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Using FRET in bulk and on single molecules, we assessed the structural role of histone acetylation in nucleosomes reconstituted on the 170 bp long Widom 601 sequence. We followed salt-induced nucleosome disassembly, using donor–acceptor pairs on the ends or in the internal part of the nucleosomal DNA, and on H2B histone for measuring H2A/H2B dimer exchange. This allowed us to distinguish the influence of acetylation on salt-induced DNA unwrapping at the entry–exit site from its effect on nucleosome core dissociation. The effect of lysine acetylation is not simply cumulative, but showed distinct histone-specificity. Both H3- and H4-acetylation enhance DNA unwrapping above physiological ionic strength; however, while H3-acetylation renders the nucleosome core more sensitive to salt-induced dissociation and to dimer exchange, H4-acetylation counteracts these effects. Thus, our data suggest, that H3- and H4-acetylation have partially opposing roles in regulating nucleosome architecture and that distinct aspects of nucleosome dynamics might be independently controlled by individual histones.

Nucleosomes play a dual role in compacting the genome and regulating the access to DNA. To unravel the underlying mechanism, we study fluorescently labeled mononucleosomes by multi-parameter FRET measurements and characterize their structural and dynamic heterogeneity upon NaCl-induced destabilization. Species-selective fluorescence lifetime analysis and dynamic photon distribution analysis reveal intermediates during nucleosome opening and lead to a coherent structural and kinetic model. In dynamic octasomes and hexasomes the interface between the H2A-H2B dimers and the (H3-H4)2 tetramer opens asymmetrically by an angle of ≈20° on a 50 and 15 µs time scale, respectively. This is followed by a slower stepwise release of the dimers coupled with DNA unwrapping. A mutation (H2A-R81A) at the interface between H2A and H3 facilitates initial opening, confirming the central role of the dimer:tetramer interface for nucleosome stability. Partially opened states such as those described here might serve as convenient nucleation sites for DNA-recognizing proteins., Nucleosomes compact the genome and regulate access to specific DNA sequences. Here the authors employ single-molecule FRET studies to characterize nucleosome dynamics at different salt concentrations and dissect nucleosome disassembly into elementary steps.

Nucleosomes are important for chromatin compaction and gene regulation; their integrity depends crucially on the structural properties of the histone tails. Recent all-atom molecular dynamics simulations revealed that removal of the N-terminal tails of histone H3, known to destabilize nucleosomes, causes a rearrangement of two arginines of histone H2A, namely R81 and R88 by altering the electrostatic environment of the H2A α3 domain. Whether this rearrangement is the cause or the effect of decreased stability, is unclear. Here, we emulate the altered electrostatic environment that was found after H3 tail clipping through charge-modifying mutations to decouple its impact on intranucleosomal interactions from that of the histone tails. Förster resonance energy transfer experiments on recombinant nucleosomes and all-atom molecular dynamics simulations reveal a compensatory role of those amino acids in nucleosome stability. The simulations indicate a weakened interface between H2A-H2B dimers and the (H3-H4)2 tetramer, as well as between dimers and DNA. These findings agree with the experimental observations of position and charge dependent decreased nucleosome stability induced by the introduced mutations. This work highlights the importance of the H2A α3 domain and suggests allosteric effects between this domain and the outer DNA gyre as well as the H3 N-terminal tail.

Opening of the nucleosome structure is essential for accessing genomic DNA. To study the mechanism of this process, we monitor the distance between various fluorescently labeled positions on mononucleosomes by single-molecule Forster resonance energy transfer (FRET). Here, we compare nucleosomes reconstituted from recombinant mouse, Xenopus, and yeast histones. As DNA sequences we compared, the effect of 5S rDNA, MMTV-B sequence, and Widom 601 DNA. The stability, as measured by the salt concentration at the opening transition midpoint, is lowest for yeast, followed by Xenopus and mouse. The 601 DNA sequence builds much more stable nucleosomes and the distribution of FRET efficiencies is narrower than for those reconstituted on 5S rDNA or MMTV-B sequences. The opening pathway through an intermediate state, as found for Xenopus histones, could be verified for the mouse and yeast systems and for the different DNA sequences, suggesting a general mechanism for accessing nucleosomal DNA. © 2013 International Society for Advancement of Cytometry

