Nucleosome Repeat Length Research Articles

Epigenetic engineering of yeast reveals dynamic molecular adaptation to methylation stress and genetic modulators of specific DNMT3 family members.

Cytosine methylation is a ubiquitous modification in mammalian DNA generated and maintained by several DNA methyltransferases (DNMTs) with partially overlapping functions and genomic targets. To systematically dissect the factors specifying each DNMT’s activity, we engineered combinatorial knock-in of human DNMT genes in Komagataella phaffii, a yeast species lacking endogenous DNA methylation. Time-course expression measurements captured dynamic network-level adaptation of cells to DNMT3B1-induced DNA methylation stress and showed that coordinately modulating the availability of S-adenosyl methionine (SAM), the essential metabolite for DNMT-catalyzed methylation, is an evolutionarily conserved epigenetic stress response, also implicated in several human diseases. Convolutional neural networks trained on genome-wide CpG-methylation data learned distinct sequence preferences of DNMT3 family members. A simulated annealing interpretation method resolved these preferences into individual flanking nucleotides and periodic poly(A) tracts that rotationally position highly methylated cytosines relative to phased nucleosomes. Furthermore, the nucleosome repeat length defined the spatial unit of methylation spreading. Gene methylation patterns were similar to those in mammals, and hypo- and hypermethylation were predictive of increased and decreased transcription relative to control, respectively, in the absence of mammalian readers of DNA methylation. Introducing controlled epigenetic perturbations in yeast thus enabled characterization of fundamental genomic features directing specific DNMT3 proteins.

The embryonic linker histone dBigH1 alters the functional state of active chromatin.

Linker histones H1 are principal chromatin components, whose contribution to the epigenetic regulation of chromatin structure and function is not fully understood. In metazoa, specific linker histones are expressed in the germline, with female-specific H1s being normally retained in the early-embryo. Embryonic H1s are present while the zygotic genome is transcriptionally silent and they are replaced by somatic variants upon activation, suggesting a contribution to transcriptional silencing. Here we directly address this question by ectopically expressing dBigH1 in Drosophila S2 cells, which lack dBigH1. We show that dBigH1 binds across chromatin, replaces somatic dH1 and reduces nucleosome repeat length (NRL). Concomitantly, dBigH1 expression down-regulates gene expression by impairing RNApol II binding and histone acetylation. These effects depend on the acidic N-terminal ED-domain of dBigH1 since a truncated form lacking this domain binds across chromatin and replaces dH1 like full-length dBigH1, but it does not affect NRL either transcription. In vitro reconstitution experiments using Drosophila preblastodermic embryo extracts corroborate these results. Altogether these results suggest that the negatively charged N-terminal tail of dBigH1 alters the functional state of active chromatin compromising transcription.

Chromatin fibers stabilize nucleosomes under torsional stress

Torsional stress generated during DNA replication and transcription has been suggested to facilitate nucleosome unwrapping and thereby the progression of polymerases. However, the propagation of twist in condensed chromatin remains yet unresolved. Here, we measure how force and torque impact chromatin fibers with a nucleosome repeat length of 167 and 197. We find that both types of fibers fold into a left-handed superhelix that can be stabilized by positive torsion. We observe that the structural changes induced by twist were reversible, indicating that chromatin has a large degree of elasticity. Our direct measurements of torque confirmed the hypothesis of chromatin fibers as a twist buffer. Using a statistical mechanics-based torsional spring model, we extracted values of the chromatin twist modulus and the linking number per stacked nucleosome that were in good agreement with values measured here experimentally. Overall, our findings indicate that the supercoiling generated by DNA-processing enzymes, predicted by the twin-supercoiled domain model, can be largely accommodated by the higher-order structure of chromatin.

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.

Modelling and DNA topology of compact 2-start and 1-start chromatin fibres.

