Conformational switching of Arp5 subunit differentially regulates INO80 chromatin remodeling

Abstract

The INO80 chromatin remodeler is a versatile enzyme capable of several functions, including spacing nucleosomes equal distances apart, precise positioning of nucleosomes based on DNA shape/sequence and exchanging histone dimers. Within INO80, the Arp5 subunit plays a central role in INO80 remodeling, evidenced by its interactions with the histone octamer, nucleosomal and extranucleosomal DNA, and its necessity in linking INO80's ATPase activity to nucleosome movement. Our investigation reveals that the grappler domain of Arp5 interacts with the acidic pocket of nucleosomes through two distinct mechanisms: an arginine anchor or a hydrophobic/acidic patch. These two modes of binding serve distinct functions within INO80 as shown in vivo by mutations in these regions resulting in varying phenotypes and in vitro by diverse effects on nucleosome mobilization. Our findings suggest that the hydrophobic/acidic patch of Arp5 is likely important for dimer exchange by INO80, while the arginine anchor is crucial for mobilizing nucleosomes.

Keywords: ARP5; DNA repair; DNA replication stress; INO80; INO80 structure; chromatin/nucleosome remodeling; nucleosome dynamics.

Figure 1.. Two regions of Arp5’s grappler domain associate with the acid pocket of nucleosomes.

(A). The domain organization is shown for the Arp5 subunit of INO80 with the actin-like portion highlighted in cyan and the grappler domain in purple. The DNA binding domain of Arp5 is indicate (dark blue. The Arp5 regions crosslinked to residue 80 of histone H3 and residue 89 of H2A are labeled. (B) The interactions of Arp5 with the histone octamer face of nucleosomes are probed by site-specific histone crosslinking. The relative efficiency of crosslinking Arp5, Ies2 and Ies6 is shown for three replicates. Error bars represent the mean ± SD.(C) The regions of Arp5 crosslinked to histones are mapped by immobilizing the C-terminus of Arp5 and partial digesting with Arg-C. Labeled proteolytic fragments of Arp5 are separated on a 4–20% SDS-PAGE and visualized by phosphorimaging. (D) The two regions are individually deleted, and the complex purified using by FLAG affinity chromatography. (E) Nucleosome mobilizing activity of the INO80 complex with Arp5 ^Δ516–564 (lanes 1–8) is compared to wild type INO80 (lanes 9–16) using EMSA. (24nM) of nucleosomes were saturated with 25nM of INO80 complex (WT or mutant) with addition of 80 μM ATP. (F) The phenotype of the yeast strain with Arp5 ^Δ516–564 was compared to wild type Arp5 (WT, Arp5 WT), N-terminal truncation of Arp8 (Arp8 ΔN) and the absence of Arp5 (ΔArp5).

Figure 2.. The arginine anchor is not required in vivo for Arp5’s role in DNA replication, DNA damage repair and transcription elongation.

(A) Various arginines mutated to alanines in Arp5 are tested in spot assays to determine their impact on the in vivo activity of Arp5. The region spanning amino acids 516–564 has several regions where the sequence is conserved and are probed by mutating to alanine. (B) An Anchor Away (AA) system is used to acutely test cell viability of various mutant Arp5 under different conditions where wild type Arp5 is required for cell proliferation. (C-D) The effect of different mutations in the grappler domain are tested using the AA system with (RAPA) and without (DMSO) added. The abbreviations are as follows: HU-hydroxy urea, 6AU – 6 amino uracil and MMS – methyl methanesulfonate.

Figure 3.. Arginines 482, 488 and 496 of Arp5 are important for Arp5 binding to the acid pocket and for INO80 mobilizing nucleosomes

(A) The graph shows the extent of nucleosomes mobilized by INO80 with wild type (WT) and mutant Arp5 (R3) that has arginine 482, 488 and 496 mutated to alanine using EMSA (see Figure S3B). 50nM of nucleosomes were incubated with 75nM of INO80 complex with addition of 80 μM ATP (B) The ATPase activity of wild type and mutant INO80 was measured using thin layer chromatography under similar condition to that in (A). (C) The interactions of Arp5 with the histone octamer face of nucleosomes are probed for WT and R3 mutant by site-specific histone crosslinking. The relative efficiency of crosslinking Arp5, Ies2 and Ies6 is shown for three replicates. (D-E) The extent of DNA movement on the (D) exit and (E) entry side is plotted versus time using a photoreactive probe attached to residue 53 of histone H2B for wild type and R3 mutant Arp5 containing INO80 complex. (E) The net loss of DNA crosslinking on the entry side is plotted versus time. Three replicates are performed for each experiment and error bars represent the mean ± SD.

