Higher-order structure of long repeat chromatin.

The higher-order structure of chromatin isolated from sea urchin sperm, which has a long nucleosomal DNA repeat length (approximately 240 bp), has been studied by electron microscopy and X-ray diffraction. Electron micrographs show that this chromatin forms 300 A filaments which are indistinguishable from those of chicken erythrocytes (approximately 212 bp repeat); X-ray diffraction patterns from partially oriented samples show that the edge-to-edge packing of nucleosomes in the direction of the 300 A filament axis, and the radial disposition of nucleosomes around it, are both similar to those of the chicken erythrocyte 300 A filament, which is described by the solenoid model. The invariance of the structure with increased linker DNA length is inconsistent with many other models proposed for the 300 A filament and, furthermore, means that the linker DNA must be bent. The low-angle X-ray scattering in the 300-400 A region both in vitro and in vivo differs from that of chicken erythrocyte chromatin. The nature of the difference suggests that 300 A filaments in sea urchin sperm in vivo are packed so tightly together that electron-density contrast between individual filaments is lost; this is consistent with electron micrographs of the chromatin in vitro.     

Higher order structure of chromatin: evidence from photochemically detected linear dichroism

We have used photochemically detected linear dichroism to measure the separate average angular orientations of nucleosomes and linker DNA in 30-nm chromatin fibers of varying linker size (20-80 base pairs). Our results indicate that the average tilt angles vary with linker size, but not in a monotonic manner, suggesting that the constancy of geometry of the 30-nm fiber is maintained by compensatory changes of nucleosomal tilt which accommodate packing of variable lengths of linker DNA. We discuss the compatibility of our results with the various classes of models that have been proposed for the 30-nm fiber, including the continuous solenoid model and models built from the basic unit of the zig-zag ribbon. Many models can be eliminated, and all have to be modified to fit our results for chromatins with very long linkers.     

A defined structure of the 30 nm chromatin fibre which accommodates different nucleosomal repeat lengths

Earlier work on the condensation of chromatins of different repeat lengths into the 30 nm fibre has been surveyed and it is shown that the external geometry of the fibre must be the same for all the chromatins. This can only be fitted by a helical coiling of nucleosomes into a solenoid with the linker DNA disposed internally. On this basis, various models were calculated and compared with published electric dichroism data. The only good fit is found with a 'reverse-loop' model, where the linker DNA forms a complete turn into the hole of the solenoid, of opposite hand to the nucleosomal DNA superhelix. This gives a topological linking number of one per nucleosome and would resolve the 'linking number paradox' if the DNA screw is the same in chromatin as in solution. The feasibility of a reverse-loop for short linkers (down to 15 base pairs) was investigated by model building and kinks of approximately 120 degrees into both DNA grooves are described, which will allow such packing. There will, however, be a 'forbidden' range for the linker DNA length, between approximately 1 and 14 bp, corresponding to nucleosomal repeats of 163 and 176 bp.     

ACF catalyses chromatosome movements in chromatin fibres

Nucleosome-remodelling factors containing the ATPase ISWI, such as ACF, render DNA in chromatin accessible by promoting the sliding of histone octamers. Although the ATP-dependent repositioning of mononucleosomes is readily observable in vitro, it is unclear to which extent nucleosomes can be moved in physiological chromatin, where neighbouring nucleosomes, linker histones and the folding of the nucleosomal array restrict mobility. We assembled arrays consisting of 12 nucleosomes or 12 chromatosomes (nucleosomes plus linker histone) from defined components and subjected them to remodelling by ACF or the ATPase CHD1. Both factors increased the access to DNA in nucleosome arrays. ACF, but not CHD1, catalysed profound movements of nucleosomes throughout the array, suggesting different remodelling mechanisms. Linker histones inhibited remodelling by CHD1. Surprisingly, ACF catalysed significant repositioning of entire chromatosomes in chromatin containing saturating levels of linker histone H1. H1 inhibited the ATP-dependent generation of DNA accessibility by only about 50%. This first demonstration of catalysed chromatosome movements suggests that the bulk of interphase euchromatin may be rendered dynamic by dedicated nucleosome-remodelling factors.     

Nucleotide sequence-directed mapping of the nucleosomes

The concept of sequence-dependent deformational anisotropy of DNA proposed earlier is further elaborated and a computational procedure is developed for the sequence-directed mapping of the nucleosomes along chromatin DNA nucleotide sequences. The deformational anisotropy is found to be nonuniform along the molecule of the nucleosomal DNA, suggesting that the DNA superhelix in the nucleosome is slightly oval rather than circular in projection. The number of superhelical turns in the nucleosome core particle is estimated to be 2.0 +/- 0.2. Preliminary mapping of the nucleosomes in various chromatin DNA sequences yields the distribution of linker lengths which shows several minima separated by about 10 base-pairs. This is explained by sterical exclusion effects due to overlapping of the nucleosomes in space when some specific linker lengths are chosen. The mapping procedure described is tested by comparing its results with all the most accurate experimental mapping data reported so far. The comparison demonstrates that the exact positions of all the nucleosomes appear to be determined exclusively by the nucleotide sequences.     

