Mesoscale Modeling of Nucleosome-Binding Antibody PL2-6: Mono- versus Bivalent Chromatin Complexes

Interactions of chromatin with bivalent immunoglobin nucleosome-binding antibodies and their monovalent (papain-derived) antigen-binding fragment analogs are useful probes for examining chromatin conformational states. To help interpret antibody-chromatin interactions and explore how antibodies might compete for interactions with chromatin components, we incorporate coarse-grained PL2-6 antibody modeling into our mesoscale chromatin model. We analyze interactions and fiber structures for the antibody-chromatin complexes in open and condensed chromatin, with and without H1 linker histone (LH). Despite minimal and transient interactions at physiological salt, we capture significant differences in antibody-chromatin complex configurations in open fibers, with more intense interactions between the bivalent antibody and chromatin compared to monovalent antigen-binding fragments. For these open chromatin fiber morphologies, antibody binding to histone tails is increased and compaction is greater for bivalent compared to monovalent and antibody-free systems. Differences between monovalent and bivalent binding result from antibody competition with internal chromatin fiber components (nucleosome core and linker DNA) for histone tail (H3, H4, H2A, H2B) interactions. This antibody competition for tail contacts reduces tail-core and tail-linker interactions and increases tail-antibody interactions. Such internal structural changes in open fibers resemble mechanisms of LH condensation, driven by charge screening and entropy changes. For condensed fibers at physiological salt, the three systems are much more similar overall, but some subtle tail interaction differences can be noted. Adding LH results in less-dramatic changes for all systems, except that the bivalent complex at physiological salt shows cooperative effects between LH and the antibodies in condensing chromatin fibers. Such dynamic interactions that depend on the internal structure and complex-stabilizing interactions within the chromatin fiber have implications for gene regulation and other chromatin complexes such as with LH, remodeling proteins, and small molecular chaperones that bind and modulate chromatin structure.     

Linker histone tails and N-tails of histone H3 are redundant: scanning force microscopy studies of reconstituted fibers.

The mechanisms responsible for organizing linear arrays of nucleosomes into the three-dimensional structure of chromatin are still largely unknown. In a companion paper (Leuba, S. H., et al. 1998. Biophys. J. 74:2823-2829), we study the contributions of linker histone domains and the N-terminal tail of core histone H3 to extended chromatin fiber structure by scanning force microscopy imaging of mildly trypsinized fibers. Here we complement and extend these studies by scanning force microscopy imaging of selectively reconstituted chromatin fibers, which differ in subtle but distinctive ways in their histone composition. We demonstrate an absolute requirement for the globular domain of the linker histones and a structural redundancy of the tails of linker histones and of histone H3 in determining conformational stability.     

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.     

Modeling Studies of Chromatin Fiber Structure as a Function of DNA Linker Length

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.     

HMG-D and histone H1 interplay during chromatin assembly and early embryogenesis

HMG-D is an abundant chromosomal protein associated with condensed chromatin during the first nuclear cleavage cycles of the developing Drosophila embryo. We previously suggested that HMG-D might substitute for the linker histone H1 in the preblastoderm embryo and that this substitution might result in the characteristic less compacted chromatin. We have now studied the association of HMG-D with chromatin using a cell-free system for chromatin reconstitution derived from Drosophila embryos. Association of HMG-D with chromatin, like that of histone H1, increases the nucleosome spacing indicative of binding to the linker DNA between nucleosomes. HMG-D interacts with DNA during the early phases of nucleosome assembly but is gradually displaced as chromatin matures. By contrast, purified chromatin can be loaded with stoichiometric amounts of HMG-D, and this can be displaced upon addition of histone H1. A direct physical interaction between HMG-D and histone H1 was observed in a Far Western analysis. The competitive nature of this interaction is reminiscent of the apparent replacement of HMG-D by H1 during mid-blastula transition. These data are consistent with the hypothesis that HMG-D functions as a specialized linker protein prior to appearance of histone H1.     

The Dynamic Influence of Linker Histone Saturation within the Poly-Nucleosome Array

Linker histones bind to nucleosomes and modify chromatin structure and dynamics as a means of epigenetic regulation. Biophysical studies have shown that chromatin fibers can adopt a plethora of conformations with varying levels of compaction. Linker histone condensation, and its specific binding disposition, has been associated with directly tuning this ensemble of states. However, the atomistic dynamics and quantification of this mechanism remains poorly understood. Here, we present molecular dynamics simulations of octa-nucleosome arrays, based on a cryo-EM structure of the 30-nm chromatin fiber, with and without the globular domains of the H1 linker histone to determine how they influence fiber structures and dynamics. Results show that when bound, linker histones inhibit DNA flexibility and stabilize repeating tetra-nucleosomal units, giving rise to increased chromatin compaction. Furthermore, upon the removal of H1, there is a significant destabilization of this compact structure as the fiber adopts less strained and untwisted states. Interestingly, linker DNA sampling in the octa-nucleosome is exaggerated compared to its mono-nucleosome counterparts, suggesting that chromatin architecture plays a significant role in DNA strain even in the absence of linker histones. Moreover, H1-bound states are shown to have increased stiffness within tetra-nucleosomes, but not between them. This increased stiffness leads to stronger long-range correlations within the fiber, which may result in the propagation of epigenetic signals over longer spatial ranges. These simulations highlight the effects of linker histone binding on the internal dynamics and global structure of poly-nucleosome arrays, while providing physical insight into a mechanism of chromatin compaction.     

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.     

