Dynamics of the nucleosomal histone H3 N-terminal tail revealed by high precision single-molecule FRET.

Chromatin compaction and gene accessibility are orchestrated by assembly and disassembly of nucleosomes. Although the disassembly process was widely studied, little is known about the structure and dynamics of the disordered histone tails, which play a pivotal role for nucleosome integrity. This is a gap filling experimental FRET study from the perspective of the histone H3 N-terminal tail (H3NtT) of reconstituted mononucleosomes. By systematic variation of the labeling positions we monitored the motions of the H3NtT relative to the dyad axis and linker DNA. Single-molecule FRET unveiled that H3NtTs do not diffuse freely but follow the DNA motions with multiple interaction modes with certain permitted dynamic transitions in the µs to ms time range. We also demonstrate that the H3NtT can allosterically sense charge-modifying mutations within the histone core (helix α3 of histone H2A (R81E/R88E)) resulting in increased dynamic transitions and lower rate constants. Those results complement our earlier model on the NaCl induced nucleosome disassembly as changes in H3NtT configurations coincide with two major steps: unwrapping of one linker DNA and weakening of the internal DNA - histone interactions on the other side. This emphasizes the contribution of the H3NtT to the fine-tuned equilibrium between overall nucleosome stability and DNA accessibility.

High precision FRET studies reveal reversible transitions in nucleosomes between microseconds and minutes.

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.

Random Motion of Chromatin Is Influenced by Lamin A Interconnections.

Using fluorescence correlation spectroscopy in single-plane illumination microscopy, we investigated the dynamics of chromatin in interphase mouse adult fibroblast cell nuclei under the influence of the intermediate filament protein lamin A. We find that 1) lamin A-eGFP and histone H2A-mRFP show significant comobility, indicating that their motions are clearly interconnected in the nucleus, and 2) that the random motion of histones H2A within the chromatin network is subdiffusive, i.e., the effective diffusion coefficient decreases for slow timescales. Knocking out lamin A changes the diffusion back to normal. Thus, lamin A influences the dynamics of the entire chromatin network. Our conclusion is that lamin A plays a central role in determining the viscoelasticity of the chromatin network and helping to maintain local ordering of interphase chromosomes.

Widefield High Frame Rate Single-Photon SPAD Imagers for SPIM-FCS.

Photon-counting sensors based on standard complementary metal-oxide-semiconductor single-photon avalanche diodes (SPADs) represent an emerging class of imagers that enable the counting and/or timing of single photons at zero readout noise (better than high-speed electron-multiplying charge-coupling devices) and over large arrays. They have seen substantial progress over the last 15 years, increasing their spatial resolution, timing accuracy, and sensitivity while reducing spurious signals such as afterpulsing and dark counts. They are increasingly being applied for time-resolved applications with the added advantage of enabling real-time options such as autocorrelation. We report in this study on the use of such a state-of-the-art 512 × 128 SPAD array, capable of a time resolution of 10-5-10-6 s for full frames while retaining acceptable photosensitivity thanks to the use of dedicated microlenses, in a selective plane illumination-fluorescence correlation spectroscopy setup. The latter allows us to perform thousands of fluorescence-correlation spectroscopy measurements simultaneously in a two-dimensional slice of the sample. This high-speed SPAD imager enables the measurement of molecular motion of small fluorescent particles such as single chemical dye molecules. Inhomogeneities in the molecular detection efficiency were compensated for by means of a global fit of the auto- and cross-correlation curves, which also made a calibration-free measurement of various samples possible. The afterpulsing effect could also be mitigated, making the measurement of the diffusion of Alexa-488 possible, and the overall result quality was further improved by spatial binning. The particle concentrations in the focus tend to be overestimated by a factor of 1.7 compared to a confocal setup; a calibration is thus required if absolute concentrations need to be measured. The first high-speed selective plane illumination-fluorescence correlation spectroscopy in vivo measurements to our knowledge were also recorded: although two-component fit models could not be employed because of noise, the diffusion of eGFP oligomers in HeLa cells could be measured. Sensitivity and noise will be further improved in the next generation of SPAD-based widefield sensors, which are currently under testing.

Assembly Kinetics of Vimentin Tetramers to Unit-Length Filaments: A Stopped-Flow Study.