Effect of the length of internucleosome regions of DNA on the conformation of chromatid fibrils

Models of chromatin fibers structures with linear regions of linker DNA were analysed. Limitations put by end dimensions of linker DNA and nucleosomes are considered. Good agreement between the structural properties of model and real chromatin fibers was obtained. It has been shown that the models with three and more configurations of closely located nucleosomes have linker DNA of 19-53 base pairs length, which is characteristic of real chromatin of the majority of somatic cells.     

Chromatin fibers are left-handed double helices with diameter and mass per unit length that depend on linker length

Four classes of models have been proposed for the internal structure of eukaryotic chromosome fibers--the solenoid, twisted-ribbon, crossed-linker, and superbead models. We have collected electron image and x-ray scattering data from nuclei, and isolated chromatin fibers of seven different tissues to distinguish between these models. The fiber diameters are related to the linker lengths by the equation: D(N) = 19.3 + 0.23 N, where D(N) is the external diameter (nm) and N is the linker length (base pairs). The number of nucleosomes per unit length of the fibers is also related to linker length. Detailed studies were done on the highly regular chromatin from erythrocytes of Necturus (mud puppy) and sperm of Thyone (sea cucumber). Necturus chromatin fibers (N = 48 bp) have diameters of 31 nm and have 7.5 +/- 1 nucleosomes per 10 nm along the axis. Thyone chromatin fibers (N = 87 bp) have diameters of 39 nm and have 12 +/- 2 nucleosomes per 10 nm along the axis. Fourier transforms of electron micrographs of Necturus fibers showed left-handed helical symmetry with a pitch of 25.8 +/- 0.8 nm and pitch angle of 32 +/- 3 degrees, consistent with a double helix. Comparable conclusions were drawn from the Thyone data. The data do not support the solenoid, twisted-ribbon, or supranucleosomal particle models. The data do support two crossed-linker models having left-handed double-helical symmetry and conserved nucleosome interactions.     

Local geometry and elasticity in compact chromatin structure

The hierarchical packaging of DNA into chromatin within a eukaryotic nucleus plays a pivotal role in both the accessibility of genomic information and the dynamics of replication. Our work addresses the role of nanoscale physical and geometric properties in determining the structure of chromatin at the mesoscale level. We study the packaging of DNA in chromatin fibers by optimization of regular helical morphologies, considering the elasticity of the linker DNA as well as steric packing of the nucleosomes and linkers. Our model predicts a broad range of preferred helix structures for a fixed linker length of DNA; changing the linker length alters the predicted ensemble. Specifically, we find that the twist registry of the nucleosomes, as set by the internucleosome repeat length, determines the preferred angle between the nucleosomes and the fiber axis. For moderate to long linker lengths, we find a number of energetically comparable configurations with different nucleosome-nucleosome interaction patterns, indicating a potential role for kinetic trapping in chromatin fiber formation. Our results highlight the key role played by DNA elasticity and local geometry in regulating the hierarchical packaging of the genome.     

A critical role for linker DNA in higher-order folding of chromatin fibers

Nucleosome-nucleosome interactions drive the folding of nucleosomal arrays into dense chromatin fibers. A better physical account of the folding of chromatin fibers is necessary to understand the role of chromatin in regulating DNA transactions. Here, we studied the unfolding pathway of regular chromatin fibers as a function of single base pair increments in linker length, using both rigid base-pair Monte Carlo simulations and single-molecule force spectroscopy. Both computational and experimental results reveal a periodic variation of the folding energies due to the limited flexibility of the linker DNA. We show that twist is more restrictive for nucleosome stacking than bend, and find the most stable stacking interactions for linker lengths of multiples of 10 bp. We analyzed nucleosomes stacking in both 1- and 2-start topologies and show that stacking preferences are determined by the length of the linker DNA. Moreover, we present evidence that the sequence of the linker DNA also modulates nucleosome stacking and that the effect of the deletion of the H4 tail depends on the linker length. Importantly, these results imply that nucleosome positioning in vivo not only affects the phasing of nucleosomes relative to DNA but also directs the higher-order structure of chromatin.     

