Nucleosomal Arrays Research Articles

Engineered Chromatin Remodeling Proteins for Precise Nucleosome Positioning

SUMMARYRegulation of chromatin structure is essential for controlling access of DNA to factors that require association with specific DNA sequences. Here we describe the development and validation of engineered chromatin remodeling proteins (E-ChRPs) for inducing programmable changes in nucleosome positioning by design. We demonstrate that E-ChRPs function both in vitro and in vivo to specifically reposition target nucleosomes and entire nucleosomal arrays. We show that induced, systematic positioning of nucleosomes over yeast Ume6 binding sites leads to Ume6 exclusion, hyperacetylation, and transcriptional induction at target genes. We also show that programmed global loss of nucleosome-free regions at Reb1 targets is generally inhibitory with mildly repressive transcriptional effects. E-ChRPs are compatible with multiple targeting modalities, including the SpyCatcher and dCas9 moieties, resulting in high versatility and enabling diverse future applications. Thus, engineered chromatin remodeling proteins represent a simple and robust means to probe and disrupt DNA-dependent processes in different chromatin contexts.

CTCF-dependent chromatin boundaries formed by asymmetric nucleosome arrays with decreased linker length.

The CCCTC-binding factor (CTCF) organises the genome in 3D through DNA loops and in 1D by setting boundaries isolating different chromatin states, but these processes are not well understood. Here we investigate chromatin boundaries in mouse embryonic stem cells, defined by the regions with decreased Nucleosome Repeat Length (NRL) for ∼20 nucleosomes near CTCF sites, affecting up to 10% of the genome. We found that the nucleosome-depleted region (NDR) near CTCF is asymmetrically located >40 nucleotides 5′-upstream from the centre of CTCF motif. The strength of CTCF binding to DNA and the presence of cohesin is correlated with the decrease of NRL near CTCF, and anti-correlated with the level of asymmetry of the nucleosome array. Individual chromatin remodellers have different contributions, with Snf2h having the strongest effect on the NRL decrease near CTCF and Chd4 playing a major role in the symmetry breaking. Upon differentiation, a subset of preserved, common CTCF sites maintains asymmetric nucleosome pattern and small NRL. The sites which lost CTCF upon differentiation are characterized by nucleosome rearrangement 3′-downstream, with unchanged NDR 5′-upstream of CTCF motifs. Boundaries of topologically associated chromatin domains frequently contain several inward-oriented CTCF motifs whose effects, described above, add up synergistically.

CTCF confers local nucleosome resiliency after DNA replication and during mitosis.

The access of Transcription Factors (TFs) to their cognate DNA binding motifs requires a precise control over nucleosome positioning. This is especially important following DNA replication and during mitosis, both resulting in profound changes in nucleosome organization over TF binding regions. Using mouse Embryonic Stem (ES) cells, we show that the TF CTCF displaces nucleosomes from its binding site and locally organizes large and phased nucleosomal arrays, not only in interphase steady-state but also immediately after replication and during mitosis. Correlative analyses suggest this is associated with fast gene reactivation following replication and mitosis. While regions bound by other TFs (Oct4/Sox2), display major rearrangement, the post-replication and mitotic nucleosome positioning activity of CTCF is not unique: Esrrb binding regions are also characterized by persistent nucleosome positioning. Therefore, selected TFs such as CTCF and Esrrb act as resilient TFs governing the inheritance of nucleosome positioning at regulatory regions throughout the cell-cycle.

Histone Modifications Regulate Chromatin Compartmentalization by Contributing to a Phase Separation Mechanism.

Eukaryotic chromosomes contain compartments of various functions, which are marked by and enriched with specific histone modifications. However, the molecular mechanisms by which these histone marks function in chromosome compartmentalization are poorly understood. Constitutive heterochromatin is a largely silent chromosome compartment characterized in part by H3K9me2 and 3. Here, we show that heterochromatin protein 1 (HP1), an H3K9me2 and 3 "reader," interacts with SUV39H1, an H3K9me2 and 3 "writer," and with TRIM28, an abundant HP1 scaffolding protein, to form complexes with increased multivalent engagement of H3K9me2 and 3-modified chromatin. H3K9me2 and 3-marked nucleosomal arrays and associated complexes undergo phase separation to form macromolecule-enriched liquid droplets. The droplets are reminiscent of heterochromatin as they are highly dense chromatin-containing structures that are resistant to DNase and exclude the general transcription factor TFIIB. Our data suggest a general mechanism by which histone marks regulate chromosome compartmentalization by promoting phase separation.

