Synergy and antagonism in regulation of recombinant human INO80 chromatin remodeling complex

Abstract

We have purified a minimal core human Ino80 complex from recombinant protein expressed in insect cells. The complex comprises one subunit each of an N-terminally truncated Ino80, actin, Arp4, Arp5, Arp8, Ies2 and Ies6, together with a single heterohexamer of the Tip49a and Tip49b proteins. This core complex has nucleosome sliding activity that is similar to that of endogenous human and yeast Ino80 complexes and is also inhibited by inositol hexaphosphate (IP6). We show that IP6 is a non-competitive inhibitor that acts by blocking the stimulatory effect of nucleosomes on the ATPase activity. The IP6 binding site is located within the C-terminal region of the Ino80 subunit. We have also prepared complexes lacking combinations of Ies2 and Arp5/Ies6 subunits that reveal regulation imposed by each of them individually and synergistically that couples ATP hydrolysis to nucleosome sliding. This coupling between Ies2 and Arp5/Ies6 can be overcome in a bypass mutation of the Arp5 subunit that is active in the absence of Ies2. These studies reveal several underlying mechanisms for regulation of ATPase activity involving a complex interplay between these protein subunits and IP6 that in turn controls nucleosome sliding.

Figure 1.

(A) Coomassie-stained SDS gel of protein complexes used in this study. Subunit identities are as marked. Western blot analysis for Ies2 is shown below and the band is situated as indicated by the arrow. (B) SEC-MALS analysis of hIno80 core complex. The expected mass of a complex containing one subunit each of actin, Arp4, Arp5, Arp8, Ies2, Ies6 and affinity tagged Ino80 together with three copies each of Tip49a and Tip49b is 743 583 Da. The molecular weight determined by SEC-MALS is 802 000 ± 10 400 Da.

Figure 2.

Mononucleosome sliding assays. (A) Gel-based assay with nucleosomes containing H2A. (B) as (A) but with nucleosomes containing H2AZ. (C) Quantification of the gels in (A) and (B). (D) FRET-based sliding assays using the same nucleosome substrates. (E) Rates of nucleosome sliding determined by each assay. (F) Inhibition of nucleosome sliding by IP₆.

Figure 3.

IP₆ inhibition mechanism. (A) ATPase activity of Ino80 over a range of IP₆ and ATP concentrations. Nucleosome concentration was kept constant at 100 nM for all of these assays. (B) V_max values from (A) plotted as % inhibition versus IP₆ concentration, with 0% inhibition equal to where [IP₆] is 0 μM. (C) Single point inhibition of Ino80 and SC2. Assays were conducted using 250 μM IP₆, 1 mM ATP and 100 nM nucleosome. (D) ATPase activity of Ino80 over a range of nucleosome and IP₆ concentrations. The ATP concentration was 1 mM for these assays. (E) MST binding data of Ino80 binding nucleosome in the presence and absence of different ligands. The ATP concentration was 1 mM and the IP₆ concentration was 250 μM. These binding experiments were conducted in the presence of 5 mM MgCl₂.

Figure 4.

FRET-based assay of nucleosome sliding activity of mutant complexes.

Figure 5.

Arp5/Ies6 couple ATPase to sliding activity. FRET-based sliding assay of complexes after supplementation with increasing levels (0, 30, 100, 300 nM) of tagged-Arp5/Ies6, at a fixed Ino80 complex concentration (100 nM). (A) Wild-type Ino80 core complex (symbols are the same in panels A–C). (B) Ino80ΔArp5Ies6, (C) Ino80ΔIes2Arp5Ies6, (D) ATPase rates for Ino80ΔArp5Ies6 complex in the same ratios as (B) with 300 nM nucleosomes (dark grey) or 600 nM nucleosomes (light grey), (E) similar data for hIno80ΔIes2Arp5Ies6, (F) coupling ratios for the complexes using the data in panels (B–E). Relative coupling = (Sliding rate/min)/(ATP/complex/min). These values were then scaled for the assay with the ratio of 0.0 Arp5+Ies6 : Ino80 core to have a relative coupling value of 1 within each complex data set.