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. Although it is well established that SNF2 family ATPases have evolved to play critical roles as ATPdependent motors that drive many types of chromatin transactions, in most cases how their intrinsic catalytic activities are regulated by their diverse array of associated proteins is poorly understood.The INO80 complex was first identified in Saccharomyces cerevisiae, where it was shown to be composed of roughly 15 subunits (1, 9). Subsequent purification of the human INO80 complex revealed that it shares with its S. cerevisiae counterpart a set of subunits including the Ino80 SNF2 ATPase, the AAA+ ATPases Tip49a and Tip49b, actin-related proteins Arp4, Arp5, and Arp8, and the Ies2 and Ies6 proteins (2, 10-12). The human INO80 complex lacks orthologs of the remaining subunits of the S. cerevisiae INO80 complex and contains instead several apparently metazoan-specific subunits including the deubiquitinating enzyme Uch37, the gene altered in glioma (GLI)-Kruppel family zinc finger transcription factor YY1, the forkhead-associated domain (FHA) containing Mcrs1, nuclear factor related to kB (Nfrkb), and the Amida, Ino80D (FLJ20309), and Ino80E (FLJ90652) proteins.The INO80 complex is capable of regulating chromatin structure in at least two ways. First, it catalyzes ATP-dependent sliding of histone octamers along DNA (1, 2). Second, it catalyzes the replacement of histone H2AZ/histone H2AB dimers in nucleosomes with histone H2A/histone H2B dimers in a reaction that in yeast has been shown to contribute to genome-wide histone H2AZ localization (13).In a recent study dissecting mechanism(s) by which the human INO80 complex catalyzes chromatin remodeling, we found that it is composed of at least three modules that assemble with three distinct domains of the Ino80 protein (14) (Fig. 1A). One module is composed of an Ino80 N-terminal domain and all of the metazoan-specific subunits except YY1. A second, the helicase-SANT-associated (HSA) module, is composed of the Ino80 HSA domain, the actin-related Arp4/Baf53a and ...

Assembly of the Arp5 (Actin-related Protein) Subunit Involved in Distinct INO80 Chromatin Remodeling Activities

“…Indeed, the insertion domain of Ino80, which is required for Ies2 and Arp5-Ies6 assembly, is less conserved (14% identical; 24% similar) than the full protein (27% identical; 35% similar) or functional domains such as the helicase-SANT-associated domain (37% identical; 54% similar) and the ATPase domain (67% identical; 75% similar). In particular, the two regions of human INO80 that were reported as crucial for Arp5-Ies6 binding (25) are relatively less conserved (9 and 14% identical; 9 and 22% similar) and may provide a binding platform for Arp5-Ies6 assembly with human INO80 that is distinct from yeast. Likewise, S. cerevisiae Arp5 insertion regions 2 and 3 ( Table 2), which are critical for INO80-mediated nucleosome sliding, are relatively less conserved among mammals than other species and may also reflect divergent modes of chromatin remodeling that have evolved in higher eukaryotes.…”

“…It was recently reported that siRNA-mediated depletion of INO80B (yeast Ies2) in HEK293T cells results in minimal loss of ACTR5-INO80C (yeast Arp5-Ies6) with the INO80 complex (25). Furthermore, INO80B is required for nucleosome and DNA-stimulated ATPase activity, whereas ACTR5 and INO80C are not.…”

“…The insertion region is evolutionarily conserved and necessary for the recruitment of mammalian RuvBL1-RuvBL2 (Rvb1-Rvb2 in yeast) (25), which are AAAϩ helicases with homology to the bacterial RuvB helicase involved in Holliday Junction migration (26), yet their roles in chromatin remodeling remain unclear. In addition, the insertion region of mammalian Ino80 is needed for ACTR5-INO80C module (Arp5-Ies6 in yeast) assembly within the INO80 complex (25), although its requirement for the yeast complex has not previously been reported. However, Rvb1-Rvb2 module is necessary for Arp5-Ies6 association with the INO80 complex (27).…”

Regulation of DNA Translocation Efficiency within the Chromatin Remodeler RSC/Sth1 Potentiates Nucleosome Sliding and Ejection

“…ARPs interact physically and functionally with the remodeler ATPase subunit (Chen et al, 2013; Shen et al, 2003; Yang et al, 2007; Zhao et al, 1998) but important aspects regarding their mechanistic relationship to DNA translocation, nucleosome sliding and ejection are poorly understood. ARPs are essential in yeast, however genome-wide screens for arp Δ suppressors yielded mutations in STH1 itself, which mapped to two small domains: one located just after the HSA domain (termed Post-HSA domain), and one located centrally within the ATPase/translocase domain (termed Protrusion 1) (Flaus et al, 2006).…”

The INO80 Complex Requires the Arp5-Ies6 Subcomplex for Chromatin Remodeling and Metabolic Regulation

“…In addition, the Arp5 and INO80 subunit 6 (Ies6) subunits comprise a separate module that is particularly interesting because of its structural placement at the "enzymatic center" of the complex, between the ATPase domain of Ino80 and the large Rvb1-Rvb2 helicase module (21). Indeed, assembly of the Arp5-Ies6 module into the INO80 complex requires the Ies2 subunit (28), Rvb1-Rvb2 (29), and the Ino80 ATPase domain (28,30). Deletion of ARP5 or IES6 diminishes INO80 activity in vitro (21,23,31), although their precise role in nucleosome positioning, particularly in vivo, has not been well established.…”