Chromatin Structure and the Pioneering Transcription Factor FOXA1 Regulate TDG-Mediated Removal of 5-Formylcytosine from DNA.

Although a functional relationship between active DNA demethylation and chromatin structure is often implied, direct experimental evidence is lacking. We investigated the relationship between chromatin structure and thymine DNA glycosylase (TDG) using chemically defined nucleosome arrays containing site-specifically positioned 5-formylcytosine (5fC) residues. We show that the extent of array compaction, as well as nucleosome positioning, dramatically influence the ability of TDG to excise 5fC from DNA, indicating that the chromatin structure is likely a key determinant of whether 5fC is removed from the genome or retained as an epigenetic mark. Furthermore, the H2A.Z/H3.3 double-variant nucleosome and the pioneering transcription factor forkhead box A1 (FOXA1), both of which are implicated in shaping the chromatin landscape during demethylation of tissue-specific enhancers, differentially regulate TDG activity on chromatin. Together, this work provides the first direct evidence that the higher order chromatin structure regulates active DNA demethylation through TDG and provides novel insights into the mechanism of 5fC turnover at enhancers.

Rouse model with transient intramolecular contacts on a timescale of seconds recapitulates folding and fluctuation of yeast chromosomes.

DNA folding and dynamics along with major nuclear functions are determined by chromosome structural properties, which remain, thus far, elusive in vivo. Here, we combine polymer modeling and single particle tracking experiments to determine the physico-chemical parameters of chromatin in vitro and in living yeast. We find that the motion of reconstituted chromatin fibers can be recapitulated by the Rouse model using mechanical parameters of nucleosome arrays deduced from structural simulations. Conversely, we report that the Rouse model shows some inconsistencies to analyze the motion and structural properties inferred from yeast chromosomes determined with chromosome conformation capture techniques (specifically, Hi-C). We hence introduce the Rouse model with Transient Internal Contacts (RouseTIC), in which random association and dissociation occurs along the chromosome contour. The parametrization of this model by fitting motion and Hi-C data allows us to measure the kinetic parameters of the contact formation reaction. Chromosome contacts appear to be transient; associated to a lifetime of seconds and characterized by an attractive energy of –0.3 to –0.5 kBT. We suggest attributing this energy to the occurrence of histone tail-DNA contacts and notice that its amplitude sets chromosomes in ‘theta’ conditions, in which they are poised for compartmentalization and phase separation.

Genome-wide reconstitution of chromatin transactions reveals that RSC preferentially disrupts H2AZ-containing nucleosomes.

Chromatin transactions are typically studied in vivo, or in vitro using artificial chromatin lacking the epigenetic complexity of the natural material. Attempting to bridge the gap between these approaches, we established a system for isolating the yeast genome as a library of mononucleosomes harboring the natural epigenetic signature, suitable for biochemical manipulation. Combined with deep sequencing, this library was used to investigate the stability of individual nucleosomes and, as proof of principle, the nucleosome preference of the chromatin remodeling complex, RSC. This approach uncovered a distinct preference of RSC for nucleosomes derived from regions with a high density of histone variant H2AZ, and this preference is indeed markedly diminished using nucleosomes from cells lacking H2AZ. The preference for H2AZ remodeling/nucleosome ejection can also be reconstituted with recombinant nucleosome arrays. Together, our data indicate that, despite being separated from their genomic context, individual nucleosomes can retain their original identity as promoter- or transcription start site (TSS)-nucleosomes. Besides shedding new light on substrate preference of the chromatin remodeler RSC, the simple experimental system outlined here should be generally applicable to the study of chromatin transactions.

Chromatin structures condensed by linker histones.

