ISWI remodelers slide nucleosomes with coordinated multi-base-pair entry steps and single-base-pair exit steps

Abstract

ISWI-family enzymes remodel chromatin by sliding nucleosomes along DNA, but the nucleosome translocation mechanism remains unclear. Here we use single-molecule FRET to probe nucleosome translocation by ISWI-family remodelers. Distinct ISWI-family members translocate nucleosomes with a similar stepping pattern maintained by the catalytic subunit of the enzyme. Nucleosome remodeling begins with a 7 bp step of DNA translocation followed by 3 bp subsequent steps toward the exit side of nucleosomes. These multi-bp, compound steps are comprised of 1 bp substeps. DNA movement on the entry side of the nucleosome occurs only after 7 bp of exit-side translocation, and each entry-side step draws in a 3 bp equivalent of DNA that allows three additional base pairs to be moved to the exit side. Our results suggest a remodeling mechanism with well-defined coordination at different nucleosomal sites featuring DNA translocation toward the exit side in 1 bp steps preceding multi-bp steps of DNA movement on the entry side.

Figure 1. Probing DNA translocation on the exit side of the nucleosome by single-molecule FRET

(A) Schematic of FRET detection for DNA translocation on the exit side of the nucleosome. The nucleosomes are labeled with the FRET donor, Cy3 (green star), and acceptor, Cy5 (red star). The histone octamer and DNA are depicted as a yellow cylinder and a blue line, respectively. (B) Representative Cy3 (green) and Cy5 (red) fluorescence and FRET (blue) time traces showing translocation of a single nucleosome after addition of the enzyme and ATP at time zero. See also Table S1 and Figure S1.

Figure 2. Identical step sizes of DNA translocation on the exit side of the nucleosome induced by different ISWI-family enzymes

(A) Remodeling of nucleosomes with initial exit linker DNA length of n = 3 bp or n = −3 bp by ISW2. Left: FRET time trace showing ISW2-induced translocation of a single n = 3 bp nucleosome. 6.2 nM ISW2 and 2 μM ATP were added at time zero. Middle: Histogram of the FRET values at translocation pauses constructed from n = 3 bp nucleosomes. Right: FRET histograms of the translocation pauses constructed from n = −3 bp nucleosomes. (B) Remodeling of the n = 3 bp and n = −3 bp nucleosomes by ISW1b. FRET histograms of the pauses for n = 3 bp (left) and n = −3 bp (right) nucleosomes in the presence of 8.8 nM ISW1b and 10-150 μM ATP. (C) Remodeling of the n = 3 bp and n = −3 bp nucleosomes by Isw2p. FRET distribution of the pauses for the n = 3 bp (left) and n = −3 bp (right) nucleosomes in the presence of 69 nM Isw2p and 1 mM ATP. The nucleosome schemes display the footprint of the histone octamer (yellow oval) on the DNA (blue line). Numbers above double-headed arrows shown in the histograms represent mean step sizes. See also Figure S2.

Figure 3. The multi-bp translocation steps on the exit side of the nucleosome are comprised of 1-bp elementary steps

(A) FRET time trace of a nucleosome indicating the dwell times of the first two translocation phases (t₁ and t₂). (B) Histogram of t₁ and t₂ values constructed from many nucleosomes. Fits to the Γ-distribution At^N−1exp(−kt) (black lines) yield N = 6.5 ± 0.4 and k = 0.52 ± 0.04 s⁻¹ for the t₁ phase (left), and N = 3.4 ± 0.4 and k = 0.67 ± 0.11 s⁻¹ for the t₂ phase (right). (C) FRET time trace, before (grey) and after (blue) 5-point averaging, of a single n = 3 bp nucleosome in the presence of 6.2 nM ISW2, 2 μM ATP and 2 mM ATP-γ-S. A HMM fit is shown by the red line. The horizontal orange dotted lines indicate 1-bp intervals (derived from the calibration in Figure S1D). (D) Histograms of FRET plateaus from many nucleosomes determined using the HMM analysis. Numbers above double-headed arrows represent mean step sizes. See also Figure S3.

