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Tandemly repeated DNA is highly mutable and causes at least 31 diseases, but it is hard to detect pathogenic repeat expansions genome-wide. Here, we report robust detection of human repeat expansions from careful alignments of long but error-prone (PacBio and nanopore) reads to a reference genome. Our method is robust to systematic sequencing errors, inexact repeats with fuzzy boundaries, and low sequencing coverage. By comparing to healthy controls, we prioritize pathogenic expansions within the top 10 out of 700,000 tandem repeats in whole genome sequencing data. This may help to elucidate the many genetic diseases whose causes remain unknown.

Chromosomal translocations are hallmarks of various types of cancers and leukemias. However, the molecular mechanisms of chromosome translocations remain largely unknown. The ataxia-telangiectasia mutated (ATM) protein, a DNA damage signaling regulator, facilitates DNA repair to prevent chromosome abnormalities. Previously, we showed that ATM deficiency led to the 11q23 chromosome translocation, the most frequent chromosome abnormalities in secondary leukemia. Here, we show that ARP8, a subunit of the INO80 chromatin remodeling complex, is phosphorylated after etoposide treatment. The etoposide-induced phosphorylation of ARP8 is regulated by ATM and ATR, and attenuates its interaction with INO80. The ATM-regulated phosphorylation of ARP8 reduces the excessive loading of INO80 and RAD51 onto the breakpoint cluster region. These findings suggest that the phosphorylation of ARP8, regulated by ATM, plays an important role in maintaining the fidelity of DNA repair to prevent the etoposide-induced 11q23 abnormalities., Competing Interests: JS, LS, AK, AF, YH, YO, MH, MI, TI, RK, ST No competing interests declared, (© 2018, Sun et al.)

Nuclear actin family proteins, comprising of actin and actin-related proteins (Arps), are essential functional components of the multiple chromatin remodeling complexes. The INO80 chromatin remodeling complex, which is evolutionarily conserved and has roles in transcription, DNA replication and repair, consists of actin and actin-related proteins Arp4, Arp5, and Arp8. We generated Arp5 knockout (KO) and Arp8 KO cells from the human Nalm-6 pre-B cell line and used these KO cells to examine the roles of Arp5 and Arp8 in the transcriptional regulation mediated by the INO80 complex. In both of Arp5 KO and Arp8 KO cells, the oxidative stress-induced expression of HMOX1 gene, encoding for heme oxygenase-1 (HO-1), was significantly impaired. Consistent with these observations, chromatin immunoprecipitation (ChIP) assay revealed that oxidative stress caused an increase in the binding of the INO80 complex to the regulatory sites of HMOX1 in wild-type cells. The binding of INO80 complex to chromatin was reduced in Arp8 KO cells compared to that in the wild-type cells. On the other hand, the binding of INO80 complex to chromatin in Arp5 KO cells was similar to that in the wild-type cells even under the oxidative stress condition. However, both remodeling of chromatin at the HMOX1 regulatory sites and binding of a transcriptional activator to these sites were impaired in Arp5 KO cells, indicating that Arp5 is required for the activation of the INO80 complex. Collectively, these results suggested that these nuclear Arps play indispensable roles in the function of the INO80 chromatin remodeling complex.

