Nanoscale nucleosome dynamics assessed with time-lapse AFM

A fundamental challenge associated with chromosomal gene regulation is accessibility of DNA within nucleosomes. Recent studies performed by various techniques, including single-molecule approaches, led to the realization that nucleosomes are dynamic structures rather than static systems, as was once believed. Direct data are required in order to understand the dynamics of nucleosomes more clearly and to answer fundamental questions, including: What is the range of nucleosome dynamics? Does a non-ATP-dependent unwrapping process of nucleosomes exist? What are the factors facilitating the large-scale opening and unwrapping of nucleosomes? This review summarizes the results of nucleosome dynamics obtained with time-lapse AFM, including a high-speed version (HS-AFM) capable of visualizing molecular dynamics on the millisecond time scale. With HS-AFM, the dynamics of nucleosomes at a sub-second time scale was observed, allowing one to visualize various pathways of nucleosome dynamics, such as sliding and unwrapping, including complete dissociation. Overall, these findings reveal new insights into the dynamics of nucleosomes and the novel mechanisms controlling spontaneous chromatin dynamics.     

Nucleosomes structure and dynamics: effect of CHAPS

Dynamics of nucleosomes and spontaneous unwrapping of DNA are fundamental property of the chromatin enabling access to nucleosomal DNA for regulatory proteins. Probing of such dynamics of nucleosomes performed by single molecule techniques revealed a large scale dynamics of nucleosomes including their spontaneous unwrapping. Dissociation of nucleosomes at low concentrations is a complicating issue for studies with single molecule techniques. In this paper, we tested the ability of 3-[(3-Cholamidopropyl)dimethylammonio]-l-propanesulfonate (CHAPS) to prevent dissociation of nucleosomes. The study was performed with mononucleosome system assembled with human histones H2A, H2B, H3 and H4 on the DNA substrate containing sequence 601 that provides the sequencespecific assembly of nucleosomes. We used Atomic Force Microscopy (AFM) to directly identify nucleosomes and analyze their structure at the nanometer level. These studies showed that in the presence of CHAPS at millimolar concentrations, nucleosomes, even at sub-nanomolar concentrations, remain intact over days compared to a complete dissociation of the same nucleosome sample over 10 min in the absence of CHAPS. Importantly, CHAPS does not change the conformation of nucleosomes as confirmed by the AFM analysis. Moreover, 16 µM CHAPS stabilizes nucleosomes in over one hour incubation in the solution containing as low as 0.4 nM in nucleosomes. The stability of nucleosomes is slightly reduced at physiological conditions (150 mM NaCl), although the nucleosomes dissociate rapidly at 300 mM NaCl. The sequence specificity of the nucleosome in the presence of CHAPS decreased suggesting that the histone core translocates along the DNA substrate utilizing sliding mechanism.     

The dynamics of the nucleosome: thermal effects, external forces and ATP

With nucleosomes being tightly associated with the majority of eukaryotic DNA, it is essential that mechanisms are in place that can move nucleosomes 'out of the way'. A focus of current research comprises chromatin remodeling complexes, which are ATP-consuming protein complexes that, for example, pull or push nucleosomes along DNA. The precise mechanisms used by those complexes are not yet understood. Hints for possible mechanisms might be found among the various spontaneous fluctuations that nucleosomes show in the absence of remodelers. Thermal fluctuations induce the partial unwrapping of DNA from the nucleosomes and introduce twist or loop defects in the wrapped DNA, leading to nucleosome sliding along DNA. In this minireview, we discuss nucleosome dynamics from two angles. First, we describe the dynamical modes of nucleosomes in the absence of remodelers that are experimentally fairly well characterized and theoretically understood. Then, we discuss remodelers and describe recent insights about the possible schemes that they might use.     