Nucleosomes are multi-component macromolecular assemblies which present a formidable obstacle to enzymatic activities that require access to the DNA, e.g. DNA and RNA polymerases. The mechanism and pathway(s) by which nucleosomes disassemble to allow DNA access are not well understood. Here we present evidence from single molecule FRET experiments for a previously uncharacterized intermediate structural state before H2A-H2B dimer release, which is characterized by an increased distance between H2B and the nucleosomal dyad. This suggests that the first step in nucleosome disassembly is the opening of the (H3-H4)(2) tetramer/(H2A-H2B) dimer interface, followed by H2A-H2B dimer release from the DNA and, lastly, (H3-H4)(2) tetramer removal. We estimate that the open intermediate state is populated at 0.2-3% under physiological conditions. This finding could have significant in vivo implications for factor-mediated histone removal and exchange, as well as for regulating DNA accessibility to the transcription and replication machinery.

The nucleosome has a central role in the compaction of genomic DNA and the control of DNA accessibility for transcription and replication. To help understanding the mechanism of nucleosome opening and closing in these processes, we studied the disassembly of mononucleosomes by quantitative single-molecule FRET with high spatial resolution, using the SELEX-generated “Widom 601” positioning sequence labeled with donor and acceptor fluorophores. Reversible dissociation was induced by increasing NaCl concentration. At least 3 species with different FRET were identified and assigned to structures: ( i ) the most stable high-FRET species corresponding to the intact nucleosome, ( ii ) a less stable mid-FRET species that we attribute to a first intermediate with a partially unwrapped DNA and less histones, and ( iii ) a low-FRET species characterized by a very broad FRET distribution, representing highly unwrapped structures and free DNA formed at the expense of the other 2 species. Selective FCS analysis indicates that even in the low-FRET state, some histones are still bound to the DNA. The interdye distance of 54.0 Å measured for the high-FRET species corresponds to a compact conformation close to the known crystallographic structure. The coexistence and interconversion of these species is first demonstrated under non-invasive conditions. A geometric model of the DNA unwinding predicts the presence of the observed FRET species. The different structures of these species in the disassembly pathway map the energy landscape indicating major barriers for 10-bp and minor ones for 5-bp DNA unwinding steps.

DNA accessibility in the cell nucleus depends on its packaging. The stability of the basic compaction unit, the nucleosome, is regulated through the interactions between DNA and histones. Natural sequence variation along the DNA strongly modulates nucleosome stability. Histone variations, mutations and posttranslational modifications alter intra- and internucleosomal interactions. We are investigating interactions which hold nucleosomes together by measuring distances in reconstituted samples using single molecule Forster energy transfer.

