CGG repeat expansion in LRP12 in amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder characterized by the degeneration of motor neurons. Although repeat expansion in C9orf72 is its most common cause, the pathogenesis of ALS isn't fully clear. In this study, we show that repeat expansion in LRP12, a causative variant of oculopharyngodistal myopathy type 1 (OPDM1), is a cause of ALS. We identify CGG repeat expansion in LRP12 in five families and two simplex individuals. These ALS individuals (LRP12-ALS) have 61-100 repeats, which contrasts with most OPDM individuals with repeat expansion in LRP12 (LRP12-OPDM), who have 100-200 repeats. Phosphorylated TDP-43 is present in the cytoplasm of iPS cell-derived motor neurons (iPSMNs) in LRP12-ALS, a finding that reproduces the pathological hallmark of ALS. RNA foci are more prominent in muscle and iPSMNs in LRP12-ALS than in LRP12-OPDM. Muscleblind-like 1 aggregates are observed only in OPDM muscle. In conclusion, CGG repeat expansions in LRP12 cause ALS and OPDM, depending on the length of the repeat. Our findings provide insight into the repeat length-dependent switching of phenotypes.

Insufficiency of ciliary cholesterol in hereditary Zellweger syndrome.

Primary cilia are antenna-like organelles on the surface of most mammalian cells that receive sonic hedgehog (Shh) signaling in embryogenesis and carcinogenesis. Cellular cholesterol functions as a direct activator of a seven-transmembrane oncoprotein called Smoothened (Smo) and thereby induces Smo accumulation on the ciliary membrane where it transduces the Shh signal. However, how cholesterol is supplied to the ciliary membrane remains unclear. Here, we report that peroxisomes are essential for the transport of cholesterol into the ciliary membrane. Zellweger syndrome (ZS) is a peroxisome-deficient hereditary disorder with several ciliopathy-related features and cells from these patients showed a reduced cholesterol level in the ciliary membrane. Reverse genetics approaches revealed that the GTP exchange factor Rabin8, the Rab GTPase Rab10, and the microtubule minus-end-directed kinesin KIFC3 form a peroxisome-associated complex to control the movement of peroxisomes along microtubules, enabling communication between peroxisomes and ciliary pocket membranes. Our findings suggest that insufficient ciliary cholesterol levels may underlie ciliopathies.

YAP is essential for tissue tension to ensure vertebrate 3D body shape.

Vertebrates have a unique 3D body shape in which correct tissue and organ shape and alignment are essential for function. For example, vision requires the lens to be centred in the eye cup which must in turn be correctly positioned in the head. Tissue morphogenesis depends on force generation, force transmission through the tissue, and response of tissues and extracellular matrix to force. Although a century ago D'Arcy Thompson postulated that terrestrial animal body shapes are conditioned by gravity, there has been no animal model directly demonstrating how the aforementioned mechano-morphogenetic processes are coordinated to generate a body shape that withstands gravity. Here we report a unique medaka fish (Oryzias latipes) mutant, hirame (hir), which is sensitive to deformation by gravity. hir embryos display a markedly flattened body caused by mutation of YAP, a nuclear executor of Hippo signalling that regulates organ size. We show that actomyosin-mediated tissue tension is reduced in hir embryos, leading to tissue flattening and tissue misalignment, both of which contribute to body flattening. By analysing YAP function in 3D spheroids of human cells, we identify the Rho GTPase activating protein ARHGAP18 as an effector of YAP in controlling tissue tension. Together, these findings reveal a previously unrecognised function of YAP in regulating tissue shape and alignment required for proper 3D body shape. Understanding this morphogenetic function of YAP could facilitate the use of embryonic stem cells to generate complex organs requiring correct alignment of multiple tissues.

