INO80 Chromatin Remodeling Complex Research Articles

Subunit Composition and Substrate Specificity of a MOF-containing Histone Acetyltransferase Distinct from the Male-specific Lethal (MSL) Complex

Human MOF (MYST1), a member of the MYST (Moz-Ybf2/Sas3-Sas2-Tip60) family of histone acetyltransferases (HATs), is the human ortholog of the Drosophila males absent on the first (MOF) protein. MOF is the catalytic subunit of the male-specific lethal (MSL) HAT complex, which plays a key role in dosage compensation in the fly and is responsible for a large fraction of histone H4 lysine 16 (H4K16) acetylation in vivo. MOF was recently reported to be a component of a second HAT complex, designated the non-specific lethal (NSL) complex (Mendjan, S., Taipale, M., Kind, J., Holz, H., Gebhardt, P., Schelder, M., Vermeulen, M., Buscaino, A., Duncan, K., Mueller, J., Wilm, M., Stunnenberg, H. G., Saumweber, H., and Akhtar, A. (2006) Mol. Cell 21, 811-823). Here we report an analysis of the subunit composition and substrate specificity of the NSL complex. Proteomic analyses of complexes purified through multiple candidate subunits reveal that NSL is composed of nine subunits. Two of its subunits, WD repeat domain 5 (WDR5) and host cell factor 1 (HCF1), are shared with members of the MLL/SET family of histone H3 lysine 4 (H3K4) methyltransferase complexes, and a third subunit, MCRS1, is shared with the human INO80 chromatin-remodeling complex. In addition, we show that assembly of the MOF HAT into MSL or NSL complexes controls its substrate specificity. Although MSL-associated MOF acetylates nucleosomal histone H4 almost exclusively on lysine 16, NSL-associated MOF exhibits a relaxed specificity and also acetylates nucleosomal histone H4 on lysines 5 and 8.

Genome-wide association data reveal a global map of genetic interactions among protein complexes.

This work demonstrates how gene association studies can be analyzed to map a global landscape of genetic interactions among protein complexes and pathways. Despite the immense potential of gene association studies, they have been challenging to analyze because most traits are complex, involving the combined effect of mutations at many different genes. Due to lack of statistical power, only the strongest single markers are typically identified. Here, we present an integrative approach that greatly increases power through marker clustering and projection of marker interactions within and across protein complexes. Applied to a recent gene association study in yeast, this approach identifies 2,023 genetic interactions which map to 208 functional interactions among protein complexes. We show that such interactions are analogous to interactions derived through reverse genetic screens and that they provide coverage in areas not yet tested by reverse genetic analysis. This work has the potential to transform gene association studies, by elevating the analysis from the level of individual markers to global maps of genetic interactions. As proof of principle, we use synthetic genetic screens to confirm numerous novel genetic interactions for the INO80 chromatin remodeling complex.

Involvement of a chromatin remodeling complex in damage tolerance during DNA replication

ATP-dependent chromatin remodeling complexes have been shown to participate in DNA replication in addition to transcription and DNA repair. However, the mechanisms of their involvement in DNA replication remain unclear. Here, we reveal a specific function of the yeast INO80 chromatin remodeling complex in the DNA damage tolerance pathways. Whereas INO80 is necessary for the resumption of replication at forks stalled by methyl methane sulfonate (MMS), it is not required for replication fork collapse after treatment with hydroxyurea (HU). Mechanistically, INO80 regulates DNA damage tolerance during replication through modulation of PCNA (proliferating cell nuclear antigen) ubiquitination and Rad51-mediated processing of recombination intermediates at impeded replication forks. Our findings establish a mechanistic link between INO80 and DNA damage tolerance pathways, indicating that chromatin remodeling is important for accurate DNA replication.

