RCC1 Uses a Conformationally Diverse Loop Region to Interact with the Nucleosome: A Model for the RCC1-Nucleosome Complex

The binding of RCC1 (regulator of chromosome condensation 1) to chromatin is critical for cellular processes such as mitosis, nucleocytoplasmic transport, and nuclear envelope formation because RCC1 recruits the small GTPase Ran (Ras-related nuclear protein) to chromatin and sets up a Ran-GTP gradient around the chromosomes. However, the molecular mechanism by which RCC1 binds to nucleosomes, the repeating unit of chromatin, is not known. We have used biochemical approaches to test structural models for how the RCC1 β-propeller protein could bind to the nucleosome. In contrast to the prevailing model, RCC1 does not appear to use the β-propeller face opposite to its Ran-binding face to interact with nucleosomes. Instead, we find that RCC1 uses a conformationally flexible loop region we have termed the switchback loop in addition to its N-terminal tail to bind to the nucleosome. The juxtaposition of the RCC1 switchback loop to its Ran binding surface suggests a novel mechanism for how nucleosome-bound RCC1 recruits Ran to chromatin. Furthermore, this model accounts for previously unexplained observations for how Ran can interact with the nucleosome both dependent and independent of RCC1 and how binding of the nucleosome can enhance RCC1's Ran nucleotide exchange activity.     

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.     

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.     

Conserved Substitution Patterns around Nucleosome Footprints in Eukaryotes and Archaea Derive from Frequent Nucleosome Repositioning through Evolution

Nucleosomes, the basic repeat units of eukaryotic chromatin, have been suggested to influence the evolution of eukaryotic genomes, both by altering the propensity of DNA to mutate and by selection acting to maintain or exclude nucleosomes in particular locations. Contrary to the popular idea that nucleosomes are unique to eukaryotes, histone proteins have also been discovered in some archaeal genomes. Archaeal nucleosomes, however, are quite unlike their eukaryotic counterparts in many respects, including their assembly into tetramers (rather than octamers) from histone proteins that lack N- and C-terminal tails. Here, we show that despite these fundamental differences the association between nucleosome footprints and sequence evolution is strikingly conserved between humans and the model archaeon Haloferax volcanii. In light of this finding we examine whether selection or mutation can explain concordant substitution patterns in the two kingdoms. Unexpectedly, we find that neither the mutation nor the selection model are sufficient to explain the observed association between nucleosomes and sequence divergence. Instead, we demonstrate that nucleosome-associated substitution patterns are more consistent with a third model where sequence divergence results in frequent repositioning of nucleosomes during evolution. Indeed, we show that nucleosome repositioning is both necessary and largely sufficient to explain the association between current nucleosome positions and biased substitution patterns. This finding highlights the importance of considering the direction of causality between genetic and epigenetic change.     

Predicting nucleosome positions on the DNA: combining intrinsic sequence preferences and remodeler activities

Nucleosome positions on the DNA are determined by the intrinsic affinities of histone proteins to a given DNA sequence and by the ATP-dependent activities of chromatin remodeling complexes that can translocate nucleosomes with respect to the DNA. Here, we report a theoretical approach that takes into account both contributions. In the theoretical analysis two types of experiments have been considered: in vitro experiments with a single reconstituted nucleosome and in vivo genome-scale mapping of nucleosome positions. The effect of chromatin remodelers was described by iteratively redistributing the nucleosomes according to certain rules until a new steady state was reached. Three major classes of remodeler activities were identified: (i) the establishment of a regular nucleosome spacing in the vicinity of a strong positioning signal acting as a boundary, (ii) the enrichment/depletion of nucleosomes through amplification of intrinsic DNA-sequence-encoded signals and (iii) the removal of nucleosomes from high-affinity binding sites. From an analysis of data for nucleosome positions in resting and activated human CD4⁺ T cells [Schones et al., Cell 132, p. 887] it was concluded that the redistribution of a nucleosome map to a new state is greatly facilitated if the remodeler complex translocates the nucleosome with a preferred directionality.     

Domain architecture of the catalytic subunit in the ISW2-nucleosome complex

ATP-dependent chromatin remodeling has an important role in the regulation of cellular differentiation and development. For the first time, a topological view of one of these complexes has been revealed, by mapping the interactions of the catalytic subunit Isw2 with nucleosomal and extranucleosomal DNA in the complex with all four subunits of ISW2 bound to nucleosomes. Different domains of Isw2 were shown to interact with the nucleosome near the dyad axis, another near the entry site of the nucleosome, and another with extranucleosomal DNA. The conserved DEXD or ATPase domain was found to contact the superhelical location 2 (SHL2) of the nucleosome, providing a direct physical connection of ATP hydrolysis with this region of nucleosomes. The C terminus of Isw2, comprising the SLIDE (SANT-like domain) and HAND domains, was found to be associated with extranucleosomal DNA and the entry site of nucleosomes. It is thus proposed that the C-terminal domains of Isw2 are involved in anchoring the complex to nucleosomes through their interactions with linker DNA and that they facilitate the movement of DNA along the surface of nucleosomes.     

