spFRET using alternating excitation and FCS reveals progressive DNA unwrapping in nucleosomes

Abstract

Accessibility to DNA wrapped in nucleosomes is essential for nuclear processes such as DNA transcription. Large conformational changes in nucleosome structure are required to facilitate protein binding to target sites within nucleosomal DNA. Transient unwrapping of DNA from nucleosome ends can provide an intrinsic exposure of wrapped DNA, allowing proteins to bind DNA that would otherwise be occluded in the nucleosome. The molecular details underlying these mechanisms remain to be resolved. Here we show how DNA unwrapping occurs progressively from both nucleosome ends. We performed single-pair fluorescence resonance energy transfer (spFRET) spectroscopy with alternating laser excitation (ALEX) on nucleosomes either in free solution or confined in a gel after PAGE separation. We combined ALEX-spFRET with a correlation analysis on selected bursts of fluorescence, to resolve a variety of unwrapped nucleosome conformations. The experiments reveal that nucleosomes are unwrapped with an equilibrium constant of approximately 0.2-0.6 at nucleosome ends and approximately 0.1 at a location 27 basepairs inside the nucleosome, but still remain stably associated. Our findings, obtained using a powerful combination of single-molecule fluorescence techniques and gel electrophoresis, emphasize the delicate interplay between DNA accessibility and condensation in chromatin.

Figure 1

Experimental system and setup. (a) A FRET-labeled nucleosome. X, Y, and Z indicate the locations of various FRET labeling positions used in this work. (b) ALEX-FRET fluorescence microscope. DM, dichroic mirror; AOM, acousto-optical modulator; PH, pinhole; EF, emission filter; and SPAD, single-photon avalanche diode. (c) Typical fluorescence intensity time-traces of the four different photon streams acquired with the setup in panel b.

Figure 2

E, S footprint of nucleosomes. (a) Two-dimensional E, S histogram of reconstituted nucleosomes, label position Y. (b) FRET histogram of all bursts (shaded) and of doubly labeled bursts (solid). The low FRET population is reduced considerably by filtering out D-only species. (c) D-only filtered E-histograms for label positions X, Y, and Z in the nucleosome. Note the considerable population at intermediate FRET efficiency.

Figure 3

Nucleosome disassembly kinetics for several salt concentrations in time. The fraction of intact nucleosomes (E > 0.25) in time for different monovalent salt concentrations, buffered with 10 mM Tris.HCl (pH 8). The lines are linear (0 mM NaCl) or exponential (50 and 100 mM NaCl) fits to the data. (Inset) The fraction of doubly labeled (0.2 < S < 0.8) molecules in time. For each salt concentration tested, this fraction is constant over time.

Figure 4

Correlation curves. (Inset) Unscaled correlation curves of selected bursts in a nucleosome sample, and of a free DNA sample. The nucleosomes show an increased diffusion time compared to the DNA. Nucleosome fractions can only be separated based on FRET; the diffusion time in free solution is unaffected by conformational changes within the nucleosome.

Figure 5

ALEX-spFRET spectroscopy on gel-separated nucleosomes (a) X, (b) Y, (c) Z, and (d) DNA from template Z. (Left panels) Fluorescence image (acceptor excitation) of PAGE analysis of reconstituted nucleosomes and the corresponding DNA templates. N, nucleosome band, D, DNA band. (Middle panels) E, S-histograms of ALEX-spFRET experiments in gel in the nucleosome bands, and DNA band Z depicted. A low FRET peak can be observed in all nucleosome bands, which points at progressive DNA unwrapping from the nucleosome ends. (Right panels) Burstwise FCS analysis on nucleosome populations in gel. For nucleosomes X and Z, a clear difference in correlation time can be seen for different FRET efficiencies, reflecting different conformations. For nucleosome Y, the difference is smaller. All nucleosome populations diffuse significantly slower than the DNA.

Figure 6

D-only corrected E-histograms for nucleosomes X, Y, and Z in gel. A low FRET population can clearly be observed at all three labeling positions. The low FRET population is 38% for X and Z, and 10% for Y, indicating progressive and pronounced nucleosome unwrapping from both ends.