A novel CDK-independent function of p27Kip1 in preciliary vesicle trafficking during ciliogenesis.

p27Kip1, a member of the Cip/Kip family of cyclin-dependent kinase (CDK) inhibitors, is now known as a multifunctional protein that plays crucial roles in cell architecture and migration by regulating rearrangements of the actin cytoskeleton and microtubules. The intracellular level of p27Kip1 is increased by anti-proliferative stimuli, such as mitogen deprivation and contact inhibition, which also induce formation of primary cilia, microtubule-based membranous organelles that protrude from the cell surface. However, it remains unknown whether p27Kip1 is associated with ciliogenesis. Here, we have generated p27Kip1-knockout hTERT-immortalized human retinal pigment epithelial cells, and found that ciliogenesis is almost completely disrupted in p27Kip1-knockout cells. The defect of ciliogenesis is rescued by the exogenous expression of wild-type p27Kip1 and, surprisingly, its 86-140 amino acid region, which is neither responsible for CDK inhibition nor remodeling of the actin cytoskeleton and microtubules. Moreover, transmission electron microscopy and immunofluorescence analyses reveal that p27Kip1 abrogation impairs one of the earliest events of ciliogenesis, docking of the Ehd1-associated preciliary vesicles to the distal appendages of the basal body. Our findings identify a novel CDK-independent function of p27Kip1 in primary cilia formation.

PLK1-mediated phosphorylation of WDR62/MCPH2 ensures proper mitotic spindle orientation.

Primary microcephaly (MCPH) is an autosomal recessive disorder characterized by congenital reduction of head circumference. Here, we identified compound heterozygous mutations c.731 C > T (p.Ser 244 Leu) and c.2413 G > T (p.Glu 805 X) in the WDR62/MCPH2 gene, which encodes the mitotic centrosomal protein WDR62, in two siblings in a Japanese family with microcephaly using whole-exome sequencing. However, the molecular and cellular pathology of microcephaly caused by WDR62/MCPH2 mutation remains unclear. To clarify the physiological role of WDR62, we used the CRISPR/Cas9 system and single-stranded oligonucleotides as a point-mutation-targeting donor to generate human cell lines with knock-in of WDR62/MCPH2 c.731 C > T (p.Ser 244 Leu) missense mutation. In normal metaphase, the mitotic spindle forms parallel to the substratum to ensure symmetric cell division, while WDR62/MCPH2-mutated cells exhibited a randomized spindle orientation caused by the impaired astral microtubule assembly. It was shown that a mitotic kinase, Polo-like kinase 1 (PLK1), is required for the maintenance of spindle orientation through astral microtubule development. In this study, we demonstrated that WDR62 is a PLK1 substrate that is phosphorylated at Ser 897, and that this phosphorylation at the spindle poles promotes astral microtubule assembly to stabilize spindle orientation. Our findings provide insights into the role of the PLK1-WDR62 pathway in the maintenance of proper spindle orientation.

Albatross/FBF1 contributes to both centriole duplication and centrosome separation.

The centrosome is a small but important organelle that participates in centriole duplication, spindle formation, and ciliogenesis. Each event is regulated by key enzymatic reactions, but how these processes are integrated remains unknown. Recent studies have reported that ciliogenesis is controlled by distal appendage proteins such as FBF1, also known as Albatross. However, the precise role of Albatross in the centrosome cycle, including centriole duplication and centrosome separation, remains to be determined. Here, we report a novel function for Albatross at the proximal ends of centrioles. Using Albatross monospecific antibodies, full-length constructs, and siRNAs for rescue experiments, we found that Albatross mediates centriole duplication by recruiting HsSAS-6, a cartwheel protein of centrioles. Moreover, Albatross participates in centrosome separation during mitosis by recruiting Plk1 to residue S348 of Albatross after its phosphorylation. Taken together, our results show that Albatross is a novel protein that spatiotemporally integrates different aspects of centrosome function, namely ciliogenesis, centriole duplication, and centrosome separation.

NBS1 recruits RAD18 via a RAD6-like domain and regulates Pol η-dependent translesion DNA synthesis.

