Latest Articles Include:
- The life of proteins: the good, the mostly good and the ugly
- Nat Struct Mol Biol 18(1):1-4 (2011)
Nature Structural & Molecular Biology | Meeting Report The life of proteins: the good, the mostly good and the ugly * Richard I Morimoto1 Contact Richard I Morimoto Search for this author in: * NPG journals * PubMed * Google Scholar * Arnold J M Driessen2 Search for this author in: * NPG journals * PubMed * Google Scholar * Ramanujan S Hegde3 Search for this author in: * NPG journals * PubMed * Google Scholar * Thomas Langer4 Search for this author in: * NPG journals * PubMed * Google ScholarJournal name:Nature Structural & Molecular BiologyVolume: 18,Pages:1–4Year published:(2011)DOI:doi:10.1038/nsmb0111-1Published online06 January 2011 Read the full article * Instant access to this article: US$32Buy now * Subscribe to Nature Structural & Molecular Biology for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Login via Athens * Purchase a site license * Institutional access * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. The health of the proteome in the face of multiple and diverse challenges directly influences the health of the cell and the lifespan of the organism. A recent meeting held in Nara, Japan, provided an exciting platform for scientific exchange and provocative discussions on the biology of proteins and protein homeostasis across multiple scales of analysis and model systems. View full text Author information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Affiliations * Richard I. Morimoto is in the Department of Molecular Biosciences, Northwestern University, Evanston, Illinois, USA and at the Rice Institute for Biomedical Research, Evanston, Illinois, USA. * Arnold J. M. Driessen is in the Department of Molecular Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute, Zernike Institute for Advanced Materials, University of Groningen, Groningen, The Netherlands. * Ramanujan S. Hegde is at the National Institute of Child Health and Human Development, US National Institutes of Health, Bethesda, Maryland, USA. * Thomas Langer is at the Institute for Genetics, University of Cologne, Cologne, Germany. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Richard I Morimoto Read the full article * Instant access to this article: US$32Buy now * Subscribe to Nature Structural & Molecular Biology for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Login via Athens * Purchase a site license * Institutional access * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Additional data - Research highlights
- Nat Struct Mol Biol 18(1):5 (2011)
Nature Structural & Molecular Biology | Research Highlights Research highlights Journal name:Nature Structural & Molecular BiologyVolume: 18,Page:5Year published:(2011)DOI:doi:10.1038/nsmb0111-5Published online06 January 2011 Read the full article * Instant access to this article: US$32Buy now * Subscribe to Nature Structural & Molecular Biology for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Login via Athens * Purchase a site license * Institutional access * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Sweet perturbations Glycosylation—the attachment of polysaccharide chains or glycans to proteins—is one of the most common and important protein modifications and occurs in the endoplasmic reticulum while the polypeptide is still being biosynthesized and is partially unfolded. This suggests that glycosylation plays a role in protein folding and stability, but investigating this using natively glycosylated proteins is challenging because these proteins are usually large, multisubunit and/or membrane associated. Most studies thus focus on defined, chemically synthesized glycopeptides. In an effort to develop general rules to define and predict glycosylation-induced effects on protein folding and structure, Chen et al. used protein semisynthesis to create variants of the bacterial immunity protein Im7 that were site-specifically modified with the chitobiose disaccharide (GlcNAc-GlcNAc). Im7 is ideal for studying this modification because it is a small, globular, 87-amino-acid protein whose fol! ding pathway is well defined. Likewise, the chitobiose disaccharide was previously shown to serve as a proxy for larger glycans. The authors created seven Im7 variants glycosylated at solvent-exposed sites and studied the kinetic and thermodynamic consequences of these modifications. Glycans