Arabidopsis actin-related protein ARP5 in multicellular development and DNA repair

Actin-related protein 5 (ARP5) is a conserved subunit of the INO80 chromatin-remodeling complex in yeast and mammals. We have characterized the expression and subcellular distribution of Arabidopsis thaliana ARP5 and explored its role in the epigenetic control of multicellular development and DNA repair. ARP5-specific monoclonal antibodies localized ARP5 protein to the nucleoplasm of interphase cells in Arabidopsis and Nicotiana tabacum. ARP5 promoter-reporter fusions and the ARP5 protein are ubiquitously expressed. A null mutant and a severe knockdown allele produced moderately dwarfed plants with all organs smaller than the wild type. The small and slightly deformed organs such as leaves and hypocotyls were composed of small-sized cells. The ratio of leaf stomata to epidermal cells was high in the mutant, which also exhibited a delayed stomatal development compared with the wild type. Mutant plants were hypersensitive to DNA-damaging reagents including hydroxyurea, methylmethane sulfonate, and bleocin, demonstrating a role for ARP5 in DNA repair. Interestingly, the hypersensitivity phenotype of ARP5 null allele arp5-1 is stronger than the severe knockdown allele arp5-2. Moreover, a wild-type transgene fully complemented all developmental and DNA repair mutant phenotypes. Despite the common participation of both ARP4 and ARP5 in the INO80 complex, ARP4- and ARP5-deficient plants displayed only a small subset of common phenotypes and each displayed novel phenotypes, suggesting that in Arabidopsis they have both shared and unique functions.

Functions of the Uch37 deubiquitinating enzyme in the proteasome and the INO80 chromatin remodeling complex

Deubiquitinating enzymes (DUBs) are proteases that can antagonize ubiquitin‐mediated signalling by disassembling ubiquitin‐protein conjugates. How DUBs are regulated in vivo and how their substrate specificities are achieved are largely unknown. The conserved DUB Uch37 is found on proteasomes in organisms ranging from fission yeast to humans. We recently reported that nuclear Uch37 is also associated with the human INO80 chromatin remodeling complex (hINO80). Whereas deubiquitination by Uch37 is activated by proteasomal binding, Uch37 is inactive within hINO80. Interestingly, it can be activated by transient interactions of hINO80 wih the 26S proteasome. Thus, DUB activities can be modulated both positively and negatively via dynamic interactions with partner proteins. We will present evidence that, for Uch37, this regulation plays a role in control of histone H2B ubiquitination. This work was supported in part by National Institutes of Health grants R01 GM37666 (R.E.C.) and R37 GM41628 (R.C.C.). T.Y. is a fellow of the Leukemia and Lymphoma Society.

Nucleosome Remodeling Complexes Increase Subnuclear Dynamics Of Chromatin In Yeast

We have described the mobility of internal loci on yeast interphase chromosomes by live fluorescence microscopy with high precision. Based on single particle tracking and mathematical simulations of random walks in a confined volume, we could define the constraints exerted by the chromatin fiber. We find that local recruitment of the transcriptional activator VP16 and components of the Ino80 chromatin remodeling complex significantly increases diffusion rate, large rapid steps and the radius of constraint of a given locus. Importantly, the Ino80 induced mobility increase is largely dependent on its ATPase activity. Inhibition of transcription did not alter chromatin mobility and we find no correlation between transcriptional elongation and increased mobility. In analogy to experiments in mammalian cells, this indicates that transcription per se does not directly influence chromatin dynamics. These are the first mechanistic indications that chromatin remodelers can indeed alter mobility of the chromatin fiber - a process can facilitate chromatin contacts to distinct subnuclear regions.To examine the function of increased chromatin mobility, we have monitored the recombination rate of substrates to which Ino80 is targeted. We find that Ino80 enhances the spontaneous rate of gene conversion. This is not achieved by targeting the VP16 transactivation domain. The mode of Ino80 action at DSBs and at collapsed replication forks will be discussed both with respect to the physical characteristics of the chromatin fiber, and with respect to local nucleosome eviction.

Distinct Modes of Regulation of the Uch37 Deubiquitinating Enzyme in the Proteasome and in the Ino80 Chromatin-Remodeling Complex

Deubiquitinating enzymes (DUBs) are proteases that can antagonize ubiquitin-mediated signaling by disassembling ubiquitin-protein conjugates. How DUBs are regulated in vivo and how their substrate specificities are achieved are largely unknown. The conserved DUB Uch37 is found on proteasomes in organisms ranging from fission yeast to humans. Deubiquitination by Uch37 is activated by proteasomal binding, which enables Uch37 to process polyubiquitin chains. Here we show that in the nucleus Uch37 is also associated with the human Ino80 chromatin-remodeling complex (hINO80). In hINO80, Uch37 is held in an inactive state; however, it can be activated by transient interaction of the Ino80 complex with the proteasome. Thus, DUB activities can be modulated both positively and negatively via dynamic interactions with partner proteins. In addition, our findings suggest that the proteasome and the hINO80 chromatin-remodeling complex may cooperate to regulate transcription or DNA repair, processes in which both complexes have been implicated.