Figure 4. Dependence of the stepping kinetics on the concentrations of ATP and ATP-γ-S

(A) The pause duration after the first, 7-bp compound step (t_p,7) at various ATP-γ-S concentrations and 2 μM ATP. (B) The pause duration between each 1-bp elementary step (t_p) at various ATP-γ-S concentrations and 2 μM ATP. All pauses except for the ones after 6, 7, and 8 bp of translocation were pooled to determine t_p. As shown in Figure S4, in addition to the 7^th pause, the 6^th and 8^th pauses also appear longer than the remaining ones, likely due to errors in pause identification. The value of t_p at 0 μM ATP-γ-S was derived from 1 / k value obtained from the Γ-distribution in Figure 3B. (C) Dependence of t_p on the ATP concentration at 2 mM ATP-γ-S. At this saturating concentration of ATP-γ-S, all pauses, including the 7^th one, had approximately equal durations and were pooled to determine t_p. All data are shown as the mean ± SEM (N = 15 - 100 events). See also Figure S4.

Figure 5. DNA movement on the entry side of the nucleosome precedes that on the exit side

(A) Schematic of the nucleosome construct used for measuring DNA movement at the entry side. (B) Donor signal (green), acceptor signal (red), and FRET (blue) time traces showing ISW2-induced remodeling after adding 12 nM ISW2 and 2 μM ATP at time zero. The dashed line indicates the onset of FRET change after an initial wait time, t_wait. (C) Comparison of t_wait on the entry (yellow bars) and exit sides (purple bars) of the nucleosome under identical enzyme and ATP concentrations. Data are shown as the mean ± SEM (N = 80 - 220 events). See also Figure S5.

Figure 6. Entry-side DNA movement occurs after 7 bp of DNA translocation towards the exit side and proceeds in 3-bp steps

(A) Schematic of the nucleosome constructs used to monitor DNA movement at the entry side when exit-side translocation is restricted by a 2-nt ssDNA gap. The gap is located m bp away from the SHL2 site (shown as a purple line) such that m bp of DNA can be translocated to the exit side. (B) FRET time traces of single m = 0, 7, and 8 bp nucleosomes (blue, green, and orange line, respectively) after addition of 12 nM ISW2 and 2 μM ATP at time zero. (C) FRET values before (red bar) and after (blue bars) remodeling by ISW2 as a function of the distance m to the SHL2 site. Because the DNA path on the entry side may involve bending and/or twisting due to the direct interaction with the remodeling enzyme, we do not expect a similar linear dependence of FRET on the linker DNA length as on the exit-side where the linker DNA is largely free of enzyme-induced distortion. Data are shown as the mean ± SEM (N = 80 - 150 nucleosomes). See also Figure S6.

Figure 7. A model for nucleosome translocation by ISWI-family remodelers

DNA, histone octamer and remodeler are shown in black/grey/red, yellow and blue/green, respectively. The upper and lower DNA gyres are depicted as a solid black and dashed grey line, respectively. Each base pair of DNA translocated to the exit side is shown by a red dot. A cartoon representation of the remodeler is shown as a semi-transparent light blue or light green shape, and the locations of the ATPase and linker-DNA-binding domains as blue and green spheres, respectively. ISWI-induced remodeling starts with the ATPase domain translocating DNA from the SHL2 site towards the exit side, 1 bp at a time. The translocation by the ATPase domain generates strain on the entry-side DNA (depicted by magenta/purple coloring the DNA), which initially remains immobile. After 7 bp of DNA translocation, the accumulated strain is sufficiently strong to trigger an entry-side action, possibly a conformational change of the enzyme, which pushes a 3-bp equivalent of DNA into the nucleosome. This action partially relaxes the strain and allows three additional base pairs of DNA to be translocated to the exit side. This cycle then repeats to allow processive nucleosome translocation.