Nucleosome Positioning Research Articles

The Histone Variant H2A.Z C-Terminal Domain Has Locus-Specific Differential Effects on H2A.Z Occupancy and Nucleosome Localization.

The incorporation of histone variant H2A.Z into nucleosomes creates specialized chromatin domains that regulate DNA-templated processes, such as gene transcription. In Saccharomyces cerevisiae, the diverging H2A.Z C terminus is thought to provide the H2A.Z exclusive functions. To elucidate the roles of this H2A.Z C terminus genome-wide, we used derivatives in which the C terminus was replaced with the corresponding region of H2A (ZA protein), or the H2A region plus a transcriptional activating peptide (ZA-rII'), with the intent of regenerating the H2A.Z-dependent regulation globally. The distribution of these H2A.Z derivatives indicates that the H2A.Z C-terminal region is crucial for both maintaining the occupation level of H2A.Z and the proper positioning of targeted nucleosomes. Interestingly, the specific contribution on incorporation efficiency versus nucleosome positioning varies enormously depending on the locus analyzed. Specifically, the role of H2A.Z in global transcription regulation relies on its C-terminal region. Remarkably, however, this mostly involves genes without a H2A.Z nucleosome in the promoter. Lastly, we demonstrate that the main chaperone complex which deposits H2A.Z to gene regulatory region (SWR1-C) is necessary to localize all H2A.Z derivatives at their specific loci, indicating that the differential association of these derivatives is not due to impaired interaction with SWR1-C. IMPORTANCE We provide evidence that the Saccharomyces cerevisiae C-terminal region of histone variant H2A.Z can mediate its special function in performing gene regulation by interacting with effector proteins and chaperones. These functional interactions allow H2A.Z not only to incorporate to very specific gene regulatory regions, but also to facilitate the gene expression process. To achieve this, we used a chimeric protein which lacks the native H2A.Z C-terminal region but contains an acidic activating region, a module that is known to interact with components of chromatin-remodeling entities and/or transcription modulators. We reasoned that because this activating region can fulfill the role of the H2A.Z C-terminal region, at least in part, the role of the latter would be to interact with these activating region targets.

Analytical and therapeutic profiles of DNA methylation alterations in cancer; an overview of changes in chromatin arrangement and alterations in histone surfaces.

DNA methylation is the most important epigenetic element that activates the inhibition of gene transcription and is included in the pathogenesis of all types of malignancies. Remarkably, the effectors of DNA methylation are DNMTs (DNA methyltransferases) that catalyze denovo or keep methylation of hemimethylated DNA after the DNA replication process. DNA methylation structures incancer are altered, with three procedures by which DNA methylation helps cancer development which are including direct mutagenesis, hypomethylation of the cancer genome, and also focal hypermethylation of the promoters of TSGs (tumor suppressor genes). Conspicuously, DNA methylation, nucleosome remodeling, RNA-mediated targeting, and histone modification balance modulate many biological activities that are essential and indispensable to the genesis of cancer and also can impact many epigenetic changes including DNA methylation and histone modifications as well as adjusting of non-coding miRNAs expression in prevention and treatment of many cancers. Epigenetics points to heritable modifications in gene expression that do not comprise alterations in the DNA sequence. The nucleosome is the basic unit of chromatin, consisting of 147 base pairs (bp) of DNA bound around a histone octamer comprised of one H3/H4 tetramer and twoH2A/H2B dimers. DNA methylation is preferentially distributed over nucleosome regions and is less increased over flanking nucleosome-depleted DNA, implying a connection between nucleosome positioning and DNA methylation. In carcinogenesis, aberrations in the epigenome may also include in the progression of drug resistance. In this report, we report the rudimentary notes behind these epigenetic signaling pathways and emphasize the proofs recommending that their misregulation can conclude in cancer. These findings in conjunction with thepromising preclinical and clinical consequences observed with epigenetic drugs against chromatin regulators, confirm the important role of epigenetics in cancer therapy.

