Subunit organization of the human INO80 chromatin remodeling complex: an evolutionarily conserved core complex catalyzes ATP-dependent nucleosome remodeling

Abstract

We previously identified and purified a human ATP-dependent chromatin remodeling complex with similarity to the Saccharomyces cerevisiae INO80 complex (Jin, J., Cai, Y., Yao, T., Gottschalk, A. J., Florens, L., Swanson, S. K., Gutierrez, J. L., Coleman, M. K., Workman, J. L., Mushegian, A., Washburn, M. P., Conaway, R. C., and Conaway, J. W. (2005) J. Biol. Chem. 280, 41207-41212) and demonstrated that it is composed of (i) a Snf2 family ATPase (hIno80) related in sequence to the S. cerevisiae Ino80 ATPase; (ii) seven additional evolutionarily conserved subunits orthologous to yeast INO80 complex subunits; and (iii) six apparently metazoan-specific subunits. In this report, we present evidence that the human INO80 complex is composed of three modules that assemble with three distinct domains of the hIno80 ATPase. These modules include (i) one that is composed of the N terminus of the hIno80 protein and all of the metazoan-specific subunits and is not required for ATP-dependent nucleosome remodeling; (ii) a second that is composed of the hIno80 Snf2-like ATPase/helicase and helicase-SANT-associated/post-HSA (HSA/PTH) domain, the actin-related proteins Arp4 and Arp8, and the GLI-Kruppel family transcription factor YY1; and (iii) a third that is composed of the hIno80 Snf2 ATPase domain, the Ies2 and Ies6 proteins, the AAA(+) ATPases Tip49a and Tip49b, and the actin-related protein Arp5. Through purification and characterization of hINO80 complex subassemblies, we demonstrate that ATP-dependent nucleosome remodeling by the hINO80 complex is catalyzed by a core complex comprising the hIno80 protein HSA/PTH and Snf2 ATPase domains acting in concert with YY1 and the complete set of its evolutionarily conserved subunits. Taken together, our findings shed new light on the structure and function of the INO80 chromatin-remodeling complex.

FIGURE 1.

Schematic diagram showing the domain organization of the hIno80 ATPase and hIno80 mutants used in this study. HSA, HSA/PTH domain; Snf2N, Snf2 family N terminus; HelicC, helicase superfamily C terminus, conserved domains in the Snf2 ATPase domain of hIno80. Numbers refer to positions in the amino acid sequence of the hIno80 protein (accession number NP_060023.1). Yellow asterisk shows the position of the E653Q mutation used to inactivate the hIno80 ATPase.

FIGURE 2.

Modular organization of the hINO80 complex. A, MudPIT analysis of intact hINO80 complex and hINO80 complex subassemblies. The table shows hINO80 subunits detected by MudPIT mass spectrometry in complexes containing full-length hIno80 or the indicated hIno80 mutants. Red, subunits associating with the hIno80 NTD; blue, subunits associating with the hIno80 HSA/PTH domain; purple, subunits associating with the Snf2 ATPase domain. The subunit used as FLAG-bait for purification of each complex is indicated with an asterisk. NSAFs provide a rough estimate of the relative amounts of each protein detected in a MudPIT data set (26–29); relative NSAFs shown in the table were calculated by normalizing the NSAF for each subunit to the NSAF for hIno80 or hIno80 derivative in each complex. B, hINO80 complex and hINO80 subassemblies analyzed by SDS-PAGE and silver staining. Open circles indicate the positions of hIno80 and hIno80 derivatives. Lanes separated by black line are from separate gels. C, hINO80 complex and hINO80 subassemblies analyzed by Western blotting with anti-FLAG antibodies to detect FLAG-hIno80 fragments or with antibodies against the indicated subunits.

FIGURE 3.

Nucleosome remodeling activities of hINO80 complex and hINO80 complex subassemblies. Nucleosome sliding assays were performed with the indicated complexes as described under “Experimental Procedures.” hINO80 complexes at a relative concentration (rel. conc.) of 4 contain ∼400 fmol of Arp5, equivalent to the amount of material loaded onto the gels shown in Fig. 2, B and C. 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 (see lane 15). The faint band at the bottom of the gels is due to a small amount of free DNA in the nucleosome preparation. The data in lanes 1–15, 16–23, and 24–27 are from three separate experiments; quantitative comparisons should be made only within an individual experiment.

FIGURE 4.

Modulation of DNA-dependent ATPase activity by the hIno80 CTD. Representative DNA-dependent ATPase assays were performed as described under “Experimental Procedures.” Each reaction included ∼400 fmol of Arp5, equivalent to the amount of material loaded onto the gels shown in Fig. 2, B and C.