located in the center of α-helices negatively affected folding, whereas one located in the tight loop between two helices increased the overall rate of folding. Finally, glycosylation at the ends of α-helices and in larger loops seemed to have only a minimal effect. These results provide insight into why there is an increased probability of finding glycosylation sites between regions of secondary structure. The introduction of a glycan can thus have an effect on protein folding that critically depends on the exact position of the modification. (Proc. Natl. Acad. Sci. USA doi:10.1073/pnas.1015356107, published online 9 December 2010) BK Inhibiting factor RNA polymerase (RNAP) is tightly regulated by transcription factors such as the bacterial protein GreA or its Thermus thermophilus homolog Gfh1, which inhibit transcription initiation and elongation. Previously solved structures of RNAP in the absence of transcription factors show a crab's-claw appearance, with four separate modules—the core, shelf, clamp and jaw-lobe—that are able to move relative to each other. Between the two central modules, the core and the shelf, lies the entry point of nucleotide triphosphates (NTPs) into the catalytic site. When Gfh1 is added to RNAP, transcription is inhibited in a process whose exact mechanism has not been clear. Now Yokoyama and colleagues have revealed the structure of Gfh1 bound to RNAP in the presence of DNA and RNA. In the structure, the four-module architecture of RNAP is retained, but the two central modules are rotated into a 'ratcheted' orientation by 7° relative to their positions in the previous transcription elonga! tion complex or apo holoenzyme structures. This has the effect of opening up the nucleic acid binding channel. At the same time, the N-terminal coiled coil of Gfh1 blocks the NTP entry channel between the two central modules, with the tip of the coiled coil located within the phosphate-binding site. This structure shows that Gfh1 works by stopping NTP from binding to RNAP and holding RNAP in a ratcheted state, which perhaps might be useful for other transcription steps. (Nature doi:10.1038/nature09573, published online 1 December 2010) MH View full text Read the full article * Instant access to this article: US$32Buy now * Subscribe to Nature Structural & Molecular Biology for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Login via Athens * Purchase a site license * Institutional access * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Additional data - Substrate binding on the APC/C occurs between the coactivator Cdh1 and the processivity factor Doc1
- Nat Struct Mol Biol 18(1):6-13 (2011)
Nature Structural & Molecular Biology | Article Substrate binding on the APC/C occurs between the coactivator Cdh1 and the processivity factor Doc1 * Bettina A Buschhorn1, 5 Search for this author in: * NPG journals * PubMed * Google Scholar * Georg Petzold1, 5 Search for this author in: * NPG journals * PubMed * Google Scholar * Marta Galova1 Search for this author in: * NPG journals * PubMed * Google Scholar * Prakash Dube2 Search for this author in: * NPG journals * PubMed * Google Scholar * Claudine Kraft1, 4 Search for this author in: * NPG journals * PubMed * Google Scholar * Franz Herzog1, 4 Search for this author in: * NPG journals * PubMed * Google Scholar * Holger Stark2, 3 Contact Holger Stark Search for this author in: * NPG journals * PubMed * Google Scholar * Jan-Michael Peters1 Contact Jan-Michael Peters Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorsJournal name:Nature Structural & Molecular BiologyVolume: 18,Pages:6–13Year published:(2011)DOI:doi:10.1038/nsmb.1979Received27 July 2010Accepted17 November 2010Published online26 December 2010 Abstract * Abstract * Accession codes * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg The anaphase-promoting complex/cyclosome (APC/C) is a 22S ubiquitin ligase complex that initiates chromosome segregation and mitotic exit. We have used biochemical and electron microscopic analyses of Saccharomyces cerevisiae and human APC/C to address how the APC/C subunit Doc1 contributes to recruitment and processive ubiquitylation of APC/C substrates, and to understand how APC/C monomers interact to form a 36S dimeric form. We show that Doc1 interacts with Cdc27, Cdc16 and Apc1 and is located in the vicinity of the cullin–RING module Apc2–Apc11 in the inner cavity of the APC/C. Substrate proteins also bind in the