Structures of Native-like Nucleosomes: One Step Closer toward Understanding the Structure and Function of Chromatin

Genomic DNA in eukaryotes is organized into chromatin through association with core histone proteins to form nucleosomes. To understand the structure and function of chromatin, we must determine the structures of nucleosomes containing native DNA sequences. However, to date, our knowledge of nucleosome structures is mainly based on the crystallographic studies of the nucleosomes containing non-native DNA sequences. Here, we discuss the technical issues related to the determination of the nucleosome structures and review the few structural studies on native-like nucleosomes. We show how an antibody fragment-aided single-particle cryo-EM can be a useful method to determine the structures of nucleosomes containing genomic DNA. Finally, we provide a perspective for future structural studies of some native-like nucleosomes that play critical roles in chromatin functions.     

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.     

Specialized RSC: Substrate Specificities for a Conserved Chromatin Remodeler

The remodel the structure of chromatin (RSC) nucleosome remodeling complex is a conserved chromatin regulator with roles in chromatin organization, especially over nucleosome depleted regions therefore functioning in gene expression. Recent reports in Saccharomyces cerevisiae have identified specificities in RSC activity toward certain types of nucleosomes. RSC has now been shown to preferentially evict nucleosomes containing the histone variant H2A.Z in vitro. Furthermore, biochemical activities of distinct RSC complexes has been found to differ when their nucleosome substrate is partially unraveled. Mammalian BAF complexes, the homologs of yeast RSC and SWI/SNF complexes, are also linked to nucleosomes with H2A.Z, but this relationship may be complex and extent of conservation remains to be determined. The interplay of remodelers with specific nucleosome substrates and regulation of remodeler outcomes by nucleosome composition are tantalizing questions given the wave of structural data emerging for RSC and other SWI/SNF family remodelers.     

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.     

Effects of histone acetylation and CpG methylation on the structure of nucleosomes

Nucleosomes are the fundamental packing units of the eukaryotic genome. A nucleosome core particle comprises an octameric histone core wrapped around by ~147bp DNA. Histones and DNA are targets for covalent modifications mediated by various chromatin modification enzymes. These modifications play crucial roles in various gene regulation activities. A group of common hypotheses for the mechanisms of gene regulation involves changes in the structure and structural dynamics of chromatin induced by chromatin modifications. We employed single molecule fluorescence methods to test these hypotheses by monitoring the structure and structural dynamics of nucleosomes before and after histone acetylation and DNA methylation, two of the best-conserved chromatin modifications throughout eukaryotes. Our studies revealed that these modifications induce changes in the structure and structural dynamics of nucleosomes that may contribute directly to the formation of open or repressive chromatin conformation.     

Multiple Nhp6 molecules are required to recruit Spt16-Pob3 to form yFACT complexes and to reorganize nucleosomes

The Saccharomyces cerevisiae Nhp6 protein contains a DNA-binding motif that is similar to those found in the high mobility group B family of chromatin proteins. Nhp6 bound to nucleosomes and made at least two changes in them: the nucleosomal DNA became more sensitive to DNase I at specific sites, and the nucleosomes became competent to bind Spt16-Pob3 to form yFACT.nucleosome complexes. Both changes occurred at similar concentrations of Nhp6, suggesting that they reflect the same structural reorganization of the nucleosome. Nucleosomes have multiple binding sites for Nhp6, and structural reorganization was associated with a concentration of Nhp6 about 10-fold higher than that needed for simple binding. We propose that the coordinated action of multiple Nhp6 molecules is required to convert nucleosomes to an alternative form as the first step in a two-step reorganization of nucleosomes with the second step being dependent on Spt16-Pob3. The presence of linker DNA had only subtle effects on these processes, indicating that both Nhp6 and yFACT act on core nucleosome structure rather than on the interaction between nucleosomes and adjacent DNA. These results suggest that Nhp6 and the related high mobility group B proteins may have a general role in promoting rearrangements of chromatin by initiating the destabilization of core nucleosomal structure.     

Chromatin pattern consisting of repeating bipartite structures in WI-38 cells infected with human cytomegalovirus.

A novel chromatin structural pattern displaying bipartite and oblate ellipsoid structures orderly arranged along a fiber axis has been observed in WI-38 cells infected with human cytomegalovirus. This chromatin type coexists with chromatin fibers showing conventional nucleosomes. Each bipartite-oblate structure is 40 nm in length, about four times as long as an ordinary nucleosome, and the number of these structures per micrometer (11/micrometer) is clearly less than that of typical cellular nucleosomes (32/micrometer).     

