Calnexin co-expression and the use of weaker promoters increase the expression of correctly assembled Shaker potassium channel in insect cells

AbstractVoltage-gated potassium channels control the membrane potential of excitable cells. To understand their function, knowledge of their structure is essential. However, these channels are scarce in natural sources, and overexpression is necessary to generate material for structural studies. We have compared functional expression of the Drosophila Shaker H4 potassium channel in stable insect cell lines and in baculovirus-infected insect cells, using three different baculovirus promoters. Stable insect cell lines expressed correctly assembled channel, which was glycosylated and found predominantly at, or close to, the cell surface. In comparison, the majority of baculovirus-overexpressed Shaker was intracellular and incorrectly assembled. The proportion of functional Shaker increased, however, if the weaker basic protein promoter was used rather than the stronger p10 or polyhedrin promoters. In addition, co-expression of the molecular chaperone, calnexin, increased the quantity of correctly assembled channel protein, suggesting that calnexin can be used to increase the efficiency of channel expression in insect cells

Chemical inhibition of NAT10 corrects defects of laminopathic cells.

Down-regulation and mutations of the nuclear-architecture proteins lamin A and C cause misshapen nuclei and altered chromatin organization associated with cancer and laminopathies, including the premature-aging disease Hutchinson-Gilford progeria syndrome (HGPS). Here, we identified the small molecule "Remodelin" that improved nuclear architecture, chromatin organization, and fitness of both human lamin A/C-depleted cells and HGPS-derived patient cells and decreased markers of DNA damage in these cells. Using a combination of chemical, cellular, and genetic approaches, we identified the acetyl-transferase protein NAT10 as the target of Remodelin that mediated nuclear shape rescue in laminopathic cells via microtubule reorganization. These findings provide insights into how NAT10 affects nuclear architecture and suggest alternative strategies for treating laminopathies and aging.We thank Imagif and Institut de Chimie des Substances Naturelles, centre de recherche CNRS de Gif-sur-Yvette, France, for proteomic analysis. Research in the Jackson laboratory is funded by Cancer Research UK program grant 11 C6/A11224, the European Research Council, and the European Community Seventh Framework Programme grant agreement no. HEALTH-F2-2010-259893 (DDR). Core funding is provided by CRUK (C6946/A14492) and the Wellcome Trust (WT092096). S.P.J. receives his salary from the University of Cambridge, UK, supplemented by CRUK. D.L is funded by an EMBO Long-term fellowship ALTF 834-2011 and by a Project Grant from the Medical Research Council, UK MR/L019116/1, S.B. was funded by an EMBO Long-Term fellowship ALTF 93-2010 and Cancer Research UK. R.R. is supported by the Centre National de la Recherche Scientifique. M.D. is supported by the European Research Council grant DDREAM.

CRISPR-Cas9^D10A nickase-based genotypic and phenotypic screening to enhance genome editing