In some neurological diseases caused by repeat expansions such as myotonic dystrophy, the RNA-binding protein muscleblind-like 1 (MBNL1) accumulates in intranuclear inclusions containing mutant repeat RNA. The interaction between MBNL1 and mutant RNA in the nucleus is a key event leading to loss of MBNL function, yet the details of this effect have been elusive. Here, we investigated the mechanism and significance of MBNL1 nuclear localization. We found that MBNL1 contains two classes of nuclear localization signal (NLS), a classical bipartite NLS and a novel conformational NLS. Alternative splicing of exon 7 acts as a switch between these NLS types and couples MBNL1 activity and intracellular localization. Depending on its nuclear localization, MBNL1 promoted nuclear accumulation of mutant RNA containing a CUG or CAG repeat, some of which produced proteins containing homopolymeric tracts such as polyglutamine. Furthermore, MBNL1 repressed the expression of these homopolymeric proteins including those presumably produced through repeat-associated non-ATG (RAN) translation. These results suggest that nuclear retention of expanded RNA reflects a novel role of MBNL proteins in repressing aberrant protein expression and may provide pathological and therapeutic implications for a wide range of repeat expansion diseases associated with nuclear RNA retention and/or RAN translation., (© The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Actin and actin-related proteins (Arps), which are members of the actin family, are essential components of many of these remodeling complexes. Actin, Arp4, Arp5, and Arp8 are found to be evolutionarily conserved components of the INO80 chromatin remodeling complex, which is involved in transcriptional regulation, DNA replication, and DNA repair. A recent report showed that Arp8 forms a module in the INO80 complex and this module can directly capture a nucleosome. In the present study, we showed that recombinant human Arp8 binds to DNAs, and preferentially binds to single-stranded DNA. Analysis of the binding of adenine nucleotides to Arp8 mutants suggested that the ATP-binding pocket, located in the evolutionarily conserved actin fold, plays a regulatory role in the binding of Arp8 to DNA. To determine the cellular function of Arp8, we derived tetracycline-inducible Arp8 knockout cells from a cultured human cell line. Analysis of results obtained after treating these cells with aphidicolin and camptothecin revealed that Arp8 is involved in DNA repair. Together with the previous observation that Arp8, but not γ-H2AX, is indispensable for recruiting INO80 complex to DSB in human, results of our study suggest an individual role for Arp8 in DNA repair.

Persistent DNA double-strand breaks (DSBs) are recruited to the nuclear periphery in budding yeast. Both the Nup84 pore subcomplex and Mps3, an inner nuclear membrane (INM) SUN domain protein, have been implicated in DSB binding. It was unclear what, if anything, distinguishes the two potential sites of repair. Here, we characterize and distinguish the two binding sites. First, DSB-pore interaction occurs independently of cell-cycle phase and requires neither the chromatin remodeler INO80 nor recombinase Rad51 activity. In contrast, Mps3 binding is S and G2 phase specific and requires both factors. SWR1-dependent incorporation of Htz1 (H2A.Z) is necessary for break relocation to either site in both G1- and S-phase cells. Importantly, functional assays indicate that mutations in the two sites have additive repair defects, arguing that the two perinuclear anchorage sites define distinct survival pathways., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Myotonic dystrophy type 1 (DM1) is the most common muscular dystrophy in adults and as yet no cure for DM1. Here, we report the potential of manumycin A for a novel DM1 therapeutic reagent. DM1 is caused by expansion of CTG repeat. Mutant transcripts containing expanded CUG repeats lead to aberrant regulation of alternative splicing. Myotonia (delayed muscle relaxation) is the most commonly observed symptom in DM1 patients and is caused by aberrant splicing of the skeletal muscle chloride channel (CLCN1) gene. Identification of small-molecule compounds that correct aberrant splicing in DM1 is attracting much attention as a way of improving understanding of the mechanism of DM1 pathology and improving treatment of DM1 patients. In this study, we generated a reporter screening system and searched for small-molecule compounds. We found that manumycin A corrects aberrant splicing of Clcn1 in cell and mouse models of DM1.

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.

Chromosome translocations induced by DNA damaging agents, such as ionizing radiation and certain chemotherapies, alter genetic information resulting in malignant transformation. Abrogation or loss of the ataxia-telangiectasia mutated (ATM) protein, a DNA damage signaling regulator, increases the incidence of chromosome translocations. However, how ATM protects cells from chromosome translocations is still unclear. Chromosome translocations involving the MLL gene on 11q23 are the most frequent chromosome abnormalities in secondary leukemias associated with chemotherapy employing etoposide, a topoisomerase II poison. Here we show that ATM deficiency results in the excessive binding of the DNA recombination protein RAD51 at the translocation breakpoint hotspot of 11q23 chromosome translocation after etoposide exposure. Binding of Replication protein A (RPA) and the chromatin remodeler INO80, which facilitate RAD51 loading on damaged DNA, to the hotspot were also increased by ATM deficiency. Thus, in addition to activating DNA damage signaling, ATM may avert chromosome translocations by preventing excessive loading of recombinational repair proteins onto translocation breakpoint hotspots.

