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Showing papers on "Mutant published in 2017"


Journal ArticleDOI
02 Aug 2017-Nature
TL;DR: The efficiency, accuracy and safety of the approach presented suggest that it has potential to be used for the correction of heritable mutations in human embryos by complementing preimplantation genetic diagnosis.
Abstract: Genome editing has potential for the targeted correction of germline mutations. Here we describe the correction of the heterozygous MYBPC3 mutation in human preimplantation embryos with precise CRISPR-Cas9-based targeting accuracy and high homology-directed repair efficiency by activating an endogenous, germline-specific DNA repair response. Induced double-strand breaks (DSBs) at the mutant paternal allele were predominantly repaired using the homologous wild-type maternal gene instead of a synthetic DNA template. By modulating the cell cycle stage at which the DSB was induced, we were able to avoid mosaicism in cleaving embryos and achieve a high yield of homozygous embryos carrying the wild-type MYBPC3 gene without evidence of off-target mutations. The efficiency, accuracy and safety of the approach presented suggest that it has potential to be used for the correction of heritable mutations in human embryos by complementing preimplantation genetic diagnosis. However, much remains to be considered before clinical applications, including the reproducibility of the technique with other heterozygous mutations.

691 citations


Journal ArticleDOI
TL;DR: It is reported that IDH1/2 mutations induce a homologous recombination defect that renders tumor cells exquisitely sensitive to poly(adenosine 5′-diphosphate–ribose) polymerase (PARP) inhibitors, and an unexpected link between oncometabolites, altered DNA repair, and genetic instability is uncovered.
Abstract: 2-Hydroxyglutarate (2HG) exists as two enantiomers, (R)-2HG and (S)-2HG, and both are implicated in tumor progression via their inhibitory effects on α-ketoglutarate (αKG)-dependent dioxygenases. The former is an oncometabolite that is induced by the neomorphic activity conferred by isocitrate dehydrogenase 1 (IDH1) and IDH2 mutations, whereas the latter is produced under pathologic processes such as hypoxia. We report that IDH1/2 mutations induce a homologous recombination (HR) defect that renders tumor cells exquisitely sensitive to poly(adenosine 5'-diphosphate-ribose) polymerase (PARP) inhibitors. This "BRCAness" phenotype of IDH mutant cells can be completely reversed by treatment with small-molecule inhibitors of the mutant IDH1 enzyme, and conversely, it can be entirely recapitulated by treatment with either of the 2HG enantiomers in cells with intact IDH1/2 proteins. We demonstrate mutant IDH1-dependent PARP inhibitor sensitivity in a range of clinically relevant models, including primary patient-derived glioma cells in culture and genetically matched tumor xenografts in vivo. These findings provide the basis for a possible therapeutic strategy exploiting the biological consequences of mutant IDH, rather than attempting to block 2HG production, by targeting the 2HG-dependent HR deficiency with PARP inhibition. Furthermore, our results uncover an unexpected link between oncometabolites, altered DNA repair, and genetic instability.

394 citations


Journal ArticleDOI
TL;DR: This work generated loss-of-function mutant alleles of a tomato gene, SlDML2, which is a close homolog of the Arabidopsis DNA demethylase gene ROS1, and discovered that SlD ML2 is required for the demethylation and activation of genes important for fruit ripening, including genes involved in fruit pigment and flavor synthesis, ethylene synthesis and signaling, and cell wall hydrolysis.
Abstract: DNA methylation is a conserved epigenetic mark important for genome integrity, development, and environmental responses in plants and mammals Active DNA demethylation in plants is initiated by a family of 5-mC DNA glycosylases/lyases (ie, DNA demethylases) Recent reports suggested a role of active DNA demethylation in fruit ripening in tomato In this study, we generated loss-of-function mutant alleles of a tomato gene, SlDML2, which is a close homolog of the Arabidopsis DNA demethylase gene ROS1 In the fruits of the tomato mutants, increased DNA methylation was found in thousands of genes These genes included not only hundreds of ripening-induced genes but also many ripening-repressed genes Our results show that SlDML2 is critical for tomato fruit ripening and suggest that active DNA demethylation is required for both the activation of ripening-induced genes and the inhibition of ripening-repressed genes