inner cavity, in close proximity to Doc1 and the coactivator Cdh1, and induce conformational changes in Apc2–Apc11. Our results suggest that substrates are recruited to the APC/C by binding to a bipartite substrate receptor composed of a coactivator protein and Doc1. View full text Figures at a glance * Figure 1: Incorporation of a photo-cross-linker into Doc1 results in cross-link products between Doc1 and APC/C subunits. () Composition of wild-type APC/C (WT) and APC/C lacking Doc1 (ΔDoc1) affinity-purified via a TAP tag on Apc4. Apc4–CBP indicates calmodulin-binding protein that remains on Apc4 after TEV cleavage. Asterisk marks contaminating band. () APC/C ubiquitylation of [35S]methionine-labeled Hsl1667–872 substrate is impaired when Doc1 is absent as compared to when the wild-type form of APC/C is present. () Cross-linker incorporation into in vitro–translated Doc1. Full-length Doc1 contains the photoactivatable amino acid. () Overview of cross-link products obtained with 35S-labeled Doc1 amber mutants carrying the photo-cross-linker at six different sites. The two consecutive sites Ser128 and Lys129 each created two cross-link products of different sizes. () Doc1 crystal structure of S. cerevisiae. Cross-linker incorporation sites are indicated. Predominant cross-links of APC/C subunits to respective Doc1 sites are color coded (green, Cdc27; blue, Cdc16, orange, Apc1). * Figure 2: Identification of APC/C subunits that interact with Doc1. () Cdc16-myc6 incorporated into the APC/C complex caused a mobility shift of the cross-link product (arrow) when incubated with radiolabeled Doc1 carrying the photo-cross-linker at the position of Lys129, which indicates an interaction between Doc1-Lys129 and Cdc16. () Doc1 residue Arg182 contacts Cdc16. Cdc27 interacts with Doc1 via residue Phe244. () The large subunit Apc1 contacts the Doc1 protein at residues Ser128, Lys154 and Asn205. Arrows indicate the mobility shift of the cross-link product (asterisk). * Figure 3: Purification and 3D reconstruction of budding yeast APC/C. () SDS-PAGE of APC/C TAP tag purified via Cdc16. APC/C subunits were identified from their characteristic electrophoretic mobility and by MS. () EM analysis of yeast APC/C. Top row shows selected EM raw images of yeast APC/C in different orientations. Class averages were obtained by alignment, multivariate statistical analysis and classification of the raw images and are shown in the second row. The third row shows the surface representation of the computed yeast APC/C 3D structure in the corresponding orientations. The fourth row shows re-projections of the yeast APC/C 3D structure in angular directions determined for the views in the third row. () Surface representations of yeast and human APC/C 3D structures in three different orientations. Indicated rotations refer to the top structures. The location of APC/C's head, arc lamp and platform domain is indicated. Size bars, 10 nm. * Figure 4: Localization of Doc1 and Swm1 in the yeast APC/C 3D structure by subunit deletion and td2 labeling. () 3D models illustrating Doc1 localization. Density not present in APC/CΔDoc1 compared to APC/CWT is labeled as main density difference in the APC/C WT – ΔDoc1 difference map. Subunit deletion and td2 labeling converge at identical locations. () Labeling and EM analysis of td2-Doc1 compared to wild-type APC/C. Class averages of wild-type and Doc1-td2–labeled APC/C are shown in two different orientations. The additional density caused by the td2 tag is indicated with arrows in the class averages. 3D models computed from the corresponding datasets are depicted in similar orientations. () Localization of Swm1 using subunit deletion and td2 labeling. Swm1 is located in the head domain. The asterisks mark the face of APC/C's central cavity. Size bars, 10 nm. * Figure 5: APC/C subunit localization using td2 labeling. () SDS-PAGE analysis of td2-labeled Cdc16 or Cdc27 subunits incorporated into yeast APC/C. () Class averages of APC/C-Cdc27-td2 and APC/C-Cdc16-td2 compared to wild-type APC/C. Extra densities caused by the td2 label are highlighted in the class averages and marked in red in the respective 3D models. Size bar, 10 nm. () Results of all labeling experiments carried out in this study superimposed onto the structure of yeast APC/C. * Figure 6: Analysis of APC/C dimers. () Class averages of dimeric APC/C with different orientations. Size bar, 10 nm. () 3D model of dimeric