Translesion DNA synthesis, a process orchestrated by monoubiquitinated PCNA, is critical for DNA damage tolerance. While the ubiquitin-conjugating enzyme RAD6 and ubiquitin ligase RAD18 are known to monoubiquitinate PCNA, how they are regulated by DNA damage is not fully understood. We show that NBS1 (mutated in Nijmegen breakage syndrome) binds to RAD18 after UV irradiation and mediates the recruitment of RAD18 to sites of DNA damage. Disruption of NBS1 abolished RAD18-dependent PCNA ubiquitination and Polη focus formation, leading to elevated UV sensitivity and mutation. Unexpectedly, the RAD18-interacting domain of NBS1, which was mapped to its C terminus, shares structural and functional similarity with the RAD18-interacting domain of RAD6. These domains of NBS1 and RAD6 allow the two proteins to interact with RAD18 homodimers simultaneously and are crucial for Polη-dependent UV tolerance. Thus, in addition to chromosomal break repair, NBS1 plays a key role in translesion DNA synthesis.

Updated summary of genome editing technology in human cultured cells linked to human genetics studies.

Current deep-sequencing technology provides a mass of nucleotide variations associated with human genetic disorders to accelerate the identification of causative mutations. To understand the etiology of genetic disorders, reverse genetics in human cultured cells is a useful approach for modeling a disease in vitro. However, gene targeting in human cultured cells is difficult because of their low activity of homologous recombination. Engineered endonucleases enable enhancement of the local activation of DNA repair pathways at the human genome target site to rewrite the desired sequence, thereby efficiently generating disease-modeling cultured cell clones. These edited cells can be used to explore the molecular functions of a causative gene product to uncover the etiological mechanisms. The correction of mutations in patient cells using genome editing technology could contribute to the development of unique gene therapies. This technology can also be applied to screening causative mutations. Rare genetic disorders and non-exonic mutation-caused diseases remain frontier in the field of human genetics as it is difficult to validate whether the extracted nucleotide variants are mutation or polymorphism. When isogenic human cultured cells with a candidate variant reproduce the pathogenic phenotypes, it is confirmed that the variant is a causative mutation.

TALEN-mediated single-base-pair editing identification of an intergenic mutation upstream of BUB1B as causative of PCS (MVA) syndrome.

Cancer-prone syndrome of premature chromatid separation with mosaic variegated aneuploidy [PCS (MVA) syndrome] is a rare autosomal recessive disorder characterized by constitutional aneuploidy and a high risk of childhood cancer. We previously reported monoallelic mutations in the BUB1B gene (encoding BUBR1) in seven Japanese families with the syndrome. No second mutation was found in the opposite allele of any of the families studied, although a conserved BUB1B haplotype and a decreased transcript were identified. To clarify the molecular pathology of the second allele, we extended our mutational search to a candidate region surrounding BUB1B. A unique single nucleotide substitution, G > A at ss802470619, was identified in an intergenic region 44 kb upstream of a BUB1B transcription start site, which cosegregated with the disorder. To examine whether this is the causal mutation, we designed a transcription activator-like effector nuclease-mediated two-step single-base pair editing strategy and biallelically introduced this substitution into cultured human cells. The cell clones showed reduced BUB1B transcripts, increased PCS frequency, and MVA, which are the hallmarks of the syndrome. We also encountered a case of a Japanese infant with PCS (MVA) syndrome carrying a homozygous single nucleotide substitution at ss802470619. These results suggested that the nucleotide substitution identified was the causal mutation of PCS (MVA) syndrome.

