Nucleosome Repeat Length Research Articles

In silico design of DNA sequences for in vivo nucleosome positioning.

The computational design of synthetic DNA sequences with designer in vivo properties is gaining traction in the field of synthetic genomics. We propose here a computational method which combines a kinetic Monte Carlo framework with a deep mutational screening based on deep learning predictions. We apply our method to build regular nucleosome arrays with tailored nucleosomal repeat lengths (NRL) in yeast. Our design was validated in vivo by successfully engineering and integrating thousands of kilobases long tandem arrays of computationally optimized sequences which could accommodate NRLs much larger than the yeast natural NRL (namely 197 and 237bp, compared to the natural NRL of ∼165bp). RNA-seq results show that transcription of the arrays can occur but is not driven by the NRL. The computational method proposed here delineates the key sequence rules for nucleosome positioning in yeast and should be easily applicable to other sequence properties and other genomes.

Proteasome-dependent degradation of histone H1 subtypes is mediated by its C-terminal domain.

Histone H1 is involved in chromatin compaction and dynamics. In human cells, the H1 complement is formed by different amounts of somatic H1 subtypes, H1.0-H1.5 and H1X. The amount of each variant depends on the cell type, the cell cycle phase, and the time of development and can be altered in disease. However, the mechanisms regulating H1 protein levels have not been described. We have analyzed the contribution of the proteasome to the degradation of H1 subtypes in human cells using two different inhibitors: MG132 and bortezomib. H1 subtypes accumulate upon treatment with both drugs, indicating that the proteasome is involved in the regulation of H1 protein levels. Proteasome inhibition caused a global increase in cytoplasmatic H1, with slight changes in the composition of H1 bound to chromatin and chromatin accessibility and no alterations in the nucleosome repeat length. The analysis of the proteasome degradation pathway showed that H1 degradation is ubiquitin-independent. The whole protein and its C-terminal domain can be degraded directly by the 20S proteasome in vitro. Partial depletion of PA28γ revealed that this regulatory subunit contributes to H1 degradation within the cell. Our study shows that histone H1 protein levels are under tight regulation to prevent its accumulation in the nucleus. We revealed a new regulatory mechanism for histone H1 degradation, where the C-terminal disordered domain is responsible for its targeting and degradation by the 20S proteasome, a process enhanced by the regulatory subunit PA28γ.

Nucleosome reorganisation in breast cancer tissues

BackgroundNucleosome 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.ResultsWe 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.ConclusionsOur 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.

Aging clock based on nucleosome reorganisation derived from cell-free DNA.

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 distributionsallowed predicting person's age with the median absolute error of 3-3.5 years.

Linker histone H1 drives heterochromatin condensation via phase separation in Arabidopsis.

In the eukaryotic nucleus, heterochromatin forms highly condensed, visible foci known as heterochromatin foci (HF). These HF are enriched with linker histone H1, a key player in heterochromatin condensation and silencing. However, it is unknown how H1 aggregates HF and condenses heterochromatin. In this study, we established that H1 facilitates heterochromatin condensation by enhancing inter- and intrachromosomal interactions between and within heterochromatic regions of the Arabidopsis (Arabidopsis thaliana) genome. We demonstrated that H1 drives HF formation via phase separation, which requires its C-terminal intrinsically disordered region (C-IDR). A truncated H1 lacking the C-IDR fails to form foci or recover HF in the h1 mutant background, whereas C-IDR with a short stretch of the globular domain (18 out of 71 amino acids) is sufficient to rescue both defects. In addition, C-IDR is essential for H1's roles in regulating nucleosome repeat length and DNA methylation in Arabidopsis, indicating that phase separation capability is required for chromatin functions of H1. Our data suggest that bacterial H1-like proteins, which have been shown to condense DNA, are intrinsically disordered and capable of mediating phase separation. Therefore, we propose that phase separation mediated by H1 or H1-like proteins may represent an ancient mechanism for condensing chromatin and DNA.

