Direct observation of coordinated DNA movements on the nucleosome during chromatin remodelling

Abstract

ATP-dependent chromatin remodelling enzymes (remodellers) regulate DNA accessibility in eukaryotic genomes. Many remodellers reposition (slide) nucleosomes, however, how DNA is propagated around the histone octamer during this process is unclear. Here we examine the real-time coordination of remodeller-induced DNA movements on both sides of the nucleosome using three-colour single-molecule FRET. During sliding by Chd1 and SNF2h remodellers, DNA is shifted discontinuously, with movement of entry-side DNA preceding that of exit-side DNA. The temporal delay between these movements implies a single rate-limiting step dependent on ATP binding and transient absorption or buffering of at least one base pair. High-resolution cross-linking experiments show that sliding can be achieved by buffering as few as 3 bp between entry and exit sides of the nucleosome. We propose that DNA buffering ensures nucleosome stability during ATP-dependent remodelling, and provides a means for communication between remodellers acting on opposite sides of the nucleosome.

Fig. 1

Simultaneous detection of entry- and exit-side movements by three-colour FRET. a Cartoon representations of the three-colour labelling scheme. The ATPase motor of the remodeller is shown in brown and Cy3, Cy5, and Alexa750 are depicted by green, red, and purple stars, respectively. The entry side is on the TA-poor side of the 601 sequence, and the exit side is on the TA-rich side. b Schematic of FRET detection with alternating 532 nm and 638 nm laser excitation of nucleosomes labelled with Cy3, Cy5, and Alexa750. Entry-side (c) and exit-side (d) FRET histograms constructed from 1543 traces before (solid bars) and 507 traces after (open bars) remodelling by 300 nM Chd1 in the presence of 1 mM ATP. e Representative Cy3 (green), Cy5 (red), and Alexa750 (purple) fluorescence and FRET time traces (entry-side, light blue; exit-side, dark blue) showing sliding of a single nucleosome after addition of 300 nM Chd1 and 1 mM ATP. Time traces were recorded with alternating 532 nm and 638 nm laser excitation, as indicated by the areas shaded in green and red, respectively. The dashed lines indicate the onset of changes in entry-side and exit-side FRET. Entry- and exit-side FRET values, determined simultaneously in the same experiment using alternating laser excitation, resembled those separately determined using corresponding two-colour FRET nucleosomes possessing Cy5-H2B and either an exit- or entry-side DNA fluorophore, respectively (Supplementary Fig. 2). Source data are provided as a Source Data file

Fig. 2

Entry-side movement of nucleosomal DNA precedes exit-side movement. a Cy3 (green), Cy5 (red), and Alexa750 (purple) fluorescence and FRET time traces (exit-side: dark blue; entry-side: light blue) showing sliding of a single nucleosome, after addition of 300 nM Chd1 and 100 μM ATP. The lag time between the onset of entry- and exit-side DNA movements is denoted as t_lag. Time traces were recorded with alternating 532 nm and 638 nm laser excitation, as indicated by the areas shaded in green and red, respectively. b Histograms of t_lag values (red bars) observed with 100 or 500 μM ATP, fit with a single-exponential distribution (N = 185–220 events). c Dependence of 1/t_lag on the ATP concentration. Data are shown as the mean ± SEM (N = 40–340 events). d Left: Cartoon schematic showing the 601-flip nucleosome, with short exit DNA on the TA-poor side and long entry DNA on the TA-rich side of the 601 sequence. Middle: Two representative FRET time traces (exit-side: dark blue; entry-side: light blue) showing sliding of single 601-flip nucleosomes after addition of 300 nM Chd1 and 500 μM ATP. Right: Histogram of t_lag values (red bars) from many 601-flip nucleosomes observed with 500 μM ATP, fit with a single-exponential distribution (N = 190 events). e Nucleosome sliding by SNF2h shows the same coordination of entry/exit DNA movements. Middle: Two representative FRET time traces (exit-side: dark blue; entry-side: light blue) showing sliding of single nucleosomes after addition of 1 μM SNF2h and 10 μM ATP. Right: Histogram of t_lag values (red bars) constructed from 226 events, fit to a single-exponential distribution (black line). Source data are provided as a Source Data file

Fig. 3

Buffering 1 to 3 bp is sufficient for allowing nucleosome repositioning. a Model of the nucleosome-Chd1 structure (pdb code: 5O9G [ref. .]) showing the position of the ATPase motor (brown) relative to 2 nt ssDNA gaps (black) that limit nucleosome sliding. b Native gel electrophoretic mobility assay comparing sliding of nucleosomes with ssDNA gaps at m = 3, m = 5, or m = 8 nt from the entry-side SHL2. Reactions contained 150 nM nucleosome, 200 nM Chd1 and 1 mM ATP. The supershifted bands at the top of each gel represent Chd1-nucleosome complexes. Gels are representative of two or more independent replicates. c Schematic depicting site-specific cross-linking positions on the nucleosome. Using nucleosomes containing two cysteine substitutions, three regions were simultaneously followed. Nucleosome sliding by Chd1, which shifts the histone core toward the side with longer linker DNA (to the right), can be observed by corresponding shifts of DNA cross-linking sites. d, e Time-course experiments showing Chd1-dependent changes in DNA positioning of nucleosomes containing gaps at m = 5 bp (d) and m = 8 bp (e). At each cross-linking site, stacked intensity plots showing time-dependent changes in the distribution of cross-links are given on the left, and the relative intensities of starting versus shifted cross-linking products are plotted on the right. Cross-linking products that rapidly appeared and differed depending on the location of the gap are indicated in orange. Products that formed more slowly over time, which correspond to the preferred phase of the 601 sequence, are shown in blue. Note that consistent with previous work, cross-linking on the exit side did not show a nucleosome population shifted by +11 bp (blue), likely due to sequence bias in cross-linking. Reactions contained 150 nM nucleosome, 200 nM Chd1, and 1 mM ATP. The time-course for these experiments was 0”, 7”, 15”, 30”, 45”, 60”, 90”, 2’, 4’, 8’, 16’, and 32’. Results are consistent across three separate experiments, two of which use similar time courses. Gel images are shown in Supplementary Fig. 6. Source data are provided as a Source Data file

Fig. 4

A model for nucleosome sliding by Chd1 and ISWI. The remodeller ATPase (brown), located at SHL2, initiates nucleosome sliding on un-shifted DNA (blue). Cycles of hydrolysis translocate DNA at the SHL2 site, drawing DNA onto the entry side of the nucleosome and pushing DNA towards the exit side in ~1 bp steps (shifted DNA shown in red). While some segments of nucleosomal DNA immediately twist to convey the additional bp, other segments absorb the translocated DNA, resulting in local twist defects (+) on the nucleosome. Creation of these twist defects delays the movement of DNA to the exit side