NucleoMap: A computational tool for identifying nucleosomes in ultra-high resolution contact maps

Although poorly positioned nucleosomes are ubiquitous in the eukaryotic genome, they are difficult to identify with existing nucleosome identification methods. Recently available enhanced high-throughput chromatin conformation capture techniques such as Micro-C, DNase Hi-C, and Hi-CO characterize nucleosome-level chromatin proximity, probing the positions of mono-nucleosomes and the spacing between nucleosome pairs at the same time, enabling nucleosome profiling in poorly positioned regions. Here we develop a novel computational approach, NucleoMap, to identify nucleosome positioning from ultra-high resolution chromatin contact maps. By integrating nucleosome read density, contact distances, and binding preferences, NucleoMap precisely locates nucleosomes in both prokaryotic and eukaryotic genomes and outperforms existing nucleosome identification methods in both precision and recall. We rigorously characterize genome-wide association in eukaryotes between the spatial organization of mono-nucleosomes and their corresponding histone modifications, protein binding activities, and higher-order chromatin functions. We also find evidence of two tetra-nucleosome folding structures in human embryonic stem cells and analyze their association with multiple structural and functional regions. Based on the identified nucleosomes, nucleosome contact maps are constructed, reflecting the inter-nucleosome distances and preserving the contact distance profiles in original contact maps.     

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.     

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.     

Chromatin remodeling activities act on UV-damaged nucleosomes and modulate DNA damage accessibility to photolyase

Nucleosomes inhibit DNA repair in vitro, suggesting that chromatin remodeling activities might be required for efficient repair in vivo. To investigate how structural and dynamic properties of nucleosomes affect damage recognition and processing, we investigated repair of UV lesions by photolyase on a nucleosome positioned at one end of a 226-bp-long DNA fragment. Repair was slow in the nucleosome but efficient outside. No disruption or movement of the nucleosome was observed after UV irradiation and during repair. However, incubation with the nucleosome remodeling complex SWI/SNF and ATP altered the conformation of nucleosomal DNA as judged by UV photo-footprinting and promoted more homogeneous repair. Incubation with yISW2 and ATP moved the nucleosome to a more central position, thereby altering the repair pattern. This is the first demonstration that two different chromatin remodeling complexes can act on UV-damaged nucleosomes and modulate repair. Similar activities might relieve the inhibitory effect of nucleosomes on DNA repair processes in living cells.     

Nucleosome gaping supports a functional structure for the 30nm chromatin fiber

The biological functions played by the nucleus of eukaryotic cells and especially those involved in cellular differentiation not only depend on the genomic sequence but also on all the proteins which form the nucleo-protein complex named chromatin. The tridimensional organization of this huge polymer involves many structural levels, the most basic one being the nucleosome. Nucleosomes further organize into the so-called 30nm fiber, which, according to recent works, is likely to be the main functional level of chromatin. We wish here to propose a plausible structure for the 30nm chromatin fiber that could explain its functional role. In our model, silenced chromatin is locked by nucleosome stacking interactions. This is achieved by a conformational transition within the nucleosome core particle (NCP) which allows nucleosomes to stack along two helices without bending the DNA linkers. We used molecular modeling to check that this conformational transition was plausible. Then we proposed to modify the well-known two-angle model according to these atomic level results. The emerging picture is an allosteric behavior of the nucleosomes induced by their collective organization within the 30nm chromatin fiber.     

ISWI is an ATP-dependent nucleosome remodeling factor

The ATPase ISWI is a subunit of several distinct nucleosome remodeling complexes that increase the accessibility of DNA in chromatin. We found that the isolated ISWI protein itself was able to carry out nucleosome remodeling, nucleosome rearrangement, and chromatin assembly reactions. The ATPase activity of ISWI was stimulated by nucleosomes but not by free DNA or free histones, indicating that ISWI recognizes a specific structural feature of nucleosomes. Nucleosome remodeling, therefore, does not require a functional interaction between ISWI and the other subunits of ISWI complexes. The role of proteins associated with ISWI may be to regulate the activity of the remodeling engine or to define the physiological context within which a nucleosome remodeling reaction occurs.