Intermediate filaments (IFs) are principal components of the cytoskeleton, a dynamic integrated system of structural proteins that provides the functional architecture of metazoan cells. They are major contributors to the elasticity of cells and tissues due to their high mechanical stability and intrinsic flexibility. The basic building block for the assembly of IFs is a rod-like, 60-nm-long tetrameric complex made from two antiparallel, half-staggered coiled coils. In low ionic strength, tetramers form stable complexes that rapidly assemble into filaments upon raising the ionic strength. The first assembly products, "frozen" by instantaneous chemical fixation and viewed by electron microscopy, are 60-nm-long "unit-length" filaments (ULFs) that apparently form by lateral in-register association of tetramers. ULFs are the active elements of IF growth, undergoing longitudinal end-to-end annealing with one another and with growing filaments. Originally, we have employed quantitative time-lapse atomic force and electron microscopy to analyze the kinetics of vimentin-filament assembly starting from a few seconds to several hours. To obtain detailed quantitative insight into the productive reactions that drive ULF formation, we now introduce a "stopped-flow" approach in combination with static light-scattering measurements. Thereby, we determine the basic rate constants for lateral assembly of tetramers to ULFs. Processing of the recorded data by a global fitting procedure enables us to describe the hierarchical steps of IF formation. Specifically, we propose that tetramers are consumed within milliseconds to yield octamers that are obligatory intermediates toward ULF formation. Although the interaction of tetramers is diffusion controlled, it is strongly driven by their geometry to mediate effective subunit targeting. Importantly, our model conclusively reflects the previously described occurrence of polymorphic ULF and mature filaments in terms of their number of tetramers per cross section.

Protein Flexibility and Synergy of HMG Domains Underlie U-Turn Bending of DNA by TFAM in Solution.

Human mitochondrial transcription factor A (TFAM) distorts DNA into a U-turn, as shown by crystallographic studies. The relevance of this U-turn is associated with transcription initiation at the mitochondrial light strand promoter (LSP). However, it has not been yet discerned whether a tight U-turn or an alternative conformation, such as a V-shape, is formed in solution. Here, single-molecule FRET experiments on freely diffusing TFAM/LSP complexes containing different DNA lengths show that a DNA U-turn is induced by progressive and cooperative binding of the two TFAM HMG-box domains and the linker between them. SAXS studies further show compaction of the protein upon complex formation. Finally, molecular dynamics simulations reveal that TFAM/LSP complexes are dynamic entities, and the HMG boxes induce the U-turn against the tendency of the DNA to adopt a straighter conformation. This tension is resolved by reversible unfolding of the linker, which is a singular mechanism that allows a flexible protein to stabilize a tight bending of DNA.

Effects of charge-modifying mutations in histone H2A α3-domain on nucleosome stability assessed by single-pair FRET and MD simulations.

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.

Defining the epichromatin epitope.

Epichromatin is identified by immunostaining fixed and permeabilized cells with particular bivalent anti-nucleosome antibodies (mAbs PL2-6 and 1H6). During interphase, epichromatin resides adjacent to the inner nuclear membrane; during mitosis, at the outer surface of mitotic chromosomes. By STED (stimulated emission depletion) microscopy, PL2-6 stained interphase epichromatin is ∼76 nm thick and quite uniform; mitotic epichromatin is more variable in thickness, exhibiting a "wrinkled" surface with an average thickness of ∼78 nm. Co-immunostaining with anti-Ki-67 demonstrates Ki-67 deposition between the PL2-6 "ridges" of mitotic epichromatin. Monovalent papain-derived Fab fragments of PL2-6 yield a strikingly different punctate "chromomeric" immunostaining pattern throughout interphase nuclei and along mitotic chromosome arms. Evidence from electrophoretic mobility shift assay (EMSA) and from analytical ultracentrifugation characterize the Fab/mononucleosome complex, supporting the concept that there are two binding sites per nucleosome. The peptide sequence of the Hv3 region (heavy chain variable region 3) of the PL2-6 antibody binding site strongly resembles other nucleosome acidic patch binding proteins (especially, LANA and CENPC), supporting that the nucleosome acidic patch is included within the epichromatin epitope. It is speculated that the interphase epichromatin epitope is "exposed" with favorable geometric arrangements for binding bivalent PL2-6 at the surface chromatin; whereas, the epitope is "hidden" within internal chromatin. Furthermore, it is suggested that the "exposed" nucleosome surface of mitotic epichromatin may play a role in post-mitotic nuclear envelope reformation.

Single plane illumination microscopy as a tool for studying nucleome dynamics.

