Dependence of the Linker Histone and Chromatin Condensation on the Nucleosome Environment.
ABSTRACT: The linker histone (LH), an auxiliary protein that can bind to chromatin and interact with the linker DNA to form stem motifs, is a key element of chromatin compaction. By affecting the chromatin condensation level, it also plays an active role in gene expression. However, the presence and variable concentration of LH in chromatin fibers with different DNA linker lengths indicate that its folding and condensation are highly adaptable and dependent on the immediate nucleosome environment. Recent experimental studies revealed that the behavior of LH in mononucleosomes markedly differs from that in small nucleosome arrays, but the associated mechanism is unknown. Here we report a structural analysis of the behavior of LH in mononucleosomes and oligonucleosomes (2-6 nucleosomes) using mesoscale chromatin simulations. We show that the adapted stem configuration heavily depends on the strength of electrostatic interactions between LH and its parental DNA linkers, and that those interactions tend to be asymmetric in small oligonucleosome systems. Namely, LH in oligonucleosomes dominantly interacts with one DNA linker only, as opposed to mononucleosomes where LH has similar interactions with both linkers and forms a highly stable nucleosome stem. Although we show that the LH condensation depends sensitively on the electrostatic interactions with entering and exiting DNA linkers, other interactions, especially by nonparental cores and nonparental linkers, modulate the structural condensation by softening LH and thus making oligonucleosomes more flexible, in comparison to to mono- and dinucleosomes. We also find that the overall LH/chromatin interactions sensitively depend on the linker length because the linker length determines the maximal nucleosome stem length. For mononucleosomes with DNA linkers shorter than LH, LH condenses fully, while for DNA linkers comparable or longer than LH, the LH extension in mononucleosomes strongly follows the length of DNA linkers, unhampered by neighboring linker histones. Thus, LH is more condensed for mononucleosomes with short linkers, compared to oligonucleosomes, and its orientation is variable and highly environment-dependent. More generally, the work underscores the agility of LH whose folding dynamics critically controls genomic packaging and gene expression.
Project description: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.
Project description:Linker histones are an integral component of chromatin but how these proteins promote assembly of chromatin fibers and higher order structures and regulate gene expression remains an open question. Using Förster resonance energy transfer (FRET) approaches we find that association of a linker histone with oligonucleosomal arrays induces condensation of the intrinsically disordered H1 CTD in a manner consistent with adoption of a defined fold or ensemble of folds in the bound state. However, H1 CTD structure when bound to nucleosomes in arrays is distinct from that induced upon H1 association with mononucleosomes or bare double stranded DNA. Moreover, the H1 CTD becomes more condensed upon condensation of extended nucleosome arrays to the contacting zig-zag form found in moderate salts, but does not detectably change during folding to fully compacted chromatin fibers. We provide evidence that linker DNA conformation is a key determinant of H1 CTD structure and that constraints imposed by neighboring nucleosomes cause linker DNAs to adopt distinct trajectories in oligonucleosomes compared to H1-bound mononucleosomes. Finally, inter-molecular FRET between H1s within fully condensed nucleosome arrays suggests a regular spatial arrangement for the H1 CTD within the 30 nm chromatin fiber.
Project description:Chromatin fibers encountered in various species and tissues are characterized by different nucleosome repeat lengths (NRLs) of the linker DNA connecting the nucleosomes. While single cellular organisms and rapidly growing cells with high protein production have short NRL ranging from 160 to 189 bp, mature cells usually have longer NRLs ranging between 190 and 220 bp. Recently, various experimental studies have examined the effect of NRL on the internal organization of chromatin fiber. Here, we investigate by mesoscale modeling of oligonucleosomes the folding patterns for different NRL, with and without linker histone (LH), under typical monovalent salt conditions using both one-start solenoid and two-start zigzag starting configurations. We find that short to medium NRL chromatin fibers (173 to 209 bp) with LH condense into zigzag structures and that solenoid-like features are viable only for longer NRLs (226 bp). We suggest that medium NRLs are more advantageous for packing and various levels of chromatin compaction throughout the cell cycle than their shortest and longest brethren; the former (short NRLs) fold into narrow fibers, while the latter (long NRLs) arrays do not easily lead to high packing ratios due to possible linker DNA bending. Moreover, we show that the LH has a small effect on the condensation of short-NRL arrays but has an important condensation effect on medium-NRL arrays, which have linker lengths similar to the LH lengths. Finally, we suggest that the medium-NRL species, with densely packed fiber arrangements, may be advantageous for epigenetic control because their histone tail modifications can have a greater effect compared to other fibers due to their more extensive nucleosome interaction network.