293 citations


Journal ArticleDOI
TL;DR: The findings demonstrate a mechanism of immune evasion in IDH-MUT gliomas and suggest that specific inhibitors of mutant IDH may improve the efficacy of immunotherapy in patients with IDH -MUTgliomas.
Abstract: Mutations in the isocitrate dehydrogenase genes IDH1 and IDH2 are among the first genetic alterations observed during the development of lower-grade glioma (LGG). LGG-associated IDH mutations confer gain-of-function activity by converting α-ketoglutarate to the oncometabolite R-2-hydroxyglutarate (2HG). Clinical samples and gene expression data from The Cancer Genome Atlas (TCGA) demonstrate reduced expression of cytotoxic T lymphocyte-associated genes and IFN-γ-inducible chemokines, including CXCL10, in IDH-mutated (IDH-MUT) tumors compared with IDH-WT tumors. Given these findings, we have investigated the impact of IDH mutations on the immunological milieu in LGG. In immortalized normal human astrocytes (NHAs) and syngeneic mouse glioma models, the introduction of mutant IDH1 or treatment with 2HG reduced levels of CXCL10, which was associated with decreased production of STAT1, a regulator of CXCL10. Expression of mutant IDH1 also suppressed the accumulation of T cells in tumor sites. Reductions in CXCL10 and T cell accumulation were reversed by IDH-C35, a specific inhibitor of mutant IDH1. Furthermore, IDH-C35 enhanced the efficacy of vaccine immunotherapy in mice bearing IDH-MUT gliomas. Our findings demonstrate a mechanism of immune evasion in IDH-MUT gliomas and suggest that specific inhibitors of mutant IDH may improve the efficacy of immunotherapy in patients with IDH-MUT gliomas.

280 citations


Journal ArticleDOI
TL;DR: This work has identified SNPs that either cause or destroy PAM motifs critical for CRISPR-selective editing of one allele versus the other in cells from HD patients and in a transgenic HD model harboring the human allele.

226 citations


Journal ArticleDOI
TL;DR: Selective, targeted mutagenesis of the three delta‐12‐desaturase (FAD2) genes was achieved by CRISPR‐Cas9 gene editing, leading to reduced levels of polyunsaturated fatty acids and increased accumulation of oleic acid in the oil.
Abstract: In many plant species, gene dosage is an important cause of phenotype variation. Engineering gene dosage, particularly in polyploid genomes, would provide an efficient tool for plant breeding. The hexaploid oilseed crop Camelina sativa, which has three closely related expressed subgenomes, is an ideal species for investigation of the possibility of creating a large collection of combinatorial mutants. Selective, targeted mutagenesis of the three delta-12-desaturase (FAD2) genes was achieved by CRISPR-Cas9 gene editing, leading to reduced levels of polyunsaturated fatty acids and increased accumulation of oleic acid in the oil. Analysis of mutations over four generations demonstrated the presence of a large variety of heritable mutations in the three isologous CsFAD2 genes. The different combinations of single, double and triple mutants in the T3 generation were isolated, and the complete loss-of-function mutants revealed the importance of delta-12-desaturation for Camelina development. Combinatorial association of different alleles for the three FAD2 loci provided a large diversity of Camelina lines with various lipid profiles, ranging from 10% to 62% oleic acid accumulation in the oil. The different allelic combinations allowed an unbiased analysis of gene dosage and function in this hexaploid species, but also provided a unique source of genetic variability for plant breeding.

198 citations


Journal ArticleDOI
TL;DR: In this article, the photoreceptor genes COP1/2, COP3 (encoding channelrhodopsin 1 [ChR1]), COP4, COP5, PHOT, UVR8, VGCC, MAT3, and aCRY were used for gene editing in Chlamydomonas reinhardtii.
Abstract: The fast-growing biflagellated single-celled chlorophyte Chlamydomonas reinhardtii is the most widely used alga in basic research. The physiological functions of the 18 sensory photoreceptors are of particular interest with respect to Chlamydomonas development and behavior. Despite the demonstration of gene editing in Chlamydomonas in 1995, the isolation of mutants lacking easily ascertained newly acquired phenotypes remains problematic due to low DNA recombination efficiency. We optimized gene-editing protocols for several Chlamydomonas strains (including wild-type CC-125) using zinc-finger nucleases (ZFNs), genetically encoded CRISPR/associated protein 9 (Cas9) from Staphylococcus aureus and Streptococcus pyogenes, and recombinant Cas9 and developed protocols for rapidly isolating nonselectable gene mutants. Using this technique, we disrupted the photoreceptor genes COP1/2, COP3 (encoding channelrhodopsin 1 [ChR1]), COP4 (encoding ChR2), COP5, PHOT, UVR8, VGCC, MAT3, and aCRY and created the chr1 chr2 and uvr8 phot double mutants. Characterization of the chr1, chr2, and mat3 mutants confirmed the value of photoreceptor mutants for physiological studies. Genes of interest were disrupted in 5 to 15% of preselected clones (∼1 out of 4000 initial cells). Using ZFNs, genes were edited in a reliable, predictable manner via homologous recombination, whereas Cas9 primarily caused gene disruption via the insertion of cotransformed DNA. These methods should be widely applicable to research involving green algae.