Single plane illumination microscopy (SPIM) is a new optical method that has become extremely important in recent years. It is based on the formation of a "light slice" in the specimen in which fluorescently tagged molecules are observed. The spatial resolution is close to that of confocal optics, but without the disadvantages inherent to scanning or high laser irradiation doses. A recent development is light sheet fluctuation microscopy, which exploits the dynamic information contained in the fluorescence intensity fluctuations of each image pixel. Here we review the principles of this method and show some recent applications to the dynamics of transcription factors and chromatin. We show that the dimerization of Fos and Jun proteins is directly linked to their binding to DNA; that nuclear receptor activation changes their intranuclear dynamics; and that the viscoelastic behavior of interphase chromatin strongly depends on the presence of lamin A.

Nucleosome repositioning during differentiation of a human myeloid leukemia cell line.

Cell differentiation is associated with changes in chromatin organization and gene expression. In this study, we examine chromatin structure following differentiation of the human myeloid leukemia cell line (HL-60/S4) into granulocytes with retinoic acid (RA) or into macrophage with phorbol ester (TPA). We performed ChIP-seq of histone H3 and its modifications, analyzing changes in nucleosome occupancy, nucleosome repeat length, eu-/heterochromatin redistribution and properties of epichromatin (surface chromatin adjacent to the nuclear envelope). Nucleosome positions changed genome-wide, exhibiting a specific class of alterations involving nucleosome loss in extended (∼1kb) regions, pronounced in enhancers and promoters. Genes that lost nucleosomes at their promoters showed a tendency to be upregulated. On the other hand, nucleosome gain did not show simple effects on transcript levels. The average genome-wide nucleosome repeat length (NRL) did not change significantly with differentiation. However, we detected an approximate 10 bp NRL decrease around the haematopoietic transcription factor (TF) PU.1 and the architectural protein CTCF, suggesting an effect on NRL proximal to TF binding sites. Nucleosome occupancy changed in regions associated with active promoters in differentiated cells, compared with untreated HL-60/S4 cells. Epichromatin regions revealed an increased GC content and high nucleosome density compared with surrounding chromatin. Epichromatin showed depletion of major histone modifications and revealed enrichment with PML body-associated genes. In general, chromatin changes during HL-60/S4 differentiation appeared to be more localized to regulatory regions, compared with genome-wide changes among diverse cell types studied elsewhere.

Insights into the molecular architecture and histone H3-H4 deposition mechanism of yeast Chromatin assembly factor 1.

How the very first step in nucleosome assembly, deposition of histone H3-H4 as tetramers or dimers on DNA, is accomplished remains largely unclear. Here, we report that yeast chromatin assembly factor 1 (CAF1), a conserved histone chaperone complex that deposits H3-H4 during DNA replication, binds a single H3-H4 heterodimer in solution. We identify a new DNA-binding domain in the large Cac1 subunit of CAF1, which is required for high-affinity DNA binding by the CAF1 three-subunit complex, and which is distinct from the previously described C-terminal winged-helix domain. CAF1 binds preferentially to DNA molecules longer than 40 bp, and two CAF1-H3-H4 complexes concertedly associate with DNA molecules of this size, resulting in deposition of H3-H4 tetramers. While DNA binding is not essential for H3-H4 tetrasome deposition in vitro, it is required for efficient DNA synthesis-coupled nucleosome assembly. Mutant histones with impaired H3-H4 tetramerization interactions fail to release from CAF1, indicating that DNA deposition of H3-H4 tetramers by CAF1 requires a hierarchical cooperation between DNA binding, H3-H4 deposition and histone tetramerization.

Transcriptomes reflect the phenotypes of undifferentiated, granulocyte and macrophage forms of HL-60/S4 cells.

To understand the chromatin changes underlying differential gene expression during induced differentiation of human leukemic HL-60/S4 cells, we conducted RNA-Seq analysis on quadruplicate cultures of undifferentiated, granulocytic- and macrophage-differentiated cell forms. More than half of mapped genes exhibited altered transcript levels in the differentiated cell forms. In general, more genes showed increased mRNA levels in the granulocytic form and in the macrophage form, than showed decreased levels. The majority of Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were significantly enriched in genes that exhibited differential transcript levels after either RA or TPA treatment. Changes in transcript levels for groups of genes with characteristic protein phenotypes, such as genes encoding cytoplasmic granular proteins, nuclear envelope and cytoskeletal proteins, cell adhesion proteins, and proteins involved in the cell cycle and apoptosis illustrate the profound differences among the various cell states. In addition to the transcriptome analyses, companion karyotyping by M-FISH of undifferentiated HL-60/S4 cells revealed a plethora of chromosome alterations, compared with normal human cells. The present mRNA profiling provides important information related to nuclear shape changes (e.g., granulocyte lobulation), deformability of the nuclear envelope and linkage between the nuclear envelope and cytoskeleton during induced myeloid chromatin differentiation.

