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- Materials and Methods
- Discussion and Summary
- Literature Cited
- Supporting Information
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 Förster 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
The “bead-string” morphology of eukaryotic chromatin has been an established view for 40 years [1, 2]. Its basic folding unit is the nucleosome, in which about 150 bp of DNA are wrapped around a protein core consisting of two copies each of H2A, H2B, H3, and H4 histones.
In addition to their packaging role, nucleosomes regulate the accessibility of the DNA to proteins. Chromatin accessibility depends on nucleosome stability through local epigenetic modifications to histones and DNA, which are associated with cell development, differentiation, and cellular dysfunction such as cancer. Thus, exploring nucleosome dynamics is central to our understanding of chromatin structural variations and their role in biology. These aspects may be studied in molecular detail in vitro on mononucleosome samples.
The first X-ray crystallographic determination of the nucleosome structure to atomic resolution  has stimulated the field enormously and has given rise to a large number of follow-up studies to higher resolution, with histones from different origin, and different DNA sequences (for a review, see ). The two DNA sequences from which successful crystal structures have been obtained—the strongly positioning alpha-satellite and the Widom 601 sequence—yielded very similar nucleosome structures . This does not exclude possible strong sequence effects on nucleosomes in other contexts. Histone protein variation seems to influence the crystal structure more strongly, e.g., the dimer–dimer contacts in nucleosomes from recombinant histones seem to be tighter for Xenopus than for yeast histones  and the internucleosomal interactions are different between nucleosomes prepared from yeast or metazoan histones [7, 8].
The structure obtained from crystallography is static and represents only one particular conformation. While nucleosomes in a crystal reconstituted from perfectly identical pieces of a very particular DNA with recombinant histones can be viewed like cultured pearls, nucleosomes in the real genome would be like genuine pearls with a practically infinite variety of DNA sequences together with epigenetic variations of histones. At first glance (e.g., in electron microscopic images), these “natural pearls” all seem to be the same, because these variations affect only the fine structure, not the overall shape. The fine structure of nucleosomes is the key to their function, allowing control of DNA accessibility or chromatin packaging.
The consequence is that for getting access to nucleosome fine structure and dynamics, adequate experimental techniques must be used. There also exists a large body of experimental data on the solution structure of nucleosomes by solution scattering and analytical ultracentrifugation [9-12]. The samples for these studies are often a mixture of naturally diverse nucleosomes isolated from a cell type. Such solution studies show that nucleosomes are highly dynamic under in vivo conditions and that structure fluctuations play an important role in their function.
Structural studies on nucleosomes in solution have been advanced in particular by using Förster resonance energy transfer (FRET) on fluorescently labeled samples. FRET uses a donor and an acceptor fluorophore label on the molecules under study. The donor dye is excited by a laser, the longer wavelength acceptor dye may then be excited by nonradiative energy transfer from the donor. The efficiency E of this transfer increases with decreasing interdye distance R (E ∝ R−6) and, therefore, allows determination of the distance on the nanometer scale . Fluorescent labeling requires the use of a homogeneous population of reconstituted nucleosomes, similar to crystallization. However, two important differences exist: First, not all nucleosomes that can be reconstituted will also crystallize. Second, in a crystal, only particular conformations are selected and a particular ionic environment is present. Nucleosomes in solution, on the other hand, are free to explore all their accessible conformational space and solution conditions can be varied over a much wider range.
Bulk solution FRET as measured by fluorescence emission intensity spectra measures an average over many molecules. While some deeper-going information can be obtained through fluorescence lifetime measurements, distinguishing species of different FRET efficiencies by their lifetimes is not a very selective method: Estimating a distribution of exponential decay times is a known ill-posed problem . For detecting dynamics and conformational heterogeneity, molecules must be observed individually. To this aim, single-pair FRET (spFRET) has been developed, where the sample fluorescence is excited and observed in a high-resolution microscope with confocal optics. At sufficiently low fluorophore concentrations (<0.1 nM), one observes fluorescence bursts from single molecules passing through the laser focus. These bursts can then be analyzed statistically to create a FRET efficiency distribution and to characterize eventual time-dependent conformational changes [15, 16]. We note that lifetime analysis of single molecule bursts can improve the quality of the data considerably .
A number of FRET and spFRET studies on nucleosomes exist: for review, see . With the establishment of these techniques, the literature on FRET and spFRET application to the structure and dynamics of nucleosomes, chromatin, and chromatin-associated proteins has shown significant growth in the last 5 years. In our own work, we measured the average distance between the ends of different length nucleosomal DNA and established that the DNA ends of mononucleosomes in solution do not cross . Furthermore, we characterized the salt- and linker histone H1-dependent structure of mono-  and tri-nucleosomes .
Under physiological conditions, nucleosomes open by thermal fluctuations that expose DNA sites [22, 23]. These opening events are only transient and sparsely populated, but they allow proteins such as chromatin remodelers, transcription factors, or replication factories to bind and stabilize the open state. Without additional protein factors, characterization of the open state requires to increase its equilibrium fraction artificially, e.g., by thermal denaturation, denaturating agents, or change in salt concentration. We have chosen the latter as a convenient means to destabilize the nucleosome structure and thereby increase the opening probability.