Project description:The basic and intrinsically disordered C-terminal domain (CTD) of the linker histone (LH) is essential for chromatin compaction. However, its conformation upon nucleosome binding and its impact on chromatin organization remain unknown. Our mesoscale chromatin model with a flexible LH CTD captures a dynamic, salt-dependent condensation mechanism driven by charge neutralization between the LH and linker DNA. Namely, at low salt concentration, CTD condenses, but LH only interacts with the nucleosome and one linker DNA, resulting in a semi-open nucleosome configuration; at higher salt, LH interacts with the nucleosome and two linker DNAs, promoting stem formation and chromatin compaction. CTD charge reduction unfolds the domain and decondenses chromatin, a mechanism in consonance with reduced counterion screening in vitro and phosphorylated LH in vivo. Divalent ions counteract this decondensation effect by maintaining nucleosome stems and expelling the CTDs to the fiber exterior. Additionally, we explain that the CTD folding depends on the chromatin fiber size, and we show that the asymmetric structure of the LH globular head is responsible for the uneven interaction observed between the LH and the linker DNAs. All these mechanisms may impact epigenetic regulation and higher levels of chromatin folding.
Project description: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.
Project description:Packing of about two meters of the human genome DNA into chromatin occupying a several micron-sized cell nucleus requires a high degree of compaction in a manner that allows the information encoded on DNA to remain easily accessible. This packing is mediated by repeated coiling of DNA double helix around histones to form nucleosome arrays that are further folded into higher-order structures. Relatively straight DNA linkers separate the nucleosomes and the spacing between consecutive nucleosome varies between different cells and between different chromosomal loci. In a recent work ( 1) our group used a biochemically defined in vitro reconstituted system to explore how do various DNA linkers mediate nucleosome array packing into higher-order chromatin structures. For long nucleosome linkers (about 60 bp) we observed a more open chromatin structure and no effect of small linker length alterations (±2-4 bp) on chromatin folding. In striking contrast, for shorter linkers (20-32 bp) we found more compact packing with strong periodical dependence upon the linker DNA lengths. Our data together with high-resolution nucleosome position mapping provide evidence for the natural nucleosome repeats to support a chromatin architecture that, by default, restricts spontaneous folding of nucleosome arrays into compact chromatin fibers. We suggest that incomplete folding of the nucleosome arrays may promote global inter-array interactions that lead to chromatin condensation in metaphase chromosomes and heterochromatin.
Project description:We describe a new mesoscopic model of oligonucleosomes that incorporates flexible histone tails. The nucleosome cores are modeled using the discrete surface-charge optimization model, which treats the nucleosome as an electrostatic surface represented by hundreds of point charges; the linker DNAs are treated using a discrete elastic chain model; and the histone tails are modeled using a bead/chain hydrodynamic approach as chains of connected beads where each bead represents five protein residues. Appropriate charges and force fields are assigned to each histone chain so as to reproduce the electrostatic potential, structure, and dynamics of the corresponding atomistic histone tails at different salt conditions. The dynamics of resulting oligonucleosomes at different sizes and varying salt concentrations are simulated by Brownian dynamics with complete hydrodynamic interactions. The analyses demonstrate that the new mesoscopic model reproduces experimental results better than its predecessors, which modeled histone tails as rigid entities. In particular, our model with flexible histone tails: correctly accounts for salt-dependent conformational changes in the histone tails; yields the experimentally obtained values of histone-tail mediated core/core attraction energies; and considers the partial shielding of electrostatic repulsion between DNA linkers as a result of the spatial distribution of histone tails. These effects are crucial for regulating chromatin structure but are absent or improperly treated in models with rigid histone tails. The development of this model of oligonucleosomes thus opens new avenues for studying the role of histone tails and their variants in mediating gene expression through modulation of chromatin structure.