APC/C. The purple APC/C monomer is shown in a side view orientation with the TPR-rich arc lamp domain in the front. The gray APC/C monomer is shown in a bottom view orientation with the bottom of the platform domain in the front. Asterisks mark the face of APC/C's central cavity. The middle ellipse marks a unique contact lying on the c2 symmetry axis, whereas the other ellipses show contact points that each exist as an asymmetric pair. () APC/C dimerization causes conformational rearrangements in the platform domain as well as the head domain compared to monomeric APC/C (yellow). Black lines and asterisk indicate conformational changes between monomeric and dimeric APC/C. * Figure 7: In vitro reconstitution of human APC/C bound to a substrate molecule. () Off-rate determination of Hsl1 and/or CDH1. () Reconstituted APC/CCDH1 complexes bound to either sororin or securin were subjected to off-rate experiments as described in . () Human APC/C incubated with wild-type (WT) or D box–KEN box mutant (dkm) form of His-Flag-td2-Hsl1667–872 in absence or presence of purified CDH1. Re-immunoprecipitation experiments were used to purify stoichiometric APC/CCDH1–Hsl1 complexes. () APC/CCDH1–Hsl1 incubated with pre-formed UBCH10–ubiquitin complexes results in formation of Hsl1–ubiquitin conjugates. () 3D model of human APC/C bound to its coactivator protein CDH1 (red). In the APC/CCDH1 structure, the APC2–APC11 module contacts the platform domain (arrow). () 3D model of human APC/CCDH1–Hsl1. The density attributed to the Hsl1 substrate molecule (blue) is intercalated between CDH1 and DOC1. In the APC/CCDH1–Hsl1 structure, the contact between APC2–APC11 and the platform is resolved to form a new connection (blue) to t! he coactivator protein (arrow). * Figure 8: Topological comparison of human and budding yeast APC/C. Human and yeast APC/C models are shown in either front or back view orientation at a resolution of ~25 Å. The positions of mapped subunits are indicated. Note that human APC/C carries a marked extra mass within the TPR-rich arc lamp domain, which might represent a subunit specific for vertebrate APC/C, such as Apc7. Accession codes * Abstract * Accession codes * Author information * Supplementary information Referenced accessions EMBL Nucleotide Sequence Database * EMD-1820 * EMD-1822 * EMD-1821 Author information * Abstract * Accession codes * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Bettina A Buschhorn & * Georg Petzold Affiliations * Research Institute of Molecular Pathology (IMP), Vienna, Austria. * Bettina A Buschhorn, * Georg Petzold, * Marta Galova, * Claudine Kraft, * Franz Herzog & * Jan-Michael Peters * Max Planck Institute for Biophysical Chemistry, Göttingen, Germany. * Prakash Dube & * Holger Stark * Department of Molecular 3D Electron Cryomicroscopy, Institute of Microbiology and Genetics, Georg-August Universität Göttingen, Göttingen, Germany. * Holger Stark * Present addresses: Institute of Biochemistry, Eidgenössische Technische Hochschule Zürich, Zürich, Switzerland (C.K.); Institute of Molecular Systems Biology, Eidgenössische Technische Hochschule Zürich, Zürich, Switzerland (F.H.). * Claudine Kraft & * Franz Herzog Contributions H.S. and J.-M.P. planned and supervised the project. B.A.B., G.P., C.K. and F.H. designed the experiments. B.A.B. performed most of the photo-cross-linking and biochemical experiments on yeast APC/C. G.P. performed the experiments on substrate bound APC/C. M.G. and C.K. generated yeast strains and performed growth assays and yeast APC/C purifications. F.H. performed antibody labeling on human APC/C. P.D. performed EM. H.S. calculated and analyzed the 3D EM structures. B.A.B., G.P. and J.-M.P. wrote the paper. Competing financial interests The authors declare no competing financial interests. Corresponding authors Correspondence to: * Holger Stark or * Jan-Michael Peters Supplementary information * Abstract * Accession codes * Author information * Supplementary information PDF files * Supplementary Text and Figures (4M) Supplementary Methods, Supplementary Figures 1–5 and Supplementary Tables 1–5 Additional data - Crystal structure of the open conformation of the mammalian chaperonin CCT in complex with tubulin
- Nat Struct Mol Biol 18(1):14-19 (2011)