Mutations of optineurin in amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) has its onset in middle age and is a progressive disorder characterized by degeneration of motor neurons of the primary motor cortex, brainstem and spinal cord. Most cases of ALS are sporadic, but about 10% are familial. Genes known to cause classic familial ALS (FALS) are superoxide dismutase 1 (SOD1), ANG encoding angiogenin, TARDP encoding transactive response (TAR) DNA-binding protein TDP-43 (ref. 4) and fused in sarcoma/translated in liposarcoma (FUS, also known as TLS). However, these genetic defects occur in only about 20-30% of cases of FALS, and most genes causing FALS are unknown. Here we show that there are mutations in the gene encoding optineurin (OPTN), earlier reported to be a causative gene of primary open-angle glaucoma (POAG), in patients with ALS. We found three types of mutation of OPTN: a homozygous deletion of exon 5, a homozygous Q398X nonsense mutation and a heterozygous E478G missense mutation within its ubiquitin-binding domain. Analysis of cell transfection showed that the nonsense and missense mutations of OPTN abolished the inhibition of activation of nuclear factor kappa B (NF-kappaB), and the E478G mutation revealed a cytoplasmic distribution different from that of the wild type or a POAG mutation. A case with the E478G mutation showed OPTN-immunoreactive cytoplasmic inclusions. Furthermore, TDP-43- or SOD1-positive inclusions of sporadic and SOD1 cases of ALS were also noticeably immunolabelled by anti-OPTN antibodies. Our findings strongly suggest that OPTN is involved in the pathogenesis of ALS. They also indicate that NF-kappaB inhibitors could be used to treat ALS and that transgenic mice bearing various mutations of OPTN will be relevant in developing new drugs for this disorder.

Zinc-finger nuclease-mediated targeted insertion of reporter genes for quantitative imaging of gene expression in sea urchin embryos.

To understand complex biological systems, such as the development of multicellular organisms, it is important to characterize the gene expression dynamics. However, there is currently no universal technique for targeted insertion of reporter genes and quantitative imaging in multicellular model systems. Recently, genome editing using zinc-finger nucleases (ZFNs) has been reported in several models. ZFNs consist of a zinc-finger DNA-binding array with the nuclease domain of the restriction enzyme FokI and facilitate targeted transgene insertion. In this study, we successfully inserted a GFP reporter cassette into the HpEts1 gene locus of the sea urchin, Hemicentrotus pulcherrimus. We achieved this insertion by injecting eggs with a pair of ZFNs for HpEts1 with a targeting donor construct that contained ∼1-kb homology arms and a 2A-histone H2B-GFP cassette. We increased the efficiency of the ZFN-mediated targeted transgene insertion by in situ linearization of the targeting donor construct and cointroduction of an mRNA for a dominant-negative form of HpLig4, which encodes the H. pulcherrimus homolog of DNA ligase IV required for error-prone nonhomologous end joining. We measured the fluorescence intensity of GFP at the single-cell level in living embryos during development and found that there was variation in HpEts1 expression among the primary mesenchyme cells. These findings demonstrate the feasibility of ZFN-mediated targeted transgene insertion to enable quantification of the expression levels of endogenous genes during development in living sea urchin embryos.

Efficient TALEN construction and evaluation methods for human cell and animal applications.

Transcription activator-like effector nucleases (TALENs) have recently arisen as effective tools for targeted genome engineering. Here, we report streamlined methods for the construction and evaluation of TALENs based on the 'Golden Gate TALEN and TAL Effector Kit' (Addgene). We diminished array vector requirements and increased assembly rates using six-module concatemerization. We altered the architecture of the native TALEN protein to increase nuclease activity and replaced the final destination vector with a mammalian expression/in vitro transcription vector bearing both CMV and T7 promoters. Using our methods, the whole process, from initiating construction to completing evaluation directly in mammalian cells, requires only 1 week. Furthermore, TALENs constructed in this manner may be directly applied to transfection of cultured cells or mRNA synthesis for use in animals and embryos. In this article, we show genomic modification of HEK293T cells, human induced pluripotent stem cells, Drosophila melanogaster, Danio rerio and Xenopus laevis, using custom-made TALENs constructed and evaluated with our protocol. Our methods are more time efficient compared with conventional yeast-based evaluation methods and provide a more accessible and effective protocol for the application of TALENs in various model organisms.

