Showing papers by "Laboratory of Molecular Biology published in 2014"

Optimized CRISPR/Cas tools for efficient germline and somatic genome engineering in Drosophila.

Abstract: The type II clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system has emerged recently as a powerful method to manipulate the genomes of various organisms. Here, we report a toolbox for high-efficiency genome engineering of Drosophila melanogaster consisting of transgenic Cas9 lines and versatile guide RNA (gRNA) expression plasmids. Systematic evaluation reveals Cas9 lines with ubiquitous or germ-line–restricted patterns of activity. We also demonstrate differential activity of the same gRNA expressed from different U6 snRNA promoters, with the previously untested U6:3 promoter giving the most potent effect. An appropriate combination of Cas9 and gRNA allows targeting of essential and nonessential genes with transmission rates ranging from 25–100%. We also demonstrate that our optimized CRISPR/Cas tools can be used for offset nicking-based mutagenesis. Furthermore, in combination with oligonucleotide or long double-stranded donor templates, our reagents allow precise genome editing by homology-directed repair with rates that make selection markers unnecessary. Last, we demonstrate a novel application of CRISPR/Cas-mediated technology in revealing loss-of-function phenotypes in somatic cells following efficient biallelic targeting by Cas9 expressed in a ubiquitous or tissue-restricted manner. Our CRISPR/Cas tools will facilitate the rapid evaluation of mutant phenotypes of specific genes and the precise modification of the genome with single-nucleotide precision. Our results also pave the way for high-throughput genetic screening with CRISPR/Cas.

The STAT3-binding long noncoding RNA lnc-DC controls human dendritic cell differentiation.

Abstract: Long noncoding RNAs (lncRNAs) play important roles in diverse biological processes; however, few have been identified that regulate immune cell differentiation and function. Here, we identified lnc-DC, which was exclusively expressed in human conventional dendritic cells (DCs). Knockdown of lnc-DC impaired DC differentiation from human monocytes in vitro and from mouse bone marrow cells in vivo and reduced capacity of DCs to stimulate T cell activation. lnc-DC mediated these effects by activating the transcription factor STAT3 (signal transducer and activator of transcription 3). lnc-DC bound directly to STAT3 in the cytoplasm, which promoted STAT3 phosphorylation on tyrosine-705 by preventing STAT3 binding to and dephosphorylation by SHP1. Our work identifies a lncRNA that regulates DC differentiation and also broadens the known mechanisms of lncRNA action.

Identification of genomic alterations in oesophageal squamous cell cancer

Abstract: Oesophageal cancer is one of the most aggressive cancers and is the sixth leading cause of cancer death worldwide(1). Approximately 70% of global oesophageal cancer cases occur in China, with oesophageal squamous cell carcinoma (ESCC) being the histopathological form in the vast majority of cases (>90%)(2,3). Currently, there are limited clinical approaches for the early diagnosis and treatment of ESCC, resulting in a 10% five-year survival rate for patients. However, the full repertoire of genomic events leading to the pathogenesis of ESCC remains unclear. Here we describe a comprehensive genomic analysis of 158 ESCC cases, as part of the International Cancer Genome Consortium research project. We conducted whole-genome sequencing in 17 ESCC cases and whole-exome sequencing in 71 cases, of which 53 cases, plus an additional 70 ESCC cases not used in the whole-genome and whole-exome sequencing, were subjected to array comparative genomic hybridization analysis. We identified eight significantly mutated genes, of which six are well known tumour-associated genes (TP53, RB1, CDKN2A, PIK3CA, NOTCH1, NFE2L2), and two have not previously been described in ESCC (ADAM29 and FAM135B). Notably, FAM135B is identified as a novel cancer-implicated gene as assayed for its ability to promote malignancy of ESCC cells. Additionally, MIR548K, a microRNA encoded in the amplified 11q13.3-13.4 region, is characterized as a novel oncogene, and functional assays demonstrate that MIR548K enhances malignant phenotypes of ESCC cells. Moreover, we have found that several important histone regulator genes (MLL2 (also called KMT2D), ASH1L, MLL3 (KMT2C), SETD1B, CREBBP and EP300) are frequently altered in ESCC. Pathway assessment reveals that somatic aberrations are mainly involved in the Wnt, cell cycle and Notch pathways. Genomic analyses suggest that ESCC and head and neck squamous cell carcinoma share some common pathogenic mechanisms, and ESCC development is associated with alcohol drinking. This study has explored novel biological markers and tumorigenic pathways that would greatly improve therapeutic strategies for ESCC.

