H2A.Z can be dynamically incorporated into the nucleosome. However, the assembly dynamics of H2A.Z-containing nucleosomes remain elusive. Here, we demonstrate that our previous chemical kinetic model for nucleosome assembly can be extended to H2A.Z-containing nucleosome assembly processes. The efficiency of H2A.Z-containing nucleosome assembly, like that of canonical nucleosome assembly, was also positively correlated with the total histone octamer concentration, reaction rate constant, and reaction time. We expanded the kinetic model to represent the competitive dynamics of H2A and H2A.Z in nucleosome assembly, thus providing a novel method through which to assess the competitive ability of histones to assemble nucleosomes. Based on this model, we confirmed that histone H2A has a higher competitive ability to assemble nucleosomes in vitro than histone H2A.Z. Our competitive kinetic model and experimental results also confirmed that in vitro H2A.Z-containing nucleosome assembly is governed by chemical kinetic principles.

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The establishment and maintenance of nucleosome-free regions (NFRs) are prominent processes within chromatin dynamics. Transcription factors, ATP-dependent chromatin remodeling complexes (CRCs) and DNA sequences are the main factors involved. In Saccharomyces cerevisiae, CRCs such as RSC contribute to chromatin opening at NFRs, while other complexes, including ISW1a, contribute to NFR shrinking. Regarding DNA sequences, growing evidence points to poly(dA:dT) tracts as playing a direct role in active processes involved in nucleosome positioning dynamics. Intriguingly, poly(dA:dT)-tract-containing NFRs span asymmetrically relative to the location of the tract by a currently unknown mechanism. In order to obtain insight into the role of poly(dA:dT) tracts in nucleosome remodeling, we performed a systematic analysis of their influence on the activity of ISW1a and RSC complexes. Our results show that poly(dA:dT) tracts differentially affect the activity of these CRCs. Moreover, we found differences between the effects exerted by the two alternative tract orientations. Remarkably, tract-containing linker DNA is taken as exit DNA for nucleosome sliding catalyzed by RSC. Our findings show that defined DNA sequences, when present in linker DNA, can dictate in which direction a remodeling complex has to slide nucleosomes and shed light into the mechanisms underlying asymmetrical chromatin opening around poly(dA:dT) tracts.

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The organization of chromatin - including the positions of nucleosomes and the binding of other proteins to DNA - helps define transcriptional profiles in eukaryotic organisms. While techniques like ChIP-Seq and MNase-Seq can map protein-DNA and nucleosome localization separately, assays designed to simultaneously capture nucleosome positions and protein-DNA interactions can produce a detailed picture of the chromatin landscape. Most assays that monitor chromatin organization and protein binding rely on antibodies, which often exhibit nonspecific binding, and/or the addition of bulky adducts to the DNA-binding protein being studied, which can affect their expression and activity. Here, we describe SpyCatcher Linked Targeting of Chromatin Endogenous Cleavage (SpLiT-ChEC), where a 13-amino acid SpyTag peptide, appended to a protein of interest, serves as a highly-specific targeting moiety for in situ enzymatic digestion. The SpyTag/SpyCatcher system forms a covalent bond, linking the target protein and a co-expressed MNase-SpyCatcher fusion construct. SpyTagged proteins are expressed from endogenous loci, whereas MNase-SpyCatcher expression is induced immediately before harvesting cultures. MNase is activated with high concentrations of calcium, which primarily digests DNA near target protein binding sites. By sequencing the DNA fragments released by targeted MNase digestion, we found that this method recovers information on protein binding and proximal nucleosome positioning. SpLiT-ChEC provides precise temporal control that we anticipate can be used to monitor chromatin under various conditions and at distinct points in the cell cycle.

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Prime editing (PE) is a highly versatile CRISPR-Cas9 genome editing technique. The current constructs, however, have variable efficiency and may require laborious experimental optimization. This study presents statistical models for learning the salient epigenomic and sequence features of target sites modulating the editing efficiency and provides guidelines for designing optimal PEs. We found that both regional constitutive heterochromatin and local nucleosome occlusion of target sites impede editing, while position-specific G/C nucleotides in the primer-binding site (PBS) and reverse transcription (RT) template regions of PE guide RNA (pegRNA) yield high editing efficiency, especially for short PBS designs. The presence of G/C nucleotides was most critical immediately 5' to the protospacer adjacent motif (PAM) site for all designs. The effects of different last templated nucleotides were quantified and observed to depend on the length of both PBS and RT templates. Our models found AGG to be the preferred PAM and detected a guanine nucleotide four bases downstream of the PAM to facilitate editing, suggesting a hitherto-unrecognized interaction with Cas9. A neural network interpretation method based on nonextensive statistical mechanics further revealed multi-nucleotide preferences, indicating dependency among several bases across pegRNA. Our work clarifies previous conflicting observations and uncovers context-dependent features important for optimizing PE designs.