Ino80 Chromatin Remodeling Complex Promotes Recovery of Stalled Replication Forks

Chromatin remodeling complexes facilitate the access of enzymes that mediate transcription, replication or repair of DNA by modulating nucleosome position and/or composition. Ino80 is the DNA-dependent Snf2-like ATPase subunit of a complex whose nucleosome remodeling activity requires actin-related proteins, Arp4, Arp5 and Arp8, as well as two RuvB-like DNA helicase subunits. Budding yeast mutants deficient for Ino80 function are not only hypersensitive to reagents that induce DNA double-strand breaks, but also to those that impair replication fork progression. To understand why ino80 mutants are sensitive to agents that perturb DNA replication, we used chromatin immunoprecipitation to map the binding sites of the INO80 chromatin-remodeling complex on four budding yeast chromosomes. We found that Ino80 and Arp5 binding sites coincide with origins of DNA replication and tRNA genes. In addition, Ino80 was bound at 67% of the promoters of genes that are sensitive to ino80 mutation. When replication forks were arrested near origins in the presence of hydroxyurea (HU), the amount of INO80 complex at stalled forks and at unfired origins increased selectively. Importantly, the resumption of DNA replication after release from a HU block was impaired in ino80 mutants. These cells accumulated double-strand breaks as they attempted to restart replication. Consistently, ino80-deficient cells, although proficient for checkpoint activation, delay recovery from the checkpoint response. The INO80 chromatin remodeling complex is enriched at stalled replication forks, where it promotes the resumption of replication upon recovery from fork arrest.

INO80 meets a fork in the road

The INO80 chromatin-remodeling complex has roles in transcription, DNA repair and replication. By studying the role of Ino80 in DNA replication, a study now finds that the INO80 complex is essential for replication fork progression under conditions of replicative stress, stabilizes stalled forks and helps ensure proper restart.

Chromatin Responses to DNA Damage

Proper repair of DNA damage is critical in preventing genome instability and carcinogenesis. While the role of ATP‐dependent chromatin remodeling in transcription is well established, a link between chromatin remodeling and DNA repair has remained elusive. We have found that the evolutionarily conserved INO80 chromatin remodeling complex directly participates in the repair of a double strand break (DSB) in yeast. Our findings reveal a new role of ATP‐dependent chromatin remodeling in nuclear processes and suggest that an ATP‐dependent chromatin remodeling complex can read a DNA repair histone code. We have also found recently that a subunit of the INO80 complex is phosphorylated in an ATM/ATR‐dependent fashion in response to DNA damage, and the phosphorylation events modulate DNA checkpoint pathways. Thus, chromatin responses to DNA damages involve complex changes in both histones and complexes that regulate chromatin.

A YY1–INO80 complex regulates genomic stability through homologous recombination–based repair

DNA damage repair is crucial for the maintenance of genome integrity and cancer suppression. We found that loss of the mouse transcription factor YY1 resulted in polyploidy and chromatid aberrations, which are signatures of defects in homologous recombination. Further biochemical analyses identified a YY1 complex comprising components of the evolutionarily conserved INO80 chromatin-remodeling complex. Notably, RNA interference-mediated knockdown of YY1 and INO80 increased cellular sensitivity toward DNA-damaging agents. Functional assays revealed that both YY1 and INO80 are essential in homologous recombination-based DNA repair (HRR), which was further supported by the finding that YY1 preferentially bound a recombination-intermediate structure in vitro. Collectively, these observations reveal a link between YY1 and INO80 and roles for both in HRR, providing new insight into mechanisms that control the cellular response to genotoxic stress.