Human colorectal cancer-specific CCAT1-L lncRNA regulates long-range chromatin interactions at the MYC locus

Abstract: The human 8q24 gene desert contains multiple enhancers that form tissue-specific long-range chromatin loops with the MYC oncogene, but how chromatin looping at the MYC locus is regulated remains poorly understood. Here we demonstrate that a long noncoding RNA (lncRNA), CCAT1-L, is transcribed specifically in human colorectal cancers from a locus 515 kb upstream of MYC. This lncRNA plays a role in MYC transcriptional regulation and promotes long-range chromatin looping. Importantly, the CCAT1-L locus is located within a strong super-enhancer and is spatially close to MYC. Knockdown of CCAT1-L reduced long-range interactions between the MYC promoter and its enhancers. In addition, CCAT1-L interacts with CTCF and modulates chromatin conformation at these loop regions. These results reveal an important role of a previously unannotated lncRNA in gene regulation at the MYC locus.

Structure of the Yeast Mitochondrial Large Ribosomal Subunit

Abstract: Mitochondria have specialized ribosomes that have diverged from their bacterial and cytoplasmic counterparts. We have solved the structure of the yeast mitoribosomal large subunit using single-particle cryo-electron microscopy. The resolution of 3.2 angstroms enabled a nearly complete atomic model to be built de novo and refined, including 39 proteins, 13 of which are unique to mitochondria, as well as expansion segments of mitoribosomal RNA. The structure reveals a new exit tunnel path and architecture, unique elements of the E site, and a putative membrane docking site.

The structure of the ribosome with elongation factor G trapped in the posttranslocational state.

Abstract: Elongation factor G (EF-G) is a guanosine triphosphatase (GTPase) that plays a crucial role in the translocation of transfer RNAs (tRNAs) and messenger RNA (mRNA) during translation by the ribosome. We report a crystal structure refined to 3.6 angstrom resolution of the ribosome trapped with EF-G in the posttranslocational state using the antibiotic fusidic acid. Fusidic acid traps EF-G in a conformation intermediate between the guanosine triphosphate and guanosine diphosphate forms. The interaction of EF-G with ribosomal elements implicated in stimulating catalysis, such as the L10-L12 stalk and the L11 region, and of domain IV of EF-G with the tRNA at the peptidyl-tRNA binding site (P site) and with mRNA shed light on the role of these elements in EF-G function. The stabilization of the mobile stalks of the ribosome also results in a more complete description of its structure.