Insufficiency of BUBR1, a mitotic spindle checkpoint regulator, causes impaired ciliogenesis in vertebrates.

Budding uninhibited by benzimidazole-related 1 (BUBR1) is a central molecule of the spindle assembly checkpoint. Germline mutations in the budding uninhibited by benzimidazoles 1 homolog beta gene encoding BUBR1 cause premature chromatid separation (mosaic variegated aneuploidy) [PCS (MVA)] syndrome, which is characterized by constitutional aneuploidy and a high risk of childhood cancer. Patients with the syndrome often develop Dandy-Walker complex and polycystic kidneys; implying a critical role of BUBR1 in morphogenesis. However, little is known about the function of BUBR1 other than mitotic control. Here, we report that BUBR1 is essential for the primary cilium formation, and that the PCS (MVA) syndrome is thus a novel ciliopathy. Morpholino knockdown of bubr1 in medaka fish also caused ciliary dysfunction characterized by defects in cerebellar development and perturbed left-right asymmetry of the embryo. Biochemical analyses demonstrated that BUBR1 is required for ubiquitin-mediated proteasomal degradation of cell division cycle protein 20 in the G0 phase and maintains anaphase-promoting complex/cyclosome-CDC20 homolog 1 activity that regulates the optimal level of dishevelled for ciliogenesis.

Imaging of the Ciliary Cholesterol Underlying the Sonic Hedgehog Signal Transduction.

Primary cilia are antenna-like structures that develop on the surface of quiescent G0-phase cells and receive extracellular signals including sonic hedgehog (Shh) for embryogenesis and adult tissue homeostasis. In mammalian cells, cholesterol activates the seven-transmembrane protein Smoothened to transduce the Shh signal. Germline mutations of the DHCR7 gene encoding the cholesterol biogenesis enzyme 7-dehydrocholesterol reductase cause Smith-Lemli-Opitz syndrome with ciliopathy-related symptoms such as polycystic kidney and polydactyly, implying that cholesterol is indeed involved in ciliary functions. Notably, it has been reported that the cholesterol in ciliary membranes is significantly more abundant than that in the rest of the plasma membrane. However, several studies have failed to image the enriched ciliary cholesterol. Here, we propose a set of protocols for the sensitive imaging of ciliary cholesterol using the fluorescent small compound Filipin III and the green fluorescent protein tagged Domain 4 of the exotoxin Perfringolysin O derived from the anaerobic bacterium Clostridium perfringens. These cholesterol probes should be powerful tools for understanding the physiological and pathological roles of ciliary cholesterol in the context of Shh signaling in mammalian cells.

iPSC reprogramming-mediated aneuploidy correction in autosomal trisomy syndromes.

Trisomy 21, 18, and 13 are the major autosomal aneuploidy disorders in humans. They are mostly derived from chromosome non-disjunction in maternal meiosis, and the extra trisomic chromosome can cause several congenital malformations. Various genes on the trisomic chromosomes are intricately involved in the development of disease, and fundamental treatments have not yet been established. However, chromosome therapy has been developed to correct the extra chromosome in cultured patient cells, and it was recently reported that during reprogramming into iPSCs, fibroblasts from a Down syndrome patient lost the extra chromosome 21 due to a phenomenon called trisomy-biased chromosome loss. To gain preliminary insights into the underlying mechanism of trisomy rescue during the early stages of reprogramming, we reprogrammed skin fibroblasts from patients with trisomy syndromes 21, 18, 13, and 9 to iPSC, and evaluated the genomes of the individual iPSC colonies by molecular cytogenetic techniques. We report the spontaneous correction from trisomy to disomy upon cell reprogramming in at least one cell line examined from each of the trisomy syndromes, and three possible combinations of chromosomes were selected in the isogenic trisomy-rescued iPSC clones. Single nucleotide polymorphism analysis showed that the trisomy-rescued clones exhibited either heterodisomy or segmental uniparental isodisomy, ruling out the possibility that two trisomic chromosomes were lost simultaneously and the remaining one was duplicated, suggesting instead that one trisomic chromosome was lost to generate disomic cells. These results demonstrated that trisomy rescue may be a phenomenon with random loss of the extra chromosome and subsequent selection for disomic iPSCs, which is analogous to the karyotype correction in early preimplantation embryos. Our finding is relevant for elucidating the mechanisms of autonomous karyotype correction and future application in basic and clinical research on aneuploidy disorders.

