Nuclear Actin-related Proteins Research Articles

Structure of the full-length yeast Arp7–Arp9 heterodimer

The nuclear actin-related proteins Arp7 and Arp9 are components of the yeast SWI/SNF and RSC chromatin-remodelling complexes. The 3.1 Å resolution crystal structure reported here shows that the full-length Arp7 and Arp9 proteins exist as a dimer without a requirement for additional polypeptides. Of the 11 actin-related proteins, Arp7 and Arp9 are the only two directly demonstrated to form a dimer within this family. The Arp7-Arp9 heterodimer is unlikely to form an actin-like filament based on modelling using the structure. The Arp7-Arp9 structure reveals that its dimerization interface is not altered when bound in a complex with the SWI/SNF Snf2 HSA domain and the regulatory protein Rtt102.

Interactions of Nuclear Actin-Related Proteins with SWI/SNF Chromatin-Remodeling Complexes

ATP-dependent chromatin-remodeling complexes (remodelers) modulate chromatin structure and thus play important roles in transcription, DNA replication, and DNA repair. Nuclear actin and actin-related proteins (Arps) are subunits of several such complexes, where they are commonly found in pairs. However, the role of actin/Arps and the mechanism through which they bind the catalytic subunits of their host complexes are unknown. Here, we investigated how budding yeast Arp7 and Arp9 are incorporated into SWI/SNF-family chromatin-remodeling complexes. We present the first biochemical evidence that Arp7 and Arp9 must heterodimerize to bind ATP and interact with the catalytic subunits of the SWI/SNF and RSC complexes. In addition, we present ITC data, which define the remodeler region that interacts with Arp7/9, thus providing a mechanistic framework for understanding how actin/Arps are selectively loaded into their host remodelers.

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.

Swe1‐dependent and Swe1‐independent pathways controlling Hydroxyurea resistance in Saccharomyces cerevisiae

Exposure to Hydroxyurea (HU) slows down or stalls DNA replication fork and activates the replication checkpoint. In Saccharomyces cerevisiae the replication fork component proteins Tof1, Csm3, Ctf4 and Mrc1 as well as its regulators such as Dcc1, Ctf18 and Ctf8 confer HU resistance. HU treatment of yeast cells causes actin depolarization in which several DNA integrity checkpoint proteins as well as morphogenesis controller Swe1 have been implicated. Recently we have shown that a sphingolipid gene ISC1 also controls cell morphology under HU stress by monitoring Swe1 and Cdk1 activity.Chromatin remodeling protein complexes such as Ino80 and Swr1 that function to reorganize histones during transcription and DNA repair, have recently been implicated in genotoxic tolerance in HU and methyl methanesulfonate (MMS). We have observed that although replication checkpoint proteins Mrc1, Tof1, Csm3 and Ctf4 and certain chromatin remodeling complex proteins such as Arp6 and Arp8 are involved in replication fork stalling, genotoxic tolerance and recovery from HU and MMS, their modes of action may be different. We find that they cooperate in HU/MMS sensitivity and recovery from HU/MMS.We have also observed that HU treatment causes morphological aberrations in strains lacking replication associated genes such as CTF4 and DCC1. Interestingly, although HU causes morphological aberrations in the absence of ARP8, an INO80 complex gene, it does cause any aberrations in the absence of ARP6, a component of SWR1 complex gene. First time we are showing the functional interactions among replication checkpoint proteins, nuclear actin‐related proteins of chromatin remodelers, and actin regulators in the genotoxic tolerance and genotoxin‐induced cell morphology.

The actin family member Arp6 and the histone variant H2A.Z are required for spatial positioning of chromatin in chicken cell nuclei.

The spatial organization of chromatin in the nucleus contributes to genome function and is altered during the differentiation of normal and tumorigenic cells. Although nuclear actin-related proteins (Arps) have roles in the local alteration of chromatin structure, it is unclear whether they are involved in the spatial positioning of chromatin. In the interphase nucleus of vertebrate cells, gene-dense and gene-poor chromosome territories (CTs) are located in the center and periphery, respectively. We analyzed chicken DT40 cells in which Arp6 had been knocked out conditionally, and showed that the radial distribution of CTs was impaired in these knockout cells. Arp6 is an essential component of the SRCAP chromatin remodeling complex, which deposits the histone variant H2A.Z into chromatin. The redistribution of CTs was also observed in H2A.Z-deficient cells for gene-rich microchromosomes, but to lesser extent for gene-poor macrochromosomes. These results indicate that Arp6 and H2A.Z contribute to the radial distribution of CTs through different mechanisms. Microarray analysis suggested that the localization of chromatin to the nuclear periphery per se is insufficient for the repression of most genes.