Nature Structural & Molecular Biology | Article Crystal structure of the open conformation of the mammalian chaperonin CCT in complex with tubulin * Inés G Muñoz1, 5 Search for this author in: * NPG journals * PubMed * Google Scholar * Hugo Yébenes2, 5 Search for this author in: * NPG journals * PubMed * Google Scholar * Min Zhou3 Search for this author in: * NPG journals * PubMed * Google Scholar * Pablo Mesa1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * Marina Serna2 Search for this author in: * NPG journals * PubMed * Google Scholar * Ah Young Park3 Search for this author in: * NPG journals * PubMed * Google Scholar * Elisabeth Bragado-Nilsson1 Search for this author in: * NPG journals * PubMed * Google Scholar * Ana Beloso2 Search for this author in: * NPG journals * PubMed * Google Scholar * Guillermo de Cárcer4 Search for this author in: * NPG journals * PubMed * Google Scholar * Marcos Malumbres4 Search for this author in: * NPG journals * PubMed * Google Scholar * Carol V Robinson3 Search for this author in: * NPG journals * PubMed * Google Scholar * José M Valpuesta2 Contact José M Valpuesta Search for this author in: * NPG journals * PubMed * Google Scholar * Guillermo Montoya1 Contact Guillermo Montoya Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorsJournal name:Nature Structural & Molecular BiologyVolume: 18,Pages:14–19Year published:(2011)DOI:doi:10.1038/nsmb.1971Received02 August 2010Accepted03 November 2010Published online12 December 2010 Abstract * Abstract * Accession codes * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Protein folding is assisted by molecular chaperones. CCT (chaperonin containing TCP-1, or TRiC) is a 1-MDa oligomer that is built by two rings comprising eight different 60-kDa subunits. This chaperonin regulates the folding of important proteins including actin, α-tubulin and β-tubulin. We used an electron density map at 5.5 Å resolution to reconstruct CCT, which showed a substrate in the inner cavities of both rings. Here we present the crystal structure of the open conformation of this nanomachine in complex with tubulin, providing information about the mechanism by which it aids tubulin folding. The structure showed that the substrate interacts with loops in the apical and equatorial domains of CCT. The organization of the ATP-binding pockets suggests that the substrate is stretched inside the cavity. Our data provide the basis for understanding the function of this chaperonin. View full text Figures at a glance * Figure 1: Scheme of the overall model of CCT containing two copies of each of the eight subunits α, β, γ, ζ, ɛ, δ, θ and η. () Left, scheme of the proposed arrangement of subunits in the ring of CCT according to previous biochemical and EM studies13, 14. Right, scheme of the location of the domains in the subunits along the pseudo-eightfold axis. () Overall view of the CCT complex perpendicular (left) and parallel (right) to the pseudo-eightfold symmetry axis. The experimental electron density map is contoured at 1σ. The subunits are depicted in Cα tube representation. Each subunit is represented in a different color with a C-terminal domain of a tubulin molecule docked in the density inside the cavity for illustrative purposes (lime green). () The C-terminal domain of two tubulin molecules (magenta and lime green) docked in the positions where the extra internal densities appear. The experimental electron density map is contoured at 1σ. * Figure 2: Apical domain conformations. () Electron density map of an end-on view contoured at 1σ in the area of the apical domains. () Schematic representation of the same ring of CCT showing the relative orientations of the different apical domains (orange spheres, with the helical extension of the lid shown as red cylinders). The equatorial domains are shown as blue spheres and the sensor loops as thin blue cylinders. () Models of the polypeptide chains B (yellow), G (magenta) and H (green). These correspond to the most distant positions adopted by the apical domain H and G with respect to the central domain B. Right, a superposition of the equatorial domains of the three subunits to illustrate the different conformations adopted by their apical domains. * Figure 3: Interaction between tubulin and CCT. () Mass spectrometry analysis shows that the CCT complex has one molecule of substrate bound. Comparison between intact CCT complex (top; blue series, mass measured as 997.7 kDa) and the complex after ATP incubation and size-exclusion–chromatography purification (bottom) shows the appearance of a second charge-state series (orange, mass measured as 