The RNA-guided Cas9 nuclease is being widely employed to engineer the genomes of various cells and organisms. Despite the efficient mutagenesis induced by Cas9, off target effects have raised concerns over the system’s specificity. Recently a “doublenicking” strategy using catalytic mutant Cas9^D10A nickase has been developed to minimise off-target effects. Here, we describe a Cas9^D10A -based screening approach that combines an All-in-One Cas9^D10A nickase vector with fluorescence-activated cell sorting enrichment followed by high-throughput genotypic and phenotypic clonal screening strategies to generate isogenic knockouts and knock-ins highly efficiently, with minimal off-target effects. We validated this approach by targeting genes for the DNA-damage response proteins MDC1, 53BP1, RIF1 and P53, plus the nuclear architecture proteins Lamin A/C, in three different human cell lines. We also efficiently obtained biallelic knock-in clones, using single-stranded oligodeoxynucleotides as homologous templates, for insertion of an EcoRI recognition site at the RIF1 locus and introduction of a point mutation at the histone H2AFX locus to abolish assembly of DDR factors at sites of DNA double-strand breaks. This versatile screening approach should facilitate research aimed at defining gene functions, modelling of cancers and other diseases underpinned by genetic factors, and exploring new therapeutic opportunities.We thank all members of the S.P.J. laboratory for helpful discussions. We are especially grateful to Y. Galanty, R. Belotserkovskaya and J. Forment for valuable ideas and suggestions, and for critically reading the manuscript, and K. Dry for editorial assistance. In addition, we thank A. Riddell (Flow Cytometry Core Facility at the University of Cambridge Stem Cell Institute) for flow cytometry cell sorting support, and Roger Grand (University of Birmingham, Birmingham, UK) for the gift of HEK293FT cells. The Jackson laboratory is funded by Cancer Research UK program grant C6/A18796 and the European Research Council. Core infrastructure funding is provided by CRUK (C6946/A14492) and the Wellcome Trust (WT092096). S.P.J. receives his salary from the University of Cambridge, UK, supplemented by CRUK. T-W.C. is supported by a Cambridge International Scholarship. C.S. and M.D. are funded by ERC Advanced Researcher Grant DDREAM. D.L is funded by a Project Grant from the Medical Research Council, UK MR/L019116/1.This is the final version of the article. It first appeared from Nature Publishing Group via https://doi.org/10.1038/srep2435

CRISPR-Cas9(D10A) nickase-based genotypic and phenotypic screening to enhance genome editing.

The RNA-guided Cas9 nuclease is being widely employed to engineer the genomes of various cells and organisms. Despite the efficient mutagenesis induced by Cas9, off-target effects have raised concerns over the system's specificity. Recently a "double-nicking" strategy using catalytic mutant Cas9(D10A) nickase has been developed to minimise off-target effects. Here, we describe a Cas9(D10A)-based screening approach that combines an All-in-One Cas9(D10A) nickase vector with fluorescence-activated cell sorting enrichment followed by high-throughput genotypic and phenotypic clonal screening strategies to generate isogenic knockouts and knock-ins highly efficiently, with minimal off-target effects. We validated this approach by targeting genes for the DNA-damage response (DDR) proteins MDC1, 53BP1, RIF1 and P53, plus the nuclear architecture proteins Lamin A/C, in three different human cell lines. We also efficiently obtained biallelic knock-in clones, using single-stranded oligodeoxynucleotides as homologous templates, for insertion of an EcoRI recognition site at the RIF1 locus and introduction of a point mutation at the histone H2AFX locus to abolish assembly of DDR factors at sites of DNA double-strand breaks. This versatile screening approach should facilitate research aimed at defining gene functions, modelling of cancers and other diseases underpinned by genetic factors, and exploring new therapeutic opportunities.We thank all members of the S.P.J. laboratory for helpful discussions. We are especially grateful to Y. Galanty, R. Belotserkovskaya and J. Forment for valuable ideas and suggestions, and for critically reading the manuscript, and K. Dry for editorial assistance. In addition, we thank A. Riddell (Flow Cytometry Core Facility at the University of Cambridge Stem Cell Institute) for flow cytometry cell sorting support, and Roger Grand (University of Birmingham, Birmingham, UK) for the gift of HEK293FT cells. The Jackson laboratory is funded by Cancer Research UK program grant C6/A18796 and the European Research Council. Core infrastructure funding is provided by CRUK (C6946/A14492) and the Wellcome Trust (WT092096). S.P.J. receives his salary from the University of Cambridge, UK, supplemented by CRUK. T-W.C. is supported by a Cambridge International Scholarship. C.S. and M.D. are funded by ERC Advanced Researcher Grant DDREAM. D.L is funded by a Project Grant from the Medical Research Council, UK MR/L019116/1.This is the final version of the article. It first appeared from Nature Publishing Group via https://doi.org/10.1038/srep2435

MDC1 PST-repeat region promotes histone H2AX-independent chromatin association and DNA damage tolerance