In eukaryotic cells, genomic DNA exists in the form of chromatin through association with histone proteins, which consist of four core histone (H2A, H2B, H3, and H4) families and one linker histone (H1) family. The core histones bind to DNA to form the nucleosome, the recurring structural unit of chromatin. The linker histone binds to the nucleosome to form the next structural unit of chromatin, the chromatosome, which occurs dominantly in metazoans. Linker histones also play an essential role in condensing chromatin to form higher order structures. Unlike the core histones in the formation of the nucleosome, the role of linker histone in the formation of the chromatosome and high-order chromatin structure is not well understood. Nevertheless, exciting progress in the structural studies of chromatosomes and nucleosome arrays condensed by linker histones has been made in the last several years. In this mini-review, we discuss these recent experimental results and provide some perspectives for future studies.

Post-translational modifications and chromatin dynamics.

The dynamic structure of chromatin is linked to gene regulation and many other biological functions. Consequently, it is of importance to understand the factors that regulate chromatin dynamics. While the in vivo analysis of chromatin has verified that histone post-translational modifications play a role in modulating DNA accessibility, the complex nuclear environment and multiplicity of modifications prevents clear conclusions as to how individual modifications influence chromatin dynamics in the cell. For this reason, in vitro analyses of model reconstituted nucleosomal arrays has been pivotal in understanding the dynamic nature of chromatin compaction and the affects that specific post-translational modifications can have on the higher order chromatin structure. In this mini-review, we briefly describe the dynamic chromatin structures that have been observed in vitro and the environmental conditions that give rise to these various conformational states. Our focus then turns to a discussion of the specific histone post-translational modifications that have been shown to alter formation of these higher order chromatin structures in vitro and how this may relate to the biological state and accessibility of chromatin in vivo.

Nucleosome spacing periodically modulates nucleosome chain folding and DNA topology in circular nucleosome arrays

The length of linker DNA that separates nucleosomes is highly variable, but its mechanistic role in modulating chromatin structure and functions remains unknown. Here, we established an experimental system using circular arrays of positioned nucleosomes to investigate whether variations in nucleosome linker length could affect nucleosome folding, self-association, and interactions. We conducted EM, DNA topology, native electrophoretic assays, and Mg2+-dependent self-association assays to study intrinsic folding of linear and circular nucleosome arrays with linker DNA length of 36 bp and 41 bp (3.5 turns and 4 turns of DNA double helix, respectively). These experiments revealed that potential artifacts arising from open DNA ends and full DNA relaxation in the linear arrays do not significantly affect overall chromatin compaction and self-association. We observed that the 0.5 DNA helical turn difference between the two DNA linker lengths significantly affects DNA topology and nucleosome interactions. In particular, the 41-bp linkers promoted interactions between any two nucleosome beads separated by one bead as expected for a zigzag fiber, whereas the 36-bp linkers promoted interactions between two nucleosome beads separated by two other beads and also reduced negative superhelicity. Monte Carlo simulations accurately reproduce periodic modulations of chromatin compaction, DNA topology, and internucleosomal interactions with a 10-bp periodicity. We propose that the nucleosome spacing and associated chromatin structure modulations may play an important role in formation of different chromatin epigenetic states, thus suggesting implications for how chromatin accessibility to DNA-binding factors and the RNA transcription machinery is regulated.

Nucleosome positioning and spacing: from genome-wide maps to single arrays.

The positioning of nucleosomes relative to DNA and their neighboring nucleosomes represents a fundamental layer of chromatin organization. Changes in nucleosome positioning and spacing affect the accessibility of DNA to regulatory factors and the formation of higher order chromatin structures. Sequencing of mononucleosomal fragments allowed mapping nucleosome positions on a genome-wide level in many organisms. This revealed that successions of evenly spaced and well-positioned nucleosomes-so called phased nucleosome arrays-occur at the 5' end of many active genes and in the vicinity of transcription factor and other protein binding sites. Phased arrays arise from the interplay of barrier elements on the DNA, which position adjacent nucleosomes, and the nucleosome spacing activity of ATP-dependent chromatin remodelers. A shortcoming of classic mononucleosomal mapping experiments is that they only reveal nucleosome spacing and array regularity at select sites in the genome with well-positioned nucleosomes. However, new technological approaches elucidate nucleosome array structure throughout the genome and with single-cell resolution. In the future, it will be interesting to see whether changes in nucleosome array regularity and spacing contribute to the formation of higher order chromatin structures and the spatial organization of the genome in vivo.