Nuclear actin-related protein is required for chromosome segregation in Toxoplasma gondii

Apicomplexa parasites use complex cell cycles to replicate that are not well understood mechanistically. We have established a robust forward genetic strategy to identify the essential components of parasite cell division. Here we describe a novel temperature sensitive Toxoplasma strain, mutant 13-20C2, which growth arrests due to a defect in mitosis. The primary phenotype is the mis-segregation of duplicated chromosomes with chromosome loss during nuclear division. This defect is conditional-lethal with respect to temperature, although relatively mild in regard to the preservation of the major microtubule organizing centers. Despite severe DNA loss many of the physical structures associated with daughter budding and the assembly of invasion structures formed and operated normally at the non-permissive temperature before completely arresting. These results suggest there are coordinating mechanisms that govern the timing of these events in the parasite cell cycle. The defect in mutant 13-20C2 was mapped by genetic complementation to Toxoplasma chromosome III and to a specific mutation in the gene encoding an ortholog of nuclear actin-related protein 4. A change in a conserved isoleucine to threonine in the helical structure of this nuclear actin related protein leads to protein instability and cellular mis-localization at the higher temperature. Given the age of this protist family, the results indicate a key role for nuclear actin-related proteins in chromosome segregation was established very early in the evolution of eukaryotes.

Nuclear actin-related proteins take shape

The function of nuclear actin is poorly understood. It is known to be a discrete component of several chromatin-modifying complexes. Nevertheless, filamentous forms of actin are important for various nuclear processes as well. Nuclear actin is often associated with nuclear actin-related protein Arp4 and other actin-related proteins like Arp8 in the INO80 chromatin remodeler. We recently determined the crystal structure of S. cerevisiae Arp4 that explains why Arp4 is unable to form actin like filaments and shows that it is constitutively bound to an ATP nucleotide. More interestingly, in vitro activities of Arp4 and Arp8 seem to be directed towards stabilizing monomeric actin and to integrate it stoichiometrically into the INO80 complex. Based on this activity, we discuss possible roles of nuclear Arps in chromatin modifying complexes and in regulating more general aspects of nuclear actin dynamics.

Arp you ready for actin in the nucleus?

Actin and actin‐related proteins (Arps) are critical components of chromatin remodelling and modifying complexes, where they are commonly found in pairs. Yet, little is known about their role in these complexes. In this issue of The EMBO Journal , Fenn et al (2011) report the first structural investigation of two nuclear Arps, the INO80 complex subunits Arp4 and Arp8. Importantly, their results also shed light on the critical role that the nucleotide state of actin may have on its interactions with the Arps.

Structural biochemistry of nuclear actin-related proteins 4 and 8 reveals their interaction with actin.

Nuclear actin and actin-related proteins (Arps) are integral components of various chromatin-remodelling complexes. Actin in such nuclear assemblies does not form filaments but associates in defined complexes, for instance with Arp4 and Arp8 in the INO80 remodeller. To understand the relationship between nuclear actin and its associated Arps and to test the possibility that Arp4 and Arp8 help maintain actin in defined states, we structurally analysed Arp4 and Arp8 from Saccharomyces cerevisiae and tested their biochemical effects on actin assembly and disassembly. The solution structures of isolated Arp4 and Arp8 indicate them to be monomeric and the crystal structure of ATP-Arp4 reveals several differences to actin that explain why Arp4 does not form filaments itself. Remarkably, Arp4, assisted by Arp8, influences actin polymerization in vitro and is able to depolymerize actin filaments. Arp4 likely forms a complex with monomeric actin via the barbed end. Our data thus help explaining how nuclear actin is held in a discrete complex within the INO80 chromatin remodeller.

Actin-related proteins localized in the nucleus: from discovery to novel roles in nuclear organization.

The actin family consists of conventional actin and actin-related proteins (ARPs), and the members show moderate similarity and share the same basal structure. Following the finding of various ARPs in the cytoplasm in the 1990s, multiple subfamilies that are localized predominantly in the nucleus were identified. Consistent with these cytological observations, subsequent biochemical analyses revealed the involvement of the nuclear ARPs in ATP-dependent chromatin-remodeling and histone acetyltransferase complexes. In addition to their contribution to chromatin remodeling, recent studies have shown that nuclear ARPs have roles in the organization of the nucleus that are independent of the activity of the above-mentioned complexes. Therefore, nuclear ARPs are recognized as novel key regulators of genome function, and affect not only the remodeling of chromatin but also the spatial arrangement and dynamics of chromatin within the nucleus.