Spontaneous access to DNA target sites in folded chromatin fibers

DNA wrapped in nucleosomes is sterically occluded from many protein complexes that must act on it; how such complexes gain access to nucleosomal DNA is not known. In vitro studies on isolated nucleosomes show that they undergo spontaneous partial unwrapping conformational transitions, which make the wrapped nucleosomal DNA transiently accessible. Thus, site exposure might provide a general mechanism allowing access of protein complexes to nucleosomal DNA. However, existing quantitative analyses of site exposure focused on single nucleosomes, while the presence of neighbor nucleosomes and concomitant chromatin folding might significantly influence site exposure. In this work, we carried out quantitative studies on the accessibility of nucleosomal DNA in homogeneous nucleosome arrays. Two striking findings emerged. Organization into chromatin fibers changes the accessibility of nucleosomal DNA only modestly, from ∼ 3-fold decreases to ∼ 8-fold increases in accessibility. This means that nucleosome arrays are intrinsically dynamic and accessible even when they are visibly condensed. In contrast, chromatin folding decreases the accessibility of linker DNA by as much as ∼ 50-fold. Thus, nucleosome positioning dramatically influences the accessibility of target sites located inside nucleosomes, while chromatin folding dramatically regulates access to target sites in linker DNA.     

UV Damage in DNA Promotes Nucleosome Unwrapping

The association of DNA with histones in chromatin impedes DNA repair enzymes from accessing DNA lesions. Nucleosomes exist in a dynamic equilibrium in which portions of the DNA molecule spontaneously unwrap, transiently exposing buried DNA sites. Thus, nucleosome dynamics in certain regions of chromatin may provide the exposure time and space needed for efficient repair of buried DNA lesions. We have used FRET and restriction enzyme accessibility to study nucleosome dynamics following DNA damage by UV radiation. We find that FRET efficiency is reduced in a dose-dependent manner, showing that the presence of UV photoproducts enhances spontaneous unwrapping of DNA from histones. Furthermore, this UV-induced shift in unwrapping dynamics is associated with increased restriction enzyme accessibility of histone-bound DNA after UV treatment. Surprisingly, the increased unwrapping dynamics is even observed in nucleosome core particles containing a single UV lesion at a specific site. These results highlight the potential for increased “intrinsic exposure” of nucleosome-associated DNA lesions in chromatin to repair proteins.     

Repair of UV lesions in nucleosomes--intrinsic properties and remodeling

Nucleotide excision repair and reversal of pyrimidine dimers by photolyase (photoreactivation) are two major pathways to remove UV-lesions from DNA. Here, it is discussed how lesions are recognized and removed when the DNA is condensed into nucleosomes. During the recent years it was shown that nucleosomes inhibit photolyase and excision repair in vitro and slow down repair in vivo. The correlation of DNA-repair rates with nucleosome positions in yeast suggests that intrinsic properties of nucleosomes such as mobility and transient unwrapping of nucleosomal DNA facilitate damage recognition. Moreover, it was shown that nucleosome remodeling activities can act on UV-damaged DNA in vitro and facilitate repair suggesting that random remodeling of chromatin might contribute to damage recognition in vivo. Recent work on nucleosome structure and mobility is included to evaluate how nucleosomes accommodate DNA lesions and how nucleosome mobility and remodeling can take place on damaged DNA.     

Rapid spontaneous accessibility of nucleosomal DNA

DNA wrapped in nucleosomes is sterically occluded, creating obstacles for proteins that must bind it. How proteins gain access to DNA buried inside nucleosomes is not known. Here we report measurements of the rates of spontaneous nucleosome conformational changes in which a stretch of DNA transiently unwraps off the histone surface, starting from one end of the nucleosome, and then rewraps. The rates are rapid. Nucleosomal DNA remains fully wrapped for only approximately 250 ms before spontaneously unwrapping; unwrapped DNA rewraps within approximately 10-50 ms. Spontaneous unwrapping of nucleosomal DNA allows any protein rapid access even to buried stretches of the DNA. Our results explain how remodeling factors can be recruited to particular nucleosomes on a biologically relevant timescale, and they imply that the major impediment to entry of RNA polymerase into a nucleosome is rewrapping of nucleosomal DNA, not unwrapping.     