Abstract: Histone H2AX and MDC1 are key DNA repair and DNA-damage signalling proteins. When DNA double-strand breaks (DSBs) occur, H2AX is phosphorylated and then recruits MDC1, which in turn serves as a docking platform to promote the localization of other factors, including 53BP1, to DSB sites. Here, by using CRISPR-Cas9 engineered human cell lines, we identify a hitherto unknown, H2AX-independent, function of MDC1 mediated by its PST-repeat region. We show that the PST-repeat region directly interacts with chromatin via the nucleosome acidic patch and mediates DNA damage-independent association of MDC1 with chromatin. We find that this region is largely functionally dispensable when the canonical γH2AX-MDC1 pathway is operative but becomes critical for 53BP1 recruitment to DNA-damage sites and cell survival following DSB induction when H2AX is not available. Consequently, our results suggest a role for MDC1 in activating the DDR in areas of the genome lacking or depleted of H2AX

Targeting of NAT10 enhances healthspan in a mouse model of human accelerated aging syndrome.

Hutchinson-Gilford Progeria Syndrome (HGPS) is a rare, but devastating genetic disease characterized by segmental premature aging, with cardiovascular disease being the main cause of death. Cells from HGPS patients accumulate progerin, a permanently farnesylated, toxic form of Lamin A, disrupting the nuclear shape and chromatin organization, leading to DNA-damage accumulation and senescence. Therapeutic approaches targeting farnesylation or aiming to reduce progerin levels have provided only partial health improvements. Recently, we identified Remodelin, a small-molecule agent that leads to amelioration of HGPS cellular defects through inhibition of the enzyme N-acetyltransferase 10 (NAT10). Here, we show the preclinical data demonstrating that targeting NAT10 in vivo, either via chemical inhibition or genetic depletion, significantly enhances the healthspan in a Lmna G609G HGPS mouse model. Collectively, the data provided here highlights NAT10 as a potential therapeutic target for HGPS

CtIP tetramer assembly is required for DNA-end resection and repair.

Mammalian CtIP protein has major roles in DNA double-strand break (DSB) repair. Although it is well established that CtIP promotes DNA-end resection in preparation for homology-dependent DSB repair, the molecular basis for this function has remained unknown. Here we show by biophysical and X-ray crystallographic analyses that the N-terminal domain of human CtIP exists as a stable homotetramer. Tetramerization results from interlocking interactions between the N-terminal extensions of CtIP's coiled-coil region, which lead to a 'dimer-of-dimers' architecture. Through interrogation of the CtIP structure, we identify a point mutation that abolishes tetramerization of the N-terminal domain while preserving dimerization in vitro. Notably, we establish that this mutation abrogates CtIP oligomer assembly in cells, thus leading to strong defects in DNA-end resection and gene conversion. These findings indicate that the CtIP tetramer architecture described here is essential for effective DSB repair by homologous recombination.We thank M. Kilkenny for help with the collection of X-ray diffraction data, A. Sharff and P. Keller for help with X-ray data processing and J.D. Maman for assistance with SEC-MALS. This work was supported by a Wellcome Trust Senior Research Fellowship award in basic biomedical sciences (L.P.), an Isaac Newton Trust research grant (L.P. and O.R.D.) and a Cambridge Overseas Trust PhD studentship (M.D.S.). Research in the laboratory of S.P.J. is funded by Cancer Research UK (CRUK; programme grant C6/A11224), the European Research Council and the European Community Seventh Framework Programme (grant agreement no. HEALTH-F2-2010-259893 (DDResponse)). Core funding is provided by Cancer Research UK (C6946/A14492) and the Wellcome Trust (WT092096). S.P.J. receives his salary from the University of Cambridge, supplemented by CRUK. J.V.F. is funded by Cancer Research UK programme grant C6/A11224 and the Ataxia Telangiectasia Society. R.B. and J.C. are funded by Cancer Research UK programme grant C6/A11224. Y.G. and M.D. are funded by the European Research Council grant DDREAM.This is the accepted manuscript of a paper published in Nature Structural & Molecular Biology, 22, 150–157 (2015) doi: 10.1038/nsmb.293

Quality of Immature and Mature Pepper (Capsicum annuum L.) Seeds in Relation to Bio-Priming with Endophytic Pseudomonas and Bacillus spp.