Nuclear actin-related proteins at the core of epigenetic control

Nuclear Actin-Related Proteins (ARPs) and actin combine as heterodimers to bind a large helicase subunit and form a core complex essential to the assembly and function of most chromatin remodeling and modifying machines. They are the most common shared subunits of these large and diverse assemblies in eukaryotes. We recently argued that most nuclear ARPs evolved directly from actin prior to the divergence of the eukaryotic kingdoms and did not evolve from pre-existing ARPs.2 Arabidopsis plants defective in nuclear ARP4, ARP5, ARP6, or ARP7 have extreme developmental phenotypes. Our recent publication demonstrates that ARP5-defective plants are not only dwarfed and have aberrant cell sizes, but are also hypersensitive to mutagenic agents that cause double strand DNA breaks.5 In Smith et al.6 we show that ARP6-defective plants, in addition to their extreme developmental phenotypes like small organs and early flowering, present an apparent “Phosphate Starvation Response” with strong morphological and molecular phenotypes. Herein, we interpret our latest data in the light of a hypothesis stating that in addition to their roles in overcoming DNA compaction that affects basal gene expression and silencing, nuclear ARP-containing chromatin complexes exert primary epigenetic control over high-level regulatory factors.

Chapter 5 Nuclear Actin-Related Proteins in Epigenetic Control

The nuclear actin-related proteins (ARPs) share overall structure and low-level sequence homology with conventional actin. They are indispensable subunits of macromolecular machines that control chromatin remodeling and modification leading to dynamic changes in DNA structure, transcription, and DNA repair. Cellular, genetic, and biochemical studies suggest that the nuclear ARPs are essential to the epigenetic control of the cell cycle and cell proliferation in all eukaryotes, while in plants and animals they also exert epigenetic controls over most stages of multicellular development including organ initiation, the switch to reproductive development, and senescence and programmed cell death. A theme emerging from plants and animals is that in addition to their role in controlling the general compaction of DNA and gene silencing, isoforms of nuclear ARP-containing chromatin complexes have evolved to exert dynamic epigenetic control over gene expression and different phases of multicellular development. Herein, we explore this theme by examining nuclear ARP phylogeny, activities of ARP-containing chromatin remodeling complexes that lead to epigenetic control, expanding developmental roles assigned to several animal and plant ARP-containing complexes, the evidence that thousands of ARP complex isoforms may have evolved in concert with multicellular development, and ARPs in human disease.

Nuclear actin in plants

Actins constitute a wide family of proteins that are major components of the cytoskeleton. Animal cells have nuclear G-actin forms that assemble into several nuclear macromolecular complexes and are substrates for myosin I beta (NMI). The nuclear actin related proteins (ARPs) are part of the chromatin-remodelling complex, while nuclear acting binding proteins (nABPs) comprise either nuclear forms of cytoplasmic ABPs (as NMI) or specific nABPs. No evidence of the presence of nuclear actin exists in plants, which lack orthologues of the main animal structural nABPs. Here we prove the presence of actin forms with different solubility, and their associated protein NMI in the plant nucleus, as components of the transcription complexes and the nucleoskeleton. For this, WB and confocal immunofluorescence with antibodies against human actin and NMI were used.

ACTIN-RELATED PROTEIN8 Encodes an F-Box Protein Localized to the Nucleolus in Arabidopsis

Arabidopsis encodes six nuclear actin-related proteins (ARPs), among them ARP8 is unique in having an F-box domain and an actin homology domain. Analysis of the ARP8 promoter-beta-glucuronidase (GUS) fusion suggests that ARP8 is ubiquitously expressed in all organs and cell types. Immunocytochemical analysis with ARP8-specific monoclonal antibodies revealed that ARP8 protein is localized to the nucleolus in interphase cells and dispersed in the cytoplasm in mitotic cells. The cell cycle-dependent subcellular patterns of distribution of ARP8 are conserved in other members of Brassicaceae. Our findings provide the first insight into the possible contributions of plant ARP8 to nucleolar functions.

The HSA domain binds nuclear actin-related proteins to regulate chromatin-remodeling ATPases

We identify the helicase-SANT-associated (HSA) domain as the primary binding platform for nuclear actin-related proteins (ARPs) and actin. Individual HSA domains from chromatin remodelers (RSC, yeast SWI-SNF, human SWI-SNF, SWR1 and INO80) or modifiers (NuA4) reconstitute their respective ARP-ARP or ARP-actin modules. In RSC, the HSA domain resides on the catalytic ATPase subunit Sth1. The Sth1 HSA is essential in vivo, and its omission causes the specific loss of ARPs and a moderate reduction in ATPase activity. Genetic selections for arp suppressors yielded specific gain-of-function mutations in two new domains in Sth1, the post-HSA domain and protrusion 1, which are essential for RSC function in vivo but not ARP association. Taken together, we define the role of the HSA domain and provide evidence for a regulatory relationship involving the ARP-HSA module and two new functional domains conserved in remodeler ATPases that contain ARPs.