Histone Acetylation Regulates Chromatin Accessibility: Role of H4K16 in Inter-nucleosome Interaction.

The N-terminal tail of histone H4 is an indispensable mediator for inter-nucleosome interaction, which is required for chromatin fiber condensation. H4K16 acetylation (H4K16Ac) activates gene transcription by influencing both chromatin structure and interplay with nonhistone proteins. To understand the influence of H4K16Ac on inter-nucleosome interaction, we performed a simulation study for the H4 tail in the context of two nucleosomes in neighboring unit cells in the crystal structure. The binding conformation of H4 tail with/without K16Ac was sampled by replica exchange with solute tempering, and the free energy landscape was explored by metadynamics. The results indicate two important features of H4K16: 1) it is the first button to anchor the H4 tail on the adjacent nucleosome; and 2) it is the only acetylation site interacting with the acidic patch. H4K16Ac disrupts the electrostatic interactions of K16, weakens H4 tail-acidic patch binding, and significantly increases H4 tail conformation diversity. Our study suggests that H4K16Ac directly reduces the inter-nucleosome interaction mediated by the H4 tail, which might further encourage the binding of nonhistone proteins on the acidic patch.

EGFP oligomers as natural fluorescence and hydrodynamic standards.

EGFP oligomers are convenient standards for experiments on fluorescent protein-tagged biomolecules. In this study, we characterized their hydrodynamic and fluorescence properties. Diffusion coefficients D of EGFP1-4 were determined by analytical ultracentrifugation with fluorescence detection and by fluorescence correlation spectroscopy (FCS), yielding 83.4…48.2 µm(2)/s and 97.3…54.8 µm(2)/s from monomer to tetramer. A "barrels standing in a row" model agreed best with the sedimentation data. Oligomerization red-shifted EGFP emission spectra without any shift in absorption. Fluorescence anisotropy decreased, indicating homoFRET between the subunits. Fluorescence lifetime decreased only slightly (4%) indicating insignificant quenching by FRET to subunits in non-emitting states. FCS-measured D, particle number and molecular brightness depended on dark states and light-induced processes in distinct subunits, resulting in a dependence on illumination power different for monomers and oligomers. Since subunits may be in "on" (bright) or "off" (dark) states, FCS-determined apparent brightness is not proportional to that of the monomer. From its dependence on the number of subunits, the probability of the "on" state for a subunit was determined to be 96% at pH 8 and 77% at pH 6.38, i.e., protonation increases the dark state. These fluorescence properties of EGFP oligomeric standards can assist interpreting results from oligomerized EGFP fusion proteins of biological interest.

In Vitro Assembly Kinetics of Cytoplasmic Intermediate Filaments: A Correlative Monte Carlo Simulation Study.

Intermediate filament (IF) elongation proceeds via full-width "mini-filaments", referred to as "unit-length" filaments (ULFs), which instantaneously form by lateral association of extended coiled-coil complexes after assembly is initiated. In a comparatively much slower process, ULFs longitudinally interact end-to-end with other ULFs to form short filaments, which further anneal with ULFs and with each other to increasingly longer filaments. This assembly concept was derived from time-lapse electron and atomic force microscopy data. We previously have quantitatively verified this concept through the generation of time-dependent filament length-profiles and an analytical model that describes assembly kinetics well for about the first ten minutes. In this time frame, filaments are shorter than one persistence length, i.e. ~1 µm, and thus filaments were treated as stiff rods associating via their ends. However, when filaments grow several µm in length over hours, their flexibility becomes a significant factor for the kinetics of the longitudinal annealing process. Incorporating now additional filament length distributions that we have recorded after extended assembly times by total internal reflection fluorescence microscopy (TIRFM), we developed a Monte Carlo simulation procedure that accurately describes the underlying assembly kinetics for large time scales.

Imaging fluorescence (cross-) correlation spectroscopy in live cells and organisms.