947.5 kDa) that corresponds to the CCT complex alone. () Top, section across the cryo-EM non-symmetrized 3D reconstruction at 30 Å of the purified CCT (the same material used for crystallization) showing the two extra densities, one in each cavity. Bottom, CCT at 30 Å resolution without the extra densities after ATP treatment. Right, galleries of representative views from the 2D single-particle analysis of the CCT–tubulin complex and CCT after ATP treatment (no symmetry imposed). The first column represents the 3D averages of the images in the corresponding class. () Detailed view of the apical and sensor loops interacting w! ith the tubulin density. The experimental electron density map is contoured at 1σ. * Figure 4: Detailed view of the ATP binding pockets. (,) Experimental electron density map contoured at 1σ in the ATP binding site depicting differences among the equatorial domains. The location of the nucleotide was obtained after superposition with the thermosome equatorial domains (PDB 1Q3Q). The position of the nucleotide is shown (see Supplementary Fig. 9). () Model of the CCT–tubulin contacts and major CCT structural variability. The subunits of CCT that interact with tubulin (gray) through the sensor loop (colors as in Fig. 1a, right) and the apical loop. The different conformations observed on the crystal structure could be interpreted as potential local movements of certain regions, mainly involving the sensor loop and the intermediate-apical domain (both 'motions' represented as blue arrows). The sensor loop extends to contact the substrate. The movement of the apical domain can be seen as an angular motion with its main pivot point fixed at the connection between the equatorial and intermediate domains. Addition! al flexibility is seen at the intermediate-apical domain, which is related to the extra pivot point between these domains and the different conformations displayed by the helical protrusion. The substrate-binding regions and the structural flexibility could both be related and connected through the nucleotide-binding site (red oval) of the equatorial domain, as part of the sensor loop and the intermediate domain form the pocket. * Figure 5: Silencing and complementation assays to analyze the function of the sensor and apical loops in the CCT-β subunit in HEK293 cells. () Western blot showing the time course of CCT-β expression in cells after transfection with two different shRNA vectors: pRCTL (scrambled sequences) and pRβ (targeting the 3′ UTR of endogenous CCT-β mRNA). In the latter case, optimum silencing was reached on the third day after transfection. () Histogram of the effect on cell growth (t = 72 h) of transient co-transfections, each performed with two kinds of vector: a silencing shRNA vector (pRβ or pRCTL) and a protein expression plasmid (for exogenous expression of wild-type or mutant CCT-β or for expression of CCT-α as control). Data are grouped into controls (gray bars), CCT-β silencing (black bar), CCT-β complementation (white bar) and CCTβ mutant testing (sensor and apical loop mutants, blue bars; nucleotide-binding–site mutants, red bars; see Supplementary Table 1 for mutation details). The plotted data show mean ± s.d. from five independent experiments. All mutants except β-D392A show a significant defec! t (P < 0.01) with respect to the CCT-β wild-type complementation assay. Bottom, CCT-β protein levels (wild-type or mutant) at 72 h compared with those of GAPDH as a loading control. Detailed growth curves are shown in Supplementary Figure 11. Accession codes * Abstract * Accession codes * Author information * Supplementary information Primary accessions Protein Data Bank * 2XSM * 2XSM Referenced accessions Protein Data Bank * 1A6D * 1Q3Q * 1A6D * 1Q3Q Author information * Abstract * Accession codes * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Inés G Muñoz & * Hugo Yébenes Affiliations * Macromolecular Crystallography Group, Structural Biology and Biocomputing Programme, Spanish National Cancer Research Centre (CNIO), Madrid, Spain. * Inés G Muñoz, * Pablo Mesa, * Elisabeth Bragado-Nilsson & * Guillermo Montoya * Centro Nacional de Biotecnología, CSIC, Campus de la Universidad Autónoma de Madrid, Madrid, Spain. * Hugo Yébenes, * Pablo Mesa, * Marina Serna, * Ana Beloso & * José M Valpuesta * Department of Chemistry, Physical and Theoretical Chemistry Laboratory, Oxford, UK. * Min Zhou, * Ah Young Park & * Carol V Robinson * Cell Division & Cancer Group, Molecular Oncology Programme, Spanish National Cancer Research Centre (CNIO), Madrid, Spain. * Guillermo de Cárcer & * Marcos Malumbres Contributions H.Y. and A.B. isolated the complex, I.G.M. and H.Y. obtained the crystals, I.G.M. and G.M. solved the structure, H.Y. and J.M.V. performed the cryo-EM, H.Y. and M.Z. carried out the protease digestion and substrate cleaning experiments, M.Z., A.Y.P. and C.V.R. performed the mass spectrometry and proteomic analysis, P.M., G.d.C., E.B.