In vitro co-expression chromatin assembly and remodeling platform for plant histone variants

Histone variants play a central role in shaping the chromatin landscape in plants, yet, how their distinct combinations affect nucleosome properties and dynamics is still largely elusive. To address this, we developed a novel chromatin assembly platform for Arabidopsis thaliana, using wheat germ cell-free protein expression. Four canonical histones and five reported histone variants were used to assemble twelve A. thaliana nucleosome combinations. Seven combinations were successfully reconstituted and confirmed by supercoiling and micrococcal nuclease (MNase) assays. The effect of the remodeling function of the CHR11-DDR4 complex on these seven combinations was evaluated based on the nucleosome repeat length and nucleosome spacing index obtained from the MNase ladders. Overall, the current study provides a novel method to elucidate the formation and function of a diverse range of nucleosomes in plants.

Nucleosome repositioning in chronic lymphocytic leukemia.

The location of nucleosomes in the human genome determines the primary chromatin structure and regulates access to regulatory regions. However, genome-wide information on deregulated nucleosome occupancy and its implications in primary cancer cells is scarce. Here, we conducted a genome-wide comparison of high-resolution nucleosome maps in peripheral blood B cells from patients with chronic lymphocytic leukemia (CLL) and healthy individuals at single-base-pair resolution. Our investigation uncovered significant changes of nucleosome positioning in CLL. Globally, the spacing between nucleosomes-the nucleosome repeat length (NRL)-is shortened in CLL. This effect is stronger in the more aggressive IGHV-unmutated CLL subtype than in the IGHV-mutated CLL subtype. Changes in nucleosome occupancy at specific sites are linked to active chromatin remodeling and reduced DNA methylation. Nucleosomes lost or gained in CLL marks differential binding of 3D chromatin organizers such as CTCF as well as immune response-related transcription factors and delineated mechanisms of epigenetic deregulation. The principal component analysis of nucleosome occupancy in cancer-specific regions allowed the classification of samples between cancer subtypes and normal controls. Furthermore, patients could be better assigned to CLL subtypes according to differential nucleosome occupancy than based on DNA methylation or gene expression. Thus, nucleosome positioning constitutes a novel readout to dissect molecular mechanisms of disease progression and to stratify patients. Furthermore, we anticipate that the global nucleosome repositioning detected in our study, such as changes in the NRL, can be exploited for liquid biopsy applications based on cell-free DNA to stratify patients and monitor disease progression.

Cryo-ET study from in vitro to in vivo revealed a general folding mode of chromatin with two-start helical architecture

The organization and dynamics of chromatin fiber play crucial roles in regulating DNA accessibility for gene expression. Here we combine cryoelectron tomography (cryo-ET), sub-volume averaging, and 3D segmentation to visualize the invitro and invivo chromatin fibers folding by linker histone. We discover that an increased nucleosome repeat length and prolonged fiber length do not change the two-start helical architecture in reconstituted chromatin of homogeneous composition. Additionally, an isolated chromatin fiber with heterogeneous composition was observed, which includes short-range regions compatible with two-start helix. Invivo, sub-volume averaging reveals similar subunits of two-start helical architecture in transcriptionally inactive chromatin in frog erythrocyte nuclei. Strikingly, unambiguous DNA trajectories that displayed a zigzag pattern universally between alternate N/N+2 nucleosomes were further determined by cryo-ET with voltage phase plate. Therefore, these structural similarities suggest a general folding mode of chromatin induced by linker histone, and heterogeneous compositions mainly affect local conformation rather than changing the overall architecture.

Till SMARCA5 loss do nucleosomes part.

In this issue of Molecular Cell, Bomber etal. demonstrate that acute loss of SMARCA5 in human cells leads to eviction of CTCF and an increase in nucleosome repeat length without direct impact on transcriptional activity.

Human SMARCA5 is continuously required to maintain nucleosome spacing

Genetic models suggested that SMARCA5 was required for DNA-templated events including transcription, DNA replication, and DNA repair. We engineered a degron tag into the endogenous alleles of SMARCA5, a catalytic component of the imitation switch complexes in three different human cell lines to define the effects of rapid degradation of this key regulator. Degradation of SMARCA5 was associated with a rapid increase in global nucleosome repeat length, which may allow greater chromatin compaction. However, there were few changes in nascent transcription within the first 6h of degradation. Nevertheless, we demonstrated a requirement for SMARCA5 to control nucleosome repeat length at G1/S and during the S phase. SMARCA5 co-localized with CTCF and H2A.Z, and we found a rapid loss of CTCF DNA binding and disruption of nucleosomal phasing around CTCF binding sites. This spatiotemporal analysis indicates that SMARCA5 is continuously required for maintaining nucleosomal spacing.