Bioorthogonal Reactions for Labeling Proteins

Abstract: O the past 15 years a great deal of progress has been made on the discovery, rediscovery, and invention of bioorthogonal reactions between functional groups that do not react with biological entities under physiological conditions but selectively react with each other. Strategies for labeling different classes of biomolecules have been developed by coopting the biosynthetic machinery of cells to introduce molecules containing bioorthogonal functional groups. Tagging approaches have allowed some additional functional groups to be attached to proteins, and genetic code expansion and reprogramming have facilitated the site-specific incorporation of unnatural amino acids bearing bioorthogonal functional groups into proteins in bacteria, mammalian cells, and animals via the discovery and synthetic evolution of orthogonal aminoacyl-tRNA synthetase/tRNA pairs and orthogonal ribosomes. In addition, selective pressure incorporation and its derivatives have allowed the statistical labeling of proteins and proteomes with analogues of natural amino acids. The incorporation of unnatural amino acids bearing bioorthogonal functional groups and their chemoselective labeling has great potential for imaging and controlling individual proteins and labeling proteomes, but the ability of investigators to leverage these approaches for biological discovery will be crucially dependent on the properties of the chemical reactions used. The reactants in a bioorthogonal reaction should be kinetically, thermodynamically, and metabolically stable before the reaction takes place and not toxic to living systems. The reaction should yield stable covalent linkages with no or innocuous byproducts. Moreover, the two bioorthogonal moieties have to react selectively with each other under physiological conditions (ambient temperature and pressure, neutral pH, aqueous conditions), without either of them crossreacting with the plethora of chemical functionalities found in living cells. Despite the challenges of meeting these criteria, a number of reactions have been developed that show good biocompatibility and selectivity in living systems (see Figure 1). Some of these reactions are chemoselective with respect to many but not all biological functionalities and have been used to label proteins in vitro and on the cell surface, while other reactions have additionally been used for the more challenging task of labeling proteins inside cells or living animals. Most bioorthogonal reactions follow second-order kinetics, and their rates depend directly on the concentrations of both reaction partners as well as on the intrinsic second-order rate constant k2 [M −1 s−1] of the reaction. Rapid reactions with high second-order rate constants are therefore advantageous for labeling during biological processes that occur on a very short time scale or for the labeling of low abundance proteins. Lower abundance proteins can sometimes be labeled with a large excess of labeling reagent, but this strategy may be practically limited by solubility, off target reactions and toxicity. Bioorthogonal reactions for which one partner can be installed into proteins are summarized in Figure 1. Their second-order rate constants span 9 orders of magnitude with the fastest bioorthogonal labeling reactions reaching rates up to 10 M−1 s−1, which approaches the rate constants for many enzymatic labeling approaches. Here we briefly introduce the bioorthogonal chemistries used for labeling proteins and comment on their utility for protein labeling before providing a perspective on future directions. Amongst the first functionalities to be explored as bioorthogonal reporters were ketones and aldehydes. Under acidic conditions (pH 4−6) their carbonyl groups react with strong α-effect nucleophiles such as hydrazines and alkoxyamines. Ketone/aldehyde condensations show rather slow kinetics with second-order rate constants in the range of 10−4 to 10−3 M−1 s−1, necessitating high concentrations of labeling reagent in order to achieve good labeling, which might be problematic in terms of toxicity and background signal. In general ketone/aldehyde condensations are best suited for in vitro or cell-surface labeling, because the reaction requires an acidic pH, which is difficult to obtain inside most cellular compartments. Furthermore, inside living cells, α-effect nucleophiles may undergo side-reactions with carbonyl-bearing metabolites. A functionality that is essentially absent from biological systems and truly orthogonal in its reactivity to the majority of biological functionalities is the azide group. Azide-bearing unnatural amino acids have been incorporated into proteins and used in a variety of chemical reactions. One potential limitation of the use of azides for protein labeling is that some unnatural amino acids bearing azides appear to be reduced in some proteins examined. Azide-modified proteins have been reacted with phosphines in Staudinger ligations. This reaction has been used to label biomolecules in living cells and animals. The Staudinger ligation, however, has slow kinetics: the reaction proceeds with second-order rate constants in the low 10−3 M−1 s−1 range. In addition many of the phosphine reagents are oxidized by air or metabolic enzymes. Azides can also react with terminal alkynes in [3 + 2] cycloadditions, catalyzed by Cu salts. The CuAAC (Cucatalyzed alkyne−azide cycloaddition) reaction proceeds considerably faster than the Staudinger ligation in physiological settings. However, its reliance on the Cu catalyst is not without problems, since Cu may be toxic to living systems, and decreasing the copper concentration is generally accompanied by a large decrease in reaction rate. The development of tailored water-soluble Cu ligands and/or