-N., M.M. and M.S. performed the shRNA assay, all the authors analyzed the data and J.M.V. and G.M. wrote the manuscript with input from all authors. Competing financial interests The authors declare no competing financial interests. Corresponding authors Correspondence to: * Guillermo Montoya or * José M Valpuesta Supplementary information * Abstract * Accession codes * Author information * Supplementary information Movies * Supplementary Movie 1 (1 MB) This movie summarizes the relative movements that take place inside one of the CCT rings (only the equatorial domain and part of the intermediate domain are shown here). One of the CCT subunits alternates between the different conformations present in the ring (all the other subunits were fitted on that position superimposing their structurally conserved equatorial domains). A dashed circle is depicted as a reference to display the variable sensor loop conformations, which apparently produce a retractile movement towards the centre of the CCT inner cavity (the frame sequence has been ordered to show this "movement"). This movie also depicts the relative movement of the intermediate domain with respect the equatorial domain, which produces the opening and closure of the nucleotide-binding site (here represented with an AMPPNP molecule modelled from the thermosome structure 1Q3Q). * Supplementary Movie 2 (7 MB) A combination of the different conformation of the CCT subunits has been mounted in a random sequence (each subunit varies between eight different resolved conformations; see Supplementary Movie 1) depicting a possible mechanism for CCT mediated folding. In this representation, the piston-like sensor loops modify the shape of a white rubber band that represents a theoretical substrate. If this substrate is bound to several sensor loops at the same time, the substrate could experience local compressions, expansions and torsions that force its folding process. This input of kinetic energy, driven by the ATP binding-hydrolysis cycles, would be used to overcome folding-pathway barriers. Eventually a properly folded product is obtained. PDF files * Supplementary Text and Figures (2 MB) Supplementary Notes, Supplementary Figures 1–11, Supplementary Table 1 and Supplementary Methods Additional data - Conformational changes in Dnm1 support a contractile mechanism for mitochondrial fission
- Nat Struct Mol Biol 18(1):20-26 (2011)
Nature Structural & Molecular Biology | Article Conformational changes in Dnm1 support a contractile mechanism for mitochondrial fission * Jason A Mears1 Search for this author in: * NPG journals * PubMed * Google Scholar * Laura L Lackner2 Search for this author in: * NPG journals * PubMed * Google Scholar * Shunming Fang1 Search for this author in: * NPG journals * PubMed * Google Scholar * Elena Ingerman2, 3 Search for this author in: * NPG journals * PubMed * Google Scholar * Jodi Nunnari2 Search for this author in: * NPG journals * PubMed * Google Scholar * Jenny E Hinshaw1 Contact Jenny E Hinshaw Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature Structural & Molecular BiologyVolume: 18,Pages:20–26Year published:(2011)DOI:doi:10.1038/nsmb.1949Received23 April 2010Accepted06 October 2010Published online19 December 2010 Abstract * Abstract * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Mitochondria are dynamic organelles that undergo cycles of fission and fusion. The yeast dynamin-related protein Dnm1 has been localized to sites of mitochondrial division. Using cryo-EM, we have determined the three-dimensional (3D) structure of Dnm1 in a GTP-bound state. The 3D map showed that Dnm1 adopted a unique helical assembly when compared with dynamin, which is involved in vesicle scission during endocytosis. Upon GTP hydrolysis, Dnm1 constricted liposomes and subsequently dissociated from the lipid bilayer. The magnitude of Dnm1 constriction was substantially larger than the decrease in diameter