Cannabinoids Transmogrify Cancer Metabolic Phenotype via Epigenetic Reprogramming and a Novel CBD Biased G Protein-Coupled Receptor Signaling Platform

The concept of epigenetic reprogramming predicts long-term functional health effects. This reprogramming can be activated by exogenous or endogenous insults, leading to altered healthy and different disease states. The exogenous or endogenous changes that involve developing a roadmap of epigenetic networking, such as drug components on epigenetic imprinting and restoring epigenome patterns laid down during embryonic development, are paramount to establishing youthful cell type and health. This epigenetic landscape is considered one of the hallmarks of cancer. The initiation and progression of cancer are considered to involve epigenetic abnormalities and genetic alterations. Cancer epigenetics have shown extensive reprogramming of every component of the epigenetic machinery in cancer development, including DNA methylation, histone modifications, nucleosome positioning, non-coding RNAs, and microRNA expression. Endocannabinoids are natural lipid molecules whose levels are regulated by specific biosynthetic and degradative enzymes. They bind to and activate two primary cannabinoid receptors, type 1 (CB1) and type 2 (CB2), and together with their metabolizing enzymes, form the endocannabinoid system. This review focuses on the role of cannabinoid receptors CB1 and CB2 signaling in activating numerous receptor tyrosine kinases and Toll-like receptors in the induction of epigenetic landscape alterations in cancer cells, which might transmogrify cancer metabolism and epigenetic reprogramming to a metastatic phenotype. Strategies applied from conception could represent an innovative epigenetic target for preventing and treating human cancer. Here, we describe novel cannabinoid-biased G protein-coupled receptor signaling platforms (GPCR), highlighting putative future perspectives in this field.

Effect of nucleosome density, nucleosome positions and cohesin on the structure of chromatin studied with Monte Carlo simulations.

The structure of chromatin is important for the regulation of transcriptional activity in the nucleus. It is influenced by several factors, such as nucleosome density, which varies over a wide range, the position of nucleosomes on DNA, which is actively regulated by the cell, and the binding of cohesin and CTCF by looping. We applied Monte Carlo computer simulations to study different systems up to 2 Mbp in size under different conditions, and compared the results with experimental data from conformation capture experiments, high-resolution microscopy, and electron microscopy. We identified cell-to-cell variability in the number of bound nucleosomes, linker histones, and internucleosomal interaction strength as a function of a region's transcriptional activity. Chromatin looping mediated by CTCF and cohesin increased the number of contacts between distant sites. These contacts were dependent on nucleosome positions. The structure of chromatin under density conditions, as observed in the nucleus modeled by periodic boundary conditions, is determined by the distribution of nucleosome positions. It controls accessibility by modulating the diffusion behavior of factors in the chromatin network.

Circulating Histones to Detect and Monitor the Progression of Cancer.

Liquid biopsies have emerged as a minimally invasive cancer detection and monitoring method, which could identify cancer-related alterations in nucleosome or histone levels and modifications in blood, saliva, and urine. Histones, the core component of the nucleosome, are essential for chromatin compaction and gene expression modulation. Increasing evidence suggests that circulating histones and histone complexes, originating from cell death or immune cell activation, could act as promising biomarkers for cancer detection and management. In this review, we provide an overview of circulating histones as a powerful liquid biopsy approach and methods for their detection. We highlight current knowledge on circulating histones in hematologic malignancies and solid cancer, with a focus on their role in cancer dissemination, monitoring, and tumorigenesis. Last, we describe recently developed strategies to identify cancer tissue-of-origin in blood plasma based on nucleosome positioning, inferred from nucleosomal DNA fragmentation footprint, which is independent of the genetic landscape.

Universal NicE-Seq: A Simple and Quick Method for Accessible Chromatin Detection in Fixed Cells.

Genome-wide accessible chromatin sequencing and identification has enabled deciphering the epigenetic information encoded in chromatin, revealing accessible promoters, enhancers, nucleosome positioning, transcription factor occupancy, and other chromosomal protein binding. The starting biological materials are often fixed using formaldehyde crosslinking. Here, we describe accessible chromatin library preparation from low numbers of formaldehyde-crosslinked cells using a modified nick translation method, where a nicking enzyme nicks one strand of DNA and DNA polymerase incorporates biotin-conjugated dATP, dCTP, and methyl-dCTP. Once the DNA is labeled, it can be isolated for NGS library preparation. We termed this method as universalNicE-seq (nicking enzyme-assisted sequencing). We also demonstrate a single tube method that enables direct NGS library preparation from low cell numbers without DNA purification. Furthermore, we demonstrated universalNicE-seq on FFPE tissue section sample.

NicE-viewSeq: An Integrative Visualization and Genomics Method to Detect Accessible Chromatin in Fixed Cells.