196 citations


Journal ArticleDOI
TL;DR: Interestingly, palmitoylated monomers of ANCL CSP α mutants were shown to be short-lived compared with wild-type CSPα, suggesting that the mutants either have a faster rate of depalmitoylation or that they are consumed in a time-dependent manner into high molecular weight aggregates.
Abstract: Autosomal-dominant adult-onset neuronal ceroid lipofuscinosis (ANCL) is caused by mutation of the DNAJC5 gene encoding cysteine string protein alpha (CSPα). The disease-causing mutations, which result in substitution of leucine-115 with an arginine (L115R) or deletion of the neighbouring leucine-116 (∆L116) in the cysteine-string domain cause CSPα to form high molecular weight SDS-resistant aggregates, which are also present in post-mortem brain tissue from patients. Formation and stability of these mutant aggregates is linked to palmitoylation of the cysteine-string domain, however the regions of the mutant proteins that drive aggregation have not been determined. The importance of specific residues in the cysteine-string domain was investigated, revealing that a central core of palmitoylated cysteines is essential for aggregation of ANCL CSPα mutants. Interestingly, palmitoylated monomers of ANCL CSPα mutants were shown to be short-lived compared with wild-type CSPα, suggesting that the mutants either have a faster rate of depalmitoylation or that they are consumed in a time-dependent manner into high molecular weight aggregates. These findings provide new insight into the features of CSPα that promote aggregation in the presence of L115R/∆L116 mutations and reveal a change in the lifetime of palmitoylated monomers of the mutant proteins.

184 citations



Journal ArticleDOI
TL;DR: The feasibility of curing genetic disease in human somatic cells and embryos by base editor system is demonstrated and base editor variants, with narrowed deamination window, could promote G-to-A conversion at HBB −28 site precisely in human embryos.
Abstract: β-Thalassemia is a global health issue, caused by mutations in the HBB gene. Among these mutations, HBB -28 (A>G) mutations is one of the three most common mutations in China and Southeast Asia patients with β-thalassemia. Correcting this mutation in human embryos may prevent the disease being passed onto future generations and cure anemia. Here we report the first study using base editor (BE) system to correct disease mutant in human embryos. Firstly, we produced a 293T cell line with an exogenous HBB -28 (A>G) mutant fragment for gRNAs and targeting efficiency evaluation. Then we collected primary skin fibroblast cells from a β-thalassemia patient with HBB -28 (A>G) homozygous mutation. Data showed that base editor could precisely correct HBB -28 (A>G) mutation in the patient's primary cells. To model homozygous mutation disease embryos, we constructed nuclear transfer embryos by fusing the lymphocyte or skin fibroblast cells with enucleated in vitro matured (IVM) oocytes. Notably, the gene correction efficiency was over 23.0% in these embryos by base editor. Although these embryos were still mosaic, the percentage of repaired blastomeres was over 20.0%. In addition, we found that base editor variants, with narrowed deamination window, could promote G-to-A conversion at HBB -28 site precisely in human embryos. Collectively, this study demonstrated the feasibility of curing genetic disease in human somatic cells and embryos by base editor system.

171 citations


Journal ArticleDOI
TL;DR: TACCA combines lossless genome-complexity reduction via chromosome flow sorting with Chicago long-range linkage to assemble complex genomes and was applied to produce a high-quality de novo chromosome assembly of the wheat line CH Campala Lr22a in only 4 months.
Abstract: Cereal crops such as wheat and maize have large repeat-rich genomes that make cloning of individual genes challenging Moreover, gene order and gene sequences often differ substantially between cultivars of the same crop species A major bottleneck for gene cloning in cereals is the generation of high-quality sequence information from a cultivar of interest In order to accelerate gene cloning from any cropping line, we report 'targeted chromosome-based cloning via long-range assembly' (TACCA) TACCA combines lossless genome-complexity reduction via chromosome flow sorting with Chicago long-range linkage to assemble complex genomes We applied TACCA to produce a high-quality (N50 of 976 Mb) de novo chromosome assembly of the wheat line CH Campala Lr22a in only 4 months Using this assembly we cloned the broad-spectrum Lr22a leaf-rust resistance gene, using molecular marker information and ethyl methanesulfonate (EMS) mutants, and found that Lr22a encodes an intracellular immune receptor homologous to the Arabidopsis thaliana RPM1 protein

Journal ArticleDOI
TL;DR: This study demonstrates the application of CRISPR-Cas9/Cpf1 to precisely target genomic locations and develop transgene-free homozygous heritable gene edits and confirms that the loss of function analysis of the candidate genes emerging from different systems biology based approaches, could be performed, and therefore, this system adds value in the validation of gene function studies.
Abstract: CRISPR-Cas9/Cpf1 system with its unique gene targeting efficiency, could be an important tool for functional study of early developmental genes through the generation of successful knockout plants. The introduction and utilization of systems biology approaches have identified several genes that are involved in early development of a plant and with such knowledge a robust tool is required for the functional validation of putative candidate genes thus obtained. The development of the CRISPR-Cas9/Cpf1 genome editing system has provided a convenient tool for creating loss of function mutants for genes of interest. The present study utilized CRISPR/Cas9 and CRISPR-Cpf1 technology to knock out an early developmental gene EPFL9 (Epidermal Patterning Factor like-9, a positive regulator of stomatal development in Arabidopsis) orthologue in rice. Germ-line mutants that were generated showed edits that were carried forward into the T2 generation when Cas9-free homozygous mutants were obtained. The homozygous mutant plants showed more than an eightfold reduction in stomatal density on the abaxial leaf surface of the edited rice plants. Potential off-target analysis showed no significant off-target effects. This study also utilized the CRISPR-LbCpf1 (Lachnospiracae bacterium Cpf1) to target the same OsEPFL9 gene to test the activity of this class-2 CRISPR system in rice and found that Cpf1 is also capable of genome editing and edits get transmitted through generations with similar phenotypic changes seen with CRISPR-Cas9. This study demonstrates the application of CRISPR-Cas9/Cpf1 to precisely target genomic locations and develop transgene-free homozygous heritable gene edits and confirms that the loss of function analysis of the candidate genes emerging from different systems biology based approaches, could be performed, and therefore, this system adds value in the validation of gene function studies.