We have investigated the structure of the most compact 30-nm chromatin fibres by modelling those with 2-start or 1-start crossed-linker organisations. Using an iterative procedure we obtained possible structural solutions for fibres of the highest possible compaction permitted by physical constraints, including the helical repeat of linker DNA. We find that this procedure predicts a quantized nucleosome repeat length (NRL) and that only fibres with longer NRLs (≥197 bp) can more likely adopt the 1-start organisation. The transition from 2-start to 1-start fibres is consistent with reported differing binding modes of the linker histone. We also calculate that in 1-start fibres the DNA constrains more torsion (as writhe) than 2-start fibres with the same NRL and that the maximum constraint obtained is in accord with previous experimental results. We posit that the coiling of the fibre is driven by overtwisting of linker DNA which, in the most compact forms - for example, in echinoderm sperm and avian erythrocytes - could adopt a helical repeat of ∼10 bp/turn. We argue that in vivo the total twist of linker DNA could be modulated by interaction with other abundant chromatin-associated proteins and by epigenetic modifications of the C-terminal tail of linker histones.

Sensitive effect of linker histone binding mode and subtype on chromatin condensation.

The complex role of linker histone (LH) on chromatin compaction regulation has been highlighted by recent discoveries of the effect of LH binding variability and isoforms on genome structure and function. Here we examine the effect of two LH variants and variable binding modes on the structure of chromatin fibers. Our mesoscale modeling considers oligonucleosomes with H1C and H1E, bound in three different on and off-dyad modes, and spanning different LH densities (0.5–1.6 per nucleosome), over a wide range of physiologically relevant nucleosome repeat lengths (NRLs). Our studies reveal an LH-variant and binding-mode dependent heterogeneous ensemble of fiber structures with variable packing ratios, sedimentation coefficients, and persistence lengths. For maximal compaction, besides dominantly interacting with parental DNA, LHs must have strong interactions with nonparental DNA and promote tail/nonparental core interactions. An off-dyad binding of H1E enables both; others compromise compaction for bendability. We also find that an increase of LH density beyond 1 is best accommodated in chromatosomes with one on-dyad and one off-dyad LH. We suggest that variable LH binding modes and concentrations are advantageous, allowing tunable levels of chromatin condensation and DNA accessibility/interactions. Thus, LHs add another level of epigenetic regulation of chromatin.

Structure and Dynamics of the Telomeric Nucleosome and Chromatin

Eukaryotic genomic DNA is condensed into chromatin: 146bp DNA wraps around the histone octamer (HO) comprising two copies each of the core histones H2A, H2B, H3 and H4 forming the nucleosome core particle (NCP). A string of nucleosomes folds into the so-called called “30nm” chromatin fibre, which compacts into metaphase chromosomes. Human telomeres constitute the protective structure at the end of the chromosomes with repetitive TTAGGG sequences that are about 10kbp. Little is known about the consequences of this unique sequence for chromatin structural and dynamic properties. Although it is established that telomeric DNA is packaged in chromatin with an unusually short (157 bp) nucleosome repeat length (NRL) and can form nucleosomes and chromatin, almost nothing is known about the telomeric nucleosome core and telomeric chromatin structure at the detailed molecular level. We have characterised the telomeric human HO 145 bp DNA NCP (Telo-NCP), using biophysical methods and determined its structure at 2.1 Å resolution with X-ray crystallography. Although the structure is largely similar to the well-known published atomic resolution NCP structures, several differences can be identified, e.g. DNA stretching on the Telo-NCP is different from that of the “601” (high-affinity nucleosome positioning sequence) and alpha satellite NCPs of the same length. We have designed and prepared 20×157 bp telomeric DNA template repeats, reconstituted these with human HO and performed characterisation of the structural and dynamic properties of the telomeric chromatin fibres with EM and single molecule magnetic tweezer experiments. Telomeric chromatin fibres are considerably more homogeneous than corresponding 157 bp repeat fibres reconstituted from 601-DNA array templates. However, the structural and dynamic properties of individual telomeric arrays saturated with 20 nucleosomes, appear similar to equivalent 601 chromatin fibres under the influence of force in single molecule pulling experiment.

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.

Genome-wide measurement of local nucleosome array regularity and spacing by nanopore sequencing.

The nature of chromatin as regular succession of nucleosomes has gained iconic status. However, since most nucleosomes in metazoans are poorly positioned it is unknown to which extent bulk genomic nucleosome repeat length reflects the regularity and spacing of nucleosome arrays at individual loci. We describe a new approach to map nucleosome array regularity and spacing through sequencing oligonucleosome-derived DNA by Illumina sequencing and emergent nanopore technology. In Drosophila cells, this revealed modulation of array regularity and nucleosome repeat length depending on functional chromatin states independently of nucleosome positioning and even in unmappable regions. We also found that nucleosome arrays downstream of silent promoters are considerably more regular than those downstream of highly expressed ones, despite more extensive nucleosome phasing of the latter. Our approach is generally applicable and provides an important parameter of chromatin organization that so far had been missing.

