Quantitative analysis of single-molecule force spectroscopy on folded chromatin fibers

Single-molecule techniques allow for picoNewton manipulation and nanometer accuracy measurements of single chromatin fibers. However, the complexity of the data, the heterogeneity of the composition of individual fibers and the relatively large fluctuations in extension of the fibers complicate a structural interpretation of such force-extension curves. Here we introduce a statistical mechanics model that quantitatively describes the extension of individual fibers in response to force on a per nucleosome basis. Four nucleosome conformations can be distinguished when pulling a chromatin fiber apart. A novel, transient conformation is introduced that coexists with single wrapped nucleosomes between 3 and 7 pN. Comparison of force-extension curves between single nucleosomes and chromatin fibers shows that embedding nucleosomes in a fiber stabilizes the nucleosome by 10 k_BT. Chromatin fibers with 20- and 50-bp linker DNA follow a different unfolding pathway. These results have implications for accessibility of DNA in fully folded and partially unwrapped chromatin fibers and are vital for understanding force unfolding experiments on nucleosome arrays.     

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.     

On the structure and dynamics of the complex of the nucleosome and the linker histone

Several different models of the linker histone (LH)–nucleosome complex have been proposed, but none of them has unambiguously revealed the position and binding sites of the LH on the nucleosome. Using Brownian dynamics-based docking together with normal mode analysis of the nucleosome to account for the flexibility of two flanking 10 bp long linker DNAs (L-DNA), we identified binding modes of the H5-LH globular domain (GH5) to the nucleosome. For a wide range of nucleosomal conformations with the L-DNA ends less than 65 Å apart, one dominant binding mode was identified for GH5 and found to be consistent with fluorescence recovery after photobleaching (FRAP) experiments. GH5 binds asymmetrically with respect to the nucleosomal dyad axis, fitting between the nucleosomal DNA and one of the L-DNAs. For greater distances between L-DNA ends, docking of GH5 to the L-DNA that is more restrained and less open becomes favored. These results suggest a selection mechanism by which GH5 preferentially binds one of the L-DNAs and thereby affects DNA dynamics and accessibility and contributes to formation of a particular chromatin fiber structure. The two binding modes identified would, respectively, favor a tight zigzag chromatin structure or a loose solenoid chromatin fiber.     

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.     

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.     

Structure and dynamics of nucleosomal DNA

The last five years have seen exciting advances in our understanding of the structure of the nucleosome core particle, the basic repeating unit in all eukaryotic chromatin. A picture emerges in which nucleosomal DNA, while distorted and compacted fivefold by tight interactions with the histone octamer core, is at the same time highly dynamic and adaptable. Here, we summarize the salient features from recent structural studies of nucleosome core particles (both published and unpublished) that concern the structure and dynamics of nucleosomal DNA, and the nature of protein-DNA interactions. Current mechanisms for chromatin remodeling and nucleosome sliding are discussed in light of new structural evidence. Finally, techniques to study nucleosome stability and ultimately dynamics are introduced.     

Nucleosome dynamics

In the 30 years since the discovery of the nucleosome, our picture of it has come into sharp focus. The recent high-resolution structures have provided a wealth of insight into the function of the nucleosome, but they are inherently static. Our current knowledge of how nucleosomes can be reconfigured dynamically is at a much earlier stage. Here, recent advances in the understanding of chromatin structure and dynamics are highlighted. The ways in which different modes of nucleosome reconfiguration are likely to influence each other are discussed, and some of the factors likely to regulate the dynamic properties of nucleosomes are considered.     