Journal ArticleDOI
TL;DR: BA 1436032 has been shown to significantly prolong survival of mice intracerebrally transplanted with human astrocytoma carrying the IDH1R132H mutation and is developed a pan-inhibitor targeting tumors with different IDH 1R132 mutations.
Abstract: Mutations in codon 132 of isocitrate dehydrogenase (IDH) 1 are frequent in diffuse glioma, acute myeloid leukemia, chondrosarcoma and intrahepatic cholangiocarcinoma. These mutations result in a neomorphic enzyme specificity which leads to a dramatic increase of intracellular D-2-hydroxyglutarate (2-HG) in tumor cells. Therefore, mutant IDH1 protein is a highly attractive target for inhibitory drugs. Here, we describe the development and properties of BAY 1436032, a pan-inhibitor of IDH1 protein with different codon 132 mutations. BAY 1436032 strongly reduces 2-HG levels in cells carrying IDH1-R132H, -R132C, -R132G, -R132S and -R132L mutations. Cells not carrying IDH mutations were unaffected. BAY 1436032 did not exhibit toxicity in vitro or in vivo. The pharmacokinetic properties of BAY 1436032 allow for oral administration. In two independent experiments, BAY 1436032 has been shown to significantly prolong survival of mice intracerebrally transplanted with human astrocytoma carrying the IDH1R132H mutation. In conclusion, we developed a pan-inhibitor targeting tumors with different IDH1R132 mutations.

Journal ArticleDOI
TL;DR: The data provide the first functional evidence that dmrt1 is a male determining gene in tongue sole, and TALEN of dmRT1 efficiently induced mutations of this gene.
Abstract: Chinese tongue sole is a marine fish with ZW sex determination. Genome sequencing suggested that the Z-linked dmrt1 is a putative male determination gene, but direct genetic evidence is still lacking. Here we show that TALEN of dmrt1 efficiently induced mutations of this gene. The ZZ dmrt1 mutant fish developed ovary-like testis, and the spermatogenesis was disrupted. The female-related genes foxl2 and cyp19a1a were significantly increased in the gonad of the ZZ dmrt1 mutant. Conversely, the male-related genes Sox9a and Amh were significantly decreased. The dmrt1 deficient ZZ fish grew much faster than ZZ male control. Notably, we obtained an intersex ZW fish with a testis on one side and an ovary on the other side. This fish was chimeric for a dmrt1 mutation in the ovary, and wild-type dmrt1 in the testis. Our data provide the first functional evidence that dmrt1 is a male determining gene in tongue sole.

Journal ArticleDOI
TL;DR: This study identified cold tolerance phenotype of T1 mutant lines from T0 biallelic mutants using the 4∼6°C for 3 days cold treatment and suggested that OsAnn3 was involved in cold tolerance of rice.
Abstract: Plant annexins are Ca2+-dependent phospholipid-binding proteins and exist as multigene families in plants. They are implicated in the regulation of plant development as well as protection from environmental stresses. In this study, the rice annexin gene OsAnn3 knockout was performed via the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR associated proteins) mediated genome editing. Thus, mutant plantlets were successfully obtained. We identified cold tolerance phenotype of T1 mutant lines from T0 biallelic mutants using the 4∼6°C for 3 days cold treatment. The results showed that REC (the relative electrical conductivity) of T1 mutant lines was increased, and the survival ratio of T1 mutant lines was decreased dramatically compared with the wild type after the exposure to cold treatment. It was suggested that OsAnn3 was involved in cold tolerance of rice.

Journal ArticleDOI
TL;DR: It is shown that LEC1 transcriptionally regulates genes involved in photosynthesis and other developmental processes in early and maturation genes in late seed development, and strong conservation in the developmental processes and gene networks regulated by L EC1 is demonstrated in two dicotyledonous plants that diverged ∼92 Mya.
Abstract: LEAFY COTYLEDON1 (LEC1), an atypical subunit of the nuclear transcription factor Y (NF-Y) CCAAT-binding transcription factor, is a central regulator that controls many aspects of seed development including the maturation phase during which seeds accumulate storage macromolecules and embryos acquire the ability to withstand desiccation. To define the gene networks and developmental processes controlled by LEC1, genes regulated directly by and downstream of LEC1 were identified. We compared the mRNA profiles of wild-type and lec1-null mutant seeds at several stages of development to define genes that are down-regulated or up-regulated by the lec1 mutation. We used ChIP and differential gene-expression analyses in Arabidopsis seedlings overexpressing LEC1 and in developing Arabidopsis and soybean seeds to identify globally the target genes that are transcriptionally regulated by LEC1 in planta. Collectively, our results show that LEC1 controls distinct gene sets at different developmental stages, including those that mediate the temporal transition between photosynthesis and chloroplast biogenesis early in seed development and seed maturation late in development. Analyses of enriched DNA sequence motifs that may act as cis-regulatory elements in the promoters of LEC1 target genes suggest that LEC1 may interact with other transcription factors to regulate distinct gene sets at different stages of seed development. Moreover, our results demonstrate strong conservation in the developmental processes and gene networks regulated by LEC1 in two dicotyledonous plants that diverged ∼92 Mya.