Cathepsin L proteolytically processes histone H3 during mouse embryonic stem cell differentiation

Chromatin undergoes developmentally-regulated structural and chemical changes as cells differentiate, which subsequently lead to differences in cellular function by altering patterns of gene expression. To gain insight into chromatin alterations that occur during mammalian differentiation, we turned to a mouse embryonic stem cell (ESC) model. Here we show that histone H3 is proteolytically cleaved at its N-terminus during ESC differentiation. We map the sites of H3 cleavage and identify Cathepsin L as a protease responsible for proteolytically processing the N-terminal H3 tail. In addition, our data suggest that H3 cleavage may be regulated by covalent modifications present on the histone tail itself. Our studies underscore the intriguing possibility that histone proteolysis, brought about by Cathepsin L and potentially other family members, plays a role in development and differentiation that was not previously recognized.     

Linker histone H1 per se can induce three-dimensional folding of chromatin fiber

Higher-order architectures of chromosomes play important roles in the regulation of genome functions. To understand the molecular mechanism of genome packing, an in vitro chromatin reconstitution method and a single-molecule imaging technique (atomic force microscopy) were combined. In 50 mM NaCl, well-stretched beads-on-a-string chromatin fiber was observed. However, in 100 mM NaCl, salt-induced interaction between nucleosomes caused partial aggregation. Addition of histone H1 promoted a further folding of the fiber into thicker fibers 20-30 nm in width. Micrococcal nuclease digestion of these thicker fibers produced an approximately 170 bp fragment of nucleosomal DNA, which was approximately 20 bp longer than in the absence of histone H1 ( approximately 150 bp), indicating that H1 is correctly placed at the linker region. The width of the fiber depended on the ionic strength. Widths of 20 nm in 50 mM NaCl became 30 nm as the ionic strength was changed to 100 mM. On the basis of these results, a flexible model of chromatin fiber formation was proposed, where the mode of the fiber compaction changes depending both on salt environment and linker histone H1. The biological significance of this property of the chromatin architecture will be apparent in the closed segments ( approximately 100 kb) between SAR/MAR regions.     

Toward convergence of experimental studies and theoretical modeling of the chromatin fiber

Understanding the structural organization of eukaryotic chromatin and its control of gene expression represents one of the most fundamental and open challenges in modern biology. Recent experimental advances have revealed important characteristics of chromatin in response to changes in external conditions and histone composition, such as the conformational complexity of linker DNA and histone tail domains upon compact folding of the fiber. In addition, modeling studies based on high-resolution nucleosome models have helped explain the conformational features of chromatin structural elements and their interactions in terms of chromatin fiber models. This minireview discusses recent progress and evidence supporting structural heterogeneity in chromatin fibers, reconciling apparently contradictory fiber models.     

Conserved organization of centromeric chromatin in flies and humans

Recent studies have highlighted the importance of centromere-specific histone H3-like (CENP-A) proteins in centromere function. We show that Drosophila CID and human CENP-A appear at metaphase as a three-dimensional structure that lacks histone H3. However, blocks of CID/CENP-A and H3 nucleosomes are linearly interspersed on extended chromatin fibers, and CID is close to H3 nucleosomes in polynucleosomal preparations. When CID is depleted by RNAi, it is replaced by H3, demonstrating flexibility of centromeric chromatin organization. Finally, contrary to models proposing that H3 and CID/CENP-A nucleosomes are replicated at different times in S phase, we show that interspersed H3 and CID/CENP-A chromatin are replicated concurrently during S phase in humans and flies. We propose that the unique structural arrangement of CID/CENP-A and H3 nucleosomes presents centromeric chromatin to the poleward face of the condensing mitotic chromosome.     

Dynamic condensation of linker histone C-terminal domain regulates chromatin structure

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.     

The type of interaction of histone H5 wo ith DNA changes significantly at various stages of chromatin condensation

Histones were covalently bound to DNA by dimethylsulfate-induced crosslinking and DNA-contacting peptides of histone H5, thus modified, were mapped by a combination of peptide cleavage reactions and peptide gel electrophoresis. In the nucleosome, the only strong crosslinking point is His-25 which resides near the ends of nucleosomal DNA. This contact point persists throughout different steps of chromatin condensation--decondensation. In decondensed chromatin, it is supplemented by the contact with DNA of the N-terminus of the histone H5 molecule. The high level of chromatin condensation existing in the nuclei or induced by bivalent cations results in a new and considerably stronger crosslinking point His-62, which is also characteristic for cooperative H5-DNA complexes. This structural change is observed only on oligonucleosomal chains containing no less than 3 contiguous nucleosomes, and is absent in isolated mono- or dinucleosomes. We propose that the formation of the 30-nm chromatin fibre, typical for the nuclei, is accomplished in part by the histone H5-linker DNA cooperative interactions, manifested by strong His-62--linker DNA contact.     

30 nm chromatin fibre decompaction requires both H4-K16 acetylation and linker histone eviction

The mechanism by which chromatin is decondensed to permit access to DNA is largely unknown. Here, using a model nucleosome array reconstituted from recombinant histone octamers, we have defined the relative contribution of the individual histone octamer N-terminal tails as well as the effect of a targeted histone tail acetylation on the compaction state of the 30 nm chromatin fiber. This study goes beyond previous studies as it is based on a nucleosome array that is very long (61 nucleosomes) and contains a stoichiometric concentration of bound linker histone, which is essential for the formation of the 30 nm chromatin fiber. We find that compaction is regulated in two steps: Introduction of H4 acetylated to 30% on K16 inhibits compaction to a greater degree than deletion of the H4 N-terminal tail. Further decompaction is achieved by removal of the linker histone.