Chromatin on a membrane: accessibility of histone H5 for antibodies in the supernucleosomal structure

Histone H5 accessibility for the antibodies in chromatin was studied. Chromatin was immobilised on the nitrocellulose membrane in conditions which provide different levels of its compactization. Antiserum specific to the globular domain of histone H5 was used. It was shown, that for establishing real protection of histone H5 in the supernucleosomal structure it is necessary to use long fibers of chromatin. Their linking to the membrane must occur by a minimum quantity of points. It was established, that histone H5 is 5 times more accessive in the preparations of dispersed chromatin (low ionic strength) then in chromatin with the supernucleosomal organization (physiological ionic strength). We suppose that the small level of accessibility of histone H5 for the antibodies in the compact chromatin can be explained by some disruptions in the supernucleosomal organization. On the contrary, the long equable solenoid of nucleosomes provides complete protection of histone H5. In accordance with the results obtained, the model of ordered packaging of nucleosomes in the solenoid is discussed. In this model the point of entrance and exit of DNA on the nucleosomes, fixed by globular region of histone H5, is localized inside the solenoid.     

Characterization of Dnmt1 Binding and DNA Methylation on Nucleosomes and Nucleosomal Arrays

The packaging of DNA into nucleosomes and the organisation into higher order structures of chromatin limits the access of sequence specific DNA binding factors to DNA. In cells, DNA methylation is preferentially occuring in the linker region of nucleosomes, suggesting a structural impact of chromatin on DNA methylation. These observations raise the question whether DNA methyltransferases are capable to recognize the nucleosomal substrates and to modify the packaged DNA. Here, we performed a detailed analysis of nucleosome binding and nucleosomal DNA methylation by the maintenance DNA methyltransferase Dnmt1. Our binding studies show that Dnmt1 has a DNA length sensing activity, binding cooperatively to DNA, and requiring a minimal DNA length of 20 bp. Dnmt1 needs linker DNA to bind to nucleosomes and most efficiently recognizes nucleosomes with symmetric DNA linkers. Footprinting experiments reveal that Dnmt1 binds to both DNA linkers exiting the nucleosome core. The binding pattern correlates with the efficient methylation of DNA linkers. However, the enzyme lacks the ability to methylate nucleosomal CpG sites on mononucleosomes and nucleosomal arrays, unless chromatin remodeling enzymes create a dynamic chromatin state. In addition, our results show that Dnmt1 functionally interacts with specific chromatin remodeling enzymes to enable complete methylation of hemi-methylated DNA in chromatin.     

Changing Chromatin Fiber Conformation by Nucleosome Repositioning

Chromatin conformation is dynamic and heterogeneous with respect to nucleosome positions, which can be changed by chromatin remodeling complexes in the cell. These molecular machines hydrolyze ATP to translocate or evict nucleosomes, and establish loci with regularly and more irregularly spaced nucleosomes as well as nucleosome-depleted regions. The impact of nucleosome repositioning on the three-dimensional chromatin structure is only poorly understood. Here, we address this issue by using a coarse-grained computer model of arrays of 101 nucleosomes considering several chromatin fiber models with and without linker histones, respectively. We investigated the folding of the chain in dependence of the position of the central nucleosome by changing the length of the adjacent linker DNA in basepair steps. We found in our simulations that these translocations had a strong effect on the shape and properties of chromatin fibers: i), Fiber curvature and flexibility at the center were largely increased and long-range contacts between distant nucleosomes on the chain were promoted. ii), The highest destabilization of the fiber conformation occurred for a nucleosome shifted by two basepairs from regular spacing, whereas effects of linker DNA changes of ∼10 bp in phase with the helical twist of DNA were minimal. iii), A fiber conformation can stabilize a regular spacing of nucleosomes inasmuch as favorable stacking interactions between nucleosomes are facilitated. This can oppose nucleosome translocations and increase the energetic costs for chromatin remodeling. Our computational modeling framework makes it possible to describe the conformational heterogeneity of chromatin in terms of nucleosome positions, and thus advances theoretical models toward a better understanding of how genome compaction and access are regulated within the cell.     

Spontaneous access to DNA target sites in folded chromatin fibers

DNA wrapped in nucleosomes is sterically occluded from many protein complexes that must act on it; how such complexes gain access to nucleosomal DNA is not known. In vitro studies on isolated nucleosomes show that they undergo spontaneous partial unwrapping conformational transitions, which make the wrapped nucleosomal DNA transiently accessible. Thus, site exposure might provide a general mechanism allowing access of protein complexes to nucleosomal DNA. However, existing quantitative analyses of site exposure focused on single nucleosomes, while the presence of neighbor nucleosomes and concomitant chromatin folding might significantly influence site exposure. In this work, we carried out quantitative studies on the accessibility of nucleosomal DNA in homogeneous nucleosome arrays. Two striking findings emerged. Organization into chromatin fibers changes the accessibility of nucleosomal DNA only modestly, from ∼ 3-fold decreases to ∼ 8-fold increases in accessibility. This means that nucleosome arrays are intrinsically dynamic and accessible even when they are visibly condensed. In contrast, chromatin folding decreases the accessibility of linker DNA by as much as ∼ 50-fold. Thus, nucleosome positioning dramatically influences the accessibility of target sites located inside nucleosomes, while chromatin folding dramatically regulates access to target sites in linker DNA.     