Fruit maturity for seed production can occur at various times because of the continual flowering of pepper plants. Accordingly, seeds with different maturity are acquired as the fruits are collected in a single harvest. Immature seeds obtained in this harvest may lead to a decrease in the quality of seed lots. Therefore, this research aimed to evaluate the influence of four different endophytic bacteria strains (Pseudomonas fluorescens strain L5b, Pseudomonas gessardii strain L13, Bacillus subtilis strain Bs1 and Bacillus mojavensis strain ApBm) on germination and seedling vigor of immature and mature bell pepper seeds. To obtain seeds with different maturity levels, fruits were collected 45–49 days after flowering for immature seeds and 65–69 days for mature seeds. The effectiveness of these bacteria strains was examined by coating seeds with four different endophytic bacteria strains separately. Additionally, to see the activity of endophytic bacteria more clearly, a mock treatment with sterile water was added to the experiment as a control (+) group. Bio-priming (especially strain Bs1 and L13) improved germination and seedling emergence characteristics of both immature and mature seed lots compared to control groups (p < 0.05). The results demonstrate that bio-priming with beneficial endophytic bacteria can be used to stimulate the quality of both immature and mature seeds from the pepper

Systematic E2 screening reveals a UBE2D–RNF138–CtIP axis promoting DNA repair

This is the author accepted manuscript. The final version is available from Nature Publishing Group via http://dx.doi.org/10.1038/ncb3260Ubiquitylation is crucial for proper cellular responses to DNA double-strand breaks (DSBs). If unrepaired, these highly cytotoxic lesions cause genome instability, tumourigenesis, neurodegeneration or premature ageing. Here, we conduct a comprehensive, multilayered screen to systematically profile all human ubiquitin E2- enzymes for impacts on cellular DSB responses. Applying a widely applicable approach, we use an exemplary E2 family, UBE2Ds, to identify ubiquitylation-cascade components downstream of E2s. Thus, we uncover the nuclear E3-ligase RNF138 as a key homologous recombination (HR)-promoting factor that functions with UBE2Ds in cells. Mechanistically, UBE2Ds and RNF138 accumulate at DNA-damage sites and act at early resection stages by promoting CtIP ubiquitylation and accrual. This work supplies insights into regulation of DSB repair by HR. Moreover, it provides a rich information resource on E2s that can be exploited by follow-on studies.Alex Sossick, Nicola Lawrence and Richard Butler. Research in the S.P.J. lab is funded by Cancer Research UK Program Grant C6/A11224, the European Research Council (DDREAM), the European Community Seventh Framework Programme grant agreement no. HEALTH-F2- 2010-259893 (DDResponse). Core infrastructure funding was provided by Cancer Research UK Grant C6946/A14492 and Wellcome Trust Grant WT092096. S.P.J. receives a salary from the University of Cambridge, supplemented by Cancer Research UK. C.K.S. was funded by a FEBS Return-to-Europe fellowship. P.B. is supported by the Emmy Noether Programme of the German Research Foundation (DFG, BE 5342/1-1)

Research data supporting "Systematic E2 screening reveals a UBE2D–RNF138–CtIP axis promoting DNA repair"

Complete set of graphs for gammaH2AX, 53BP1 and ubiquitin (FK2 antibody) foci/cell kinetics in response to ionising radiation (IR)- and mock-treated human U2OS cells treated with siRNAs targeting ubiquitin E2s (screening module 1; refers to Fig. 1). Complete set of DDR signalling immunoblots, generated from whole cell extracts of IR-, camptothecin- or mock-treated human U2OS cells treated with siRNAs targeting ubiquitin E2s in screening module 3 (refers to Fig. 3 and Supplementary Fig. 2).This work was supported by the European Community Seventh Framework Programme [grant number HEALTH-F2-2010-259893], the European Research Council (DDREAM), CRUK [grant number C6946/A14492], Wellcome Trust [grant number WT092096], the German Research Foundation (DFG) [BE 5342/1-1], and the Return-to-Europe Fellowship (FEBS)