AFM for analysis of structure and dynamics of DNA and protein-DNA complexes

This paper describes protocols for studies of structure and dynamics of DNA and protein-DNA complexes with atomic force microscopy (AFM) utilizing the surface chemistry approach. The necessary specifics for the preparation of functionalized surfaces and AFM probes with the use of silanes and silatranes, including the protocols for synthesis of silatranes are provided. The methodology of studies of local and global conformations DNA with the major focus on the time-lapse imaging of DNA in aqueous solutions is illustrated by the study of dynamics of Holliday junctions including branch migration. The analysis of nucleosome dynamics is selected as an example to illustrate the application of the time-lapse AFM to studies of dynamics of protein-DNA complexes. The force spectroscopy is the modality of AFM with a great importance to various fields of biomedical studies. The AFM force spectroscopy approach for studies of specific protein-DNA complexes is illustrated by the data on analysis of dynamics of synaptic SfiI-DNA complexes. When necessary, additional specifics are added to the corresponding example.     

Quantifying epigenetic modulation of nucleosome breathing by high-throughput AFM imaging

Nucleosomes are the basic units of chromatin and critical for storage and expression of eukaryotic genomes. Chromatin accessibility and gene readout are heavily regulated by epigenetic marks, in which post-translational modifications of histones play a key role. However, the mode of action and the structural implications at the single-molecule level of nucleosomes is still poorly understood. Here we apply a high-throughput atomic force microscopy imaging and analysis pipeline to investigate the conformational landscape of the nucleosome variants three additional methyl groups at lysine 36 of histone H3 (H3K36me3), phosphorylation of H3 histones at serine 10 (H3S10phos), and acetylation of H4 histones at lysines 5, 8, 12, and 16 (H4K5/8/12/16ac). Our data set of more than 25,000 nucleosomes reveals nucleosomal unwrapping steps corresponding to 5-bp DNA. We find that H3K36me3 nucleosomes unwrap significantly more than wild-type nucleosomes and additionally unwrap stochastically from both sides, similar to centromere protein A (CENP-A) nucleosomes and in contrast to the highly anticooperative unwrapping of wild-type nucleosomes. Nucleosomes with H3S10phos or H4K5/8/12/16ac modifications show unwrapping populations similar to wild-type nucleosomes and also retain the same level of anticooperativity. Our findings help to put the mode of action of these modifications into context. Although H3K36me3 likely acts partially by directly affecting nucleosome structure on the single-molecule level, H3S10phos and H4K5/8/12/16ac must predominantly act through higher-order processes. Our analysis pipeline is readily applicable to other nucleosome variants and will facilitate future high-resolution studies of the conformational landscape of nucleoprotein complexes.     

Telomeric nucleosomes are intrinsically mobile

Nucleosomes are no longer considered only static basic units that package eukaryotic DNA but they emerge as dynamic players in all chromosomal processes. Regulatory proteins can gain access to recognition sequences hidden by the histone octamer through the action of ATP-dependent chromatin remodeling complexes that cause nucleosome sliding. In addition, it is known that nucleosomes are able to spontaneously reposition along the DNA due to intrinsic dynamic properties, but it is not clear yet to what extent sequence-dependent dynamic properties contribute to nucleosome repositioning. Here, we study mobility of nucleosomes formed on telomeric sequences as a function of temperature and ionic strength. We find that telomeric nucleosomes are highly intrinsically mobile under physiological conditions, whereas nucleosomes formed on an average DNA sequence mostly remain in the initial position. This indicates that DNA sequence affects not only the thermodynamic stability and the positioning of nucleosomes but also their dynamic properties. Moreover, our findings suggest that the high mobility of telomeric nucleosomes may be relevant to the dynamics of telomeric chromatin.     

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.     

Nucleosome occupancy landscape and dynamics at mouse recombination hotspots

During meiosis, paternal and maternal homologous chromosomes recombine at specific recombination sites named hotspots. What renders 2% of the mammalian genomes permissive to meiotic recombination by allowing Spo11 endonuclease to initiate double‐strand breaks is largely unknown. Work in yeast has shown that chromatin accessibility seems to be important for this activity. Here, we define nucleosome profiles and dynamics at four mouse recombination hotspots by purifying highly enriched fractions of meiotic cells. We found that nucleosome occupancy is generally stable during meiosis progression. Interestingly, the cores of recombination hotspots have largely open chromatin structure, and the localization of the few nucleosomes present in these cores correlates precisely with the crossover‐free zones in recombinogenic domains. Collectively, these high‐resolution studies suggest that nucleosome occupancy seems to direct, at least in part, how meiotic recombination events are processed.