Multiple modes of regulation of the human Ino80 SNF2 ATPase by subunits of the INO80 chromatin-remodeling complex

Abstract

SNF2 family ATPases are ATP-dependent motors that often function in multisubunit complexes to regulate chromatin structure. Although the central role of SNF2 ATPases in chromatin biology is well established, mechanisms by which their catalytic activities are regulated by additional subunits of chromatin-remodeling complexes are less well understood. Here we present evidence that the human Inositol auxotrophy 80 (Ino80) SNF2 ATPase is subject to regulation at multiple levels in the INO80 chromatin-remodeling complex. The zinc finger histidine triad domain-containing protein Ies2 (Ino Eighty Subunit 2) functions as a potent activator of the intrinsic catalytic activity of the Ino80 ATPase, whereas the YL-1 family Ies6 (Ino Eighty Subunit 6) and actin-related Arp5 proteins function together to promote binding of the Ino80 ATPase to nucleosomes. These findings support the idea that both substrate recognition and the intrinsic catalytic activities of SNF2 ATPases have evolved as important sites for their regulation.

Keywords: INO80 complex; enzyme activity.

Fig. 1.

Ino80 mutants used in this study. (A) Diagram showing modular assembly of the complete INO80 complex. (B) Ino80ΔN and derivatives. In Ino80ΔN BRGins and Ino80ΔN SRCAPins, the Ino80 insertion region, residues 824–1099 of NP_060023.1 (purple), was replaced by the Brg1 insertion, residues 1054–1078 of NP_001122316 (green) or the SRCAP insertion, residues 909–2071 of NP_006653 (yellow), respectively. (C) Insertion region deletion (DEL) constructs lack the indicated residues. Ins, insertion region; SNF2_N, SNF2 family amino-terminal.

Fig. 2.

Requirement for the Ino80 insertion region. Purified chimeric INO80 complexes (purified through FLAG-tagged Ino80ΔN, Ino80ΔN BRGins (F- Ino80ΔN BRGins), or Ino80ΔN SRCAPins (F- Ino80ΔN SRCAPins) were subjected to SDS/PAGE and silver (Ag) staining (A) or Western blotting using the indicated antibodies (B). In B, complete INO80 complexes purified through FLAG-tagged INO80E (F-INO80E) are shown in lane 1 for comparison. All lanes shown in B are derived from a single Western-blotting experiment. (C) ATP-dependent nucleosome-remodeling activities of complexes purified through the indicated FLAG-tagged Ino80 derivative. Reactions shown in lanes 2–5 and 6–9 contained ∼0.1, 0.2, 0.4, and 0.6 pmol complex, respectively, and those in lanes 10–11 contained ∼0.3 pmol. (D) Nucleosome binding was assayed by EMSA. Reactions contained 0.7, 1.1, and 1.7 pmol of the indicated complexes. Free nuc, free nucleosomes.

Fig. 3.

Isolation of INO80 subcomplexes lacking either Ies2 or Arp5 and Ies6. (A) Complete INO80 complexes (lane 1) or INO80 subcomplexes (lanes 2–5) were purified through the indicated FLAG-tagged protein and subjected to Western blotting. The data shown in this figure are derived from the same Western blots as those shown in Fig. 2B; the control lanes 1 and 2 are repeated from Fig. 2B to allow comparison with complexes carrying intact Ino80 insertion regions. (B) Subunit composition of FLAG-Ino80ΔN-associated complexes isolated by anti-FLAG immunopurification from cells treated with nontargeting (NT) siRNA (lanes 1–5) or siRNAs targeting Ies6 (lanes 6–8), Ies2 (lanes 9–11), or Arp5 (lanes 12–14). Varying amounts of each complex were analyzed by Western blotting to evaluate the efficiency of siRNA-mediated subunit depletion.

Fig. 4.

Ies2 regulates Ino80 catalytic activity. (A) Approximately 0.2 pmol of INO80 complex or subcomplexes were assayed as described in ref. for their abilities to hydrolyze ATP with (+Nuc) or without (−Nuc) a saturating concentration (∼200 nM) of HeLa cell nucleosomes. Aliquots of each reaction were removed at 15, 30, and 50 min for measurement of ATP hydrolysis. The graph depicts rates of ATP hydrolysis that were averaged from measurements made at each time point. (B) Approximately 0.2 or 0.4 pmol of the indicated complexes was assayed for nucleosome remodeling. (C) Nucleosome-remodeling assays contained ∼0.2 pmol of the indicated complexes. Reactions were supplemented with recombinant Ies2, expressed in and purified from baculovirus (bv)-infected insect cells; 1x Ies2 is ∼0.1 pmol. Percent remodeled nucleosomes is equivalent to the amount of radioactivity in the upper band, corresponding to the centrally positioned remodeled nucleosome, divided by the total amount of radioactivity in the remodeled nucleosome and the prominent lower band, which corresponds to the laterally positioned, starting nucleosome.

Fig. 5.

Differential roles of Ies2 and of Ies6/Arp5 in INO80 chromatin remodeling. (A). Approximately 0.5 pmol of INO80ΔN or INO80ΔN EQ complexes purified from cells treated with NT siRNA or siRNAs targeting Ies2, Arp5, or Ies6 were assayed for their abilities to hydrolyze ATP in the presence of ∼200 nM nucleosomes or ∼30 nM plasmid DNA. Values are based on measurements from at least three independent reactions; error bars represent SD. (B) Nucleosome-remodeling assays contained ∼0.05, 0.1, 0.2, and 0.4 pmol of INO80ΔN complexes purified from cells treated with the indicated siRNAs. (C) Nucleosome-remodeling assays performed with or without ∼0.3 pmol INO80ΔN complexes from cells treated with Ies2 siRNA. Reactions were supplemented with ∼0.05, 0.1, or 0.2 pmol of Ies2 purified from E. coli (ec Ies2) or baculovirus-infected insect cells (bv Ies2). (D) Nucleosome-remodeling assays performed with or without 0.2 pmol INO80ΔN complexes from cells treated with Ies6 siRNA. Where indicated, reactions were supplemented with ∼2 pmol of Ies6 and Arp5, expressed alone (bv Ies6 and bv Arp5) or together (bv Ies6+Arp5) in baculovirus-infected insect cells.

Fig. 6.

Arp5 and Ies6, but not Ies2, contribute to nucleosome recognition. (A) INO80ΔN complexes purified from cells that do (ΔN:shIes6) or do not (ΔN) stably express Ies6 shRNA were analyzed by Western blotting with the indicated antibodies. (B) Depletion of Arp5 and Ies6 leads to a decrease in INOΔN nucleosome-binding activity measured by EMSA. Nucleosome-binding reactions contained ∼1, 1.5, and 2 pmol of the indicated complexes. (C) INO80ΔN complexes (ΔN InsΔ3) lacking Ies2 bind nucleosomes. Reactions contained 0.4, 0.7, 1.1, and 1.7 pmol of the indicated complexes.