Preferentially quantized linker DNA lengths in Saccharomyces cerevisiae

The exact lengths of linker DNAs connecting adjacent nucleosomes specify the intrinsic three-dimensional structures of eukaryotic chromatin fibers. Some studies suggest that linker DNA lengths preferentially occur at certain quantized values, differing one from another by integral multiples of the DNA helical repeat, approximately 10 bp; however, studies in the literature are inconsistent. Here, we investigate linker DNA length distributions in the yeast Saccharomyces cerevisiae genome, using two novel methods: a Fourier analysis of genomic dinucleotide periodicities adjacent to experimentally mapped nucleosomes and a duration hidden Markov model applied to experimentally defined dinucleosomes. Both methods reveal that linker DNA lengths in yeast are preferentially periodic at the DNA helical repeat ( approximately 10 bp), obeying the forms 10n+5 bp (integer n). This 10 bp periodicity implies an ordered superhelical intrinsic structure for the average chromatin fiber in yeast.     

Changing Chromatin Fiber Conformation by Nucleosome Repositioning

Chromatin conformation is dynamic and heterogeneous with respect to nucleosome positions, which can be changed by chromatin remodeling complexes in the cell. These molecular machines hydrolyze ATP to translocate or evict nucleosomes, and establish loci with regularly and more irregularly spaced nucleosomes as well as nucleosome-depleted regions. The impact of nucleosome repositioning on the three-dimensional chromatin structure is only poorly understood. Here, we address this issue by using a coarse-grained computer model of arrays of 101 nucleosomes considering several chromatin fiber models with and without linker histones, respectively. We investigated the folding of the chain in dependence of the position of the central nucleosome by changing the length of the adjacent linker DNA in basepair steps. We found in our simulations that these translocations had a strong effect on the shape and properties of chromatin fibers: i), Fiber curvature and flexibility at the center were largely increased and long-range contacts between distant nucleosomes on the chain were promoted. ii), The highest destabilization of the fiber conformation occurred for a nucleosome shifted by two basepairs from regular spacing, whereas effects of linker DNA changes of ∼10 bp in phase with the helical twist of DNA were minimal. iii), A fiber conformation can stabilize a regular spacing of nucleosomes inasmuch as favorable stacking interactions between nucleosomes are facilitated. This can oppose nucleosome translocations and increase the energetic costs for chromatin remodeling. Our computational modeling framework makes it possible to describe the conformational heterogeneity of chromatin in terms of nucleosome positions, and thus advances theoretical models toward a better understanding of how genome compaction and access are regulated within the cell.     

MeCP2-chromatin interactions include the formation of chromatosome-like structures and are altered in mutations causing Rett syndrome

hMeCP2 (human methylated DNA-binding protein 2), mutations of which cause most cases of Rett syndrome (RTT), is involved in the transmission of repressive epigenetic signals encoded by DNA methylation. The present work focuses on the modifications of chromatin architecture induced by MeCP2 and the effects of RTT-causing mutants. hMeCP2 binds to nucleosomes close to the linker DNA entry-exit site and protects approximately 11 bp of linker DNA from micrococcal nuclease. MeCP2 mutants differ in this property; the R106W mutant gives very little extra protection beyond the approximately 146-bp nucleosome core, whereas the large C-terminal truncation R294X reveals wild type behavior. Gel mobility assays show that linker DNA is essential for proper MeCP2 binding to nucleosomes, and electron microscopy visualization shows that the protein induces distinct conformational changes in the linker DNA. When bound to nucleosomes, MeCP2 is in close proximity to histone H3, which exits the nucleosome core close to the proposed MeCP2-binding site. These findings firmly establish nucleosomal linker DNA as a crucial binding partner of MeCP2 and show that different RTT-causing mutations of MeCP2 are correspondingly defective in different aspects of the interactions that alter chromatin architecture.     

Higher order structure of chromatin: orientation of nucleosomes within the 30 nm chromatin solenoid is independent of species and spacer length

We have used electric dichroism to study the arrangement of nucleosomes in 30 nm chromatin solenoidal fibers prepared from a variety of sources (CHO cells, HeLa cells, rat liver, chicken erythrocytes, and sea urchin sperm) in which the nucleosome spacer length varies from approximately 10 to approximately 80 bp. Field-free relaxation times are consistent only with structures containing 6 +/- 1 nucleosomes for every 11 nm of solenoidal length. With very few assumptions about the arrangement of the spacer DNA, our dichroism data are consistent with the same orientation of the chromatosomes for every chromatin sample examined. This orientation, which maintains the faces of the radially arranged chromatosomes inclined at an angle between 20 degrees-33 degrees to the solenoid axis, thus appears to be a general structural feature of the higher order chromatin fiber.