Controlled DNA compaction within chromatin: the tail-bridging effect

We study the mechanism underlying the attraction between nucleosomes, the fundamental packaging units of DNA inside the chromatin complex. We introduce a simple model of the nucleosome, the eight-tail colloid, consisting of a charged sphere with eight oppositely charged, flexible, grafted chains that represent the terminal histone tails. We demonstrate that our complexes are attracted via the formation of chain bridges and that this attraction can be tuned by changing the fraction of charged monomers on the tails. This suggests a physical mechanism of chromatin compaction where the degree of DNA condensation can be controlled via biochemical means, namely the acetylation and deacetylation of lysines in the histone tails.Comment: 4 pages, 5 figures, submitte

The dynamics of individual nucleosomes controls the chromatin condensation pathway: direct AFM visualization of variant chromatin

Chromatin organization and dynamics is studied in this work at scales ranging from single nucleosome to nucleosomal array by using a unique combination of biochemical assays, single molecule imaging technique and numerical modeling. We demonstrate that a subtle modification in the nucleosome structure induced by the histone variant H2A.Bbd drastically modifies the higher order organization of the nucleosomal arrays. Importantly, as directly visualized by AFM, conventional H2A nucleosomal arrays exhibit specific local organization, in contrast to H2A.Bbd arrays, which show "beads on a string" structure. The combination of systematic image analysis and theoretical modeling allows a quantitative description relating the observed gross structural changes of the arrays to their local organization. Our results strongly suggest that higher-order organization of H1-free nucleosomal arrays is mainly determined by the fluctuation properties of individual nucleosomes. Moreover, numerical simulations suggest the existence of attractive interactions between nucleosomes to provide the degree of compaction observed for conventional chromatin fibers.Comment: Biophys J. in pres

Chromatin: a tunable spring at work inside chromosomes

This paper focuses on mechanical aspects of chromatin biological functioning. Within a basic geometric modeling of the chromatin assembly, we give for the first time the complete set of elastic constants (twist and bend persistence lengths, stretch modulus and twist-stretch coupling constant) of the so-called 30-nm chromatin fiber, in terms of DNA elastic properties and geometric properties of the fiber assembly. The computation naturally embeds the fiber within a current analytical model known as the ``extensible worm-like rope'', allowing a straightforward prediction of the force-extension curves. We show that these elastic constants are strongly sensitive to the linker length, up to 1 bp, or equivalently to its twist, and might locally reach very low values, yielding a highly flexible and extensible domain in the fiber. In particular, the twist-stretch coupling constant, reflecting the chirality of the chromatin fiber, exhibits steep variations and sign changes when the linker length is varied. We argue that this tunable elasticity might be a key feature for chromatin function, for instance in the initiation and regulation of transcription.Comment: 38 pages 15 figure

DNA folding: structural and mechanical properties of the two-angle model for chromatin

We present a theoretical analysis of the structural and mechanical properties of the 30-nm chromatin fiber. Our study is based on the two-angle model introduced by Woodcock et al. (Woodcock, C. L., S. A. Grigoryev, R. A. Horowitz, and N. Whitaker. 1993. PNAS 90:9021-9025) that describes the chromatin fiber geometry in terms of the entry-exit angle of the nucleosomal DNA and the rotational setting of the neighboring nucleosomes with respect to each other. We explore analytically the different structures that arise from this building principle, and demonstrate that the geometry with the highest density is close to the one found in native chromatin fibers under physiological conditions. On the basis of this model we calculate mechanical properties of the fiber under stretching. We obtain expressions for the stress-strain characteristics which show good agreement with the results of recent stretching experiments (Cui, Y., and C. Bustamante. 2000. PNAS 97:127-132) and computer simulations (Katritch, V., C. Bustamante, and W. K. Olson. 2000. J. Mol. Biol. 295:29-40), and which provide simple physical insights into correlations between the structural and elastic properties of chromatin.Comment: 23 pages, 6 figures, to be published in Biophys.

Crucial role of dynamic linker histone binding and divalent ions for DNA accessibility and gene regulation revealed by mesoscale modeling of oligonucleosomes.

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

Intrinsically Disordered Proteins within the Genome

The hundreds of millions of DNA base pairs within eukaryotic cells are not found free but packed inside the micrometre-sized nuclei through the formation of a macromolecular structure known as chromatin. Chromatin consists of a chain of nucleosomes – nucleoprotein complexes where the DNA makes ∼1.75 turns around a protein octamer core composed of two copies each of H2A, H2B, H3 and H4 histones. A fifth histone H1 binds on the nucleosomal surface close to the entry/exit site of DNA, interacts with linker DNA and aids in chromatin compaction. Enabling the condensation of DNA to fit into the nucleus is however only one-half of chromatin’s role. The three-dimensional spatial organization of chromatin serves a second important role in allowing the capability to exert control over gene expression. The chromatin structure thus serves as an additional layer of complexity above the genome code and permits the transcription of different proteins varying with cell lineages/cycles. The proteins that makeup, modify and read the chromatin structure are particularly enriched in `Intrinsic Disorder’ – a class of proteins lacking a well-defined structure but existing as a dynamic ensemble of rapidly interchanging states. While folded proteins with well-defined structures are amenable to be characterized through standard methods of protein structure determination, the `plasticity’ of the disordered proteins challenges the use of such ensemble averaged techniques. In this thesis, Molecular Dynamics simulations are used to characterize the disordered regions of three proteins that form the core of chromatin structure: histones, linker histones (H1) and heterochromatin protein (HP1). The carboxy-terminal domain of H1 when within the nucleosome, adopts a compact but unstructured conformation that allows its positioning between the two linker DNA strands. In contrast, the amino-terminal domain of H1 undergoes a disorder-to-order transition to an amphiphilic helical conformation. The transition to the amphiphilic helix is however subtype-dependant with the degree of condensation varying with the subtypes' nucleosomal affinity. Finally, the simulations demonstrate that the affinity of HP1 subtypes for the H3 histone is caused by the synergetic effects of both the proteins' unstructured amino-terminal domain and the structured chromodomain

