The core histone N termini function independently of linker histones during chromatin condensation

The relationships between the core histone N termini and linker histones during chromatin assembly and salt-dependent chromatin condensation were investigated using defined chromatin model systems reconstituted from tandemly repeated 5 S rDNA, histone H5, and either native "intact" core histone octamers or "tailless" histone octamers lacking their N-terminal domains. Nuclease digestion and sedimentation studies indicate that H5 binding and the resulting constraint of entering and exiting nucleosomal DNA occur to the same extent in both tailless and intact chromatin arrays. However, despite possessing a normal chromatosomal structure, tailless chromatin arrays can neither condense into extensively folded structures nor cooperatively oligomerize in MgCl(2). Tailless nucleosomal arrays lacking linker histones also are unable to either fold extensively or oligomerize, demonstrating that the core histone N termini perform the same functions during salt-dependent condensation regardless of whether linker histones are components of the array. Our results further indicate that disruption of core histone N termini function in vitro allows a linker histone-containing chromatin fiber to exist in a decondensed state under conditions that normally would promote extensive fiber condensation. These findings have key implications for both the mechanism of chromatin condensation, and the regulation of genomic function by chromatin.     

Identification of specific functional subdomains within the linker histone H10 C-terminal domain

Linker histone binding to nucleosomal arrays in vitro causes linker DNA to form an apposed stem motif, stabilizes extensively folded secondary chromatin structures, and promotes self-association of individual nucleosomal arrays into oligomeric tertiary chromatin structures. To determine the involvement of the linker histone C-terminal domain (CTD) in each of these functions, and to test the hypothesis that the functions of this highly basic domain are mediated by neutralization of linker DNA negative charge, four truncation mutants were created that incrementally removed stretches of 24 amino acids beginning at the extreme C terminus of the mouse H1(0) linker histone. Native and truncated H1(0) proteins were assembled onto biochemically defined nucleosomal arrays and characterized in the absence and presence of salts to probe primary, secondary, and tertiary chromatin structure. Results indicate that the ability of H1(0) to alter linker DNA conformation and stabilize condensed chromatin structures is localized to specific C-terminal subdomains, rather than being equally distributed throughout the entire CTD. We propose that the functions of the linker histone CTD in chromatin are linked to the characteristic intrinsic disorder of this domain.     

Laser Raman spectra of calf thymus chromatin and its constituents.

Extensive Raman measurements have been made on calf thymus chromatin, core chromatin, the (H3,H4)/DNA complex, and isolated DNA. The results indicate that the alpha-helical content of the nucleosomal histones gradually increases as they form the heterocomplexes that lead to the formation of the octameric nucleosome core. The secondary structure of the latter is not modified as it binds to DNA. The spectra indicate that the DNA essentially retains its B conformation in nucleosomes, although slight changes probably occur in the ribose-phosphate backbone. No specific interactions between the nucleosomal histones and DNA can be established from the spectra, but histone H1 possibly interacts selectively with the thymine bases.     

Nucleosomes protect DNA from DNA methylation in vivo and in vitro

Positioned nucleosomes limit the access of proteins to DNA. However, the impact of nucleosomes on DNA methylation in vitro and in vivo is poorly understood. Here, we performed a detailed analysis of nucleosome binding and nucleosomal DNA methylation by the de novo methyltransferases. We show that compared to linker DNA, nucleosomal DNA is largely devoid of CpG methylation. ATP-dependent chromatin remodelling frees nucleosomal CpG dinucleotides and renders the remodelled nucleosome a 2-fold better substrate for Dnmt3a methyltransferase compared to free DNA. These results reflect the situation in vivo, as quantification of nucleosomal DNA methylation levels in HeLa cells shows a 2-fold decrease of nucleosomal DNA methylation levels compared to linker DNA. Our findings suggest that nucleosomal positions are stably maintained in vivo and nucleosomal occupancy is a major determinant of global DNA methylation patterns in vivo.     

Base excision repair of 8-oxoG in dinucleosomes

In this work we have studied the effect of chromatin structure on the base excision repair (BER) efficiency of 8-oxoG. As a model system we have used precisely positioned dinucleosomes assembled with linker histone H1. A single 8-oxoG was inserted either in the linker or the core particle DNA within the dinucleosomal template. We found that in the absence of histone H1 the glycosylase OGG1 removed 8-oxoG from the linker DNA and cleaved DNA with identical efficiency as in the naked DNA. In contrast, the presence of histone H1 resulted in close to 10-fold decrease in the efficiency of 8-oxoG initiation of repair in linker DNA independently of linker DNA length. The repair of 8-oxoG in nucleosomal DNA was very highly impeded in both absence and presence of histone H1. Chaperone-induced uptake of H1 restored the efficiency of the glycosylase induced removal of 8-oxoG from linker DNA, but not from the nucleosomal DNA. We show, however, that removal of histone H1 and nucleosome remodelling are both necessary and sufficient for an efficient removal of 8-oxoG in nucleosomal DNA. Finally, a model for BER of 8-oxoG in chromatin templates is suggested.     

