Single-Pair FRET Microscopy Reveals Mononucleosome Dynamics
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- Koopmans, W.J.A., Brehm, A., Logie, C. et al. J Fluoresc (2007) 17: 785. doi:10.1007/s10895-007-0218-9
We applied spFRET microscopy for direct observation of intranucleosomal DNA dynamics. Mononucleosomes, reconstituted with DNA containing a FRET pair at the dyad axis and exit of the nucleosome core particle, were immobilized through a 30 bp DNA tether on a polyethyleneglycol functionalized slide and visualized using Total Internal Reflection Fluorescence microscopy. FRET efficiency time-traces revealed two types of dynamics: acceptor blinking and intramolecular rearrangements. Both Cy5 and ATTO647N acceptor dyes showed severe blinking in a deoxygenated buffer in the presence of 2% βME. Replacing the triplet quencher βME with 1 mM Trolox eliminated most blinking effects. After suppression of blinking three subpopulations were observed: 90% appeared as dissociated complexes; the remaining 10% featured an average FRET efficiency in agreement with intact nucleosomes. In 97% of these intact nucleosomes no significant changes in FRET efficiency were observed in the experimentally accessible time window ranging from 10 ms to 10’s of seconds. However, 3% of the intact nucleosomes showed intervals with reduced FRET efficiency, clearly distinct from blinking, with a lifetime of 120 ms. These fluctuations can unambiguously be attributed to DNA breathing. Our findings illustrate not only the merits but also typical caveats encountered in single-molecule FRET studies on complex biological systems.
KeywordsFRET Nucleosome Single-molecule Blinking
The nucleosome core particle consists of ∼ 50 nm DNA wrapped nearly twice around a histone-octamer protein-core . Nucleosomal DNA has to unwrap from the nucleosome core to sterically allow processes such as transcription, replication and repair. Accessibility to nucleosomal DNA is facilitated by ATP-dependent remodeling enzymes in vivo . However, it is known that spontaneous conformational changes of the nucleosome expose occluded sites in the DNA as well . DNA breathing, the transient unwrapping and rewrapping of a stretch of DNA from the nucleosome core, has recently been studied in detail with a variety of fluorescence techniques. The equilibrium constant of this process was determined with bulk FRET measurements . Unwrapping lifetimes of 10–50 ms were obtained with stopped-flow FRET measurements and Fluorescence Cross Correlation Spectroscopy (FCCS) . Interestingly, based on their single pair FRET work, Tomschik et al. concluded that unwrapping of nucleosomal DNA occurs to a much larger extent than was previously anticipated : they suggested that 30–60% of the nucleosomal DNA was unwrapped with a lifetime on the order of ∼150 ms before rewrapping.
Although the conceptual beauty of FRET studies is undisputed, there are a number of important caveats in single-molecule FRET studies of biomolecules, such as fluorophore blinking, photobleaching and sample immobilization. Here, we addressed these issues. spFRET microscopy on mononucleosomes revealed two dominant types of dynamics: acceptor blinking and intramolecular rearrangements that we attribute to DNA breathing, which only became apparent after suppression of blinking. Upon immobilization, we observed three different populations: 90% of the nucleosomes dissociated or represented donor-only species, and 10% remained intact. Of these fully wrapped nucleosomes, 97% showed stable FRET on timescales between 0.01–10 s, while 3% showed dynamics with a dwell time of 120 ms that we attribute to conformational changes in the nucleosome.
Material and methods
Recombinant histone octamers were mixed with the DNA construct at a 1:1 ratio, in TE (1 mM EDTA, 10 mM TRIS pH 8.0) and 2 M NaCl. Mononucleosomes were reconstituted by salt dialysis against 0.85, 0.65, 0.5 and finally against 0.1 M NaCl, all buffered with TE.
