A mammalian chromatin remodeling complex with similarities to the yeast INO80 complex

The mammalian Tip49a and Tip49b proteins belong to an evolutionarily conserved family of AAA+ ATPases. In Saccharomyces cerevisiae, orthologs of Tip49a and Tip49b, called Rvb1 and Rvb2, respectively, are subunits of two distinct ATP-dependent chromatin remodeling complexes, SWR1 and INO80. We recently demonstrated that the mammalian Tip49a and Tip49b proteins are integral subunits of a chromatin remodeling complex bearing striking similarities to the S. cerevisiae SWR1 complex (Cai, Y., Jin, J., Florens, L., Swanson, S. K., Kusch, T., Li, B., Workman, J. L., Washburn, M. P., Conaway, R. C., and Conaway, J. W. (2005) J. Biol. Chem. 280, 13665-13670). In this report, we identify a new mammalian Tip49a- and Tip49b-containing ATP-dependent chromatin remodeling complex, which includes orthologs of 8 of the 15 subunits of the S. cerevisiae INO80 chromatin remodeling complex as well as at least five additional subunits unique to the human INO80 (hINO80) complex. Finally, we demonstrate that, similar to the yeast INO80 complex, the hINO80 complex exhibits DNA- and nucleosome-activated ATPase activity and catalyzes ATP-dependent nucleosome sliding.     

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

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.     

Involvement of actin-related proteins in ATP-dependent chromatin remodeling

Actin-related proteins (Arps) and conventional actin are enigmatic components of many chromatin-remodeling enzyme complexes. The yeast INO80 ATP-dependent chromatin-remodeling complex contains stoichiometric amounts of Arp4, Arp5, Arp8, and actin. Here we have revealed functions of Arp5 and Arp8 by analysis of mutants. arp5 Delta and arp8 Delta mutants display an ino80 Delta phenotype. Purification of INO80 complexes from arp5 Delta and arp8 Delta cells shows that protein complexes remain intact but are compromised for INO80 ATPase activity, DNA binding, and nucleosome mobilization. The INO80 (arp8 Delta) complex is strikingly deficient, not only for the Arp8 subunit, but also for Arp4 and actin, suggesting an ordered assembly of Arps. Binding of Arp8 to the INO80 complex requires an N-terminal region of Ino80 adjacent to the conserved ATPase domain. GST-Arp8 binds preferentially to histones H3 and H4 in vitro, suggesting a histone chaperone function. These findings show direct involvement of Arps in the chromatin-remodeling process.     

The human actin-related protein hArp5: Nucleo-cytoplasmic shuttling and involvement in DNA repair

Certain actin-related proteins (Arps) of budding yeast are localized in the nucleus, and have essential roles as stoichiometric components of histone acetyltransferase (HAT) and chromatin remodeling complexes. On the other hand, identification of vertebrate nuclear Arps and their functional analyses are just beginning. We show that human Arp5 (hArp5) proteins are localized in the nucleus, and that arp5Δ yeast cells are partially complemented by hArp5. Thus, hArp5 is a novel member of the nuclear Arps of vertebrates, which possess evolutionarily conserved functions from yeast to humans. We show here that hArp5 shuttles between the nucleus and the cytoplasm. Furthermore, after the induction of DNA double strand breaks (DSB), cell growth and the accumulation of phosphorylated histone H2AX (γ-H2AX) are impaired by hArp5 depletion. Association of hArp5 with the hIno80 chromatin remodeling enzyme and decrease of chromatin-bound hIno80 by hArp5-depletion indicate that hArp5 may have a role in the recruitment of the hINO80 complex to chromatin. Overexpression of hArp5 and hIno80 enhanced γ-H2AX accumulation. These observations suggest that hArp5 is involved in the process of DSB repair through the regulation of the chromatin remodelling machinery.     