Actin-related proteins are ubiquitous components of chromatin remodelers and are conserved from yeast to man. We have examined the role of the budding yeast actin-related protein Arp6 in gene expression, both as a component of the SWR1 complex (SWR-C) and in its absence. We mapped Arp6 binding sites along four yeast chromosomes using chromatin immunoprecipitation from wild-type and swr1 deleted (swr1Delta) cells. We find that a majority of Arp6 binding sites coincide with binding sites of Swr1, the catalytic subunit of SWR-C, and with the histone H2A variant Htz1 (H2A.Z) deposited by SWR-C. However, Arp6 binding detected at centromeres, the promoters of ribosomal protein (RP) genes, and some telomeres is independent of Swr1 and Htz1 deposition. Given that RP genes and telomeres both show association with the nuclear periphery, we monitored the ability of Arp6 to mediate the localization of chromatin to nuclear pores. Arp6 binding is sufficient to shift a randomly positioned locus to nuclear periphery, even in a swr1Delta strain. Arp6 is also necessary for the pore association of its targeted RP promoters possibly through cell cycle-dependent factors. Loss of Arp6, but not Htz1, leads to an up-regulation of these RP genes. In contrast, the pore-association of GAL1 correlates with Htz1 deposition, and loss of Arp6 reduces both GAL1 activation and peripheral localization. We conclude that Arp6 functions both together with the nucleosome remodeler Swr1 and also without it, to mediate Htz1-dependent and Htz1-independent binding of chromatin domains to nuclear pores. This association is shown to have modulating effects on gene expression., Competing Interests: The authors have declared that no competing interests exist.

The expression and function of the skeletal muscle chloride channel CLCN1/ClC-1 is regulated by alternative splicing. Inclusion of the CLCN1 exon 7A is aberrantly elevated in myotonic dystrophy (DM), a genetic disorder caused by the expansion of a CTG or CCTG repeat. Increased exon 7A inclusion leads to a reduction in CLCN1 function, which can be causative of myotonia. Two RNA-binding protein families--muscleblind-like (MBNL) and CUG-BP and ETR-3-like factor (CELF) proteins--are thought to mediate the splicing misregulation in DM. Here, we have identified multiple factors that regulate the alternative splicing of a mouse Clcn1 minigene. The inclusion of exon 7A was repressed by MBNL proteins while promoted by an expanded CUG repeat or CELF4, but not by CUG-BP. Mutation analyses suggested that exon 7A and its flanking region mediate the effect of MBNL1, whereas another distinct region in intron 6 mediates that of CELF4. An exonic splicing enhancer essential for the inclusion of exon 7A was identified at the 5' end of this exon, which might be inhibited by MBNL1. Collectively, these results provide a mechanistic model for the regulation of Clcn1 splicing, and reveal novel regulatory properties of MBNL and CELF proteins.

In recent years, several novel types of disorder caused by the expansion of triplet repeats in specific genes have been characterized; in the "polyalanine diseases", these expanded repeats result in proteins with aberrantly elongated polyalanine tracts. In this study, we fused expanded polyalanine tracts to yellow fluorescent protein to examine their physical interaction with mitochondria. Tracts containing more than 23 alanine repeats were found to physically associate with mitochondria, strongly suggesting that an interaction between polyalanine tracts and mitochondria is a contributing factor in the pathology of polyalanine diseases. Furthermore, in in vitro experiments, polyalanine tracts induced release of cytochrome c from mitochondria and caspase-3 activation, independently of the mitochondrial permeability transition pore. These results suggest that oligomerized polyalanine tracts might induce the rupture of the mitochondrial membrane, the subsequent release of cytochrome c, and apoptosis. This novel mechanism for polyalanine tract cytotoxicity might be common to the pathogenesis of all polyalanine diseases. Further investigation of this mechanism might aid the development of therapies for these diseases.