A novel genome-wide accessible chromatin visualization, quantitation, and sequencing method is described, which allows in situ fluorescence visualization and sequencing of the accessible chromatin in the mammalian cell. The cells are fixed by formaldehyde crosslinking, and processed using a modified nick translation method, where a nicking enzyme nicks one strand of DNA, and DNA polymerase incorporates biotin-conjugated dCTP, 5-methyl-dCTP, Fluorescein-12-dATP or Texas Red-5-dATP, dGTP, and dTTP. This allows accessible chromatin DNA to be labeled for visualization and on bead NGS library preparation. This technology allows cellular level chromatin accessibility quantification and genomic analysis of the epigenetic information in the chromatin, particularly accessible promoter, enhancers, nucleosome positioning, transcription factor occupancy, and other chromosomal protein binding.

Mechanical codes of chemical-scale specificity in DNA motifs.

In gene transcription, certain sequences of double-stranded (ds)DNA play a vital role in nucleosome positioning and expression initiation. That dsDNA is deformed to various extents in these processes leads us to ask: Could the genomic DNA also have sequence specificity in its chemical-scale mechanical properties? We approach this question using statistical machine learning to determine the rigidity between DNA chemical moieties. What emerges for the polyA, polyG, TpA, and CpG sequences studied here is a unique trigram that contains the quantitative mechanical strengths between bases and along the backbone. In a way, such a sequence-dependent trigram could be viewed as a DNA mechanical code. Interestingly, we discover a compensatory competition between the axial base-stacking interaction and the transverse base-pairing interaction, and such a reciprocal relationship constitutes the most discriminating feature of the mechanical code. Our results also provide chemical-scale understanding for experimental observables. For example, the long polyA persistence length is shown to have strong base stacking while its complement (polyAc) exhibits high backbone rigidity. The mechanical code concept enables a direct reading of the physical interactions encoded in the sequence which, with further development, is expected to shed new light on DNA allostery and DNA-binding drugs.

Mapping Nucleosome Location Using FS-Seq.

The organization of nucleosomes in eukaryotic chromatin is thought to play a critical role in the regulation of the biological function of the chromatin. Because of this potential role in regulation, a number of techniques have been developed, which combine chromatin fragmentation around nucleosomes with next-generation sequencing to map the location of nucleosomes in chromatin. In this section, a procedure using a kit from New England Biolabs (NEB NEXT Ultra II FS DNA library prep Kit) to fragment chromatin in preparation for next-generation sequencing is described and compared to other available procedures for mapping nucleosome location.

Characterizing chromatin interactions of regulatory elements and nucleosome positions, using Hi-C, Micro-C, and promoter capture Micro-C

BackgroundRegulatory elements such as promoters, enhancers, and insulators interact each other to mediate molecular processes. To capture chromatin interactions of regulatory elements, 3C-derived methods such as Hi-C and Micro-C are developed. Here, we generated and analyzed Hi-C, Micro-C, and promoter capture Micro-C datasets with different sequencing depths to study chromatin interactions of regulatory elements and nucleosome positions in human prostate cancer cells.ResultsCompared to Hi-C, Micro-C identifies more high-resolution loops, including ones around structural variants. By evaluating the effect of sequencing depth, we revealed that more than 2 billion reads of Micro-C are needed to detect chromatin interactions at 1 kb resolution. Moreover, we found that deep-sequencing identifies additional long-range loops that are longer than 1 Mb in distance. Furthermore, we found that more than 50% of the loops are involved in insulators while less than 10% of the loops are promoter–enhancer loops. To comprehensively capture chromatin interactions that promoters are involved in, we performed promoter capture Micro-C. Promoter capture Micro-C identifies loops near promoters with a lower amount of sequencing reads. Sequencing of 160 million reads of promoter capture Micro-C resulted in reaching a plateau of identifying loops. However, there was still a subset of promoters that are not involved in loops even after deep-sequencing. By integrating Micro-C with NOMe-seq and ChIP-seq, we found that active promoters involved in loops have a more accessible region with lower levels of DNA methylation and more highly phased nucleosomes, compared to active promoters that are not involved in loops.ConclusionWe determined the required sequencing depth for Micro-C and promoter capture Micro-C to generate high-resolution chromatin interaction maps and loops. We also investigated the effect of sequencing coverage of Hi-C, Micro-C, and promoter capture Micro-C on detecting chromatin loops. Our analyses suggest the presence of distinct regulatory element groups, which are differently involved in nucleosome positions and chromatin interactions. This study does not only provide valuable insights on understanding chromatin interactions of regulatory elements, but also present guidelines for designing research projects on chromatin interactions among regulatory elements.