Nuclear Actin-Related Proteins as Epigenetic Regulators of Development

Complex regulatory networks control cell fate and the development of organs and tissues in multicellular organisms. But what mechanisms initiate the necessary global changes in patterns of gene expression? What regulates the regulators of organismal development? The nuclear actin-related proteins (

The nuclear actin‐related protein Act3p/Arp4p is involved in the dynamics of chromatin‐modulating complexes

Chromatin remodelling and histone-modifying complexes govern the modulation of chromatin structure. While components of these complexes are diverse, nuclear actin-related proteins (Arps) have been repeatedly found in these complexes from yeast to mammals. In most cases, Arps are required for functioning of the complexes, but the molecular mechanisms of nuclear Arps have as yet been largely unknown. The Arps and actin, sharing a common ancestor, are supposed to be highly similar in the three-dimensional structure of their core regions, including the ATP-binding pocket. The Arp Act3p/Arp4p of Saccharomyces cerevisiae exists within the nucleus, partly as a component of several high molecular mass complexes, including the NuA4 histone acetyltransferase (HAT) complex, and partly as uncomplexed molecules. We observed that mutations in the putative ATP-binding pocket of Act3p/Arp4p increased its concentration in the high molecular mass complexes and, conversely, that an excess of ATP or ATPgammaS led to the release of wild-type Act3p/Arp4p from the complexes. These results suggest a requirement of ATP binding by Act3p/Arp4p for its dissociation from the complexes. In accordance, a mutation in the putative ATP binding site of Act3p/Arp4p inhibited the conversion of the NuA4 complex into the smaller piccoloNuA4, which does not contain Act3p/Arp4p and exhibits HAT activity distinct from that of NuA4. Although the in vitro binding activity of ATP by recombinant Act3p/Arp4p was found to be rather weak, our observations, taken together, suggest that the ATP-binding pocket of Act3p/Arp4p is involved in the function of chromatin modulating complexes by regulating their dynamics.

BAF53/Arp4 homolog Alp5 in fission yeast is required for histone H4 acetylation, kinetochore-spindle attachment, and gene silencing at centromere.

Nuclear actin-related proteins play vital roles in transcriptional regulation; however, their biological roles remain elusive. Here, we characterize Alp5, fission yeast homolog of Arp4/BAF53. The temperature-sensitive mutant alp5-1134 contains a single amino acid substitution in the conserved C-terminal domain (S402N) and displays mitotic phenotypes, including chromosome condensation and missegregation. Alp5 forms a complex with Mst1-HAT (histone acetyltransferase). Consistently, inhibition of histone deacetylases (HDACs), by either addition of a specific inhibitor or a mutation in HDAC-encoding clr6+ gene, rescues alp5-1134. Immunoblotting with specific antibodies against acetylated histones shows that Alp5 is required for histone H4 acetylation at lysines 5, 8, and 12, but not histone H3 lysines 9 or 14, and furthermore Clr6 plays an opposing role. Mitotic arrest is ascribable to activation of the Mad2/Bub1 spindle checkpoint, in which both proteins localize to the mitotic kinetochores in alp5-1134. Intriguingly, alp5-1134 displays transcriptional desilencing at the core centromere without altering the overall chromatin structure, which also is suppressed by a simultaneous mutation in clr6+. This result shows that Alp5 is essential for histone H4 acetylation, and its crucial role lies in the establishment of bipolar attachment of the kinetochore to the spindle and transcriptional silencing at the centromere.

The nuclear actin-related proteins Arp7 and Arp9: a dimeric module that cooperates with architectural proteins for chromatin remodeling.

Nuclear actin-related proteins (ARPs) are essential components of chromatin remodeling and modifying complexes, but their functions and relationship to actin remain elusive. The yeast SWI/SNF and RSC complexes contain Arp7 and Arp9, and are shown to form a stable heterodimer with the properties of a functional module. Arp7 and Arp9 rely on their actin-related regions for heterodimerization, and their unique C-termini cooperate for assembly into RSC. We suggest that regulated ARP-ARP (and possibly ARP-beta-actin) heterodimerization might be a conserved feature of chromatin complexes. A RSC complex lacking Arp7/9 was isolated that displays robust nucleosome remodeling activity, suggesting a separate essential role for ARPs in the regulation of chromatin structure. A screen for suppressors of arp mutations yielded the DNA bending architectural transcription factor Nhp6, which interacts with RSC complex physically and functionally and shows facilitated binding to nucleosomes by RSC. We propose that Arp7/9 dimers function with DNA bending proteins to facilitate proper chromatin architecture and complex- complex interactions.