Expanding and Reprogramming the Genetic Code of Cells and Animals

Abstract: Genetic code expansion and reprogramming enable the site-specific incorporation of diverse designer amino acids into proteins produced in cells and animals. Recent advances are enhancing the efficiency of unnatural amino acid incorporation by creating and evolving orthogonal ribosomes and manipulating the genome. Increasing the number of distinct amino acids that can be site-specifically encoded has been facilitated by the evolution of orthogonal quadruplet decoding ribosomes and the discovery of mutually orthogonal synthetase/tRNA pairs. Rapid progress in moving genetic code expansion from bacteria to eukaryotic cells and animals (C. elegans and D. melanogaster) and the incorporation of useful unnatural amino acids has been aided by the development and application of the pyrrolysyl-transfer RNA (tRNA) synthetase/tRNA pair for unnatural amino acid incorporation. Combining chemoselective reactions with encoded amino acids has facilitated the installation of posttranslational modifications, as well as rapid derivatization with diverse fluorophores for imaging.

Prostaglandin D2 activates group 2 innate lymphoid cells through chemoattractant receptor-homologous molecule expressed on TH2 cells

Abstract: Background Activation of the group 2 innate lymphoid cell (ILC2) population leads to production of the classical type 2 cytokines, thus promoting type 2 immunity. Chemoattractant receptor-homologous molecule expressed on T H2 cells (CRTH2), a receptor for prostaglandin D2 (PGD2), is expressed by human ILC2s. However, the function of CRTH2 in these cells is unclear. Objectives We sought to determine the role of PGD2 and CRTH2 in human ILC2s and compare it with that of the established ILC2 activators IL-25 and IL-33. Methods The effects of PGD 2, IL-25, and IL-33 on the cell migration, cytokine production, gene regulation, and receptor expression of ILC2s were measured with chemotaxis, ELISA, Luminex, flow cytometry, quantitative RT-PCR, and QuantiGene assays. The effects of PGD2 under physiologic conditions were evaluated by using the supernatant from activated mast cells. Results PGD2 binding to CRTH2 induced ILC2 migration and production of type 2 cytokines and many other cytokines. ILC2 activation through CRTH2 also upregulated the expression of IL-33 and IL-25 receptor subunits (ST2 and IL-17RA). The effects of PGD 2 on ILC2s could be mimicked by the supernatant from activated human mast cells and inhibited by a CRTH2 antagonist. Conclusions PGD2 is an important and potent activator of ILC2s through CRTH2 mediating strong proallergic inflammatory responses. Through IgE-mediated mast cell degranulation, these innate cells can also contribute to adaptive type 2 immunity; thus CRTH2 bridges the innate and adaptive pathways in human ILC2s. © 2013 American Academy of Allergy, Asthma & Immunology.

A novel in vivo model of tau propagation with rapid and progressive neurofibrillary tangle pathology: the pattern of spread is determined by connectivity, not proximity

Abstract: Intracellular inclusions composed of hyperphosphorylated filamentous tau are a hallmark of Alzheimer’s disease, progressive supranuclear palsy, Pick’s disease and other sporadic neurodegenerative tauopathies. Recent in vitro and in vivo studies have shown that tau aggregates do not only seed further tau aggregation within neurons, but can also spread to neighbouring cells and functionally connected brain regions. This process is referred to as ‘tau propagation’ and may explain the stereotypic progression of tau pathology in the brains of Alzheimer’s disease patients. Here, we describe a novel in vivo model of tau propagation using human P301S tau transgenic mice infused unilaterally with brain extract containing tau aggregates. Infusion-related neurofibrillary tangle pathology was first observed 2 weeks post-infusion and increased in a stereotypic, time-dependent manner. Contralateral and anterior/posterior spread of tau pathology was also evident in nuclei with strong synaptic connections (efferent and afferent) to the site of infusion, indicating that spread was dependent on synaptic connectivity rather than spatial proximity. This notion was further supported by infusion-related tau pathology in white matter tracts that interconnect these regions. The rapid and robust propagation of tau pathology in this model will be valuable for both basic research and the drug discovery process.