Chromatin Folding Heterogeneity Inferred from High-Resolution Nucleosome Structures

DNA in eukaryotic cell nuclei occurs in a highly compact chromatin structure where the basic packaging unit is the nucleosome. This dynamic structure displays a wide range in compaction and provides a regulatory mechanism for many biologically relevant processes, such as gene expression. Since the first description of nucleosome-decorated DNA as beads on a string condensing into a 30 nm fiber in vitro, there have been many attempts to understand the nature of chromatin compaction and to create models at this scale. However, the proposed models for the 3D organization of the nucleosomes and intervening DNA vary quite significantly, and the very existence and relevance of a 30 nm structure in vivo has been highly debated. An analysis of the available high-resolution nucleosome core particle (NCP) structures shows subtle, yet significant differences in DNA wrapping around the histone octamer core, potentially due to DNA and histone modifications, presence of NCP-binding proteins, experimental crystallization/cryoEM conditions, intrinsic flexibility, etc. Monte Carlo simulations of regular nucleosomal arrays using a meso-scale representation of chromatin suggest that these local differences can result in large changes in global structure, comparable to the effect of changes in nucleosome spacing by about 2-3 base pairs. Our results suggest that a regular nucleosome array with a 177-base pair repeat can display either a loose three-stack or a more compact two-stack arrangement, depending on the mode of DNA wrapping in the nucleosome. Insights from our work may help bridge the significant divide in the proposed chromatin models based on various experimental results.

Transcription factor activity and nucleosome organization in mitosis.

Mitotic bookmarking transcription factors (BFs) maintain the capacity to bind to their targets during mitosis, despite major rearrangements of the chromatin. While they were thought to propagate gene regulatory information through mitosis by statically occupying their DNA targets, it has recently become clear that BFs are highly dynamic in mitotic cells. This represents both a technical and a conceptual challenge to study and understand the function of BFs: First, formaldehyde has been suggested to be unable to efficiently capture these transient interactions, leading to profound contradictions in the literature; and second, if BFs are not permanently bound to their targets during mitosis, it becomes unclear how they convey regulatory information to daughter cells. Here, comparing formaldehyde to alternative fixatives we clarify the nature of the chromosomal association of previously proposed BFs in embryonic stem cells: While ESRRB can be considered as a canonical BF that binds at selected regulatory regions in mitosis, SOX2 and POU5F1 (also known as OCT4) establish DNA sequence-independent interactions with the mitotic chromosomes, either throughout the chromosomal arms (SOX2) or at pericentromeric regions (POU5F1). Moreover, we show that ordered nucleosomal arrays are retained during mitosis at ESRRB bookmarked sites, whereas regions losing transcription factor binding display a profound loss of order. By maintaining nucleosome positioning during mitosis, ESRRB might ensure the rapid post-mitotic re-establishment of functional regulatory complexes at selected enhancers and promoters. Our results provide a mechanistic framework that reconciles dynamic mitotic binding with the transmission of gene regulatory information across cell division.

CTCF sites display cell cycle-dependent dynamics in factor binding and nucleosome positioning.

CCCTC-binding factor (CTCF) plays a key role in the formation of topologically associating domains (TADs) and loops in interphase. During mitosis TADs are absent, but how TAD formation is dynamically controlled during the cell cycle is not known. Several contradicting observations have been made regarding CTCF binding to mitotic chromatin using both genomics- and microscopy-based techniques. Here, we have used four different assays to address this debate. First, using 5C, we confirmed that TADs and CTCF loops are readily detected in interphase, but absent during prometaphase. Second, ATAC-seq analysis showed that CTCF sites display greatly reduced accessibility and lose the CTCF footprint in prometaphase, suggesting loss of CTCF binding and rearrangement of the nucleosomal array around the binding motif. In contrast, transcription start sites remain accessible in prometaphase, although adjacent nucleosomes can also become repositioned and occupy at least a subset of start sites during mitosis. Third, loss of site-specific CTCF binding was directly demonstrated using CUT&RUN. Histone modifications and histone variants are maintained in mitosis, suggesting a role in bookmarking of active CTCF sites. Finally, live-cell imaging, fluorescence recovery after photobleaching, and single molecule tracking showed that almost all CTCF chromatin binding is lost in prometaphase. Combined, our results demonstrate loss of CTCF binding to CTCF sites during prometaphase and rearrangement of the chromatin landscape around CTCF motifs. This, combined with loss of cohesin, would contribute to the observed loss of TADs and CTCF loops during mitosis and reveals that CTCF sites, key architectural cis-elements, display cell cycle stage–dependent dynamics in factor binding and nucleosome positioning.