Chromatin Liquid-Liquid Phase Separation (LLPS) Is Regulated by Ionic Conditions and Fiber Length.

The dynamic regulation of the physical states of chromatin in the cell nucleus is crucial for maintaining cellular homeostasis. Chromatin can exist in solid- or liquid-like forms depending on the surrounding ions, binding proteins, post-translational modifications and many other factors. Several recent studies suggested that chromatin undergoes liquid–liquid phase separation (LLPS) in vitro and also in vivo; yet, controversial conclusions about the nature of chromatin LLPS were also observed from the in vitro studies. These inconsistencies are partially due to deviations in the in vitro buffer conditions that induce the condensation/aggregation of chromatin as well as to differences in chromatin (nucleosome array) constructs used in the studies. In this work, we present a detailed characterization of the effects of K+, Mg2+ and nucleosome fiber length on the physical state and property of reconstituted nucleosome arrays. LLPS was generally observed for shorter nucleosome arrays (15-197-601, reconstituted from 15 repeats of the Widom 601 DNA with 197 bp nucleosome repeat length) at physiological ion concentrations. In contrast, gel- or solid-like condensates were detected for the considerably longer 62-202-601 and lambda DNA (~48.5 kbp) nucleosome arrays under the same conditions. In addition, we demonstrated that the presence of reduced BSA and acetate buffer is not essential for the chromatin LLPS process. Overall, this study provides a comprehensive understanding of several factors regarding chromatin physical states and sheds light on the mechanism and biological relevance of chromatin phase separation in vivo.

Reconstituted TAD-size chromatin fibers feature heterogeneous nucleosome clusters

Large topologically associated domains (TADs) contain irregularly spaced nucleosome clutches, and interactions between such clutches are thought to aid the compaction of these domains. Here, we reconstituted TAD-sized chromatin fibers containing hundreds of nucleosomes on native source human and lambda-phage DNA and compared their mechanical properties at the single-molecule level with shorter ‘601’ arrays with various nucleosome repeat lengths. Fluorescent imaging showed increased compaction upon saturation of the DNA with histones and increasing magnesium concentration. Nucleosome clusters and their structural fluctuations were visualized in confined nanochannels. Force spectroscopy revealed not only similar mechanical properties of the TAD-sized fibers as shorter fibers but also large rupture events, consistent with breaking the interactions between distant clutches of nucleosomes. Though the arrays of native human DNA, lambda-phage and ‘601’ DNA featured minor differences in reconstitution yield and nucleosome stability, the fibers’ global structural and mechanical properties were similar, including the interactions between nucleosome clutches. These single-molecule experiments quantify the mechanical forces that stabilize large TAD-sized chromatin domains consisting of disordered, dynamically interacting nucleosome clutches and their effect on the condensation of large chromatin domains.

Columnar structure of human telomeric chromatin.

Telomeres, the ends of eukaryotic chromosomes, play pivotal parts in ageing and cancer and are targets of DNA damage and the DNA damage response1-5. Little is known about the structure of telomeric chromatin at the molecular level. Here we used negative stain electron microscopy and single-molecule magnetic tweezers to characterize 3-kbp-long telomeric chromatin fibres. We alsoobtained the cryogenic electron microscopy structure of the condensed telomeric tetranucleosome and its dinucleosome unit. The structure displayed close stacking of nucleosomes with a columnar arrangement, and an unusually short nucleosome repeat lengththat comprised about 132 bp DNA wound in a continuous superhelix around histone octamers. This columnar structure is primarily stabilized by the H2A carboxy-terminal and histone amino-terminaltails in a synergistic manner. The columnar conformation results in exposure of the DNA helix, which may make it susceptible to both DNA damage and the DNA damage response. The conformation also exists in an alternative open state, in which one nucleosome is unstacked and flipped out, which exposes the acidic patch of the histone surface. The structuralfeatures revealed in this work suggest mechanisms by which protein factors involved in telomere maintenance can access telomeric chromatin in its compact form.