Figure.4. Mutation of Leu 567, Leu 567 and Asp5 to alanine in the grappler domain negatively impacts both the in vivo and in vitro activity of Arp 5.

(A) The sequence homology for Arp5 spanning the region from amino acid 565 to 620 of Sc Arp5 with Arp5 from other organisms is shown. Saccharomyces cerevisiae (CAA95933.1), Schizosaccharomyces pombe (CAB44762.1), Thermochaetoides thermophila (XP_006693704.1), Drosophila Melanogaster (NP_650684.1), mouse (NP_780628.3) and human (NP_079131.3). (B-C) INO80 with wild type Arp5 is compared to mut LLD with Leu 567, Leu 568 and Asp 571 mutated to Ala, mut DBD with Arg 71, Arg 73, Lys 77, Arg 93 and Arg 97 changed to Ala (DBD) to block it from binding DNA at SHL-2/-3 and mut R3 as described in Figure 3. Two sets conserved arginine residues of (1) 596 and 599 and (2) 618 and Arg 620 are mutated to Ala and screened using spot assays. In (C) The phenotype of LLD and DBD combined are examined. (D) The nucleosome remodeling activity of wild type (WT) and mutant (mut)-LLD or (mut)-DBD INO80 are measured using EMSA and the extent of mobilized nucleosome plotted versus time (see Figure S4C). 50nM of nucleosomes were incubated with 75nM of INO80 complex with addition of 80 μM ATP. (E) The ATPase activity of wild type and mutant INO80 was measured under similar condition to (D). Three replicates are performed for each experiment and error bars represent the mean ± SD.

Figure 5.. The LLD and arginine anchor (R3) are spatially separated in the grappler domain of Arp5

(A) Schematic shows the alpha helices and loops that compromise the grappler domain of Arp5 based on the closed conformation. Names for different helices are shown along with the positions of the arginine anchor (R3) and LLD region as well as the region crosslinked to H2A 89 and H3 80. (B) The structure of the crossed conformation of the grappler domain of Arp5 shown and the color coding corresponds to that in (A). The location of residue 80 of H3 and 89 of H2a are highlighted. (C) The location of the arginine anchor, DNA binding domain (DBD) and the LLD region are indicated. (D) The same orientation of the grappler domain in (C) is shown with the region crosslinked to residue 80 of H3 highlighted in green and the rest of the grappler domain in purple and the actin-like portion of Arp5 in cyan.

Figure.6. The LLD part of the Arp5 grappler domain is required for Arp5 binding to the acidic pocket of nucleosomes and can independently bind histones.

(A-B) Binding of Arp5 near the acid pocket (H2A N89C) and another lateral position on the histone octamer (H3 T80C) is probed by site-specific histone crosslinking for wild type (WT) and mutant LLD and DBD (mut-LLD and mut-DBD). (C) Pulldown assays are done with histone octamer (5μg) and one of three biotinylated peptides; LANA and two others encompassing the region of the Arg anchor and the LLD patch as shown. As a control for the specificity of the pulldown the histone octamer is preincubated with anti-H2A antibody (+). (D) Wild type or mutant LLD peptide was added to INO80 remodeling reactions at the indicated concentrations with or without ATP added and incubated for 30 min. 50nM of nucleosomes were incubated with 75nM of INO80 complex with addition of 80 μM ATP. The remodeling reactions were stopped, INO80 stripped off and analyzed by EMSA as shown. Three replicates are performed for each experiment and error bars represent the mean ± SD.

Figure 7.. DNA displacement on the entry side is reduced when the LLD patch of Arp5 is mutated.

(A-D) The extent of DNA moves (A-B) 32 bp on the exit and (D-C) 20 bp on the entry side is plotted versus time using a photoreactive probe attached to residue 53 of histone H2B for wild type (open triangle), mutant (mut)-DBD (closed square) and mutant (mut)-LLD (open circle) Arp5 complexes. The same symbols are also used in (E-F). In (C-D) the reactions are monitored for longer times. (E) The rate at which the initial DNA position disappears is shown for wild type and mutant Arp5 as in (A-D). (F) The net loss of DNA crosslinking on the entry side is plotted versus time similar to (A-D). Three replicates are performed for each condition and error bars represent the mean ± SD.