Mec1/Tel1 phosphorylation of the INO80 chromatin remodeling complex influences DNA damage checkpoint responses

The yeast Mec1/Tel1 kinases, ATM/ATR in mammals, coordinate the DNA damage response by phosphorylating proteins involved in DNA repair and checkpoint pathways. Recently, ATP-dependent chromatin remodeling complexes, such as the INO80 complex, have also been implicated in DNA damage responses, although regulatory mechanisms that direct their function remain unknown. Here, we show that the Ies4 subunit of the INO80 complex is phosphorylated by the Mec1/Tel1 kinases during exposure to DNA-damaging agents. Mutation of Ies4's phosphorylation sites does not significantly affect DNA repair processes, but does influence DNA damage checkpoint responses. Additionally, ies4 phosphorylation mutants are linked to the function of checkpoint regulators, such as the replication checkpoint factors Tof1 and Rad53. These findings establish a chromatin remodeling complex as a functional component in the Mec1/Tel1 DNA damage signaling pathway that modulates checkpoint responses and suggest that posttranslational modification of chromatin remodeling complexes regulates their involvement in distinct processes.     

The INO80 complex is required for damage-induced recombination

The budding yeast INO80 complex has a role in remodeling chromatin structure, and the SWR1 complex replaces a H2A/H2B dimer with a variant dimer, H2A.Z (Htz1)/H2B. It has been reported that these chromatin remodeling complexes contain Arp4 (actin-related protein) and actin in common and are recruited to HO endonuclease-induced DNA double-strand break (DSB) site. Reportedly, Ino80 can evict nucleosomes surrounding a HO-induced DSB; however, it has no apparent role to play in the repair of HO-induced DSB. Here we show that an essential factor for INO80 chromatin remodeling activity, Arp8, is involved in damage-induced sister chromatid recombination and interchromosomal recombination between heteroalleles. In contrast, arp6 mutants are proficient for recombination, indicating that the SWR1 complex does not promote recombination. Our data suggest that the remodeling of chromatin by the INO80 complex facilitates efficient homologous recombination upon DNA damages.     

Structure of Actin-related protein 8 and its contribution to nucleosome binding

Nuclear actin-related proteins (Arps) are subunits of several chromatin remodelers, but their molecular functions within these complexes are unclear. We report the crystal structure of the INO80 complex subunit Arp8 in its ATP-bound form. Human Arp8 has several insertions in the conserved actin fold that explain its inability to polymerize. Most remarkably, one insertion wraps over the active site cleft and appears to rigidify the domain architecture, while active site features shared with actin suggest an allosterically controlled ATPase activity. Quantitative binding studies with nucleosomes and histone complexes reveal that Arp8 and the Arp8–Arp4–actin-HSA sub-complex of INO80 strongly prefer nucleosomes and H3–H4 tetramers over H2A–H2B dimers, suggesting that Arp8 functions as a nucleosome recognition module. In contrast, Arp4 prefers free (H3–H4)₂ over nucleosomes and may serve remodelers through binding to (dis)assembly intermediates in the remodeling reaction.     

The actin-related protein hArp8 accumulates on the mitotic chromosomes and functions in chromosome alignment

The actin family consists of conventional actin and various actin-related proteins (Arps). Some of these Arps are localized in the nucleus, and a fraction of each of these nuclear Arps is functionally involved in chromatin remodeling and histone acetyltransferase complexes. On the other hand, in mitotic cells, the localization and function of the nuclear Arps are largely unknown. Human Arp8 (hArp8), an ortholog of yeast nuclear Arp8, was recently found to be associated with the hINO80-chromatin remodeling complex along with hArp5. Here we report that hArp8, but not hArp5, accumulates on mitotic chromosomes. This is the first example where a member of the actin family is found to be associated with mitotic chromosomes. Expression of truncated hArp8 proteins and depletion of endogenous hArp8 by RNA interference caused misalignment of mitotic chromosomes, suggesting that chromosome-associated hArp8 has a role in chromosome behavior. In contrast, depletion of hIno80 and hArp5 did not cause misalignment of chromosomes, suggesting that the role of hArp8 at mitotic chromosomes is independent of the activity of hINO80 complexes. These findings provide the first insight into a novel function of actin family members in mitosis.