Bulk fluorescence measurements
Single-molecule FRET measurements
Cleaned glass slides were amino functionalized with 10 μg/ml poly-d-lysine, and subsequently incubated for 4 h with an amine reactive polyethylene glycol (PEG) mixture: 20% mPEG-succinimidyl propionate 5,000 molecular weight (Nektar Therapeutics) and 0.2% biotin-PEG-n-hydroxysuccinimide 3,400 molecular weight (Nektar Therapeutics) in 0.1 M sodium carbonate buffer (pH 8.2). A flow cell was assembled by sealing a poly-dimethylsiloxane channel with a PEG functionalized slide. A 0.1 mg/ml streptavidin (Roche) solution was incubated for 5 min, and subsequently washed away. A sample, which typically consists of 10–50 pM of labeled mononucleosomes in 50–200 mM NaCl, 10 mM TRIS.HCl pH 8.0, 0.03% NP-40, and 10–50 nM unlabeled mononucleosomes, was injected in the channel and immobilized. An enzymatic oxygen scavenger system (1% glucose, 2% β-mercaptoethanol (βME) or 1 to 2 mM Trolox (Sigma), 0.2 mg/ml glucose oxidase, and 0.04 mg/ml catalase), was added to the buffer to extend the lifetime of the fluorophores before photobleaching. The buffer was degassed prior to use to further reduce the oxygen concentration. The flow cell was mounted on a microscope equipped with a 100X oil-immersion TIRF microscope objective (NA = 1.45, NIKON) and temperature-stabilized at 22°C using a water circulating bath connected to all parts of the setup in contact with the sample. The 514 nm line of an Ar+ laser (Coherent) was used to illuminate an area of ∼ 600 μm2 with a power of 0.9 mW. In the case of alternating excitation, a 636 nm diode laser (Power Technology) was used to illuminate an area of ∼900 μm2 with a power of 0.3 mW. Both beams were circularly polarized and were displaced parallel to the optical axis of the objective, so that an evanescent excitation field was generated by total internal reflection of the light at the glass-water interface. The excitation intensity at the interface in the evanescent field is ∼4 times higher than the incident beam intensity at the critical angle . We therefore estimated that the resulting excitation intensities at the interface were ∼0.6 kW/cm2 for the 514 nm excitation and ∼0.13 kW/cm2 for the 636 nm excitation respectively.
The fluorescence was collected by the objective and filtered through a custom-made dual color band pass filter (Chroma), that rejects scattered laser light, and a long pass filter (OG530, Schott). The fluorescence was further split into a donor and an acceptor channel by a custom-made dichroic wedge mirror (0.5° angle, center wavelength of 630 nm, Chroma) placed in the infinity path of the microscope . A +150 mm achromatic lens (Thorlabs) projected the separate images on a multiplication gain CCD camera (Cascade 512B, Roper Scientific) operating at a frame rate of 20 to 100 Hz.
Bulk fluorescence spectra reveal proper reconstitution of mononucleosomes
The observed average FRET efficiency in the reconstituted mononucleosomes was 0.75 ± 0.1, which was in good agreement with FRET values predicted by the position of the FRET pair in the nucleosome. From the bulk FRET experiments, and the predicted maximum FRET efficiency of ∼0.9 for a mononucleosome with the ATTO550-ATTO647N FRET-pair, we estimated the reconstitution yield to be at least 85%. Some residual donor emission can be accounted for by incomplete acceptor labeling. With absorption measurements on the DNA construct (Fig. 2b) we determined that the acceptor:donor:DNA stoichiometry was ∼0.7:0.9:1. Together, these bulk data show that the labeled DNA construct and the histone proteins properly formed mononucleosomes upon reconstitution (see Fig. 1).
spFRET microscopy reveals individual nucleosomes together with a large population of dissociated nucleosomes
In contrast, 90% of the fluorophores did not show FRET at all. This conflicts with the bulk experiments, where after correction for incomplete labeling an average FRET efficiency of 0.75 was found. As mentioned before, there was a fraction of donor only labeled species (∼30%), but this alone could not explain the observed discrepancy between bulk and single-molecule measurement. When we directly excited the acceptor fluorophores (see Fig. 3b), we found that most of the donors were colocalized with an acceptor. Therefore we conclude that FRET signal was lost during the single-molecule measurement, due to disassembly of a large fraction of the nucleosomes.