Journal ArticleDOI
TL;DR: The establishment and validation of gene panel next generation sequencing (NGS) for the molecular diagnostics of gliomas is reported and implicate gene panel NGS as a promising diagnostic technique that may facilitate integrated histological and molecular glioma classification.
Abstract: Current classification of gliomas is based on histological criteria according to the World Health Organization (WHO) classification of tumors of the central nervous system. Over the past years, characteristic genetic profiles have been identified in various glioma types. These can refine tumor diagnostics and provide important prognostic and predictive information. We report on the establishment and validation of gene panel next generation sequencing (NGS) for the molecular diagnostics of gliomas. We designed a glioma-tailored gene panel covering 660 amplicons derived from 20 genes frequently aberrant in different glioma types. Sensitivity and specificity of glioma gene panel NGS for detection of DNA sequence variants and copy number changes were validated by single gene analyses. NGS-based mutation detection was optimized for application on formalin-fixed paraffin-embedded tissue specimens including small stereotactic biopsy samples. NGS data obtained in a retrospective analysis of 121 gliomas allowed for their molecular classification into distinct biological groups, including (i) isocitrate dehydrogenase gene (IDH) 1 or 2 mutant astrocytic gliomas with frequent α-thalassemia/mental retardation syndrome X-linked (ATRX) and tumor protein p53 (TP53) gene mutations, (ii) IDH mutant oligodendroglial tumors with 1p/19q codeletion, telomerase reverse transcriptase (TERT) promoter mutation and frequent Drosophila homolog of capicua (CIC) gene mutation, as well as (iii) IDH wildtype glioblastomas with frequent TERT promoter mutation, phosphatase and tensin homolog (PTEN) mutation and/or epidermal growth factor receptor (EGFR) amplification. Oligoastrocytic gliomas were genetically assigned to either of these groups. Our findings implicate gene panel NGS as a promising diagnostic technique that may facilitate integrated histological and molecular glioma classification.

Journal ArticleDOI
TL;DR: This work demonstrates how inexpensive next-generation sequencing can be applied to generate a high-density catalog of mutations and reveals the usefulness of the resource for efficient, cost-effective identification of genes conferring specific phenotypes.
Abstract: The availability of a whole-genome sequenced mutant population and the cataloging of mutations of each line at a single-nucleotide resolution facilitate functional genomic analysis. To this end, we generated and sequenced a fast-neutron-induced mutant population in the model rice cultivar Kitaake (Oryza sativa ssp japonica), which completes its life cycle in 9 weeks. We sequenced 1504 mutant lines at 45-fold coverage and identified 91,513 mutations affecting 32,307 genes, i.e., 58% of all rice genes. We detected an average of 61 mutations per line. Mutation types include single-base substitutions, deletions, insertions, inversions, translocations, and tandem duplications. We observed a high proportion of loss-of-function mutations. We identified an inversion affecting a single gene as the causative mutation for the short-grain phenotype in one mutant line. This result reveals the usefulness of the resource for efficient, cost-effective identification of genes conferring specific phenotypes. To facilitate public access to this genetic resource, we established an open access database called KitBase that provides access to sequence data and seed stocks. This population complements other available mutant collections and gene-editing technologies. This work demonstrates how inexpensive next-generation sequencing can be applied to generate a high-density catalog of mutations.

Journal ArticleDOI
TL;DR: A forward genetics approach was used to investigate the mechanism of arsenate (As(V) tolerance and accumulation in rice and identified OsHAC4 as the causal gene for the As(V)-hypersensitive phenotype.
Abstract: Soil contamination with arsenic (As) can cause phytotoxicity and elevated As accumulation in rice grain. Here, we used a forward genetics approach to investigate the mechanism of arsenate (As(V)) tolerance and accumulation in rice. A rice mutant hypersensitive to As(V), but not to As(III), was isolated. Genomic resequencing and complementation tests were used to identify the causal gene. The function of the gene, its expression pattern and subcellular localization were characterized. OsHAC4 is the causal gene for the As(V)-hypersensitive phenotype. The gene encodes a rhodanase-like protein that shows As(V) reductase activity when expressed in Escherichia coli. OsHAC4 was highly expressed in roots and was induced by As(V). In OsHAC4pro-GUS transgenic plants, the gene was expressed exclusively in the root epidermis and exodermis. OsHAC4-eGFP was localized in the cytoplasm and the nucleus. Mutation in OsHAC4 resulted in decreased As(V) reduction in roots, decreased As(III) efflux to the external medium and markedly increased As accumulation in rice shoots. Overexpression of OsHAC4 increased As(V) tolerance and decreased As accumulation in rice plants. OsHAC4 is an As(V) reductase that is critical for As(V) detoxification and for the control of As accumulation in rice. As(V) reduction, followed by As(III) efflux, is an important mechanism of As(V) detoxification.