A Novel Method to Generate MNase Ladders Reveal Rapid Chromatin Remodeling upon Gametogenesis and Mating in Chlamydomonas

To circumvent nuclei isolation for nucleosomal mapping of wild-type (cell walled) algal cells, we developed a quick and versatile methodology, by abrasion of whole cells (Chlamydomonas, Scenedesmus and yeast), allowing Micrococcal Nuclease (MNase) direct access to nuclear chromatin, in situ. Varying parameters such as bead abrasion, vortex and incubation conditions, we optimized capture of an 'early digest' which may probe chromatin differentially, based on nucleosome accessibility. A comparison of such ladders across vegetative cells, gametes and zygotes revealed an increase in the average nucleosomal repeat length (+17-34nt) upon gametogenesis, indicating a trend of chromatin compaction. Using PCR, we compared promoter enrichment in increasing orders of fractionated nucleosomal repeats (mono-, di-, up to penta-), each differing in cleavability based on chromatin accessibility. Concordant with higher gene expression (mating locus), promoters revealed an enrichment in mono-nucleosomal fractions. Interestingly, the zygote specific gene, MT0828 displayed rapid remodelling from penta-nucleosomal enrichment when completely repressed (vegetative), to intermediate states during gametogenesis (24hrs), which finally shifted to being largely mono-nucleosomal, when induced (1h zygotes). Summarizing three candidate genes from the mating locus, we conclude that the MNase based 'Chromatin Accessibility Assay' can track a range of large-scale rapid chromatin remodelling transitions within the binaries of gene expression.

Resetting the Yeast Epigenome with Human Nucleosomes

Humans and yeast are separated by a billion years ofevolution, yet their conserved histones retain central roles in gene regulation. Here, we "reset" yeast to use core human nucleosomes in lieu of theirown (arare event taking 20days), which initially only worked with variant H3.1. The cells adapt byacquiring suppressor mutations in cell-division genes or by acquiring certain aneuploid states. Converting five histone residues to their yeast counterparts restored robust growth. We reveal that humanized nucleosomes are positioned according to endogenous yeast DNA sequence and chromatin-remodeling network, as judged by a yeast-like nucleosome repeat length. However, human nucleosomes have higher DNA occupancy, globally reduce RNA content, and slow adaptation to new conditions by delaying chromatin remodeling. These humanized yeasts (including H3.3) pose fundamental new questions about how chromatin is linked to many cell processes and provide a platform to study histone variants via yeast epigenome reprogramming.

Single-molecule force spectroscopy on histone H4 tail-cross-linked chromatin reveals fiber folding

The eukaryotic genome is highly compacted into a protein-DNA complex called chromatin. The cell controls access of transcriptional regulators to chromosomal DNA via several mechanisms that act on chromatin-associated proteins and provide a rich spectrum of epigenetic regulation. Elucidating the mechanisms that fold chromatin fibers into higher-order structures is therefore key to understanding the epigenetic regulation of DNA accessibility. Here, using histone H4-V21C and histone H2A-E64C mutations, we employed single-molecule force spectroscopy to measure the unfolding of individual chromatin fibers that are reversibly cross-linked through the histone H4 tail. Fibers with covalently linked nucleosomes featured the same folding characteristics as fibers containing wild-type histones but exhibited increased stability against stretching forces. By stabilizing the secondary structure of chromatin, we confirmed a nucleosome repeat length (NRL)-dependent folding. Consistent with previous crystallographic and cryo-EM studies, the obtained force-extension curves on arrays with 167-bp NRLs best supported an underlying structure consisting of zig-zag, two-start fibers. For arrays with 197-bp NRLs, we previously inferred solenoidal folding, which was further corroborated by force-extension curves of the cross-linked fibers. The different unfolding pathways exhibited by these two types of arrays and reported here extend our understanding of chromatin structure and its potential roles in gene regulation. Importantly, these findings imply that chromatin compaction by nucleosome stacking protects nucleosomal DNA from external forces up to 4 piconewtons.