Histone H1 binding to nucleosome arrays depends on linker DNA length and trajectory

Throughout the genome, nucleosomes often form regular arrays that differ in nucleosome repeat length (NRL), occupancy of linker histone H1 and transcriptional activity. Here, we report cryo-EM structures of human H1-containing tetranucleosome arrays with four physiologically relevant NRLs. The structures show a zig-zag arrangement of nucleosomes, with nucleosomes 1 and 3 forming a stack. H1 binding to stacked nucleosomes depends on the NRL, whereas H1 always binds to the non-stacked nucleosomes 2 and 4. Short NRLs lead to altered trajectories of linker DNA, and these altered trajectories sterically impair H1 binding to the stacked nucleosomes in our structures. As the NRL increases, linker DNA trajectories relax, enabling H1 contacts and binding. Our results provide an explanation for why arrays with short NRLs are depleted of H1 and suited for transcription, whereas arrays with long NRLs show full H1 occupancy and can form transcriptionally silent heterochromatin regions.

GC content, but not nucleosome positioning, directly contributes to intron splicing efficiency in Paramecium.

Eukaryotic genes are interrupted by introns that must be accurately spliced from mRNA precursors. With an average length of 25 nt, the more than 90,000 introns of Paramecium tetraurelia stand among the shortest introns reported in eukaryotes. The mechanisms specifying the correct recognition of these tiny introns remain poorly understood. Splicing can occur cotranscriptionally, and it has been proposed that chromatin structure might influence splice site recognition. To investigate the roles of nucleosome positioning in intron recognition, we determined the nucleosome occupancy along the P. tetraurelia genome. We show that P. tetraurelia displays a regular nucleosome array with a nucleosome repeat length of ∼151 bp, among the smallest periodicities reported. Our analysis has revealed that introns are frequently associated with inter-nucleosomal DNA, pointing to an evolutionary constraint favoring introns at the AT-rich nucleosome edge sequences. Using accurate splicing efficiency data from cells depleted for nonsense-mediated decay effectors, we show that introns located at the edge of nucleosomes display higher splicing efficiency than those at the center. However, multiple regression analysis indicates that the low GC content of introns, rather than nucleosome positioning, is associated with high splicing efficiency. Our data reveal a complex link between GC content, nucleosome positioning, and intron evolution in Paramecium.

Nucleosome Positioning on Large Tandem DNA Repeats of the ‘601’ Sequence Engineered in Saccharomyces cerevisiae

The artificial 601 DNA sequence is often used to constrain the position of nucleosomes on a DNA molecule in vitro. Although the ability of the 147 base pair sequence to precisely position a nucleosome in vitro is well documented, application of this property in vivo has been explored only in a few studies and yielded contradictory conclusions. Our goal in the present study was to test the ability of the 601 sequence to dictate nucleosome positioning in Saccharomyces cerevisiae in the context of a long tandem repeat array inserted in a yeast chromosome. We engineered such arrays with three different repeat size, namely 167, 197 and 237 base pairs. Although our arrays are able to position nucleosomes in vitro, analysis of nucleosome occupancy in vivo revealed that nucleosomes are not preferentially positioned as expected on the 601-core sequence along the repeats and that the measured nucleosome repeat length does not correspond to the one expected by design. Altogether our results demonstrate that the rules defining nucleosome positions on this DNA sequence in vitro are not valid in vivo, at least in this chromosomal context, questioning the relevance of using the 601 sequence in vivo to achieve precise nucleosome positioning on designer synthetic DNA sequences.

Histone H4 lysine 20 mono-methylation directly facilitates chromatin openness and promotes transcription of housekeeping genes