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G-quadruplexes (G4s) are gaining increasing attention as possible regulators of chromatin packaging, and robust approaches to their studies in pseudo-native context are much needed. Here, we designed a simple in vitro model of G4-prone genomic DNA and employed it to elucidate the impact of G4s and G4-stabilizing ligands on nucleosome occupancy. We obtained two 226-bp dsDNA constructs composed of the strong nucleosome positioning sequence and an internucleosomal DNA-imitating tail. The tail was G4-free in the control construct and harbored a "strong" (stable) G4 motif in the construct of interest. An additional "weak" (semi-stable) G4 motif was found within the canonical nucleosome positioning sequence. Both G4s were confirmed by optical methods and 1H NMR spectroscopy. Electrophoretic mobility assays showed that the weak G4 motif did not obstruct nucleosome assembly, while the strong G4 motif in the tail sequence diminished nucleosome yield. Atomic force microscopy data and molecular modeling confirmed that the strong G4 was maintained in the tail of the correctly assembled nucleosome structure. Using both in vitro and in silico models, we probed three known G4 ligands and detected nucleosome-disrupting effects of the least selective ligand. Our results are in line with the negative correlation between stable G4s and nucleosome density, support G4 tolerance between regularly positioned nucleosomes, and highlight the importance of considering chromatin context when targeting genomic G4s.

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Genomic DNA wraps around core histones to form nucleosomes, which provides steric constraints on how transcription factors (TFs) can interact with gene regulatory sequences. It is increasingly apparent that well-positioned, accessible nucleosomes are an inherent feature of active enhancers and can facilitate cooperative TF binding, referred to as nucleosome-mediated cooperativity. Thus, profiling chromatin and nucleosome properties (accessibility, positioning, and occupancy) on the genome is crucial to understand cell-type-specific gene regulation. Here we describe a simplified protocol to profile accessible nucleosomes in the mammalian genome using low-level and high-level micrococcal nuclease (MNase) digestion followed by genome-wide sequencing.

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5-Bromodeoxyuridine (BrdU), a thymidine analogue, is an interesting reagent that modulates various biological phenomena. BrdU, upon incorporation into DNA, causes destabilized nucleosome positioning which leads to changes in heterochromatin organization and gene expression in cells. We have previously shown that BrdU effectively induces cellular senescence, a phenomenon of irreversible growth arrest in mammalian cells. Identification of the mechanism of action of BrdU would provide a novel insight into the molecular mechanisms of cellular senescence. Here, we showed that a basic domain in the histone H2B N-terminal tail, termed the HBR (histone H2B repression) domain, is involved in the action of BrdU. Notably, deletion of the HBR domain causes destabilized nucleosome positioning and derepression of gene expression, as does BrdU. We also showed that the genes up-regulated by BrdU significantly overlapped with those by deletion of the HBR domain, the result of which suggested that BrdU and deletion of the HBR domain act in a similar way. Furthermore, we showed that decreased HBR domain function induced cellular senescence or facilitated the induction of cellular senescence. These findings indicated that the HBR domain is crucially involved in the action of BrdU, and also suggested that disordered nucleosome organization may be involved in the induction of cellular senescence.

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Nucleosome positioning is involved in diverse cellular biological processes by regulating the accessibility of DNA sequences to DNA-binding proteins and plays a vital role. Previous studies have manifested that the intrinsic preference of nucleosomes for DNA sequences may play a dominant role in nucleosome positioning. As a consequence, it is nontrivial to develop computational methods only based on DNA sequence information to accurately identify nucleosome positioning, and thus intend to verify the contribution of DNA sequences responsible for nucleosome positioning. In this work, we propose a new deep learning-based method, named DeepNup, which enables us to improve the prediction of nucleosome positioning only from DNA sequences. Specifically, we first use a hybrid feature encoding scheme that combines One-hot encoding and Trinucleotide composition encoding to encode raw DNA sequences; afterwards, we employ multiscale convolutional neural network modules that consist of two parallel convolution kernels with different sizes and gated recurrent units to effectively learn the local and global correlation feature representations; lastly, we use a fully connected layer and a sigmoid unit serving as a classifier to integrate these learned high-order feature representations and generate the final prediction outcomes. By comparing the experimental evaluation metrics on two benchmark nucleosome positioning datasets, DeepNup achieves a better performance for nucleosome positioning prediction than that of several state-of-the-art methods. These results demonstrate that DeepNup is a powerful deep learning-based tool that enables one to accurately identify potential nucleosome sequences.