We used spFRET to characterize the stability changes in nucleosomes upon histone acetylation, changes of DNA sequence, and ionic strength . Using salt-induced destabilization in spFRET, we proposed an opening mechanism of nucleosomes reconstituted on the “Widom 601” DNA  and recombinant Xenopus histones . Later, by comparing the salt-induced decrease in FRET for five different donor/acceptor pairs on the DNA, H2B, and H4 histones, we discovered a new intermediate in which the histone core opens at the interface between the H2A/H2B dimer and the (H3H4)2 tetramer, before the first H2A/H2B dimer dissociates from the DNA . We could estimate that this new “butterfly state” is populated to the order of one percent under physiological conditions.
It is now important to establish whether the mechanism found in that work is generally characteristic of nucleosome opening, and how nucleosome stability and opening pathways may vary between nucleosomes from different species and DNA sequences. For instance, earlier biochemical studies on isolated oligo- and mono-nucleosomes revealed differences in salt-induced and thermal unfolding between yeast and higher eukaryotes . To understand these biologically important variations, we studied a larger variety of nucleosomes reconstituted from histones of different origin, and on varying DNA sequences. Our data show conclusively that nucleosome stability is strongly affected by DNA sequence, whereas the origin of the histone proteins only modestly altered nucleosome stability. More importantly, we demonstrate that the pathway of nucleosome dissociation is identical for all nucleosome samples, suggesting that disassembly via the butterfly state is a general mechanism for nucleosome dissociation.
Discussion and Summary
- Top of page
- Materials and Methods
- Discussion and Summary
- Literature Cited
- Supporting Information
This work further characterizes the nucleosome opening mechanism proposed in our earlier work [26, 27], where we found that dissociation of mononucleosomes proceeds through three steps: First, the dimer-tetramer interface is destabilized and the H2A/H2B dimer moves outward, away from the center position of the DNA at the dyad axis, while still being attached to the outer DNA segment; in the next step, the dimer dissociates completely, leaving a “hexasome” or “tetrasome” structure; finally, the (H3/H4)2 tetramer dissociates, leaving the free DNA. Here, we continued these studies on nucleosomes reconstituted from Xenopus, mouse, and yeast histones on the Widom 601, 5S rDNA, and MMTV-B positioning sequences. We used salt-induced destabilization as a convenient means to create artificially such structural states as might exist during transient opening under physiological conditions. While other destabilization techniques are conceivable, such as temperature- or solvent-induced denaturation, we believe that changing the ion concentration is the least invasive of these.
As a general conclusion, we find that nucleosomes on the 601 sequence, with a 300 mM higher c1/2 value, are much more stable against salt-induced dissociation than on the 5S or MMTV-B sequences. We also see a slightly higher stability of 5S as compared with MMTV-B nucleosomes, the difference in c1/2 being 30 mM. This latter finding is qualitatively similar to the stability difference found by Kelbauskas et al.  by dilution experiments. There, the authors suggested a very pronounced stability increase of 5S over MMTV-B nucleosomes; however, their experiments were done at much lower ionic strength (10 mM Tris, no added salt, no detergent).
Mouse histones formed the most stable nucleosomes under our conditions, followed by Xenopus and yeast. It had already been shown that natively isolated nucleosomes from yeast are less stable than those from metazoa, as seen by salt dissociation analyzed on gels . Our present study suggests that this is not due to variations in post-translational modifications, but due to the differences in the histone sequence itself.
Using known strongly positioning sequences and uniform recombinant histones allowed us to generate compositionally monodisperse nucleosome preparations, so that variations in the spFRET population distribution would most probably stem from structural polydispersity of the sample. The width of the spFRET distribution then indicates the tendency of the particular nucleosome to assume structural substates. A significantly narrower spFRET peak is seen for Xenopus nucleosomes on the 601 sequence at low salt (which has been in vitro-selected for strong positioning) than for the 5S and MMTV-B sequences, of natural origin. A similar behavior was found earlier for end-labeled DNA fragments . We may conclude that the positioning power of the 601 sequence narrows down the accessible conformational space relative to the other two DNAs.
For all DNA and histone combinations studied, the general opening mechanism through the butterfly state could be confirmed. Other than postulated earlier , our data strongly suggests that nucleosome opening precedes histone dimer dissociation.
We also note that earlier spFRET studies on nucleosomes free in solution [24, 34, 35] showed disruption at much lower salt concentrations than presented here, i.e., in the range of 50–100 mM NaCl. Although a systematic study of the solution conditions that contribute to sample stability at the low concentrations necessary for spFRET is still overdue, we note here that the sample conditions in those earlier studies were different: lower nucleosome concentration, untreated sample chamber surfaces, and the absence of detergents could be responsible for the lower stability.
Concluding we can say that spFRET of mononucleosomes labeled at various donor/acceptor positions seems to be a reliable method to characterize the stability and conformational variability of chromatin from different origins. In particular, double-labeled DNAs lend themselves particularly well for systematic studies because they can be prepared rather easily and do not pose the problem of labeling ambiguity, as in the case of a histone-DNA FRET pair.