Histone lysine methylations have primarily been linked to selective recruitment of reader or effector proteins that subsequently modify chromatin regions and mediate genome functions. Here, we describe a divergent role for histone H4 lysine 20 mono-methylation (H4K20me1) and demonstrate that it directly facilitates chromatin openness and accessibility by disrupting chromatin folding. Thus, accumulation of H4K20me1 demarcates highly accessible chromatin at genes, and this is maintained throughout the cell cycle. In vitro, H4K20me1-containing nucleosomal arrays with nucleosome repeat lengths (NRL) of 187 and 197 are less compact than unmethylated (H4K20me0) or trimethylated (H4K20me3) arrays. Concordantly, and in contrast to trimethylated and unmethylated tails, solid-state NMR data shows that H4K20 mono-methylation changes the H4 conformational state and leads to more dynamic histone H4-tails. Notably, the increased chromatin accessibility mediated by H4K20me1 facilitates gene expression, particularly of housekeeping genes. Altogether, we show how the methylation state of a single histone H4 residue operates as a focal point in chromatin structure control. While H4K20me1 directly promotes chromatin openness at highly transcribed genes, it also serves as a stepping-stone for H4K20me3-dependent chromatin compaction.

Nucleosome-induced homology recognition in chromatin.

One of the least understood properties of chromatin is the ability of its similar regions to recognize each other through weak interactions. Theories based on electrostatic interactions between helical macromolecules suggest that the ability to recognize sequence homology is an innate property of the non-ideal helical structure of DNA. However, this theory does not account for the nucleosomal packing of DNA. Can homologous DNA sequences recognize each other while wrapped up in the nucleosomes? Can structural homology arise at the level of nucleosome arrays? Here, we present a theoretical model for the recognition potential well between chromatin fibres sliding against each other. This well is different from the one predicted for bare DNA; the minima in energy do not correspond to literal juxtaposition, but are shifted by approximately half the nucleosome repeat length. The presence of this potential well suggests that nucleosome positioning may induce mutual sequence recognition between chromatin fibres and facilitate the formation of chromatin nanodomains. This has implications for nucleosome arrays enclosed between CTCF-cohesin boundaries, which may form stiffer stem-like structures instead of flexible entropically favourable loops. We also consider switches between chromatin states, e.g. through acetylation/deacetylation of histones, and discuss nucleosome-induced recognition as a precursory stage of genetic recombination.

Tetranucleosome Interactions Drive Chromatin Folding.

The multiscale organizational structure of chromatin in eukaryotic cells is instrumental to DNA transcription, replication, and repair. At mesoscopic length scales, nucleosomes pack in a manner that serves to regulate gene expression through condensation and expansion of the genome. The particular structures that arise and their respective thermodynamic stabilities, however, have yet to be fully resolved. In this study, we combine molecular modeling using the 1CPN mesoscale model of chromatin with nonlinear manifold learning to identify and characterize the structure and free energy of metastable states of short chromatin segments comprising between 4- and 16-nucleosomes. Our results reveal the formation of two previously characterized tetranucleosomal conformations, the “α-tetrahedron” and the “β-rhombus”, which have been suggested to play an important role in the accessibility of DNA and, respectively, induce local chromatin compaction or elongation. The spontaneous formation of these motifs is potentially responsible for the slow nucleosome dynamics observed in experimental studies. Increases of the nucleosome repeat length are accompanied by more pronounced structural irregularity and flexibility and, ultimately, a dynamic liquid-like behavior that allows for frequent structural reorganization. Our findings indicate that tetranucleosome motifs are intrinsically stable structural states, driven by local internucleosomal interactions, and support a mechanistic picture of chromatin packing, dynamics, and accessibility that is strongly influenced by emergent local mesoscale structure.

Near-atomic resolution structures of interdigitated nucleosome fibres

Chromosome structure at the multi-nucleosomal level has remained ambiguous in spite of its central role in epigenetic regulation and genome dynamics. Recent investigations of chromatin architecture portray diverse modes of interaction within and between nucleosome chains, but how this is realized at the atomic level is unclear. Here we present near-atomic resolution crystal structures of nucleosome fibres that assemble from cohesive-ended dinucleosomes with and without linker histone. As opposed to adopting folded helical ‘30 nm’ structures, the fibres instead assume open zigzag conformations that are interdigitated with one another. Zigzag conformations obviate extreme bending of the linker DNA, while linker DNA size (nucleosome repeat length) dictates fibre configuration and thus fibre–fibre packing, which is supported by variable linker histone binding. This suggests that nucleosome chains have a predisposition to interdigitate with specific characteristics under condensing conditions, which rationalizes observations of local chromosome architecture and the general heterogeneity of chromatin structure.