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BACKGROUND: Nucleosome-mediated chromatin compaction has a direct effect on the accessibility of trans-acting activators and repressors to DNA targets and serves as a primary regulatory agent of genetic expression. Understanding the nature and dynamics of chromatin is fundamental to elucidating the mechanisms and factors that epigenetically regulate gene expression. Previous work has shown that there are three types of canonical sequences that strongly regulate nucleosome positioning and thus chromatin accessibility: putative nucleosome-positioning elements, putative nucleosome-repelling sequences, and homopolymeric runs of A/T. It is postulated that these elements can be used to remodel chromatin in C. elegans. Here we show the utility of such elements in vivo, and the extreme efficacy of a newly discovered repelling sequence, PRS-322. RESULTS: In this work, we show that it is possible to manipulate nucleosome positioning in C. elegans solely using canonical and putative positioning sequences. We have not only tested previously described sequences such as the Widom 601, but also have tested additional nucleosome-positioning sequences: the Trifonov sequence, putative repelling sequence-322 (PRS-322), and various homopolymeric runs of A and T nucleotides. CONCLUSIONS: Using each of these types of putative nucleosome-positioning sequences, we demonstrate their ability to alter the nucleosome profile in C. elegans as evidenced by altered nucleosome occupancy and positioning in vivo. Additionally, we show the effect that PRS-322 has on nucleosome-repelling and chromatin remodeling.

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The positions of nucleosomes along genomic DNA play a role in defining patterns of gene expression and chromatin organization. Determination of nucleosome positions in vivo and in vitro, as revealed by the locations of histones on DNA, has provided insight into mechanisms of nucleosome sliding, spacing, assembly, and disassembly. Here, we describe methods for the in vitro determination of histone-DNA contacts at base-pair (bp) resolution. The protocol involves the labeling of histones with ortho-phenanthroline (OP), site-specific cleavage of nucleosomal DNA, and processing and analysis of the resulting DNA fragments. This methodology provides an efficient and high-resolution means for studying kinetics and behavior of enzymes that alter nucleosome structure and/or positioning, and can be used to identify preferred distributions of nucleosomes on natural DNA sequences. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Cysteine-specific chemical modification of folded histones with ortho-phenanthroline (OP) Basic Protocol 2: Nucleosome sliding assay adapted for OP mapping of histone-DNA contacts Basic Protocol 3: OP-mediated cleavage, processing, and analysis of DNA fragments using a sequencing gel Support Protocol 1: Preparation of dideoxy sequencing ladders Support Protocol 2: Preparation and running of a denaturing DNA sequencing gel.

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Nucleosome-displacing-factors (NDFs) in yeast, similar to pioneer factors in higher eukaryotes, can open closed chromatin and generate nucleosome-depleted regions (NDRs). NDRs in yeast are also affected by ATP-dependent chromatin remodelers (CRs). However, how NDFs and CRs coordinate in nucleosome invasion and NDR formation is still unclear. Here, we design a high-throughput method to systematically study the interplay between NDFs and CRs. By combining an integrated synthetic oligonucleotide library with DNA methyltransferase-based, single-molecule nucleosome mapping, we measure the impact of CRs on NDRs generated by individual NDFs. We find that CRs are dispensable for nucleosome invasion by NDFs, and they function downstream of NDF binding to modulate the NDR length. A few CRs show high specificity toward certain NDFs; however, in most cases, CRs are recruited in a factor-nonspecific and NDR length-dependent manner. Overall, our study provides a framework to investigate how NDFs and CRs cooperate to regulate chromatin opening.

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Nucleosomes are basic units of DNA packing in eukaryotes. Their structure is well conserved from yeast to human and consists of the histone octamer core and 147 bp DNA wrapped around it. Nucleosomes are bound to a majority of the eukaryotic genomic DNA, including its regulatory regions. Hence, they also play a major role in gene regulation. For the latter, their precise positioning on DNA is essential. In the present paper, we describe Galaxy dnpatterntools-software package for nucleosome DNA sequence analysis and mapping. This software will be useful for computational biologists practitioners to conduct more profound studies of gene regulatory mechanisms.

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Nucleosome positioning plays an important role in crucial biological processes such as replication, transcription, and gene regulation. It has been widely used to predict the genome's function and chromatin organisation. So far, the studies of patterns in nucleosome positioning have been limited to transcription start sites, CTCFs binding sites, and some promoter and loci regions. The genome-wide organisational pattern remains unknown. We have developed a theoretical model to coarse-grain nucleosome positioning data in order to obtain patterns in their distribution. Using hierarchical clustering on the auto-correlation function of this coarse-grained nucleosome positioning data, a genome-wide clustering is obtained for Candida albicans. The clustering shows the existence beyond hetero- and eu-chromatin inside the chromosomes. These non-trivial clusterings correspond to different nucleosome distributions and gene densities governing differential gene expression patterns. Moreover, these distribution patterns inside the chromosome appeared to be conserved throughout the genome and within species. The pipeline of the coarse grain nucleosome positioning sequence to identify underlying genomic organisation used in our study is novel, and the classifications obtained are unique and consistent.