Journal ArticleDOI
TL;DR: The generation and characterization of genome-edited T47D and MCF7 breast cancer cell lines with the two most common ESR1 mutations are described, revealing ligand-independent growth and endocrine resistance and identifying novel target genes involved in metastasis-associated phenotypes.
Abstract: Mutations in the estrogen receptor alpha (ERα) 1 gene (ESR1) are frequently detected in ER+ metastatic breast cancer, and there is increasing evidence that these mutations confer endocrine resistance in breast cancer patients with advanced disease. However, their functional role is not well-understood, at least in part due to a lack of ESR1 mutant models. Here, we describe the generation and characterization of genome-edited T47D and MCF7 breast cancer cell lines with the two most common ESR1 mutations, Y537S and D538G. Genome editing was performed using CRISPR and adeno-associated virus (AAV) technologies to knock-in ESR1 mutations into T47D and MCF7 cell lines, respectively. Various techniques were utilized to assess the activity of mutant ER, including transactivation, growth and chromatin-immunoprecipitation (ChIP) assays. The level of endocrine resistance was tested in mutant cells using a number of selective estrogen receptor modulators (SERMs) and degraders (SERDs). RNA sequencing (RNA-seq) was employed to study gene targets of mutant ER. Cells with ESR1 mutations displayed ligand-independent ER activity, and were resistant to several SERMs and SERDs, with cell line and mutation-specific differences with respect to magnitude of effect. The SERD AZ9496 showed increased efficacy compared to other drugs tested. Wild-type and mutant cell co-cultures demonstrated a unique evolution of mutant cells under estrogen deprivation and tamoxifen treatment. Transcriptome analysis confirmed ligand-independent regulation of ERα target genes by mutant ERα, but also identified novel target genes, some of which are involved in metastasis-associated phenotypes. Despite significant overlap in the ligand-independent genes between Y537S and D538G, the number of mutant ERα-target genes shared between the two cell lines was limited, suggesting context-dependent activity of the mutant receptor. Some genes and phenotypes were unique to one mutation within a given cell line, suggesting a mutation-specific effect. Taken together, ESR1 mutations in genome-edited breast cancer cell lines confer ligand-independent growth and endocrine resistance. These biologically relevant models can be used for further mechanistic and translational studies, including context-specific and mutation site-specific analysis of the ESR1 mutations.

Journal ArticleDOI
TL;DR: A gene-indexed genome-wide mutation collection is generated through ethyl methanesulfonate mutagenesis andApplication of this mutant collection is exemplified by the identification of the ent-kaurene synthase gene, which encodes a key enzyme in the gibberellin biosynthesis pathway.

Journal ArticleDOI
21 Sep 2017-PLOS ONE
TL;DR: It is demonstrated that HWS, CUC1 and CUC2 act together to control floral organ number and loss of function of HWS is associated with larger petal size due to alterations in cell proliferation and mitotic growth, a role shared with the C UC1 gene.
Abstract: The Arabidopsis thaliana F-box gene HAWAIIAN SKIRT (HWS) affects organ growth and the timing of floral organ abscission. The loss-of-function hws-1 mutant exhibits fused sepals and increased organ size. To understand the molecular mechanisms of HWS during plant development, we mutagenized hws-1 seeds with ethylmethylsulphonate (EMS) and screened for mutations suppressing hws-1 associated phenotypes. We isolated the shs1/hws-1 (suppressor of hws-1) mutant in which hws-1 sepal fusion phenotype was suppressed. The shs1/hws-1 mutant carries a G→A nucleotide substitution in the MIR164 binding site of CUP-SHAPED COTYLEDON 1 (CUC1) mRNA. CUC1 and CUP-SHAPED COTYLEDON 2 (CUC2) transcript levels were altered in shs1, renamed cuc1-1D, and in hws-1 mutant. Genetic interaction analyses using single, double and triple mutants of cuc1-1D, cuc2-1D (a CUC2 mutant similar to cuc1-1D), and hws-1, demonstrate that HWS, CUC1 and CUC2 act together to control floral organ number. Loss of function of HWS is associated with larger petal size due to alterations in cell proliferation and mitotic growth, a role shared with the CUC1 gene.

