Lehmann LC et al. (2020), Mechanistic Insights into Regulation of the ALC...

XB-ART-58766

Cell Rep 2020 Dec 22;3312:108529. doi: 10.1016/j.celrep.2020.108529.

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Mechanistic Insights into Regulation of the ALC1 Remodeler by the Nucleosome Acidic Patch.

Lehmann LC , Bacic L , Hewitt G , Brackmann K , Sabantsev A , Gaullier G , Pytharopoulou S , Degliesposti G , Okkenhaug H , Tan S , Costa A , Skehel JM , Boulton SJ , Deindl S .

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Upon DNA damage, the ALC1/CHD1L nucleosome remodeling enzyme (remodeler) is activated by binding to poly(ADP-ribose). How activated ALC1 recognizes the nucleosome, as well as how this recognition is coupled to remodeling, is unknown. Here, we show that remodeling by ALC1 requires a wild-type acidic patch on the entry side of the nucleosome. The cryo-electron microscopy structure of a nucleosome-ALC1 linker complex reveals a regulatory linker segment that binds to the acidic patch. Mutations within this interface alter the dynamics of ALC1 recruitment to DNA damage and impede the ATPase and remodeling activities of ALC1. Full activation requires acidic patch-linker segment interactions that tether the remodeler to the nucleosome and couple ATP hydrolysis to nucleosome mobilization. Upon DNA damage, such a requirement may be used to modulate ALC1 activity via changes in the nucleosome acidic patches.

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Species referenced: Xenopus laevis
GO keywords: chromatin remodeling [+]