It is known that nucleosomes become unstable and dissociate when they are diluted to low concentrations [17, 18]. For our wide field spFRET measurements we diluted to pM fluorophore concentrations to resolve individual fluorophores. We ensured that the nucleosome concentration was always above 10–50 nM. By adding an excess of unlabeled mononucleosomes and 0.03% non-ionic detergent (NP-40) to our buffer. Thastrom et al.  reported that under these conditions nucleosomes do not dissociate in bulk solutions. We found that even 50 nM of unlabeled nucleosomes, far above the dilution-driven dissociation threshold, did not retain proper nucleosome folding, excluding dilution effects to be the cause.
It is known that H2A–H2B histone dimers can spontaneously be exchanged from the protein core , which in our case would result in a transient loss of FRET. However, we found the same amount of disassembled nucleosomes upon immobilization when the octamer protein core was crosslinked by dialysis against 0.05% glutaraldehyde in 1 mM EDTA. We confirmed that the crosslinking itself did not dissociate nucleosomes with bulk fluorescence experiments. This suggests that not the histone protein core dissociates, but rather that the wrapped DNA loosens or significantly rearranges itself around the protein core. We confirmed that mononucleosomes in free solution (in the same buffer used for single-molecule experiments) remain stable for hours at room temperature using bulk fluorescence measurements. Therefore we consider the dissociation of the nucleosomes described here to be associated with their immobilization to the functionalized cover glass. As an alternative immobilization strategy we performed experiments with biotinylated BSA-functionalized cover glasses instead of PEGs. Biotinylated BSA is often used for single-molecule studies involving nucleic acids, whereas PEGs are often used for studies involving DNA-protein complexes . Biotinylated BSA yielded even less intact nucleosomes. The exact nature of the interactions of the nucleosomes with the modified cover slides remains unclear, but the destabilizing effect of the surface forms a hurdle for obtaining large datasets of spFRET measurements.
On the 10% immobilized mononucleosomes showing FRET, irreversible loss of FRET was only found after photobleaching, implying that their nucleosomal structure remained intact after immobilization.
Single-molecule fluorescence footprint of individual nucleosomes
We found that the standard deviation in FRET efficiency of the high FRET state of the entire population (0.13) was larger than the standard deviation within individual traces (0.06), as shown in the histograms in Fig. 4c and d. This observation can be accounted for either by different nucleosome populations with slight variations in FRET efficiency, or by local differences in rotational freedom of the dyes due to immobilization.
Acceptor blinking is the dominant source of spFRET dynamics
The FRET traces shown in Fig. 4a and b are highly dynamic and fluctuate between a high FRET state (E ∼0.6, lifetime 2.5 s) and a low FRET state (E ∼0.1, lifetime 0.13 s). Interestingly, the characteristics of these fluctuations, i.e. both on and off time, and the low FRET level, are remarkably similar to those observed by Tomschik et al.  who performed analogous experiments. This similarity however, is remarkable in view of the completely different FRET-label location. Tomschik et al. labeled the nucleosome opposite to the dyad axis and probed the DNA at the most internal position of the nucleosome, whereas our substrate has labels at the most exterior position. Widom et al. have previously shown that the enzymatic accessibility of the DNA inside a nucleosome strongly reduces as the DNA is more internal in the nucleosome , suggesting a higher frequency of unwrapping events in our experiments.
Because of the nearly complete absence of acceptor emission, we investigated the nature of these fluctuations in order to exclude reversible transitions of the acceptor to an inactive state (acceptor blinking, resulting in a Förster radius of effectively zero ) as the origin of these events. By alternating donor excitation with direct acceptor excitation we could directly monitor the acceptor condition as shown in Fig. 3b. After deinterleaving the data into two time-traces, one for green excitation and one for red excitation, it became obvious that the fluorescence intensity of the acceptor upon direct excitation correlated perfectly with the enhanced emission of the acceptor due to FRET. Thus, the low FRET state must be attributed to blinking, due to a dark-state level of the acceptor. Further evidence that these fluctuations were caused by acceptor blinking was provided by experiments with alternative acceptor dye (ATTO647N, emission spectrum similar to Cy5). Alternating excitation of the acceptor dye revealed a strong positive correlation between sensitized emission of the acceptor and direct excitation of the acceptor. In this case the low FRET state was also present, but with a much shorter lifetime of 0.046 s. In conclusion, our data confirm that the fluctuations between a high and a low FRET state reflect photophysical processes in the acceptor dye rather than nucleosome conformational changes.