The transition structure of chromatin fibers at the nanoscale probed by cryogenic electron tomography.

The naked DNA inside the nucleus interacts with proteins and RNAs forming a higher order chromatin structure to spatially and temporally control transcription in eukaryotic cells. The 30 nm chromatin fiber is one of the most important determinants of the regulation of eukaryotic transcription. However, the transition of chromatin from the 30 nm inactive higher order structure to the actively transcribed lower order nucleosomal arrays is unclear, which limits our understanding of eukaryotic transcription. Using a method to extract near-native eukaryotic chromatin, we revealed the chromatin structure at the transitional state from the 30 nm chromatin to multiple nucleosomal arrays by cryogenic electron tomography (cryo-ET). Reproducible electron microscopy images revealed that the transitional structure is a branching structure that the 30 nm chromatin hierarchically branches into lower order nucleosomal arrays, indicating chromatin compaction at different levels to control its accessibility during the interphase. We further observed that some of the chromatin fibers on the branching structure have a helix ribbon structure, while the others randomly twist together. Our finding of the chromatin helix ribbon structure on the extracted native chromatin revealed by cryo-ET indicates a complex higher order chromatin organization beyond the beads-on-a-string structure. The hierarchical branching and helix ribbon structure may provide mechanistic insights into how chromatin organization plays a central role in transcriptional regulation and other DNA-related biological processes during diseases such as cancer.

SIR proteins create compact heterochromatin fibers

Heterochromatin is a silenced chromatin region essential for maintaining genomic stability and driving developmental processes. The complicated structure and dynamics of heterochromatin have rendered it difficult to characterize. In budding yeast, heterochromatin assembly requires the SIR proteins-Sir3, believed to be the primary structural component of SIR heterochromatin, and the Sir2-4 complex, responsible for the targeted recruitment of SIR proteins and the deacetylation of lysine 16 of histone H4. Previously, we found that Sir3 binds but does not compact nucleosomal arrays. Here we reconstitute chromatin fibers with the complete complement of SIR proteins and use sedimentation velocity, molecular modeling, and atomic force microscopy to characterize the stoichiometry and conformation of SIR chromatin fibers. In contrast to fibers with Sir3 alone, our results demonstrate that SIR arrays are highly compact. Strikingly, the condensed structure of SIR heterochromatin fibers requires both the integrity of H4K16 and an interaction between Sir3 and Sir4. We propose a model in which a dimer of Sir3 bridges and stabilizes two adjacent nucleosomes, while a Sir2-4 heterotetramer interacts with Sir3 associated with a nucleosomal trimer, driving fiber compaction.

Genome-wide Rules of Nucleosome Phasing in Drosophila

Regular successions of positioned nucleosomes, or phased nucleosome arrays (PNAs), are predominantly known from transcriptional start sites (TSSs). It is unclear whether PNAs occur elsewhere in the genome. To generate a comprehensive inventory of PNAs for Drosophila, we applied spectral analysis to nucleosome maps and identified thousands of PNAs throughout the genome. About half of them are not near TSSs and are strongly enriched foran uncharacterized sequence motif. Through genome-wide reconstitution of physiological chromatin in Drosophila embryo extracts, we uncovered the molecular basis of PNA formation. We identified Phaser, an unstudied zinc finger protein that positions nucleosomes flanking the motif. It also revealed how the global activity of the chromatin remodelers CHRAC/ACF, together with local barrier elements, generates islands of regular phasing throughout the genome. Our work demonstrates the potential of chromatin assembly by embryo extracts as a powerful tool to reconstitute chromatin features on a global scale invitro.