Distinct roles for SWR1 and INO80 chromatin remodeling complexes at chromosomal double-strand breaks

INO80 and SWR1 are two closely related ATP-dependent chromatin remodeling complexes that share several subunits. Ino80 was reported to be recruited to the HO endonuclease-induced double-strand break (DSB) at the budding yeast mating-type locus, MAT. We find Swr1 similarly recruited in a manner dependent on the phosphorylation of H2A (gammaH2AX). This is not unique to cleavage at MAT; both Swr1 and Ino80 bind near an induced DSB on chromosome XV. Whereas Swr1 incorporates the histone variant H2A.Z into chromatin at promoters, H2A.Z levels do not increase at DSBs. Instead, H2A.Z, gammaH2AX and core histones are coordinately removed near the break in an INO80-dependent, but SWR1-independent, manner. Mutations in INO80-specific subunits Arp8 or Nhp10 impair the binding of Mre11 nuclease, yKu80 and ATR-related Mec1 kinase at the DSB, resulting in defective end-processing and checkpoint activation. In contrast, Mre11 binding, end-resection and checkpoint activation were normal in the swr1 strain, but yKu80 loading and error-free end-joining were impaired. Thus, these two related chromatin remodelers have distinct roles in DSB repair and checkpoint activation.

Regulation of Telomere Structure and Functions by Subunits of the INO80 Chromatin Remodeling Complex

ATP-dependent chromatin remodeling complexes have been implicated in the regulation of transcription, replication, and more recently DNA double-strand break repair. Here we report that the Ies3p subunit of the Saccharomyces cerevisiae INO80 chromatin remodeling complex interacts with a conserved tetratricopeptide repeat domain of the telomerase protein Est1p. Deletion of IES3 and some other subunits of the complex induced telomere elongation and altered telomere position effect. In telomerase-negative mutants, loss of Ies3p delayed the emergence of recombinational survivors and stimulated the formation of extrachromosomal telomeric circles in survivors. Deletion of IES3 also resulted in heightened levels of telomere-telomere fusions in telomerase-deficient strains. In addition, a delay in survivor formation was observed in an Arp8p-deficient mutant. Because Arp8p is required for the chromatin remodeling activity of the INO80 complex, the complex may promote recombinational telomere maintenance by altering chromatin structure. Consistent with this notion, we observed preferential localization of multiple subunits of the INO80 complex to telomeres. Our results reveal novel functions for a subunit of the telomerase complex and the INO80 chromatin remodeling complex.

Actin-related protein Arp4 functions in kinetochore assembly

The actin-related proteins (Arps) comprise a conserved protein family. Arp4p is found in large multisubunits of the INO80 and SWR1 chromatin remodeling complexes and in the NuA4 histone acetyltransferase complex. Here we show that arp4 (arp4S23A/D159A) temperature-sensitive cells are defective in G2/M phase function. arp4 mutants are sensitive to the microtubule depolymerizing agent benomyl and arrest at G2/M phase at restrictive temperature. Arp4p is associated with centromeric and telomeric regions throughout cell cycle. Ino80p, Esa1p and Swr1p, components of the INO80, NuA4 and SWR1 complexes, respectively, also associate with centromeres. The association of many kinetochore components including Cse4p, a component of the centromere nucleosome, Mtw1p and Ctf3p is partially impaired in arp4 cells, suggesting that the G2/M arrest of arp4 mutant cells is due to a defect in formation of the chromosomal segregation apparatus.

Purification and assay of the human INO80 and SRCAP chromatin remodeling complexes

Chromatin modifying and remodeling enzymes play critical roles in many aspects of chromosome biology including transcription, replication, recombination, and repair. Our laboratory recently identified and characterized two multisubunit human chromatin remodeling enzymes designated the INO80 and SRCAP complexes. Mechanistic studies revealed that the human INO80 complex catalyzes nucleosome sliding and the SRCAP complex catalyzes ATP-dependent exchange of histone H2A/H2B dimers containing the histone variant H2A.Z into nucleosomes. Here we describe methods for purification and assay of the INO80 and SRCAP chromatin remodeling complexes.

Yeast G1 DNA damage checkpoint regulation by H2A phosphorylation is independent of chromatin remodeling

Recent studies of yeast G1 DNA damage response have identified characteristic changes in chromatin adjacent to double-strand breaks (DSBs). Histone H2A (yeast H2AX) is rapidly phosphorylated on S129 by the kinase Tel1 (ATM) over a domain extending kilobases from the DSB. The adaptor protein Rad9 (53BP1) is recruited to this chromatin domain through binding of its tudor domains to histone H3 diMe-K79. Multisite phosphorylation of Rad9 by Mec1 (ATR) then activates the signaling kinase Rad53 (CHK2) to induce a delay in G1. Here, we report a previously undescribed role for Tel1 in G1 checkpoint response and show that H2A is the likely phosphorylation target, in a much as S129 mutation to Ala confers defects in G1 checkpoint arrest, Rad9 phosphorylation, and Rad53 activation. Importantly, Rad9 fails to bind chromatin adjacent to DSBs in H2A-S129A mutants. Previous work showed that H2A phosphorylation allows binding of NuA4, SWR, and INO80 chromatin remodeling complexes, perhaps exposing H3 diMe-K79. Yet, mutants lacking SWR or INO80 remain checkpoint competent, whereas loss of NuA4-dependent histone acetylation leads to G1 checkpoint persistence, suggesting that H2A phosphorylation promotes two independent events, rapid Rad9 recruitment to DSBs and subsequent remodeling by NuA4, SWR, and INO80.

