The chromatin remodeller ACF acts as a dimeric motor to space nucleosomes

Abstract

Evenly spaced nucleosomes directly correlate with condensed chromatin and gene silencing. The ATP-dependent chromatin assembly factor (ACF) forms such structures in vitro and is required for silencing in vivo. ACF generates and maintains nucleosome spacing by constantly moving a nucleosome towards the longer flanking DNA faster than the shorter flanking DNA. How the enzyme rapidly moves back and forth between both sides of a nucleosome to accomplish bidirectional movement is unknown. Here we show that nucleosome movement depends cooperatively on two ACF molecules, indicating that ACF functions as a dimer of ATPases. Further, the nucleotide state determines whether the dimer closely engages one or both sides of the nucleosome. Three-dimensional reconstruction by single-particle electron microscopy of the ATPase-nucleosome complex in an activated ATP state reveals a dimer architecture in which the two ATPases face each other. Our results indicate a model in which the two ATPases work in a coordinated manner, taking turns to engage either side of a nucleosome, thereby allowing processive bidirectional movement. This novel dimeric motor mechanism differs from that of dimeric motors such as kinesin and dimeric helicases that processively translocate unidirectionally and reflects the unique challenges faced by motors that move nucleosomes.

Figure 1

ATP state regulates immobilization of the histone H4 tail and proximal interactions. (a) Left panels: EPR spectra of MSL labeled 0-601-60 nucleosomes. Right panels: Schematic interpretation of EPR spectra, based on data from (a), (c), & (d). Binding of Apo SNF2h to the nucleosome decreases the mobility of half the H4 tails. SNF2h binding in the presence of ADP•BeF_x decreases the mobility of both H4 tails. (b) EPR spectrum of Apo SNF2h bound to spin-labeled 60-601-60 nucleosomes reveals that only one of the two H4 tails is immobilized. (c) Hydroxyl radical foot-printing of ACF on 0-601-60 nucleosomes. Top panel: schematic of mononucleosome structure with 12 bp of flanking DNA on one side, with dyad in green, histone H4 in blue, and the region surrounding SHL (-2) and (+2) in red. Middle panel: Protection patterns for nucleosomes alone (black line, N) compared to nucleosomes bound by Apo-ACF (red line, N+ACF). Bottom panel: nucleosomes alone (black line, N) compared to nucleosomes bound by ACF in the presence of ADP·BeFx (red line, N+ACF+ ADP·BeFx). Yellow bars highlight protection in the SHL (-2) and (+2) regions. (d) Temperature dependence of probe immobilization in the Apo SNF2h-nucleosome complex. Slope of the straight line = 2.1×10^-4 ± 8×10^-4 immobilized fraction/°C. Error represents s.e.m.

Figure 2

SNF2h and ACF function as dimers of ATPases. (a) Schematic of nucleosome structure with dye attachment sites for (b) and (d). The DNA is end-labeled with Cy3 (blue) on the shorter flanking DNA. The octamer is labeled with Cy5 at H2A-120C (yellow). (b) Cy3 fluorescence intensity of the construct shown in (a) as a function of SNF2h concentration, using nucleosomes with 78bp of flanking DNA on one side. A representative replicate curve is shown. Data are fit to the general equation for cooperative binding (see Methods). Hill Coefficient (n) = 1.8 ±0.17; K_1/2 = 353±30nM. (c) Schematic of FRET-based nucleosome remodeling assay. Rate constant of remodeling is measured by following the decrease in FRET between Cy3 and Cy5 in the presence of ATP. (d) Left panel: Nucleosome remodeling rate constant as a function of SNF2h concentration for nucleosomes with 78bp of flanking DNA. Right panel: Nucleosome remodeling rate constant as a function of ACF concentration for nucleosomes with 20 bp of flanking DNA. Hill Coefficient (n) = 1.8±0.1; K’_1/2 = 281±32 nM for SNF2h and Hill Coefficient (n) = 1.9±.3; K’_1/2 = 26±3 nM for ACF. Each panel represents global fits to data obtained from three independent experiments. (e) SNF2h binds as a cooperative dimer to the nucleosome in the absence of nucleotide (black circles), and in the presence of ADP (blue squares). In the presence of ADP•BeF_x, SNF2h binds non-cooperatively (red triangles). These binding measurements were carried out with nucleosomes containing 40bp of flanking DNA on one side and a Cy3 label on the short DNA end. Binding of SNF2h to these nucleosomes is ~2-fold weaker relative to the nucleosomes used in (b),. A representative replicate curve with each nucleotide analogue is shown (left panel), and the average K_1/2 and Hill Coefficient from three replicates is shown (table). Errors represent s.e.m.

Figure 3

Visualization of SNF2h bound to the nucleosome in the presence of ADP•BeF_x using EM. (a) three different views of the 3-D reconstruction of dimeric SNF2h bound to the nucleosome (left panels) and corresponding representative 2-D class averages (right panels). The crystal structure of the core mononucleosome was placed manually into the 3D reconstruction. Histone H4 is highlighted in red. The isosurface of the 3-D reconstruction at high threshold is shown in blue, and low threshold in grey. (b) Left panel: representative 2-D class average of negative stain EM images of unbound nucleosomes. Right panel: representative 2-D class average of one SNF2h bound to a nucleosomes. (c) Representative 2-D class averages of SNF2h alone. Numbers used to calculate a particular class average shown in lower left corner.

Figure 4

Simple model for how a dimeric ACF moves nucleosomes. H4 tail is in red and the two ATPases in ACF are shown in blue and purple. Only one subunit binds ATP at a time. In the ATP state, each ATPase subunit takes turns in binding the flanking DNA. The ATPase that binds the longer flanking DNA (purple) hydrolyzes ATP faster and starts translocating DNA across the nucleosome. During and post hydrolysis, the second ATPase (blue) also engages the nucleosome, preventing loss of the DNA loop-containing intermediate (mimicked by ADP•BeF_x). In the ADP state, the non-translocating monomer disengages and the translocating monomer remains engaged with the nucleosome. ATP state may also regulate the extent of any direct contacts between the two ATPase subunits and such contacts may be substantially fewer in ADP-BeFx state than in other ATP states.