Background: 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., Results: 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., Conclusions: The INO80 chromatin remodeling complex is enriched at stalled replication forks, where it promotes the resumption of replication upon recovery from fork arrest.

Many human proteins have homopolymeric amino acid (HPAA) tracts, but their physiological functions or cellular effects are not well understood. Previously, we expressed 20 HPAAs in mammalian cells and showed characteristic intracellular localization, in that hydrophobic HPAAs aggregated strongly and caused high cytotoxicity in proportion to their hydrophobicity. In the present study, we investigated the cytotoxicity of these aggregate-prone hydrophobic HPAAs, assuming that the ubiquitin proteasome system is impaired in the same manner as other well-known aggregate-prone polyglutamine-containing proteins. Some highly hydrophobic HPAAs caused a deficiency in the ubiquitin proteasome system and excess endoplasmic reticulum stress, leading to apoptosis. These results indicate that the property of causing excess endoplasmic reticulum stress by proteasome impairment may contribute to the strong cytotoxicity of highly hydrophobic HPAAs, and proteasome impairment and the resulting excess endoplasmic reticulum stress is not a common cytotoxic effect of aggregate-prone proteins such as polyglutamine.

Many human proteins contain consecutive amino acid repeats, known as homopolymeric amino acid (HPAA) tracts. Some inherited diseases are caused by proteins in which HPAAs are expanded to an excessive length. To this day, nine polyglutamine-related diseases and nine polyalanine-related diseases have been reported, including Huntington's disease and oculopharyngeal muscular dystrophy. In this study, potential HPAA-HPAA interactions were examined by yeast two-hybrid assays using HPAAs of approximately 30 residues in length. The results indicate that hydrophobic HPAAs interact with themselves and with other hydrophobic HPAAs. Previously, we reported that hydrophobic HPAAs formed large aggregates in COS-7 cells. Here, those HPAAs were shown to have significant interactions with each other, suggesting that hydrophobicity plays an important role in aggregation. Among the observed HPAA-HPAA interactions, the Ala28-Ala29 interaction was notable because polyalanine tracts of these lengths have been established to be pathogenic in several polyalanine-related diseases. By testing several constructs of different lengths, we clarified that polyalanine self-interacts at longer lengths (>23 residues) but not at shorter lengths (six to approximately 23 residues) in a yeast two-hybrid assay and a GST pulldown assay. This self-interaction was found to be SDS sensitive in SDS-PAGE and native-PAGE assays. Moreover, the intracellular localization of these long polyalanine tracts was also observed to be disturbed. Our results suggest that long tracts of polyalanine acquire SDS-sensitive self-association properties, which may be a prerequisite event for their abnormal folding. The misfolding of these tracts is thought to be a common molecular aspect underlying the pathogenesis of polyalanine-related diseases.

Actin-related proteins (ARPs) are key players in cytoskeleton activities and nuclear functions. Two complexes, ARP2/3 and ARP1/11, also known as dynactin, are implicated in actin dynamics and in microtubule-based trafficking, respectively. ARP4 to ARP9 are components of many chromatin-modulating complexes. Conventional actins and ARPs codefine a large family of homologous proteins, the actin superfamily, with a tertiary structure known as the actin fold. Because ARPs and actin share high sequence conservation, clear family definition requires distinct features to easily and systematically identify each subfamily. In this study we performed an in depth sequence and comparative genomic analysis of ARP subfamilies. A high-quality multiple alignment of approximately 700 complete protein sequences homologous to actin, including 148 ARP sequences, allowed us to extend the ARP classification to new organisms. Sequence alignments revealed conserved residues, motifs, and inserted sequence signatures to define each ARP subfamily. These discriminative characteristics allowed us to develop ARPAnno (http://bips.u-strasbg.fr/ARPAnno), a new web server dedicated to the annotation of ARP sequences. Analyses of sequence conservation among actins and ARPs highlight part of the actin fold and suggest interactions between ARPs and actin-binding proteins. Finally, analysis of ARP distribution across eukaryotic phyla emphasizes the central importance of nuclear ARPs, particularly the multifunctional ARP4.