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	Graphical Abstract
	Figure 1. The AP Is Important for Remodeling by ALC1 (A) Left: octamer surface colored by electrostatic potential (from −5 kB T/e, indicated in red, to +5 kB T/e, indicated in blue). Right: key residues of the H2A AP. Based on PDB: 1AOI. (B) Left: schematic of FRET-labeled wild-type (WT/WT) nucleosomes and APM/APM nucleosomes with AP mutations on both faces of the octamer. Middle: ensemble remodeling of WT/WT and APM/APM nucleosomes (10 nM) in the presence of 20 μM ALC1fl R860W. Right: relative remodeling rates. Error bars represent SD (n = 3 independent experiments). (C) Ensemble remodeling of WT/WT nucleosomes (10 nM) by 80 nM ALC1fl R860W with or without LANA peptide. (D) smFRET labeling and detection. (E) ALC1fl R860W-catalyzed remodeling of individual WT/WT nucleosomes toward the longer (left) or shorter (right) linker DNA. (F) Percentages of traces with the initial remodeling direction toward longer or shorter linker DNA for WT/WT and WT/APM nucleosomes. Error bars indicate SEM (n > 100 traces).
	Figure 2. The ALC1 Linker Region Binds to the AP (A) ALC1fl-histone cross-links. Magenta lines indicate linker-histone cross-links: gray lines indicate other cross-links. (B) Sequence alignment of LANA (1–22) and ALC1 linker (601–635). (C) Fluorescence anisotropy of TAMRA-labeled ALC1RLS in the presence of various amounts of WT/WT or APM/APM nucleosomes. Error bars for WT/WT nucleosome curve represent SEM (n = 3 independent experiments). APM/APM nucleosomes: one representative curve out of two independent experiments is indicated. Error bars represent SD (n = 10 technical replicates). (D) Competition between TAMRA-labeled LANA peptide pre-bound to WT/WT nucleosomes and unlabeled ALC1RLS. See also Figure S2A. Error bars represent SEM (n = 3 independent experiments). (E) Cryo-EM map. (F) Left: cartoon representation. Top right inset: cryo-EM density around the ALC1 linker (cyan). Bottom right inset: hydrogen-bonding interactions. (G) Superposition of PDB: 1ZLA onto the linker-nucleosome complex.
	Figure 3. The Linker-AP Interaction Is Important for ALC1 Activity (A) ATPase activity for 20 μM ALC1fl R860W or ALC1fl R611A/S612A/R860W in the presence of 2 μM WT/WT nucleosomes. Error bars represent SD (n = 3 independent experiments). (B) Michaelis-Menten curves of remodeling rates for WT/WT nucleosomes (10 nM) and various ALC1fl R860W or R611A/S612A/R860W concentrations. Inset: enlarged view for ALC1fl R611A/S612A/R860W. (C) Left: remodeling of 10 nM nucleosomes by 20 μM remodeler. Right: relative remodeling rates. Error bars represent SD (n = 3 independent experiments). Data for ALC1fl R860W and WT/WT nucleosomes as well as for ALC1fl R860W and APM/APM nucleosomes: same as indicated in Figure 1B. (D) Left: remodeling of WT/WT nucleosomes (10 nM) in the presence of 20 μM ALC1fl R860W or ALC1fl R611Q. Right: relative rates for ALC1fl R860W or ALC1fl R611Q. Error bars represent SD (n = 3 independent experiments). Data for ALC1fl R860W and WT/WT nucleosomes: same as indicated in Figure 1B. (E) Representative smFRET traces indicating the remodeling of individual WT/WT nucleosomes with 3 bp of linker DNA (left) by ALC1fl R860W (middle) or ALC1fl R611A/S612A/R860W (right). Only pauses immediately following translocation phases with a FRET decrease were analyzed (shaded yellow). (F) Mean pause durations for ALC1fl R860W or ALC1fl R611A/S612A/R860W as described in (E). Error bars represent SEM (n > 170 events).
	Figure 4. ALC1RLS Mutations Alter Recruitment Dynamics (A) U2OS cells expressing R860W, R611A/S612A/R860W, or R611Q/R860W YFP-ALC1, imaged upon laser damage. Scale bar, 10 μm. (B) Kinetics of R860W, R611A/S612A/R860W, and R611Q/R860W YFP-ALC1 association with DNA breaks. Error bars represent SEM (n ≥ 131 traces from 3 independent experiments). See also Figure S4. (C) Fractional recruitment of R860W, R611A/S612A/R860W, and R611Q/R860W YFP-ALC1 to DNA breaks. Error bars represent SEM (n ≥ 131 traces from 3 independent experiments). (D) Half times from (C). Data are means ± 95% confidence intervals. (E) In the absence of DNA damage, association of the macro domain of ALC1 with its C-terminal ATPase lobe stabilizes an ATPase “OFF” state. PAR binding to the macro domain displaces it from the ATPase. ALC1RLS then tethers the remodeler to the AP, which stabilizes an active conformation (macro domain not shown) and promotes efficient coupling of ATP hydrolysis to nucleosome mobilization.