Nucleosome repositioning during differentiation of a human myeloid leukemia cell line

ABSTRACTCell differentiation is associated with changes in chromatin organization and gene expression. In this study, we examine chromatin structure following differentiation of the human myeloid leukemia cell line (HL-60/S4) into granulocytes with retinoic acid (RA) or into macrophage with phorbol ester (TPA). We performed ChIP-seq of histone H3 and its modifications, analyzing changes in nucleosome occupancy, nucleosome repeat length, eu-/heterochromatin redistribution and properties of epichromatin (surface chromatin adjacent to the nuclear envelope). Nucleosome positions changed genome-wide, exhibiting a specific class of alterations involving nucleosome loss in extended (∼1kb) regions, pronounced in enhancers and promoters. Genes that lost nucleosomes at their promoters showed a tendency to be upregulated. On the other hand, nucleosome gain did not show simple effects on transcript levels. The average genome-wide nucleosome repeat length (NRL) did not change significantly with differentiation. However, we detected an approximate 10 bp NRL decrease around the haematopoietic transcription factor (TF) PU.1 and the architectural protein CTCF, suggesting an effect on NRL proximal to TF binding sites. Nucleosome occupancy changed in regions associated with active promoters in differentiated cells, compared with untreated HL-60/S4 cells. Epichromatin regions revealed an increased GC content and high nucleosome density compared with surrounding chromatin. Epichromatin showed depletion of major histone modifications and revealed enrichment with PML body-associated genes. In general, chromatin changes during HL-60/S4 differentiation appeared to be more localized to regulatory regions, compared with genome-wide changes among diverse cell types studied elsewhere.

NucTools: analysis of chromatin feature occupancy profiles from high-throughput sequencing data

BackgroundBiomedical applications of high-throughput sequencing methods generate a vast amount of data in which numerous chromatin features are mapped along the genome. The results are frequently analysed by creating binary data sets that link the presence/absence of a given feature to specific genomic loci. However, the nucleosome occupancy or chromatin accessibility landscape is essentially continuous. It is currently a challenge in the field to cope with continuous distributions of deep sequencing chromatin readouts and to integrate the different types of discrete chromatin features to reveal linkages between them.ResultsHere we introduce the NucTools suite of Perl scripts as well as MATLAB- and R-based visualization programs for a nucleosome-centred downstream analysis of deep sequencing data. NucTools accounts for the continuous distribution of nucleosome occupancy. It allows calculations of nucleosome occupancy profiles averaged over several replicates, comparisons of nucleosome occupancy landscapes between different experimental conditions, and the estimation of the changes of integral chromatin properties such as the nucleosome repeat length. Furthermore, NucTools facilitates the annotation of nucleosome occupancy with other chromatin features like binding of transcription factors or architectural proteins, and epigenetic marks like histone modifications or DNA methylation. The applications of NucTools are demonstrated for the comparison of several datasets for nucleosome occupancy in mouse embryonic stem cells (ESCs) and mouse embryonic fibroblasts (MEFs).ConclusionsThe typical workflows of data processing and integrative analysis with NucTools reveal information on the interplay of nucleosome positioning with other features such as for example binding of a transcription factor CTCF, regions with stable and unstable nucleosomes, and domains of large organized chromatin K9me2 modifications (LOCKs). As potential limitations and problems we discuss how inter-replicate variability of MNase-seq experiments can be addressed.

Plasmodium falciparum Nucleosomes Exhibit Reduced Stability and Lost Sequence Dependent Nucleosome Positioning.

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.

Enzymatic and chemical mapping of nucleosome distribution in purified micro- and macronuclei of the ciliated model organism, Tetrahymena thermophila.

Genomic distribution of the nucleosome, the basic unit of chromatin, contains important epigenetic information. To map nucleosome distribution in structurally and functionally differentiated micronucleus (MIC) and macronucleus (MAC) of the ciliate Tetrahymena thermophila, we have purified MIC and MAC and performed micrococcal nuclease (MNase) digestion as well as hydroxyl radical cleavage. Different factors that may affect MNase digestion were examined, to optimize mono-nucleosome production. Mono-nucleosome purity was further improved by ultracentrifugation in a sucrose gradient. As MNase concentration increased, nucleosomal DNA sizes in MIC and MAC converged on 147 bp, as expected for the nucleosome core particle. Both MNase digestion and hydroxyl radical cleavage consistently showed a nucleosome repeat length of ~200 bp in MAC of Tetrahymena, supporting ~50 bp of linker DNA. Our work has systematically tested methods currently available for mapping nucleosome distribution in Tetrahymena, and provided a solid foundation for future epigenetic studies in this ciliated model organism.