NBS1 I171V variant underlies individual differences in chromosomal radiosensitivity within human populations.

Genetic information is protected against a variety of genotoxins including ionizing radiation (IR) through the DNA double-strand break (DSB) repair machinery. Genome-wide association studies and clinical sequencing of cancer patients have suggested that a number of variants in the DNA DSB repair genes might underlie individual differences in chromosomal radiosensitivity within human populations. However, the number of established variants that directly affect radiosensitivity is still limited. In this study, we performed whole-exome sequencing of 29 Japanese ovarian cancer patients and detected the NBS1 I171V variant, which is estimated to exist at a rate of approximately 0.15% in healthy human populations, in one patient. To clarify whether this variant indeed contributes to chromosomal radiosensitivity, we generated NBS1 I171V variant homozygous knock-in HCT116 cells and mice using the CRISPR/Cas9 system. Radiation-induced micronucleus formation and chromosomal aberration frequency were significantly increased in both HCT116 cells and mouse embryonic fibroblasts (MEFs) with knock-in of the NBS1 I171V variant compared with the levels in wild-type cells. These results suggested that the NBS1 I171V variant might be a genetic factor underlying individual differences in chromosomal radiosensitivity.

Applications of Genome Editing Technology in Research on Chromosome Aneuploidy Disorders.

Chromosomal segregation errors in germ cells and early embryonic development underlie aneuploidies, which are numerical chromosomal abnormalities causing fetal absorption, developmental anomalies, and carcinogenesis. It has been considered that human aneuploidy disorders cannot be resolved by radical treatment. However, recent studies have demonstrated that aneuploidies can be rescued to a normal diploid state using genetic engineering in cultured cells. Here, we summarize a series of studies mainly applying genome editing to eliminate an extra copy of human chromosome 21, the cause of the most common constitutional aneuploidy disorder Down syndrome. We also present findings on induced pluripotent stem cell reprogramming, which has been shown to be one of the most promising technologies for converting aneuploidies into normal diploidy without the risk of genetic alterations such as genome editing-mediated off-target effects.

Premature aging syndrome showing random chromosome number instabilities with CDC20 mutation.

Damage to the genome can accelerate aging. The percentage of aneuploid cells, that is, cells with an abnormal number of chromosomes, increases during aging; however, it is not clear whether increased aneuploidy accelerates aging. Here, we report an individual showing premature aging phenotypes of various organs including early hair loss, atrophic skin, and loss of hematopoietic stem cells; instability of chromosome numbers known as mosaic variegated aneuploidy (MVA); and spindle assembly checkpoint (SAC) failure. Exome sequencing identified a de novo heterozygous germline missense mutation of c.856C>A (p.R286S) in the mitotic activator CDC20. The mutant CDC20 showed lower binding affinity to BUBR1 during the formation of the mitotic checkpoint complex (MCC), but not during the interaction between MCC and the anaphase-promoting complex/cyclosome (APC/C)-CDC20 complex. While heterozygous knockout of CDC20 did not induce SAC failure, knock-in of the mutant CDC20 induced SAC failure and random aneuploidy in cultured cells, indicating that the particular missense mutation is pathogenic probably via the resultant imbalance between MCC and APC/C-CDC20 complex. We postulate that accelerated chromosome number instability induces premature aging in humans, which may be associated with early loss of stem cells. These findings could form the basis of a novel disease model of the aging of the body and organs.