INO80 Chromatin Remodeling and DNA Repair

While the role of ATP-dependent chromatin remodeling in transcription is well established, a link between chromatin remodeling and DNA repair has remained elusive. We have found that the evolutionarily conserved INO80 chromatin remodeling complex directly participates in the repair of a double strand break (DSB) in yeast. The INO80 complex is recruited to a HO endonuclease-induced DSB through a specific interaction with the DNA damage-induced phosphorylated histone H2A (?-H2AX). This interaction requires Nhp10, an HMG-like subunit of the INO80 complex. The loss of Nhp10 or ?-H2AX results in reduced INO80 recruitment to DSB. Finally, components of the INO80 complex show synthetic genetic interactions with the RAD52 DNA repair pathway, the main pathway for DSB repair in yeast. Our findings reveal a new role of ATP-dependent chromatin remodeling in nuclear processes and suggest that an ATP-dependent chromatin remodeling complex can read a DNA repair histone code. We will discuss ongoing works addressing the mechanism of INO80 chromatin remodelling in DNA repair.

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.

Plc1p, Arg82p, and Kcs1p, Enzymes Involved in Inositol Pyrophosphate Synthesis, Are Essential for Phosphate Regulation and Polyphosphate Accumulation in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, the phosphate signal transduction PHO pathway is involved in regulating several phosphate-responsive genes such as PHO5, which encodes repressible acid phosphatase. In this pathway, a cyclin-dependent kinase inhibitor (Pho81p) regulates the kinase activity of the cyclin-cyclin-dependent kinase complex Pho80p-Pho85p, which phosphorylates the transcription factor Pho4p in response to intracellular phosphate levels. However, how cells sense phosphate availability and transduce the phosphate signal to Pho81p remains unknown. To identify additional components of the PHO pathway, we have screened a collection of yeast deletion strains. We found that disruptants of PLC1, ARG82, and KCS1, which are involved in the synthesis of inositol polyphosphate, and ADK1, which encodes adenylate kinase, constitutively express PHO5. Each of these factors functions upstream of Pho81p and negatively regulates the PHO pathway independently of intracellular orthophosphate levels. Overexpression of KCS1, but not of the other genes, suppressed PHO5 expression in the wild-type strain under low phosphate conditions. These results raise the possibility that diphosphoinositol tetrakisphosphate and/or bisdiphosphoinositol triphosphate may be essential for regulation of the PHO pathway. Furthermore, the Deltaplc1, Deltaarg82, and Deltakcs1 deletion strains, but not the Deltaipk1 deletion strain, had significantly reduced intracellular polyphosphate levels, suggesting that enzymes involved in inositol pyrophosphate synthesis are also required for polyphosphate accumulation.

Binding of Chromatin-Modifying Activities to Phosphorylated Histone H2A at DNA Damage Sites

Yeast histone H2A is phosphorylated on Ser129 upon DNA damage, an event required for efficient repair. We show that phosphorylation occurs rapidly over a large region around DNA double-strand breaks (DSBs). Histone H4 acetylation is also important for DSB repair, and we found that the NuA4 HAT complex associates specifically with phospho-H2A peptides. A single NuA4 subunit, Arp4, is responsible for the interaction. The NuA4 complex is recruited to a DSB concomitantly with the appearance of H2A P-Ser129 and Arp4 is important for this binding. Arp4 is also a subunit of the Ino80 and Swr1 chromatin remodeling complexes, which also interact with H2A P-Ser129 and are recruited to DSBs. This association again requires Arp4 but also prior NuA4 recruitment and action. Thus, phosphorylation of H2A at DNA damage sites creates a mark recognized by different chromatin modifiers. This interaction leads to stepwise chromatin reconfiguration, allowing efficient DNA repair.