Single-plane illumination (SPIM) or total internal reflection fluorescence (TIRF) microscopes can be combined with fast and single-molecule-sensitive cameras to allow spatially resolved fluorescence (cross-) correlation spectroscopy (FCS or FCCS, hereafter referred to FCS/FCCS). This creates a powerful quantitative bioimaging tool that can generate spatially resolved mobility and interaction maps with hundreds to thousands of pixels per sample. These massively parallel imaging schemes also cause less photodamage than conventional single-point confocal microscopy-based FCS/FCCS. Here we provide guidelines for imaging FCS/FCCS measurements on commercial and custom-built microscopes (including sample preparation, setup calibration, data acquisition and evaluation), as well as anticipated results for a variety of in vitro and in vivo samples. For a skilled user of an available SPIM or TIRF setup, sample preparation, microscope alignment, data acquisition and data fitting, as described in this protocol, will take ∼1 d, depending on the sample and the mode of imaging.

Imaging Fos-Jun transcription factor mobility and interaction in live cells by single plane illumination-fluorescence cross correlation spectroscopy.

We collected mobility and interaction maps of c-Fos-eGFP and c-Jun-mRFP1 transcription factors within living cell nuclei. c-Fos dimerizes with c-Jun to form the transcription activator protein-1 (AP-1) which binds to the specific recognition site. To monitor this process, we used fluorescence cross-correlation spectroscopy on a single plane illumination microscope (SPIM-FCCS), which provides diffusion coefficient and protein-protein interaction data in the whole image plane simultaneously, instead of just one point on conventional confocal FCS. We find a strong correlation between diffusional mobility and interaction: regions of strong interaction show slow mobility. Controls containing either an eGFP-mRFP dimer, separately expressing eGFP and mRPF, or c-Fos-eGFP and c-Jun-mRFP1 mutants lacking dimerization and DNA-binding domains, showed no such correlation. These results extend our earlier findings from confocal FCCS to include spatial information.

The effect of DNA supercoiling on nucleosome structure and stability.

Nucleosomes have to open to allow access to DNA in transcription, replication, and DNA damage repair. Changes in DNA torsional strain (e.g. during transcription elongation) influence the accessibility of nucleosomal DNA. Here we investigated the effect of DNA supercoiling-induced torsional strain on nucleosome structure and stability by scanning force microscopy (SFM) and fluorescence correlation spectroscopy (FCS). Nucleosomes were reconstituted onto 2.7 kb DNA plasmids with varying superhelical densities. The SFM results show a clear dependence of the amount of DNA wrapped around the nucleosome core on the strength and type of supercoiling. Negative supercoiling led to smaller nucleosome opening angles as compared to relaxed or positively supercoiled DNA. FCS experiments show that nucleosomes reconstituted on negatively superhelical DNA are more resistant to salt-induced destabilization, as seen by reduced H2A-H2B dimer eviction from the nucleosome. Our results show that changes in DNA topology, e.g. during transcription elongation, affect the accessibility of nucleosomal DNA.

Opposing roles of H3- and H4-acetylation in the regulation of nucleosome structure­­a FRET study.

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 donor­acceptor 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 entry­exit 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.

The role of histone tails in the nucleosome: a computational study.

Histone tails play an important role in gene transcription and expression. We present here a systematic computational study of the role of histone tails in the nucleosome, using replica exchange molecular dynamics simulations with an implicit solvent model and different well-established force fields. We performed simulations for all four histone tails, H4, H3, H2A, and H2B, isolated and with inclusion of the nucleosome. The results confirm predictions of previous theoretical studies for the secondary structure of the isolated tails but show a strong dependence on the force field used. In the presence of the entire nucleosome for all force fields, the secondary structure of the histone tails is destabilized. Specific contacts are found between charged lysine and arginine residues and DNA phosphate groups and other binding sites in the minor and major DNA grooves. Using cluster analysis, we found a single dominant configuration of binding to DNA for the H4 and H2A histone tails, whereas H3 and H2B show multiple binding configurations with an equal probability. The leading stabilizing contribution for those binding configurations is the attractive interaction between the positively charged lysine and arginine residues and the negatively charged phosphate groups, and thus the resulting charge neutralization. Finally, we present results of molecular dynamics simulations in explicit solvent to confirm our conclusions. Results from both implicit and explicit solvent models show that large portions of the histone tails are not bound to DNA, supporting the complex role of these tails in gene transcription and expression and making them possible candidates for binding sites of transcription factors, enzymes, and other proteins.