Nucleosomes compact the genome and regulate access by wrapping and unwrapping DNA. Here we characterized NaCl-induced opening of mononucleosomes by single-molecule FRET, using donor/acceptor labels on the DNA and various histones. By species-selective fluorescence lifetime and photon distribution analysis we identified new nucleosome opening intermediates and developed a kinetic model [1]. Opening proceeds through a weakening of the H2A-H2B dimer/(H3-H4)2 tetramer interface on a 0.1 ms time scale, then by a slower two-step release of the dimers coupled to DNA unwrapping, extending from several ms to minutes. Nucleosome opening and detachment of histone dimers proceed asymmetrically depending on the DNA sequence.Mutations at the H2A/H3 interface (H2A R81A, R88A, R81A/R88A, R81E/R88E) facilitate the initial opening, confirming the importance of the dimer:tetramer interface for nucleosome stability. This is also supported by molecular dynamics (MD) simulations that show enhanced DNA fluctuations for the mutants. Partially opened states such as described here might be a convenient nucleation point for DNA-recognizing proteins.For characterizing the role of histone tails in nucleosome interaction and stability, we performed MD simulations in explicit and implicit solvent on the H4 tail interacting with the acidic patch of a neighboring nucleosome. The simulations show that H4K16 acetylation decreases the interactions between H4 tail and the acidic patch of the neighboring nucleosome, a process that is central to the regulation of chromatin compaction [2,3].References[1] A. Gansen, A. Valeri, F. Hauger, S. Felekyan, S. Kalinin, K. Toth, J. Langowski, and C. A. M. Seidel. Nucleosome disassembly intermediates characterized by single-molecule FRET. Proc Natl Acad Sci U S A, 106(36):15308-13, Sep 2009.[2] J. Erler, R. Zhang, L. Petridis, X. Cheng, J. C. Smith, and J. Langowski. The role of histone tails in the nucleosome: A computational study. Biophys. J., 107(12):2911-2922, 2014.[3] R. Zhang, J. Erler, J. Langowski. How does histone acetylation regulate chromatin accessibility? A computational study of the role of H4K16 in inter-nucleosome interaction. Biophys. J., under revision.

The TATA binding protein (TBP) is a critical transcription factor used for nucleating assembly of the RNA polymerase II machinery. TBP binds TATA box elements with high affinity and kinetic stability and in vivo is correlated with high levels of transcription activation. However, since most promoters use less stable TATA-less or TATA-like elements, while also competing with nucleosome occupancy, further mechanistic insight into TBP's DNA binding properties and ability to access chromatin is needed. Using bulk and single-molecule FRET, we find that TBP binds a minimal consensus TATA box as a two-state equilibrium process, showing no evidence for intermediate states. However, upon addition of flanking DNA sequence, we observe non-specific cooperative binding to multiple DNA sites that compete for TATA-box specificity. Thus, we conclude that TBP binding is defined by a branched pathway, wherein TBP initially binds with little sequence specificity and is thermodynamically positioned by its kinetic stability to the TATA box. Furthermore, we observed the real-time access of TBP binding to TATA box DNA located within the DNA entry–exit site of the nucleosome. From these data, we determined salt-dependent changes in the nucleosome conformation regulate TBP's access to the TATA box, where access is highly constrained under physiological conditions, but is alleviated by histone acetylation and TFIIA.

Using Forster resonance energy transfer (FRET) measurements in bulk and on single molecules, we assessed the structural role of histone acetylation and of some point mutations in nucleosomes reconstituted on the 170bp long Widom 601 DNA sequence. By measuring distances between fluorescently labeled parts of the nucleosome we could follow the salt-induced nucleosome disassembly, using donor-acceptor pairs on the ends or in the internal part of the nucleosomal DNA, and on histones. The single molecule observations helped to characterize the intermediate states as well as the distributions of the populations [1,2].

Histone tails and their posttranslational modifications play a crucial role in controlling genetic activity through alterations of nucleosome structure. Whether or not histone tails regulate DNA accessibility independently of each other or in a concerted fashion is currently under debate. Here we studied the structure-defining properties of selective histone acetylation and point mutations in the H4 tail in a combined bulk FRET - single molecule FRET assay. Nucleosome unwrapping was monitored by FRET between the linker ends of the DNA, while FRET experiments at an internal DNA site in the H2A/H2B binding region reported on nucleosome disassembly.By analysis of nucleosome unwrapping, structural heterogeneity during salt-induced disassembly and dimer exchange between nucleosomes we show that histones H3 and H4 assume significantly different roles in controlling nucleosome architecture. H4-acetylation opposes destabilization by H3-acetylation and reduces linker DNA unwrapping and dimer exchange at higher ionic strength, whereas its influence on nucleosome structure at physiological salt is minute. We found no increase in unwrapping when H3 and H4 were acetylated simultaneously, which challenges the idea of cooperativeness between tails that was observed for truncated H3 and H4. Our data suggest that the effect of lysine acetylation is not cumulative in nature but shows strong histone specificity. The specific role of the H4 tail was finally probed by comparing the effect of point mutations or acetylation of selective lysine residues at positions 5,8,12 and 16.Regardless of the state of acetylation nucleosomes disassemble via an intermediate state, which is suppressed at higher nucleosome concentration, confirming our proposed model of step-wise disassembly.