Tup1 is critical for transcriptional repression in Quiescence in S. cerevisiae

Upon glucose starvation, S. cerevisiae shows a dramatic alteration in transcription, resulting in wide-scale repression of most genes and activation of some others. This coincides with an arrest of cellular proliferation. A subset of such cells enters quiescence, a reversible non-dividing state. Here, we demonstrate that the conserved transcriptional corepressor Tup1 is critical for transcriptional repression after glucose depletion. We show that Tup1-Ssn6 binds new targets upon glucose depletion, where it remains as the cells enter the G0 phase of the cell cycle. In addition, we show that Tup1 represses a variety of glucose metabolism and transport genes. We explored how Tup1 mediated repression is accomplished and demonstrated that Tup1 coordinates with the Rpd3L complex to deacetylate H3K23. We found that Tup1 coordinates with Isw2 to affect nucleosome positions at glucose transporter HXT family genes during G0. Finally, microscopy revealed that a quarter of cells with a Tup1 deletion contain multiple DAPI puncta. Taken together, these findings demonstrate the role of Tup1 in transcriptional reprogramming in response to environmental cues leading to the quiescent state.

Manipulating chromatin architecture in C. elegans

BackgroundNucleosome-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.ResultsIn 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.ConclusionsUsing 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.

Nanopore Sensing of DNA-Histone Complexes on Nucleosome Arrays.

The location of nucleosomes in DNA and their structural stability are critical in regulating DNA compaction, site accessibility, and epigenetic gene regulation. Here, we combine the nanopore platform-based fast and label-free single-molecule detection technique with a voltage-dependent force rupture assay to detect distinct structures on nucleosomal arrays and then to induce breakdown of individual nucleosome complexes. Specifically, we demonstrate direct measurement of distinct nucleosome structures present on individual 12-mer arrays. A detailed event analysis showed that nucleosomes are present as a combination of complete and partial structures, during translocation through the pore. By comparing with the voltage-dependent translocation of the mononucleosomes, we find that the partial nucleosomes result from voltage-dependent structural disintegration of nucleosomes. High signal-to-noise detection of heterogeneous levels in translocation of 12-mer array molecules quantifies the heterogeneity and nucleosomal substructure sizes on the arrays. These results facilitate the understanding of electrostatic interactions responsible for the integrity of the nucleosome structure and possible mechanisms of its unraveling by chromatin remodeling enzymes. This study also has potential applications in chromatin profiling.

DNA Sequence-Dependent Properties of Nucleosome Positioning in Regions of Distinct Chromatin States in Mouse Embryonic Stem Cells.

Chromatin architecture is orchestrated, and plays crucial roles during the developmental process by regulating gene expression. In embryonic stem cells (ESCs), three types of chromatin states, including active, repressive and poised states, were previously identified and characterized with specific chromatin modification marks and different transcription activity, but it is largely unknown how nucleosomes are organized in these chromatin states. In this study, by using a DNA deformation energy model, we investigated the sequence-dependent nucleosome organization within the chromatin states in mouse ESCs. The results revealed that: (1) compared with poised genes, active genes are characterized with a higher level of nucleosome occupancy around their transcription start sites (TSS) and transcription termination sites (TTS), and both types of genes do not have a nucleosome-depleted region at their TTS, contrasting with the MNase-seq based result; (2) based on our previous DNA bending energy model, we developed an improved model capable of predicting both rotational positioning and nucleosome occupancy determined by a chemical mapping approach; (3) DNA bending-energy-based analyses demonstrated that the fragile nucleosomes positioned at both gene ends could be explained largely by enhanced rotational positioning signals encoded in DNA, but nucleosome phasing around the TSS of active genes was not determined by sequence preference; (4) the nucleosome occupancy landscape around the binding sites of some developmentally important transcription factors known to bind with different chromatin contexts, was also successfully predicted; (5) the difference of nucleosome occupancy around the TSS between CpG-rich and CpG-poor promoters was partly captured by our sequence-dependent model. Taken together, by developing an improved deformation-energy-based model, we revealed some sequence-dependent properties of the nucleosome arrangements in regions of distinct chromatin states in mouse ESCs.

Nucleosome Patterns in Circulating Tumor DNA Reveal Transcriptional Regulation of Advanced Prostate Cancer Phenotypes.

This study provides insights into the dynamics of nucleosome positioning and gene regulation associated with cancer phenotypes that can be ascertained from ctDNA. New methods for classification in phenotype mixtures extend the utility of ctDNA beyond assessments of somatic DNA alterations with important implications for molecular classification and precision oncology. This article is highlighted in the In This Issue feature, p. 517.