Chromatin ionic atmosphere analyzed by a mesoscale electrostatic approach

Characterizing the ionic distribution around chromatin is important for understanding the electrostatic forces governing chromatin structure and function. Here we develop an electrostatic model to handle multivalent ions and compute the ionic distribution around a mesoscale chromatin model as a function of conformation, number of nucleosome cores, and ionic strength and species using Poisson-Boltzmann theory. This approach enables us to visualize and measure the complex patterns of counterion condensation around chromatin by examining ionic densities, free energies, shielding charges, and correlations of shielding charges around the nucleosome core and various oligonucleosome conformations. We show that: counterions, especially divalent cations, predominantly condense around the nucleosomal and linker DNA, unburied regions of histone tails, and exposed chromatin surfaces; ionic screening is sensitively influenced by local and global conformations, with a wide ranging net nucleosome core screening charge (56-100e); and screening charge correlations reveal conformational flexibility and interactions among chromatin subunits, especially between the histone tails and parental nucleosome cores. These results provide complementary and detailed views of ionic effects on chromatin structure for modest computational resources. The electrostatic model developed here is applicable to other coarse-grained macromolecular complexes.     

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.     

Relationship Between Chromatin High-order Folding and Nucleosomal Linker Twist in Nuclei of Human HeLa S3 Cells

Abstract

We have used the intercalative agent ethidium bromide to examine the association between chromatin high-order folding and the twist of internucleosomal DNA regions. The analysis was carried out on intact nuclei isolated from human HeLa S3 cells. Our data shows that alterations in the nucleosomal linker twist significantly influence the way in which a chain of nucleosomes folds to form different higher-order structures. The assay used allowed us to identify the existence of two chromatin fractions differing in their extent of high-order folding. We have also found that active gene sequences are preferentially associated with the chromatin fraction corresponding to the more extended conformation. A model is proposed to account for the effect of variations in the nucleosome linker twist on the state of chromatin folding.     

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.     

A Mechanism for Histone Chaperoning Activity of Nucleoplasmin: Thermodynamic and Structural Models

Nucleoplasmin (NP), a histone chaperone, acts as a reservoir for histones H2A-H2B in Xenopus laevis eggs and can displace sperm nuclear basic proteins and linker histones from the chromatin fiber of sperm and quiescent somatic nuclei. NP has been proposed to mediate the dynamic exchange of histones during the expression of certain genes and assists the assembly of nucleosomes by modulating the interaction between histones and DNA. Here, solution structural models of full-length NP and NP complexes with the functionally distinct nucleosomal core and linker histones are presented for the first time, providing a picture of the physical interactions between the nucleosomal and linker histones with NP core and tail domains. Small-angle X-ray scattering and isothermal titration calorimetry reveal that NP pentamer can accommodate five histones, either H2A-H2B dimers or H5, and that NP core and tail domains are intimately involved in the association with histones. The analysis of the binding events, employing a site-specific cooperative model, reveals a negative cooperativity-based regulatory mechanism for the linker histone/nucleosomal histone exchange. The two histone types bind with drastically different intrinsic affinity, and the strongest affinity is observed for the NP variant that mimicks the hyperphosphorylated active protein. The different “affinity windows” for H5 and H2A-H2B might allow NP to fulfill its histone chaperone role, simultaneously acting as a reservoir for the core histones and a chromatin decondensing factor. Our data are compatible with the previously proposed model where NP facilitates nucleosome assembly by removing the linker histones and depositing H2A-H2B dimers onto DNA.     

Nonrandom induction of pyrimidine-pyrimidone (6-4) photoproducts in ultraviolet-irradiated human chromatin

Radioimmunoassays that detect pyrimidine-pyrimidone (6-4) photoproducts and cyclobutane dimers were used to determine the relative induction of these photoproducts in nucleosomal (core) and internucleosomal (linker) DNA in human cell chromatin irradiated with UV light. Cyclobutane dimers were formed in equal amounts/nucleotide in core and linker DNA, whereas (6-4) photoproducts occurred with 6-fold greater frequency/nucleotide in linker DNA.     