Journal ArticleDOI
TL;DR: It is demonstrated that in a subset of initially IDH 1 mutant gliomas IDH1 is deleted or amplified at recurrence, yielding a higher grade tumor with a reprogrammed epigenome, further suggestive of selection against the heterozygous mutant state as tumors progress.
Abstract: IDH1 mutation is the earliest genetic alteration in low-grade gliomas (LGGs), but its role in tumor recurrence is unclear. Mutant IDH1 drives overproduction of the oncometabolite d-2-hydroxyglutarate (2HG) and a CpG island (CGI) hypermethylation phenotype (G-CIMP). To investigate the role of mutant IDH1 at recurrence, we performed a longitudinal analysis of 50 IDH1 mutant LGGs. We discovered six cases with copy number alterations (CNAs) at the IDH1 locus at recurrence. Deletion or amplification of IDH1 was followed by clonal expansion and recurrence at a higher grade. Successful cultures derived from IDH1 mutant, but not IDH1 wild type, gliomas systematically deleted IDH1 in vitro and in vivo, further suggestive of selection against the heterozygous mutant state as tumors progress. Tumors and cultures with IDH1 CNA had decreased 2HG, maintenance of G-CIMP, and DNA methylation reprogramming outside CGI. Thus, while IDH1 mutation initiates gliomagenesis, in some patients mutant IDH1 and 2HG are not required for later clonal expansions.

Journal ArticleDOI
23 Feb 2017-Cell
TL;DR: A compensatory FANCA somatic mutation from an "experiment of nature" in monozygotic twins both prevented anemia and reduced HSP90 binding and provides one plausible mechanism for the variable expressivity and environmental sensitivity of genetic diseases.

Journal ArticleDOI
09 Feb 2017-Oncogene
TL;DR: The high rate of base substitution mutagenesis demonstrated by the experiments is likely to significantly contribute to the oncogenic effect of the inactivation of BRCA1 or BRCa2.
Abstract: Loss-of-function mutations in the BRCA1 and BRCA2 genes increase the risk of cancer. Owing to their function in homologous recombination repair, much research has focused on the unstable genomic phenotype of BRCA1/2 mutant cells manifest mainly as large-scale rearrangements. We used whole-genome sequencing of multiple isogenic chicken DT40 cell clones to precisely determine the consequences of BRCA1/2 loss on all types of genomic mutagenesis. Spontaneous base substitution mutation rates increased sevenfold upon the disruption of either BRCA1 or BRCA2, and the arising mutation spectra showed strong and specific correlation with a mutation signature associated with BRCA1/2 mutant tumours. To model endogenous alkylating damage, we determined the mutation spectrum caused by methyl methanesulfonate (MMS), and showed that MMS also induces more base substitution mutations in BRCA1/2-deficient cells. Spontaneously arising and MMS-induced insertion/deletion mutations and large rearrangements were also more common in BRCA1/2 mutant cells compared with the wild-type control. A difference in the short deletion phenotypes of BRCA1 and BRCA2 suggested distinct roles for the two proteins in the processing of DNA lesions, as BRCA2 mutants contained more short deletions, with a wider size distribution, which frequently showed microhomology near the breakpoints resembling repair by non-homologous end joining. An increased and prolonged gamma-H2AX signal in MMS-treated BRCA1/2 cells suggested an aberrant processing of stalled replication forks as the cause of increased mutagenesis. The high rate of base substitution mutagenesis demonstrated by our experiments is likely to significantly contribute to the oncogenic effect of the inactivation of BRCA1 or BRCA2.

Journal ArticleDOI
TL;DR: Data suggested that c‐di‐AMP modulates central metabolism at the pyruvate node to moderate citrate production and indeed, the ΔdacA mutant accumulated six times the concentration of citrate present in wild‐type bacteria.
Abstract: Cyclic diadenosine monophosphate (c-di-AMP) is a conserved nucleotide second messenger critical for bacterial growth and resistance to cell wall-active antibiotics. In Listeria monocytogenes, the sole diadenylate cyclase, DacA, is essential in rich, but not synthetic media and ΔdacA mutants are highly sensitive to the β-lactam antibiotic cefuroxime. In this study, loss of function mutations in the oligopeptide importer (oppABCDF) and glycine betaine importer (gbuABC) allowed ΔdacA mutants to grow in rich medium. Since oligopeptides were sufficient to inhibit growth of the ΔdacA mutant we hypothesized that oligopeptides act as osmolytes, similar to glycine betaine, to disrupt intracellular osmotic pressure. Supplementation with salt stabilized the ΔdacA mutant in rich medium and restored cefuroxime resistance. Additional suppressor mutations in the acetyl-CoA binding site of pyruvate carboxylase (PycA) rescued cefuroxime resistance and resulted in a 100-fold increase in virulence of the ΔdacA mutant. PycA is inhibited by c-di-AMP and these mutations prompted us to examine the role of TCA cycle enzymes. Inactivation of citrate synthase, but not down-stream enzymes suppressed ΔdacA phenotypes. These data suggested that c-di-AMP modulates central metabolism at the pyruvate node to moderate citrate production and indeed, the ΔdacA mutant accumulated six times the concentration of citrate present in wild-type bacteria.