Multi-Layered Regulations on the Chromatin Architectures: Establishing the Tight and Specific Responses of Fission Yeast fbp1 Gene Transcription

Transcriptional regulation is pivotal for all living organisms and is required for adequate response to environmental fluctuations and intercellular signaling molecules. For precise regulation of transcription, cells have evolved regulatory systems on the genome architecture, including the chromosome higher-order structure (e.g., chromatin loops), location of transcription factor (TF)-binding sequences, non-coding RNA (ncRNA) transcription, chromatin configuration (e.g., nucleosome positioning and histone modifications), and the topological state of the DNA double helix. To understand how these genome-chromatin architectures and their regulators establish tight and specific responses at the transcription stage, the fission yeast fbp1 gene has been analyzed as a model system for decades. The fission yeast fbp1 gene is tightly repressed in the presence of glucose, and this gene is induced by over three orders of magnitude upon glucose starvation with a cascade of multi-layered regulations on various levels of genome and chromatin architecture. In this review article, we summarize the multi-layered transcriptional regulatory systems revealed by the analysis of the fission yeast fbp1 gene as a model system.

Cohesin and CTCF complexes mediate contacts in chromatin loops depending on nucleosome positions

The spatial organization of the eukaryotic genome plays an important role in regulating transcriptional activity. In the nucleus, chromatin forms loops that assemble into fundamental units called topologically associating domains that facilitate or inhibit long-range contacts. These loops are formed and held together by the ring-shaped cohesin protein complex, and this can involve binding of CCCTC-binding factor (CTCF). High-resolution conformation capture experiments provide the frequency at which two DNA fragments physically associate in three-dimensional space. However, technical limitations of this approach, such as low throughput, low resolution, or noise in contact maps, make data interpretation and identification of chromatin intraloop contacts, e.g., between distal regulatory elements and their target genes, challenging. Herein, an existing coarse-grained model of chromatin at single-nucleosome resolution was extended by integrating potentials describing CTCF and cohesin. We performed replica-exchange Monte Carlo simulations with regularly spaced nucleosomes and experimentally determined nucleosome positions in the presence of cohesin-CTCF, as well as depleted systems as controls. In fully extruded loops caused by the presence of cohesin and CTCF, the number of contacts within the formed loops was increased. The number and types of these contacts were impacted by the nucleosome distribution and loop size. Microloops were observed within cohesin-mediated loops due to thermal fluctuations without additional influence of other factors, and the number, size, and shape of microloops were determined by nucleosome distribution and loop size. Nucleosome positions directly affect the spatial structure and contact probability within a loop, with presumed consequences for transcriptional activity.

DeepNup: Prediction of Nucleosome Positioning from DNA Sequences Using Deep Neural Network.

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.

Global early replication disrupts gene expression and chromatin conformation in a single cell cycle

BackgroundThe early embryonic divisions of many organisms, including fish, flies, and frogs, are characterized by a very rapid S-phase caused by high rates of replication initiation. In somatic cells, S-phase is much longer due to both a reduction in the total number of initiation events and the imposition of a temporal order of origin activation. The physiological importance of changes in the rate and timing of replication initiation in S-phase remains unclear.ResultsHere we assess the importance of the temporal control of replication initiation using a conditional system in budding yeast to drive the early replication of the majority of origins in a single cell cycle. We show that global early replication disrupts the expression of over a quarter of all genes. By deleting individual origins, we show that delaying replication is sufficient to restore normal gene expression, directly implicating origin firing control in this regulation. Global early replication disrupts nucleosome positioning and transcription factor binding during S-phase, suggesting that the rate of S-phase is important to regulate the chromatin landscape.ConclusionsTogether, these data provide new insight into the role of the temporal control of origin firing during S-phase for coordinating replication, gene expression, and chromatin establishment as occurs in the early embryo.

Multiplexing mechanical and translational cues on genes

The genetic code gives precise instructions on how to translate codons into amino acids. Due to the degeneracy of the genetic code—18 out of 20 amino acids are encoded for by more than one codon—more information can be stored in a basepair sequence. Indeed, various types of additional information have been discussed in the literature, e.g., the positioning of nucleosomes along eukaryotic genomes and the modulation of the translating efficiency in ribosomes to influence cotranslational protein folding. The purpose of this study is to show that it is indeed possible to carry more than one additional layer of information on top of a gene. In particular, we show how much translation efficiency and nucleosome positioning can be adjusted simultaneously without changing the encoded protein. We achieve this by mapping genes on weighted graphs that contain all synonymous genes, and then finding shortest paths through these graphs. This enables us, for example, to readjust the disrupted translational efficiency profile after a gene has been introduced from one organism (e.g., human) into another (e.g., yeast) without greatly changing the nucleosome landscape intrinsically encoded by the DNA molecule.