Architecture of mammalian respiratory complex I

Abstract: Complex I (NADH:ubiquinone oxidoreductase) is essential for oxidative phosphorylation in mammalian mitochondria. It couples electron transfer from NADH to ubiquinone with proton translocation across the energy-transducing inner membrane, providing electrons for respiration and driving ATP synthesis. Mammalian complex I contains 44 different nuclear- and mitochondrial-encoded subunits, with a combined mass of 1 MDa. The 14 conserved ‘core’ subunits have been structurally defined in the minimal, bacterial complex, but the structures and arrangement of the 30 ‘supernumerary’ subunits are unknown. Here we describe a 5 A resolution structure of complex I from Bos taurus heart mitochondria, a close relative of the human enzyme, determined by single-particle electron cryo-microscopy. We present the structures of the mammalian core subunits that contain eight iron–sulphur clusters and 60 transmembrane helices, identify 18 supernumerary transmembrane helices, and assign and model 14 supernumerary subunits. Thus, we considerably advance knowledge of the structure of mammalian complex I and the architecture of its supernumerary ensemble around the core domains. Our structure provides insights into the roles of the supernumerary subunits in regulation, assembly and homeostasis, and a basis for understanding the effects of mutations that cause a diverse range of human diseases. Complex I is the first enzyme of the mitochondrial electron transport chain and it is essential for oxidative phosphorylation in mammalian mitochondria; here the electron cryo-microscopy structure of complex I from bovine heart mitochondria is reported, advancing knowledge of its structure in mammals. The first enzyme of the mitochondrial electron transport chain, complex I (NADH:ubiquinone oxidoreductase), couples electron transfer from NADH to ubiquinone with proton translocation across the inner mitochondrial membrane, leading to the synthesis of ATP. This manuscript reports the electron cryo-microscopy structure of complex I from bovine heart mitochondria at 5 A resolution. The mammalian enzyme is much larger than the previously published structures of complex I from lower organisms. The authors assign the structures of 28 of the 44 subunits — the 14 conserved (core) subunits and 14 mammalian-specific subunits.

In vitro reconstitution of a highly processive recombinant human dynein complex.

Abstract: Cytoplasmic dynein is an approximately 1.4 MDa multi-protein complex that transports many cellular cargoes towards the minus ends of microtubules. Several in vitro studies of mammalian dynein have suggested that individual motors are not robustly processive, raising questions about how dynein-associated cargoes can move over long distances in cells. Here, we report the production of a fully recombinant human dynein complex from a single baculovirus in insect cells. Individual complexes very rarely show directional movement in vitro. However, addition of dynactin together with the N-terminal region of the cargo adaptor BICD2 (BICD2N) gives rise to unidirectional dynein movement over remarkably long distances. Single-molecule fluorescence microscopy provides evidence that BICD2N and dynactin stimulate processivity by regulating individual dynein complexes, rather than by promoting oligomerisation of the motor complex. Negative stain electron microscopy reveals the dynein–dynactin–BICD2N complex to be well ordered, with dynactin positioned approximately along the length of the dynein tail. Collectively, our results provide insight into a novel mechanism for coordinating cargo binding with long-distance motor movement.

Beam-induced motion correction for sub-megadalton cryo-EM particles

Abstract: In electron cryo-microscopy (cryo-EM), the electron beam that is used for imaging also causes the sample to move. This motion blurs the images and limits the resolution attainable by single-particle analysis. In a previous Research article (Bai et al., 2013) we showed that correcting for this motion by processing movies from fast direct-electron detectors allowed structure determination to near-atomic resolution from 35,000 ribosome particles. In this Research advance article, we show that an improved movie processing algorithm is applicable to a much wider range of specimens. The new algorithm estimates straight movement tracks by considering multiple particles that are close to each other in the field of view, and models the fall-off of high-resolution information content by radiation damage in a dose-dependent manner. Application of the new algorithm to four data sets illustrates its potential for significantly improving cryo-EM structures, even for particles that are smaller than 200 kDa.