	Figure S1. Related to Figure 1. The acidic patch is important for ALC1 remodeling. (A) Ensemble remodeling time courses of initially end-positioned WT/WT nucleosomes (10 nM) alone (grey) or with 80 nM (left) or 5µM (right) ALC1fl with (dark green) or without (light green) 80 nM PARP1 and 50 µM NAD+ . Representative curves from N = 3 independent experiments are shown. (B) Ensemble remodeling time courses of initially end-positioned WT/WT nucleosomes (10 nM) alone (grey) or with 80 nM (left) or 5µM (right) ALC1fl R860W with (dark orange) or without (light orange) 80 nM PARP1 and 50 µM NAD+ . Representative curves from N = 3 independent experiments are shown. (C) Ensemble remodeling time courses of initially end-positioned WT/WT nucleosomes (10 nM) by 80 nM ALC1fl, activated with 80 nM PARP1 and 50 µM NAD+ , in the presence (black) and absence (dark green) of a peptide derived from the N-terminal portion of the latency associated nuclear antigen (LANA) protein of the Kaposi’s sarcoma-associated herpesvirus (KSHV). Representative curves from N = 3 independent experiments are shown. (D) Single-molecule FRET histograms for WT/WT nucleosomes before (blue) and after (red) the addition of ALC1fl R860W alone (top left), with ATPγS (top right), or with ATP (bottom). (E) Representative donor (green) and acceptor (red) intensity (top), as well as corresponding smFRET time traces (blue, bottom) showing the ALC1fl R860Wcatalyzed remodeling of individual WT/WT (left) or WT/APM (right) nucleosomes.
	Figure S2. Related to Figure 2. LANA binding to nucleosomes and flowchart for cryo-EM data collection. (A) Fluorescence anisotropy binding measurements with TMR-labeled LANA peptide and varying amounts of WT/WT nucleosomes. The dissociation constant (Kd) is 9 nM. Error bars: S.E.M. (N = 3 independent experiments). (B) Suitable particles for cryo-EM structure determination were identified by one round of 2D classification followed by three rounds of 3D classification. (C) 3D classification identified three classes corresponding to (1) a high-resolution nucleosome, (2) a lower resolution nucleosome, and (3) a hexasome. The high-resolution nucleosome and hexasome classes were further subjected to three more rounds of 3D classification, yielding subsets of homogeneous particles suitable for refinement. Refinement protocol: 3D autorefinement, three CTF refinements (beam tilt, anisotropic magnification, per-particle defocus and permicrograph astigmatism), one 3D auto-refinement, Bayesian polishing, and a final 3D auto-refinement.
	Figure S3. Related to Figure 3. Mutations in the regulatory linker segment of ALC1 affect the pause phase duration during nucleosome remodeling. (A) ATPase rates for 1 µM ALC1fl R860W (orange) or ALC1fl R611A/S612A/R860W (light blue) in the presence of 20 µM or 50 µM dsDNA and 1 mM ATP. For comparison, the ATPase rates for 20 µM ALC1fl R860W or ALC1fl R611A/S612A/R860W in the presence of 2 µM WT/WT nucleosomes and 1 mM ATP are shown (same as shown in Figure 3A). Error bars represent standard deviation (N = 3 independent experiments). (B) Representative single-molecule FRET time traces showing the remodeling of individual WT/WT nucleosomes with 3 and 78 bp of flanking linker DNA by ALC1fl R860W (left) and by ALC1fl R611A/S612A/R860W (right). Pause phases are highlighted in yellow. (C) Histograms of pause phase durations derived from single-molecule FRET time traces for ALC1fl R860W (left) and ALC1fl R611A/S612A/R860W (right) (N = 421 or 171 plateaus from 166 or 220 time traces for ALC1fl R860W or ALC1fl R611A/S612A/R860W, respectively). (D) Single-molecule FRET histograms for WT/WT nucleosomes before (blue) and after (red) the addition of ALC1fl R860W and ATP.
	Figure S4. Related to Figure 4. Kinetics of YFP-ALC1 and YFP-ALC1 R860W association with DNA breaks. (A) CRISPR-mediated inactivation of ALC1 in U2OS cells. Immunoblot of whole cell extracts from ALC1+/+ and ALC1-/- cells, probed for ALC1. Heterogeneous Nuclear Ribonucleoprotein A1 (hnRNPA1) was probed for as a loading control. (B) Representative images of U2OS cells expressing WT or R860W YFP-ALC1, recorded at the specified time points upon laser damage. Scale bar: 10 µm. (C) Kinetics of WT (green) and R860W (orange) YFP-ALC1 association with DNA breaks. Fluorescence intensities were normalized and corrected for photobleaching (see Methods section). Error bars represent S.E.M. (N ≥ 131 traces from 3 independent experiments). (D) Fraction of maximal recruitment of WT (green) and R860W (orange) YFP-ALC1 to DNA breaks. Error bars represent S.E.M. (N ≥ 131 traces from 3 independent experiments). Solid line represents onephase association. (E) Half-time quantified from (D). Data are mean ± 95% confidence intervals.

References [+] :

Ahel, Poly(ADP-ribose)-dependent regulation of DNA repair by the chromatin remodeling enzyme ALC1. 2009, Pubmed