Project description:Linker DNA conformational variability has been proposed to direct nucleosome array folding into more or less compact chromatin fibers but direct experimental evidence for such models are lacking. Here, we tested this hypothesis by designing nucleosome arrays with A-tracts at specific locations in the nucleosome linkers to induce inward (AT-IN) and outward (AT-OUT) bending of the linker DNA. Using electron microscopy and analytical centrifugation techniques, we observed spontaneous folding of AT-IN nucleosome arrays into highly compact structures, comparable to those induced by linker histone H1. In contrast, AT-OUT nucleosome arrays formed less compact structures with decreased nucleosome interactions similar to wild-type nucleosome arrays. Adding linker histone H1 further increased compaction of the A-tract arrays while maintaining structural differences between them. Furthermore, restriction nuclease digestion revealed a strongly reduced accessibility of nucleosome linkers in the compact AT-IN arrays. Electron microscopy analysis and 3D computational Monte Carlo simulations are consistent with a profound zigzag linker DNA configuration and closer nucleosome proximity in the AT-IN arrays due to inward linker DNA bending. We propose that the evolutionary preferred positioning of A-tracts in DNA linkers may control chromatin higher-order folding and thus influence cellular processes such as gene expression, transcription and DNA repair.
Project description:Monte Carlo simulations of a mesoscale model of oligonucleosomes are analyzed to examine the role of dynamic-linker histone (LH) binding/unbinding in high monovalent salt with divalent ions, and to further interpret noted chromatin fiber softening by dynamic LH in monovalent salt conditions. We find that divalent ions produce a fiber stiffening effect that competes with, but does not overshadow, the dramatic softening triggered by dynamic-LH behavior. Indeed, we find that in typical in vivo conditions, dynamic-LH binding/unbinding reduces fiber stiffening dramatically (by a factor of almost 5, as measured by the elasticity modulus) compared with rigidly fixed LH, and also the force needed to initiate chromatin unfolding, making it consistent with those of molecular motors. Our data also show that, during unfolding, divalent ions together with LHs induce linker-DNA bending and DNA-DNA repulsion screening, which guarantee formation of heteromorphic superbeads-on-a-string structures that combine regions of loose and compact fiber independently of the characteristics of the LH-core bond. These structures might be important for gene regulation as they expose regions of the DNA selectively. Dynamic control of LH binding/unbinding, either globally or locally, in the presence of divalent ions, might constitute a mechanism for regulation of gene expression.
Project description:The length of linker DNA that separates nucleosomes is highly variable, but its mechanistic role in modulating chromatin structure and functions remains unknown. Here, we established an experimental system using circular arrays of positioned nucleosomes to investigate whether variations in nucleosome linker length could affect nucleosome folding, self-association, and interactions. We conducted EM, DNA topology, native electrophoretic assays, and Mg2+-dependent self-association assays to study intrinsic folding of linear and circular nucleosome arrays with linker DNA length of 36 bp and 41 bp (3.5 turns and 4 turns of DNA double helix, respectively). These experiments revealed that potential artifacts arising from open DNA ends and full DNA relaxation in the linear arrays do not significantly affect overall chromatin compaction and self-association. We observed that the 0.5 DNA helical turn difference between the two DNA linker lengths significantly affects DNA topology and nucleosome interactions. In particular, the 41-bp linkers promoted interactions between any two nucleosome beads separated by one bead as expected for a zigzag fiber, whereas the 36-bp linkers promoted interactions between two nucleosome beads separated by two other beads and also reduced negative superhelicity. Monte Carlo simulations accurately reproduce periodic modulations of chromatin compaction, DNA topology, and internucleosomal interactions with a 10-bp periodicity. We propose that the nucleosome spacing and associated chromatin structure modulations may play an important role in formation of different chromatin epigenetic states, thus suggesting implications for how chromatin accessibility to DNA-binding factors and the RNA transcription machinery is regulated.