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BACKGROUND: Nucleosome positioning is the precise determination of the location of nucleosomes on DNA sequence. With the continuous advancement of biotechnology and computer technology, biological data is showing explosive growth. It is of practical significance to develop an efficient nucleosome positioning algorithm. Indeed, convolutional neural networks (CNN) can capture local features in DNA sequences, but ignore the order of bases. While the bidirectional recurrent neural network can make up for CNN's shortcomings in this regard and extract the long-term dependent features of DNA sequence. RESULTS: In this work, we use word vectors to represent DNA sequences and propose three new deep learning models for nucleosome positioning, and the integrative model NP_CBiR reaches a better prediction performance. The overall accuracies of NP_CBiR on H. sapiens, C. elegans, and D. melanogaster datasets are 86.18%, 89.39%, and 85.55% respectively. CONCLUSIONS: Benefited by different network structures, NP_CBiR can effectively extract local features and bases order features of DNA sequences, thus can be considered as a complementary tool for nucleosome positioning.

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Establishing and maintaining appropriate gene repression is critical for the health and development of multicellular organisms. Histone H3 lysine 27 (H3K27) methylation is a chromatin modification associated with repressed facultative heterochromatin, but the mechanism of this repression remains unclear. We used a forward genetic approach to identify genes involved in transcriptional silencing of H3K27-methylated chromatin in the filamentous fungus Neurospora crassa. We found that the N. crassa homologs of ISWI (NCU03875) and ACF1 (NCU00164) are required for repression of a subset of H3K27-methylated genes and that they form an ACF chromatin-remodeling complex. This ACF complex interacts with chromatin throughout the genome, yet association with facultative heterochromatin is specifically promoted by the H3K27 methyltransferase, SET-7. H3K27-methylated genes that are upregulated when iswi or acf1 are deleted show a downstream shift of the +1 nucleosome, suggesting that proper nucleosome positioning is critical for repression of facultative heterochromatin. Our findings support a direct role of the ACF complex in Polycomb repression.

All the cells in an organism contain the exact same DNA, yet each type of cell performs a different role. They achieve this by turning specific genes on or off. To do this, cells wind their genetic code into structures called nucleosomes, which work a bit like spools of thread. Chemical modifications on these nucleosomes can determine whether a cell will use the genes spooled around it or not. In many organisms, cells can turn genes off using a modification called H3K27 methylation. This mark attracts a protein complex called PRC1 that packs the genes away, making them inaccessible to the proteins that would activate them. But the filamentous fungus Neurospora crassa does not produce PRC1. This suggests that this organism must keep genes with the H3K27 mark switched off in a different way. One possibility is that H3K27 methylation somehow leads to changes in the position of nucleosomes on the genome, since having nucleosomes near the beginning of gene sequences can stop the cell from reading the code. One protein complex responsible for positioning nucleosomes is known as the ATP-utilizing chromatin assembly and remodeling factor (ACF) complex, but it remained unknown whether it interacted with H3K27 methylation marks. To investigate further, Wiles et al. generated strains of Neurospora crassa that did not synthesize ACF and discovered that many of their genes, including ones marked with H3K27, were turned on. This was probably because the nucleosomes had shifted out of position, allowing the proteins responsible for activating the genes to gain access to the start of the genes' sequences. Turning genes on and off at the right time is crucial for development, cell survival, and is key in tissues and organs working properly. Understanding the role of ACF adds to what we know about this complex process, which is involved in many diseases, including cancer.

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The nucleosome is the basic structural element of genomic DNA packaging and plays a role in transcription, replication, and recombination. Poly(dA) tracts are considered major sequence determinants of nucleosome positioning, although their role is not well understood. Here, we show that the homopolymeric character and the low GC content of poly(dA)s play different roles in nucleosome formation. We found that the inherent low GC content of poly(dA) alone can account for the deep and anisotropic nucleosome depletion at structurally and functionally important regions of promoters and origins of replication. We also show that the level of nucleosome occupancy at poly(dA) is strongly related to the local nucleotide background and its high frequency of occurrence in Saccharomyces cerevisiae does not appear merely to be associated with its intrinsic nucleosome-excluding properties. In addition, we show that the GC content alone can predict more than 60% of the in vitro nucleosome map, providing further evidence that the intrinsic nucleosome positioning is more greatly determined by GC content than poly(dA) stretches. Our results are consistent with a model in which poly(dA) stretches act at two distinct levels: first, by its low GC content, which intrinsically contributes to hinder nucleosome formation, and second, by its contiguous runs of dA that selectively drive the recruitment of non-histone proteins with structural and functional roles.

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