Many human proteins have homopolymeric amino acid (HPAA) tracts, which are involved in protein-protein interactions and also have intrinsic polymerization properties. Polyglutamine or polyalanine expansions cause several neurodegenerative diseases. To examine the properties of HPAAs, we expressed 20 kinds of 30-residue HPAA fused to the C terminus of yellow fluorescent protein in mammalian cells. Specific localization was observed depending on the HPAA. Polyarginine and polylysine aggregated in the nucleus. Polyalanine, polyhistidine, polyisoleucine, polyleucine, polymethionine, polyphenylalanine, polythreonine, polytryptophan, and polyvaline localized in the cytoplasm, and some of these HPAAs formed aggregate(s). Hydrophobic HPAAs such as polyisoleucine, polyleucine, polyphenylalanine, and polyvaline were found as one major aggregate or cumulus in the perinuclear region. Western blot analysis indicated that hydrophobic HPAA tracts appear to oligomerize and form high molecular weight complexes. These results indicate that hydrophobicity itself may trigger the oligomerization and aggregation of proteins when overexpressed in cells. Our experiments provide novel insights into the nature of the HPAAs that are often seen in human and other organisms.

Actin-related proteins (Arps), which share a basal structure with actin but have distinct functions, have been found in a wide variety of organisms. While their functions are not yet clear, some Arps are localized in the nucleus and are suggested to contribute to the regulation of transcription. An essential gene of Saccharomyces cerevisiae, Act3p/Arp4, encodes the first identified nuclear Arp, which has been shown to bind to core histones in vitro. Here we have analyzed the in vivo function of Act3p/Arp4 on the his4-912delta promoter. Chromatin immunoprecipitation assays show that Act3p/Arp4 is bound to the entire his4-912delta promoter region. Conditional act3/arp4 mutations affect transcription from the his4-912delta promoter, where decreased Act3p/Arp4 binding and a change in nuclease sensitivity of chromatin were observed, showing the involvement of Act3p/Arp4 in the regulation of gene expression through the organization of chromatin structure. Taken together with the presence of Act3p/Arp4 in chromatin remodeling and histone acetyltransferase complexes, it is suggested that Act3p/Arp4 functions in transcriptional regulation to recruit chromatin remodeling and histone acetyltransferase complexes onto chromatin.

Act3p/Arp4, an essential actin-related protein of Saccharomyces cerevisiae located within the nucleus, is, according to genetic data, involved in transcriptional regulation. In addition to the basal core structure of the actin family members, which is responsible for ATPase activity, Act3p possesses two insertions, insertions I and II, the latter of which is predicted to form a loop-like structure protruding from beyond the surface of the molecule. Because Act3p is a constituent of chromatin but itself does not bind to DNA, we hypothesized that insertion II might be responsible for an Act3p-specific function through its interaction with some other chromatin protein. Far Western blot and two-hybrid analyses revealed the ability of insertion II to bind to each of the core histones, although with somewhat different affinities. Together with our finding of coimmunoprecipitation of Act3p with histone H2A, this suggests the in vivo existence of a protein complex required for correct expression of particular genes. We also show that a conditional act3 mutation affects chromatin structure of an episomal DNA molecule, indicating that the putative Act3p complex may be involved in the establishment, remodeling, or maintenance of chromatin structures.