Single-molecule nascent RNA sequencing identifies regulatory domain architecture at promoters and enhancers.

Eukaryotic RNA Polymerase II has been found at both promoters and distal enhancers, suggesting additional functions beyond mRNA production. To understand this role, we sequenced nascent RNAs at single-molecule resolution to unravel the interplay between Pol2 initiation, capping, and pausing genome-wide. Our analyses reveal two pause classes that are associated with different RNA capping profiles. More proximal pausing is associated with less complete capping, less elongation, and a more enhancer-like complement of transcription factors than later pausing. Unexpectedly, Transcription Start Sites (TSSes) are predominantly found in constellations composed of multiple divergent pairs. TSS clusters are intimately associated with precise arrays of nucleosomes, and correspond with boundaries of transcription factor binding and chromatin modification at promoters and enhancers. TSS architecture is remarkably similar after the dramatic transcriptional changes caused by heat‬ stress. Together, our results suggest that promoter- and enhancer-associated Pol2 is a regulatory nexus for integrating information across TSS ensembles.

Rigid Basepair Monte Carlo Simulations of One-Start and Two-Start Chromatin Fiber Unfolding by Force

The organization of chromatin in 30 nm fibers remains a topic of debate. Here, we quantify the mechanical properties of the linker DNA and evaluate the impact of these properties on chromatin fiber folding. We extended a rigid basepair DNA model to include (un)wrapping of nucleosomal DNA and (un)stacking of nucleosomes in one-start and two-start chromatin fibers. Monte Carlo simulations that mimic single-molecule force spectroscopy experiments of folded nucleosomal arrays reveal different stages of unfolding as a function of force and are largely consistent with a two-start folding for 167 and one-start folding for 197 nucleosome repeat length fibers. The major insight is that nucleosome unstacking and subsequent unwrapping is not necessary to obtain quantitative agreement with experimental force extension curves up to the overstretching plateau of folded chromatin fibers at 3–5 pN. Nucleosome stacking appears better accommodated in one-start than in two-start conformations, and we suggest that this difference can compensate the increased energy for bending the linker DNA. Overall, these simulations capture the dynamic structure of chromatin fibers while maintaining realistic physical properties of the linker DNA.

Principles of nucleosome organization revealed by single-cell micrococcal nuclease sequencing.

Nucleosome positioning is critical to chromatin accessibility and is associated with gene expression programs in cells1-3. Previous nucleosome mapping methods assemble profiles from cell populations and reveal a cell-averaged pattern: nucleosomes are positioned and form a phased array that surrounds the transcription start sites of active genes3-6 and DNase I hypersensitive sites7. However, even in a homogenous population of cells, cells exhibit heterogeneity in expression in response to active signalling8,9 that may be related to heterogeneity in chromatin accessibility10-12. Here we report a technique, termed single-cell micrococcal nuclease sequencing (scMNase-seq), that can be used to simultaneously measure genome-wide nucleosome positioning and chromatin accessibility in single cells. Application of scMNase-seq to NIH3T3 cells, mouse primary naive CD4 T cells and mouse embryonic stem cells reveals two principles of nucleosome organization: first, nucleosomes in heterochromatin regions, or that surround the transcription start sites of silent genes, show large variation in positioning across different cells but are highly uniformly spaced along the nucleosome array; and second, nucleosomes that surround the transcription start sites of active genes and DNase I hypersensitive sites show little variation in positioning across different cells but are relatively heterogeneously spaced along the nucleosome array. We found a bimodal distribution of nucleosome spacing at DNase I hypersensitive sites, which corresponds to inaccessible and accessible states and is associated with nucleosome variation and variation in accessibility across cells. Nucleosome variation is smaller within single cells than across cells, and smaller within the same cell type than across cell types. A large fraction of naive CD4 T cells and mouse embryonic stem cells shows depleted nucleosome occupancy at the de novo enhancers detected in their respective differentiated lineages, revealing the existence of cells primed for differentiation to specific lineages in undifferentiated cell populations.