Depletion Effects Massively Change Chromatin Properties and Influence Genome Folding

We present a Monte Carlo model for genome folding at the 30-nm scale with focus on linker-histone and nucleosome depletion effects. We find that parameter distributions from experimental data do not lead to one specific chromatin fiber structure, but instead to a distribution of structures in the chromatin phase diagram. Depletion of linker histones and nucleosomes affects, massively, the flexibility and the extension of chromatin fibers. Increasing the amount of nucleosome skips (i.e., nucleosome depletion) can lead either to a collapse or to a swelling of chromatin fibers. These opposing effects are discussed and we show that depletion effects may even contribute to chromatin compaction. Furthermore, we find that predictions from experimental data for the average nucleosome skip rate lie exactly in the regime of maximum chromatin compaction. Finally, we determine the pair distribution function of chromatin. This function reflects the structure of the fiber, and its Fourier-transform can be measured experimentally. Our calculations show that even in the case of fibers with depletion effects, the main dominant peaks (characterizing the structure and the length scales) can still be identified.     

On the role of inter-nucleosomal interactions and intrinsic nucleosome dynamics in chromatin function

Evidence is emerging that many diseases result from defects in gene functions, which, in turn, depend on the local chromatin environment of a gene. However, it still remains not fully clear how chromatin activity code is ‘translated’ to the particular ‘activating’ or ‘repressing’ chromatin structural transition. Commonly, chromatin remodeling in vitro was studied using mononucleosomes as a model. However, recent data suggest that structural reorganization of a single mononucleosome is not equal to remodeling of a nucleosome particle under multinucleosomal content – such as, interaction of nucleosomes via flexible histone termini could significantly alter the mode (and the resulting products) of nucleosome structural transitions. It is becoming evident that a nucleosome array does not constitute just a ‘polymer’ of individual ‘canonical’ nucleosomes due to multiple inter-nucleosomal interactions which affect nucleosome dynamics and structure. It could be hypothesized, that inter-nucleosomal interactions could act in cooperation with nucleosome inherent dynamics to orchestrate DNA-based processes and promote formation and stabilization of highly-dynamic, accessible structure of a nucleosome array. In the proposed paper we would like to discuss the nucleosome dynamics within the chromatin fiber mainly as it pertains to the roles of the structural changes mediated by inter-nucleosomal interactions.     

Chromatin under mechanical stress: from single 30 nm fibers to single nucleosomes

About a decade ago, the elastic properties of a single chromatin fiber and, subsequently, those of a single nucleosome started to be explored using optical and magnetic tweezers. These techniques have allowed direct measurements of several essential physical parameters of individual nucleosomes and nucleosomal arrays, including the forces responsible for the maintenance of the structure of both the chromatin fiber and the individual nucleosomes, as well as the mechanism of their unwinding under mechanical stress. Experiments on the assembly of individual chromatin fibers have illustrated the complexity of the process and the key role of certain specific components. Nevertheless a substantial disparity exists in the data reported from various experiments. Chromatin, unlike naked DNA, is a system which is extremely sensitive to environmental conditions, and studies carried out under even slightly different conditions are difficult to compare directly. In this review we summarize the available data and their impact on our knowledge of both nucleosomal structure and the dynamics of nucleosome and chromatin fiber assembly and organization.     

Nucleosomal anatomy – where are the histones?

The recent surge of discoveries concerning the structural organization of nucleosomes, together with genetic evidence of highly specialized roles for the histones in gene regulation, have brought a renewed need for a detailed understanding of nucleosomal anatomy. Here we review recent structural advances leading to a new level of understanding of the nucleosome and chromatin fibre structure. We discuss the problems and challenges for existing models of chromatin structure and, in particular, consider how linker histones may bind within the nucleosome, together with the implications of their association for the structure of the chromatin fibre.     

How does the histone code work?

Patterns of histone post-translational modifications correlate with distinct chromosomal states that regulate access to DNA, leading to the histone-code hypothesis. However, it is not clear how modification of flexible histone tails leads to changes in nucleosome dynamics and, thus, chromatin structure. The recent discovery that, like the flexible histone tails, the structured globular domain of the nucleosome core particle is also extensively modified adds a new and exciting dimension to the histone-code hypothesis, and calls for the re-examination of current models for the epigenetic regulation of chromatin structure. Here, we review these findings and other recent studies that suggest the structured globular domain of the nucleosome core particle plays a key role regulating chromatin dynamics.