Topological constraints strongly affect chromatin reconstitution in silico

The fundamental building block of chromatin, and of chromosomes, is the nucleosome, a composite material made up from DNA wrapped around a histone octamer. In this study we provide the first computer simulations of chromatin self-assembly, starting from DNA and histone proteins, and use these to understand the constraints which are imposed by the topology of DNA molecules on the creation of a polynucleosome chain. We take inspiration from the in vitro chromatin reconstitution protocols which are used in many experimental studies. Our simulations indicate that during self-assembly, nucleosomes can fall into a number of topological traps (or local folding defects), and this may eventually lead to the formation of disordered structures, characterised by nucleosome clustering. Remarkably though, by introducing the action of topological enzymes such as type I and II topoisomerase, most of these defects can be avoided and the result is an ordered 10-nm chromatin fibre. These findings provide new insight into the biophysics of chromatin formation, both in the context of reconstitution in vitro and in terms of the topological constraints which must be overcome during de novo nucleosome formation in vivo, e.g. following DNA replication or repair.     

Selective association between nucleosomes with identical DNA sequences

Self-assembly is the autonomous organization of constituents into higher order structures or assemblages and is a fundamental mechanism in biological systems. There has been an unfounded idea that self-assembly may be used in the sensing and pairing of homologous chromosomes or chromatin, including meiotic chromosome pairing, polytene chromosome formation in Diptera and transvection. Recent studies proved that double-stranded DNA molecules have a sequence-sensing property and can self-assemble, which may play a role in the above phenomena. However, to explain these processes in terms of self-assembly, it first must be proved that nucleosomes retain a DNA sequence-sensing property and can self-assemble. Here, using atomic force microscopy (AFM)-based analyses and a quantitative interaction assay, we show that nucleosomes with identical DNA sequences preferentially associate with each other in the presence of Mg²⁺ ions. Using Xenopus borealis 5S rDNA nucleosome-positioning sequence and 601 and 603 sequences, homomeric or heteromeric octa- or tetranucleosomes were reconstituted in vitro and induced to form weak intracondensates by MgCl₂. AFM clearly showed that DNA sequence-based selective association occurs between nucleosomes with identical DNA sequences. Selective association was also detected between mononucleosomes. We propose that nucleosome self-assembly and DNA self-assembly constitute the mechanism underlying sensing and pairing of homologous chromosomes or chromatin.     

Nucleosome acetylation sequencing to study the establishment of chromatin acetylation

The establishment of posttranslational chromatin modifications is a major mechanism for regulating how genomic DNA is utilized. However, current in vitro chromatin assays do not monitor histone modifications at individual nucleosomes. Here we describe a strategy, nucleosome acetylation sequencing, that allows us to read the amount of modification at each nucleosome. In this approach, a bead-bound trinucleosome substrate is enzymatically acetylated with radiolabeled acetyl CoA by the SAGA complex from Saccharomyces cerevisae. The product is digested by restriction enzymes that cut at unique sites between the nucleosomes and then counted to quantify the extent of acetylation at each nucleosomal site. We find that we can sensitively, specifically, and reproducibly follow enzyme-mediated nucleosome acetylation. Applying this strategy, when acetylation proceeds extensively, its distribution across nucleosomes is relatively uniform. However, when substrates are used that contain nucleosomes mutated at the major sites of SAGA-mediated acetylation, or that are studied under initial rate conditions, changes in the acetylation distribution can be observed. Nucleosome acetylation sequencing should be applicable to analyzing a wide range of modifications. Additionally, because our trinucleosomes synthesis strategy is highly modular and efficient, it can be used to generate nucleosomal systems in which nucleosome composition differs across the array.     

A DEK Domain-Containing Protein Modulates Chromatin Structure and Function in Arabidopsis

Chromatin is a major determinant in the regulation of virtually all DNA-dependent processes. Chromatin architectural proteins interact with nucleosomes to modulate chromatin accessibility and higher-order chromatin structure. The evolutionarily conserved DEK domain-containing protein is implicated in important chromatin-related processes in animals, but little is known about its DNA targets and protein interaction partners. In plants, the role of DEK has remained elusive. In this work, we identified DEK3 as a chromatin-associated protein in Arabidopsis thaliana. DEK3 specifically binds histones H3 and H4. Purification of other proteins associated with nuclear DEK3 also established DNA topoisomerase 1α and proteins of the cohesion complex as in vivo interaction partners. Genome-wide mapping of DEK3 binding sites by chromatin immunoprecipitation followed by deep sequencing revealed enrichment of DEK3 at protein-coding genes throughout the genome. Using DEK3 knockout and overexpressor lines, we show that DEK3 affects nucleosome occupancy and chromatin accessibility and modulates the expression of DEK3 target genes. Furthermore, functional levels of DEK3 are crucial for stress tolerance. Overall, data indicate that DEK3 contributes to modulation of Arabidopsis chromatin structure and function.