Ultrastable gold substrates for electron cryomicroscopy

Abstract: Despite recent advances, the structures of many proteins cannot be determined by electron cryomicroscopy because the individual proteins move during irradiation. This blurs the images so that they cannot be aligned with each other to calculate a three-dimensional density. Much of this movement stems from instabilities in the carbon substrates used to support frozen samples in the microscope. Here we demonstrate a gold specimen support that nearly eliminates substrate motion during irradiation. This increases the subnanometer image contrast such that α helices of individual proteins are resolved. With this improvement, we determine the structure of apoferritin, a smooth octahedral shell of α-helical subunits that is particularly difficult to solve by electron microscopy. This advance in substrate design will enable the solution of currently intractable protein structures.

TH9 cells that express the transcription factor PU.1 drive T cell-mediated colitis via IL-9 receptor signaling in intestinal epithelial cells

Abstract: The molecular checkpoints that drive inflammatory bowel diseases are incompletely understood. Here we found more T cells expressing the transcription factor PU.1 and interleukin 9 (IL-9) in patients with ulcerative colitis. In an animal model, citrine reporter mice had more IL-9-expressing mucosal T cells in experimental oxazolone-induced colitis. IL-9 deficiency suppressed acute and chronic colitis. Mice with PU.1 deficiency in T cells were protected from colitis, whereas treatment with antibody to IL-9 suppressed colitis. Functionally, IL-9 impaired intestinal barrier function and prevented mucosal wound healing in vivo. Thus, our findings suggest that the TH9 subset of helper T cells serves an important role in driving ulcerative colitis by regulating intestinal epithelial cells and that TH9 cells represent a likely target for the treatment of chronic intestinal inflammation.

High-resolution structure determination by continuous-rotation data collection in MicroED

Abstract: MicroED uses very small three-dimensional protein crystals and electron diffraction for structure determination. We present an improved data collection protocol for MicroED called 'continuous rotation'. Microcrystals are continuously rotated during data collection, yielding more accurate data. The method enables data processing with the crystallographic software tool MOSFLM, which resulted in improved resolution for the model protein lysozyme. These improvements are paving the way for the broad implementation and application of MicroED in structural biology.

Three-dimensional structure of human γ-secretase

Abstract: The γ-secretase complex, comprising presenilin 1 (PS1), PEN-2, APH-1 and nicastrin, is a membrane-embedded protease that controls a number of important cellular functions through substrate cleavage. Aberrant cleavage of the amyloid precursor protein (APP) results in aggregation of amyloid-β, which accumulates in the brain and consequently causes Alzheimer’s disease. Here we report the three-dimensional structure of an intact human γ-secretase complex at 4.5 A resolution, determined by cryo-electron-microscopy single-particle analysis. The γ-secretase complex comprises a horseshoe-shaped transmembrane domain, which contains 19 transmembrane segments (TMs), and a large extracellular domain (ECD) from nicastrin, which sits immediately above the hollow space formed by the TM horseshoe. Intriguingly, nicastrin ECD is structurally similar to a large family of peptidases exemplified by the glutamate carboxypeptidase PSMA. This structure serves as an important basis for understanding the functional mechanisms of the γ-secretase complex. The three-dimensional structure of intact human γ-secretase complex at 4.5 A resolution is revealed by cryo-electron-microscopy single-particle analysis; the complex comprises a horseshoe-shaped transmembrane domain containing 19 transmembrane segments, and a large extracellular domain from nicastrin, which sits immediately above the hollow space formed by the horseshoe. This paper reports a high-resolution cryo-electron-microscopy structure of the human γ-secretase complex, a membrane-embedded protease that controls important cellular functions and is linked to the aberrant cleavage of the amyloid precursor protein seen in Alzheimer's disease. The complex, comprising presenilin 1, PEN-2, APH-1 and nicastrin, is horseshoe-shaped, with 19 transmembrane segments. The nicastrin extracellular domain, thought to be responsible for substrate recruitment, sits immediately above the hollow space formed by the transmembrane horseshoe.