Hierarchies in eukaryotic genome organization: Insights from polymer theory and simulations

Eukaryotic genomes possess an elaborate and dynamic higher-order structure within the limiting confines of the cell nucleus. Knowledge of the physical principles and the molecular machinery that govern the 3D organization of this structure and its regulation are key to understanding the relationship between genome structure and function. Elegant microscopy and chromosome conformation capture techniques supported by analysis based on polymer models are important steps in this direction. Here, we review results from these efforts and provide some additional insights that elucidate the relationship between structure and function at different hierarchical levels of genome organization

Chromatin and epigenetics: current biophysical views

Recent advances in high-throughput sequencing experiments and their theoretical descriptions have determined fast dynamics of the "chromatin and epigenetics" field, with new concepts appearing at high rate. This field includes but is not limited to the study of DNA-protein-RNA interactions, chromatin packing properties at different scales, regulation of gene expression and protein trafficking in the cell nucleus, binding site search in the crowded chromatin environment and modulation of physical interactions by covalent chemical modifications of the binding partners. The current special issue does not pretend for the full coverage of the field, but it rather aims to capture its development and provide a snapshot of the most recent concepts and approaches. Eighteen open-access articles comprising this issue provide a delicate balance between current theoretical and experimental biophysical approaches to uncover chromatin structure and understand epigenetic regulation, allowing free flow of new ideas and preliminary results

Single-Molecule Investigation of Chromatin-Associated Factors in Genome Organization and Epigenetic Maintenance

The central dogma of biology has laid the foundation for understanding gene expression through the mechanisms of transcription and translation. However, another layer of eukaryotic gene regulation lies in the complex structure of chromatin. This scaffold of structural proteins and enzymatic regulators determines what genes are expressed at what times, leading to cell differentiation, cell fate, and often disease. Currently, the field of chromatin biology has relied on basic biochemistry and cellular assays to identify key epigenetic regulators and their role in genomic maintenance. For this thesis work, I have developed a biophysical platform to study chromatin-associated factors at the single-molecule level (Chapter 2). This methodology allows us to extract key mechanistic details often obscured by standard bulk methodologies. Using this platform, we posed the question of how epigenetic factor, Polycomb repressive complex 2 (PRC2) engages with chromatin (Chapter 3). PRC2 is a major epigenetic machinery that maintains transcriptionally silent heterochromatin in the nucleus and plays critical roles in embryonic development and oncogenesis. It is generally thought that PRC2 propagates repressive histone marks by modifying neighboring nucleosomes in a strictly linear progression. However, the behavior of PRC2 on native-like chromatin substrates remains incompletely characterized, making the precise mechanism of PRC2-mediated heterochromatin maintenance elusive. Our understanding of this process was limited by the resolution of structural techniques that fail to identify PRC2-binding modes on long chromatin substrates. In short, we found direct evidence that PRC2 can simultaneously engage nonadjacent nucleosome pairs. The demonstration of PRC2\u27s ability to bridge noncontiguous chromosomal segments furthers our understanding of how Polycomb complexes spread epigenetic modifications and compact chromatin. In addition to this single-molecule chromatin binding technology, I also created a singlemolecule platform harnessing correlative force and fluorescence microscopy to assay the material properties of phase separated condensates (Chapter 2). This assay combined methodology to visualize condensate formation at the single-molecule level, in addition to optical trapping of individual droplets to investigate their material properties. Utilizing this technology, we interrogated the role of linker histone H1 (Chapter 4). The linker histones are the most abundant group of chromatin-binding proteins that bind and organize eukaryotic chromatin. However, roles for the diverse and largely unstructured H1 proteins beyond chromatin compaction remain unclear. We used correlative single-molecule force and fluorescence microscopy to directly visualize the behavior of H1 on DNA under different tensions. Unexpectedly, our results show that H1 preferentially coalesces around nascent, relaxed singlestranded DNA. In vitro bulk assays confirmed that H1 has a higher propensity to form phaseseparated condensates with single-stranded DNA than with double-stranded DNA. Furthermore, we dissected the material properties of different H1:DNA condensates by controlled droplet fusion with optical tweezers, and found that increased DNA length and GC content result in more viscous, gel-like H1 condensates. Overall, our findings suggest a potential role for linker histones to sense and coacervate single-stranded nucleic acids in the nucleus, forming reaction hubs for genome maintenance. This work also provides a new perspective to understand how various H1 subtypes and disease-associated mutations affect chromatin structure and function. In summary, we have gained a greater understanding of the biophysical basis for chromatin regulation by both PRC2 and histone H1. Both of the biophysical platforms created for these studies can be applied to various new targets in chromatin biology. They will enable the investigation of a multiplicity of binding interactions, regulatory mechanisms, and material properties of protein-nucleic acid complexes (Chapters 5 & 6). I believe single-molecule techniques will become a major toolset to study chromatin biology, identifying the intricacies and interactions between epigenetic factors and our genome

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