Histone H1 Limits DNA Methylation in Neurospora crassa.

Histone H1 variants, known as linker histones, are essential chromatin components in higher eukaryotes, yet compared to the core histones relatively little is known about their in vivo functions. The filamentous fungus Neurospora crassa encodes a single H1 protein that is not essential for viability. To investigate the role of N. crassa H1, we constructed a functional FLAG-tagged H1 fusion protein and performed genomic and molecular analyses. Cell fractionation experiments showed that H1-3XFLAG is a chromatin binding protein. Chromatin-immunoprecipitation combined with sequencing (ChIP-seq) revealed that H1-3XFLAG is globally enriched throughout the genome with a subtle preference for promoters of expressed genes. In mammals, the stoichiometry of H1 impacts nucleosome repeat length. To determine if H1 impacts nucleosome occupancy or nucleosome positioning in N. crassa, we performed micrococcal nuclease digestion in the wild-type and the hH1 strain followed by sequencing (MNase-seq). Deletion of hH1 did not significantly impact nucleosome positioning or nucleosome occupancy. Analysis of DNA methylation by whole-genome bisulfite sequencing (MethylC-seq) revealed a modest but global increase in DNA methylation in the hH1 mutant. Together, these data suggest that H1 acts as a nonspecific chromatin binding protein that can limit accessibility of the DNA methylation machinery in N. crassa.

Correlation among DNA Linker Length, Linker Histone Concentration, and Histone Tails in Chromatin

Eukaryotic cells condense their genetic material in the nucleus in the form of chromatin, a macromolecular complex made of DNA and multiple proteins. The structure of chromatin is intimately connected to the regulation of all eukaryotic organisms, from amoebas to humans, but its organization remains largely unknown. The nucleosome repeat length (NRL) and the concentration of linker histones (ρLH) are two structural parameters that vary among cell types and cell cycles; the NRL is the number of DNA basepairs wound around each nucleosome core plus the number of basepairs linking successive nucleosomes. Recent studies have found a linear empirical relationship between the variation of these two properties for different cells, but its underlying mechanism remains elusive. Here we apply our established mesoscale chromatin model to explore the mechanisms responsible for this relationship, by investigating chromatin fibers as a function of NRL and ρLH combinations. We find that a threshold of linker histone concentration triggers the compaction of chromatin into well-formed 30-nm fibers; this critical value increases linearly with NRL, except for long NRLs, where the fibers remain disorganized. Remarkably, the interaction patterns between core histone tails and chromatin elements are highly sensitive to the NRL and ρLH combination, suggesting a molecular mechanism that could have a key role in regulating the structural state of the fibers in the cell. An estimate of the minimized work and volume associated with storage of chromatin fibers in the nucleus further suggests factors that could spontaneously regulate the NRL as a function of linker histone concentration. Both the tail interaction map and DNA packing considerations support the empirical NRL/ρLH relationship and offer a framework to interpret experiments for different chromatin conditions in the cell.

Topological Diversity of Chromatin Fibers: Interplay Between Nucleosome Repeat Length, DNA Linking Number and the Level of Transcription

Topological Diversity of Chromatin Fibers: Interplay Between Nucleosome Repeat Length, DNA Linking Number and the Level of Transcription

The construction of customized nucleosomal arrays

A simple, efficient, and reliable method is demonstrated for cloning long tandem arrays of the 601 nucleosomal positioning sequence. In addition, it is shown that such long arrays can be ligated together in vitro with high efficiency. By combining these two procedures it becomes straightforward to synthesize customized arrays that contain different (or variable) nucleosomal repeat lengths (NRLs) and monosome units bearing chemical modifications such as fluorophores, methyl groups, and reaction sites. This is, therefore, an enabling technology for the in vitro study of chromatin structure and function.