Nucleosomes, as the basic packing unit of chromatin, regulate DNA accessibility and have significant influence on gene expression. Two copies of each histone protein (H2A, H2B, H3, H4) build up the protein octamer, around which approximately 150 bp of DNA are wrapped.The N-terminal tails of the histones protrude from the nucleosome; they are important for inter- and intranucleosomal interactions. Nucleosome disassembly may be forced in vitro by increasing salt concentration and may be followed by Forster resonance energy transfer (FRET) between fluorophores. We use reconstituted Xenopus laevis nucleosomes, fluorescently labeled on different positions[2] to compare the disassembly of nucleosomes containing mutated histones or modifications to the wild type.Here we focus on the dynamics of the N-tail of H3. Mutated versions of H3 labeled with Alexa488 were used to examine whether there are interactions between the labeled H3 tail and the labeled DNA or other labeled histones. First results show an interaction of the H3 tail and the DNA in vicinity of the dyad axis.The second part is about the functional relevance of a particular region of H2A. Molecular dynamic simulations on ‘tailless’ variants of H3 and H2A have suggested conformational changes affecting two arginines of H2A. Furthermore a conformational change of the protruding part of the DNA was described, which is due to internal conformational changes of H2A[3].From those observations the question arises whether these amino acids are necessary for stable octamers and/or nucleosomes, and how they influence DNA breathing, unwrapping, and stability of entire nucleosomes. To address these questions we generated recombinant H2A proteins incorporating site-specific mutations (R81A, R88A, R81AR88A, R81E, R88E, R81ER88E). Our results show a decreasing stability associated with position (Wt > R88 > R81 > R81R88) and charge (RA > RE) of the amino acids.

Nucleosome structure and stability affect genetic accessibility by altering the local chromatin morphology. Recent FRET experiments on nucleosomes have given valuable insight into the structural transformations they can adopt. Yet, even if performed under seemingly identical conditions, experiments performed in bulk and at the single molecule level have given mixed answers due to the limitations of each technique. To compare such experiments, however, they must be performed under identical conditions. Here we develop an experimental framework that overcomes the conventional limitations of each method: single molecule FRET experiments are carried out at bulk concentrations by adding unlabeled nucleosomes, while bulk FRET experiments are performed in microplates at concentrations near those used for single molecule detection. Additionally, the microplate can probe many conditions simultaneously before expending valuable instrument time for single molecule experiments. We highlight this experimental strategy by exploring the role of selective acetylation of histone H3 on nucleosome structure and stability; in bulk, H3-acetylated nucleosomes were significantly less stable than non-acetylated nucleosomes. Single molecule FRET analysis further revealed that acetylation of histone H3 promoted the formation of an additional conformational state, which is suppressed at higher nucleosome concentrations and which could be an important structural intermediate in nucleosome regulation.

We applied fluorescence detection methods on the single-molecule level to study structural variations and dynamic processes occurring within nucleosomes. Four fluorescent nucleosome constructs were made by attaching donor and acceptor fluorophores to different positions of two nucleosome positioning sequences and reconstituting nucleosomes by salt dialysis. The photochemical and biochemical stability of nucleosomes under single-molecule conditions was optimized by adding inert protein and free radical capturing additives, allowing us to define the best experimental conditions for single-molecule spectroscopy on highly diluted solutions of nucleosome complexes. We could demonstrate for the first time the resolution of conformational subpopulations of nucleosomes by single-pair fluorescence resonance energy transfer in a freely diffusing system and could show the effect of thermally induced nucleosome repositioning.