Suppression of blinking
Recently a different approach to reduce blinking was described by Rasnik et al. . They replaced the triplet quencher βME in the oxygen scavenger system by a water-soluble analog of vitamin E, Trolox. Using this approach, Cy5 blinking in single-molecule FRET measurements on DNA constructs was eliminated. We tested the effect of Trolox in the imaging buffer on blinking of FRET pair labeled mononucleosomes. Results are shown in example traces and histograms in Fig. 5c and d. Both for Cy5 and ATTO647N blinking was dramatically suppressed in the presence of 1 to 2 mM Trolox: over 90% of the traces showed no observable blinking. Noise analyses of the intensity fluctuations in most of these traces were fully accounted for by camera noise and photon statistics (shot noise) only. Thus, within our time resolution (10 ms), no effect of short time scale blinking, or inter system crossing, was detected.
Surprisingly, less than 10% of the observed FRET pairs still showed some extent of blinking indicated by fast excursions into a FRET state below 0.2, with a typical off time of 14 ± 1 and 13 ± 1 ms respectively, as shown in the histograms in Fig. 5c and d. Because the lifetime of these blinking events was on the order of the smallest sampling time used, blinking events were not identified by alternating excitation of the acceptor dye, but only by FRET efficiencies below the noise threshold. Direct excitation of the acceptor did only reveal some occasional blinking in the acceptor traces, with the same lifetime of 13–14 ms. To confirm that a small but finite amount of fast blinking still occurred in the presence of Trolox, we performed spFRET measurements on a FRET pair that was separated by 11 basepair duplex DNA. This construct does not exhibit structural changes that affect the FRET intensity. In this case we also observed a small, but finite amount of blinking in a number of traces (data not shown), with a lifetime similar to that measured on mononucleosomes.
A fraction of the immobilized nucleosomes shows dynamics clearly distinct from blinking
Discussion and conclusion
Time-traces of spFRET microscopy on single reconstituted mononucleosomes revealed two types of dynamics: acceptor blinking and intramolecular rearrangements. Intramolecular rearrangements became only apparent after suppression of blinking. Both Cy5 and ATTO647N showed severe blinking in a deoxygenated buffer in the presence of 2% βME. Replacing the triplet quencher βME with Trolox effectively eliminated most blinking effects. The lifetime of DNA unwrapping that we obtained after rigorous elimination of blinking events (∼120 ms) was comparable to the 150–180 ms obtained by Tomschik et al. , despite the very different location of the labels in the nucleosome, and probably less important, the different DNA sequence and origin of the histones. However, we observed a very similar lifetime (∼130 ms) for Cy5 blinking under comparable buffer conditions (2% βME). The FRET efficiency of the open states in our experiments was significantly above the detection threshold, so we can explicitly exclude photophysics as the origin of the observed changes in FRET efficiency.
Our single-molecule measurements revealed at least three subpopulations in the reconstituted and immobilized nucleosome sample: 90% of the fluorophores represented dissociated nucleosomes or donor only species, 10% represented intact nucleosomes. Of these, 97% remained stable on time-scales ranging from 10 ms to 10 s of seconds, while 3% showed intervals with reduced FRET efficiency and a lifetime of 120 ms clearly distinct from blinking.