Thermodynamics of condensation of nuclear chromatin. A differential scanning calorimetry study of the salt-dependent structural transitions

We present a detailed thermodynamic investigation of the conformational transitions of chromatin in calf thymus nuclei. Differential scanning calorimetry was used as the leading method, in combination with infrared spectroscopy, electron microscopy, and techniques for the molecular characterization of chromatin components. The conformational transitions were induced by changes in the counterion concentration. In this way, it was possible to discriminate between the interactions responsible for the folding of the higher order structure and for the coiling of nucleosomal DNA. Our experiments confirm that the denaturation of nuclear chromatin at physiological ionic strength occurs at the level of discrete structural domains, the linker and the core particle, and we were able to rule out that the actual denaturation pattern might be determined by dissociation of the nucleohistone complex and successive migration of free histones toward native regions, as recently suggested. The sequence of the denaturation events is (1) the conformational change of the histone complement at 66 degrees C, (2) the unstacking of the linker DNA at 74 degrees C, and (3) the unstacking of the core particle DNA, that can be observed either at 90 or at 107 degrees C, depending on the degree of condensation of chromatin. Nuclear chromatin unfolds in low-salt buffers, and can be refolded by increasing the ionic strength, in accordance with the well-known behavior of short fragments. The process is athermal, therefore showing that the stability of the higher order structure depends on electrostatic interactions. The transition between the folded conformation and the unfolded one proceeds through an intermediate condensation state, revealed by an endotherm at 101 degrees C. The analysis of the thermodynamic parameters of denaturation of the polynucleosomal chain demonstrates that the wrapping of the DNA around the histone octamer involves a large energy change. The most striking observation concerns the linker segment, which melts a few degrees below the peak temperature of naked DNA. This finding is in line with previous thermal denaturation investigations on isolated chromatin at low ionic strength, and suggests that a progressive destabilization of the linker occurs in the course of the salt-induced coiling of DNA in the nucleosome.     

An all-atom model of the chromatin fiber containing linker histones reveals a versatile structure tuned by the nucleosomal repeat length

In the nucleus of eukaryotic cells, histone proteins organize the linear genome into a functional and hierarchical architecture. In this paper, we use the crystal structures of the nucleosome core particle, B-DNA and the globular domain of H5 linker histone to build the first all-atom model of compact chromatin fibers. In this 3D jigsaw puzzle, DNA bending is achieved by solving an inverse kinematics problem. Our model is based on recent electron microscopy measurements of reconstituted fiber dimensions. Strikingly, we find that the chromatin fiber containing linker histones is a polymorphic structure. We show that different fiber conformations are obtained by tuning the linker histone orientation at the nucleosomes entry/exit according to the nucleosomal repeat length. We propose that the observed in vivo quantization of nucleosomal repeat length could reflect nature's ability to use the DNA molecule's helical geometry in order to give chromatin versatile topological and mechanical properties.     

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.     

Novel nucleosomal particles containing core histones and linker DNA but no histone H1

Eukaryotic chromosomal DNA is assembled into regularly spaced nucleosomes, which play a central role in gene regulation by determining accessibility of control regions. The nucleosome contains ∼147 bp of DNA wrapped ∼1.7 times around a central core histone octamer. The linker histone, H1, binds both to the nucleosome, sealing the DNA coils, and to the linker DNA between nucleosomes, directing chromatin folding. Micrococcal nuclease (MNase) digests the linker to yield the chromatosome, containing H1 and ∼160 bp, and then converts it to a core particle, containing ∼147 bp and no H1. Sequencing of nucleosomal DNA obtained after MNase digestion (MNase-seq) generates genome-wide nucleosome maps that are important for understanding gene regulation. We present an improved MNase-seq method involving simultaneous digestion with exonuclease III, which removes linker DNA. Remarkably, we discovered two novel intermediate particles containing 154 or 161 bp, corresponding to 7 bp protruding from one or both sides of the nucleosome core. These particles are detected in yeast lacking H1 and in H1-depleted mouse chromatin. They can be reconstituted in vitro using purified core histones and DNA. We propose that these ‘proto-chromatosomes’ are fundamental chromatin subunits, which include the H1 binding site and influence nucleosome spacing independently of H1.     

Prothymosin α is a component of a linker histone chaperone

Linker histone H1 binds with high affinity to naked and nucleosomal DNA in vitro but is rapidly exchanged between chromatin sites in vivo suggesting the involvement of one or more linker histone chaperones. Using permeabilized cells, we demonstrate that the small acidic protein prothymosin α (ProTα) can facilitate H1 displacement from and deposition onto the native chromatin template. Depletion of ProTα levels in vivo by siRNA-mediated mRNA degradation resulted in a decreased rate of exchange of linker histones as assayed by photobleaching techniques. These results indicate that ProTα is a component of a linker histone chaperone.