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TL;DR: Cl cloning and functional characterization of maize Defective kernel 35 indicate that Dek35 encodes an PPR protein that affects the cis-splicing of mitochondrial nad4 intron 1 and is required for mitochondrial function and seed development.

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TL;DR: A unifying molecular mechanism of CO function relates it to the NF-YA paradigm, as part of a trimeric complex imparting sequence specificity to HFD/DNA interactions.
Abstract: Nuclear Factor Y (NF-Y) is a heterotrimeric transcription factor that binds CCAAT elements The NF-Y trimer is composed of a Histone Fold Domain (HFD) dimer (NF-YB/NF-YC) and NF-YA, which confers DNA sequence specificity NF-YA shares a conserved domain with the CONSTANS, CONSTANS-LIKE, TOC1 (CCT) proteins We show that CONSTANS (CO/B-BOX PROTEIN1 BBX1), a master flowering regulator, forms a trimer with Arabidopsis thaliana NF-YB2/NF-YC3 to efficiently bind the CORE element of the FLOWERING LOCUS T promoter We term this complex NF-CO Using saturation mutagenesis, electrophoretic mobility shift assays, and RNA-sequencing profiling of co, nf-yb, and nf-yc mutants, we identify CCACA elements as the core NF-CO binding site CO physically interacts with the same HFD surface required for NF-YA association, as determined by mutations in NF-YB2 and NF-YC9, and tested in vitro and in vivo The co-7 mutation in the CCT domain, corresponding to an NF-YA arginine directly involved in CCAAT recognition, abolishes NF-CO binding to DNA In summary, a unifying molecular mechanism of CO function relates it to the NF-YA paradigm, as part of a trimeric complex imparting sequence specificity to HFD/DNA interactions It is likely that members of the large CCT family participate in similar complexes with At-NF-YB and At-NF-YC, broadening HFD combinatorial possibilities in terms of trimerization, DNA binding specificities, and transcriptional regulation

Journal ArticleDOI
TL;DR: Results revealed that AtrR plays a pivotal role in a novel azole resistance mechanism by co-regulating the drug target (Cyp51A) and putative drug efflux pump (Cdr1B).
Abstract: Successful treatment of aspergillosis caused by Aspergillus fumigatus is threatened by an increasing incidence of drug resistance. This situation is further complicated by the finding that strains resistant to azoles, the major antifungal drugs for aspergillosis, have been widely disseminated across the globe. To elucidate mechanisms underlying azole resistance, we identified a novel transcription factor that is required for normal azole resistance in Aspergillus fungi including A. fumigatus, Aspergillus oryzae, and Aspergillus nidulans. This fungal-specific Zn2-Cys6 type transcription factor AtrR was found to regulate expression of the genes related to ergosterol biosynthesis, including cyp51A that encodes a target protein of azoles. The atrR deletion mutant showed impaired growth under hypoxic conditions and attenuation of virulence in murine infection model for aspergillosis. These results were similar to the phenotypes for a mutant strain lacking SrbA that is also a direct regulator for the cyp51A gene. Notably, AtrR was responsible for the expression of cdr1B that encodes an ABC transporter related to azole resistance, whereas SrbA was not involved in the regulation. Chromatin immunoprecipitation assays indicated that AtrR directly bound both the cyp51A and cdr1B promoters. In the clinically isolated itraconazole resistant strain that harbors a mutant Cyp51A (G54E), deletion of the atrR gene resulted in a hypersensitivity to the azole drugs. Together, our results revealed that AtrR plays a pivotal role in a novel azole resistance mechanism by co-regulating the drug target (Cyp51A) and putative drug efflux pump (Cdr1B).

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TL;DR: It is found that haematopoietic cells expressing mutant U2AF1(S34F), including primary patient cells, have an increased sensitivity to in vitro sudemycin treatment relative to controls, suggesting a potential for treating haem atological cancers harbouring U2 AF1 mutations with pre-mRNA splicing modulators like sudemies.
Abstract: Somatic mutations in spliceosome genes are detectable in ∼50% of patients with myelodysplastic syndromes (MDS). We hypothesize that cells harbouring spliceosome gene mutations have increased sensitivity to pharmacological perturbation of the spliceosome. We focus on mutant U2AF1 and utilize sudemycin compounds that modulate pre-mRNA splicing. We find that haematopoietic cells expressing mutant U2AF1(S34F), including primary patient cells, have an increased sensitivity to in vitro sudemycin treatment relative to controls. In vivo sudemycin treatment of U2AF1(S34F) transgenic mice alters splicing and reverts haematopoietic progenitor cell expansion induced by mutant U2AF1 expression. The splicing effects of sudemycin and U2AF1(S34F) can be cumulative in cells exposed to both perturbations-drug and mutation-compared with cells exposed to either alone. These cumulative effects may result in downstream phenotypic consequences in sudemycin-treated mutant cells. Taken together, these data suggest a potential for treating haematological cancers harbouring U2AF1 mutations with pre-mRNA splicing modulators like sudemycins.