Why most nucleosomes dissociate upon immobilization to the cover slip remains unknown. Immobilization of the molecules is necessary for extension of the available observation time. The time limit is given by photobleaching, one of the key advantages of this method with respect to, for example, Fluorescence Correlation Spectroscopy. However, the close proximity to the surface provides ample opportunity for interactions with it. Surface induced nucleosome dissociation has been reported before in Atomic Force Microscopy (AFM) studies. Using AFM in liquid, Nikova et al. observed an unwrapping of ∼25 nm of DNA from nucleosomes absorbed to a mica surface . This unwrapping was attributed to a depletion of H2A–H2B histone dimers induced by the high surface charge of the mica, resulting in unwrapping of DNA. Although PEGs are neutral polymers that are commonly used to reduce non-specific surface binding of proteins, they may affect nucleosomes in different ways: PEG molecules have been reported to interact strongly with unfolded proteins , and could therefore possibly interact with histone tails. Furthermore, histone proteins are known to be adhesive to glass or plastic .
The large fraction of dissociated nucleosomes we report here was not observed by Tomschik et al. . Because of the internal position of the labels they used, at least 50 bp of DNA had to be detached from the histone core before FRET was completely lost. We labeled the DNA at the very end of the histone bound part, and accordingly a detachment of 10–20 bp of DNA would already result in complete loss of FRET. Furthermore, the exterior part of the DNA is largely constrained by the mobile H2A–H2B dimer, whereas the labeled part of the DNA in the nucleosomes used by Tomschik et al. is mostly constrained by the more stable H3–H4 tetramer.
A labeling strategy by Li et al. [8, 9], who end-labeled a 601 nucleosome positioning element together with either histone H3 or H2A, provides a more comparable construct. Based on stopped-flow FRET and FCCS experiments they deduced an unwrapping rate of 4 s−1 an unwrapping lifetime of 10–50 ms. The 3% of our traces that showed dynamics typically featured multiple unwrapping events before photobleaching. Though photobleaching obstructs quantification of the unwrapping rate, it is of the same order of magnitude as observed by Li et al. The lifetime of the unwrapped state we observed is five to ten times larger. This discrepancy may in part be explained by differences in experimental conditions and nucleosome constructs; we can however not exclude the possibility that we overlook short-lived unwrapped states, biasing our data to a longer lifetime.
The absence of observations of DNA unwrapping in the majority of the intact nucleosomes reported in this study is in strong contrast with the extent of DNA breathing dynamics found by Li et al. [8, 9]. Two possible explanations could account for this difference: (1) The most frequently occurring DNA unwrapping occurs at a rate that exceeds the time resolution of our experiment. The rare dynamics (3%) that we observe would reflect the release of multiple histone-DNA contacts, a process that would occur less often and on longer time scales than unwrapping of only the first DNA-octamer. However, unwrapping of 10–20 bp of DNA would induce a more dramatic reduction in FRET efficiency than the reduction we observed, which is consistent with unwrapping of 10 bp or less. (2) The immobilized nucleosomes did not undergo breathing dynamics. It should be kept in mind that because of the disruption of 90% of the nucleosome upon immobilization, we only probed a subset of nucleosomes that do not dissociate upon immobilization. These nucleosomes could either be resistant to unwrapping of the DNA, or immobilized in such a way that DNA dynamics are inhibited due to interactions with the surface, while still retaining proper folding. In either case, immobilization is expected to have major impact on nucleosome dynamics, emphasizing the need for a more inert immobilization than point attachment to a PEG coated surface.
Our findings demonstrate that experimental conditions can have a profound impact on the data obtained when probing nucleosome structure and conformational dynamics. Immobilization effects and blinking dynamics have to be accounted for, and where possible suppressed in order to extract biologically relevant data from spFRET experiments. We have shown that DNA breathing kinetics obtained from carefully optimized spFRET experiments approaches values obtained from bulk experiments, opening the way to more complex single-molecule studies of chromatin dynamics.
We would like to thank Jon Widom for providing a plasmid containing the 601 sequence; Jesper Donsmark, John van Egmond and Ineke de Boer for technical assistance, and Paige Shaklee for help with the manuscript. AB, CL, and SJTvN acknowledge support from the EuroDYNA eurocores program. This work is part of the research programme of the ‘Stichting voor Fundamenteel Onderzoek der Materie (FOM)’, which is financially supported by the ‘Nederlandse Organisatie voor Wetenschappelijk Onderzoek (NWO)’.