previously reported for dynamin. We postulate that the larger conformational change is mediated by a flexible Dnm1 structure that has limited interaction with the underlying bilayer. Our structural studies support the idea that Dnm1 has a mechanochemical role during mitochondrial division. View full text Figures at a glance * Figure 1: Three-dimensional reconstructions of Dnm1 helices. () The primary sequence of Dnm1 contains four domains: GTPase, middle, B-insert and GED. () A cryo-ET reconstruction of Dnm1-lipid tubes from two orthogonal perspectives. The cylindrical shape of the helices is highlighted adjacent to a central z slice by an end view of the tomogram of Dnm1 (blue box). Scale bar, 100 nm. () The 3D structure of the Dnm1 tube with a wedge of the helix removed to show a representative cross-section. The protein is colored with a radial gradient (blue near the lipid to green at the periphery). The lipid bilayer is gray. The outer diameter (129 nm), inner lumen (89 nm), helical pitch (28.8 nm), two helical starts (labeled 1 and 2) and the spacing between each start (14.4 nm) are highlighted. The lipid bilayer, ridge and cleft features are indicated. () A representative raw image of the Dnm1 tubes. () One side of the raw image highlights the gap between lipid and protein. () An average 2D projection of the final Dnm1 reconstruction. () One side of! the 2D projection. () End view of the final 3D structure. Scale bar, 20 nm. () A cross-section of the final 3D map highlights the gap between Dnm1 and the lipid bilayer (compare with and ). Scale bar, 10 nm. * Figure 2: Analysis of helical packing of Dnm1. () Comparison between Dnm1 and ΔPRD-dynamin (Dyn-1) structures37. The helical pitch is 28.8 nm for Dnm1 and 10.6 nm for dynamin. The axial spacing between the two starts of the Dnm1 helix is 14.4 nm. The outer diameters (129 nm and 50 nm, respectively), radial path lengths (16.9 nm and 11.1 nm, respectively), ridge and cleft features are also indicated. The outer radial density (green) and the inner radial density (blue) are where the GTPase domains and the middle-GED domains, respectively, are predicted to reside. () Four GTPase domain crystal structures from dyn A (PDB ID: 1JX2, chain B) were manually fitted to one asymmetric subunit of the Dnm1 helical structure. Two dimers in separate helical starts of the asymmetric subunit are colored purple and orange, respectively. Two density thresholds are presented (low threshold, blue-green; high threshold, yellow). The dashed box highlights the region presented in (left) after a 90° rotation. () Fittings of GTPase domains are ! compared between Dnm1 (left) and Dyn-1 (right; ref. 39). A 3–4-nm gap between Dnm1 and the lipid bilayer (gray) exists where the PH domain (orange ribbon, yellow mesh) of Dyn-1 resides. * Figure 3: Dnm1-lipid tubes constrict upon addition of GTP. (–) Dnm1-lipid tubes were imaged using negative stain EM (–) and cryo-EM (–). Dnm1 tubes in the absence of nucleotide (,,), in the presence of GMP-PCP (,) and after addition of 1 mM GTP (,,,). Scale bars, 50 nm (–) and 100 nm (–). Asterisk in , bare lipid tubes; arrows in , regions where Dnm1 filaments are loosely packed; arrowheads and insets in indicate Dnm1 filaments that have dissociated from the membrane. () Distributions of tube diameters for Dnm1 tubes treated with or without 1 mM GTP for 5 s. () 90° light scattering of Dnm1 tubes decreased upon addition of 1 mM GTP (red arrowhead). * Figure 4: The diameters of Dnm1-lipid tubes recover after an initial constriction. (–) Cryo-EM images of Dnm1-lipid tubes in the absence of GTP (,) and 5 s after addition of 0.1 mM GTP (,). Scale bar, 100 nm. (–) Distribution of Dnm1 tube diameters before () and after (, 5 s; , 30 s) addition of 0.1 mM GTP. Corresponding outer tube diameters are highlighted by dashed blue lines (no GTP) in (110 nm) and (150 nm) and by dashed red lines (0.1 mM GTP, 5 s) in (65 nm) and (50 nm). * Figure 5: A model for mitochondrial fission. () An active contractile force is proposed to have a role in mitochondrial fission, in which Dnm1 is recruited to mitochondria and constricts the underlying membrane(s), which leads to fission and release of the protein. () Differences in helical packing and GTP-induced conformational changes between Dnm1 and Dyn-1. Unlike Dyn-1, Dnm1 assembles as a two-start helix and shows no axial compression upon addition of GTP. The inner lumen of Dnm1 tubes decrease from ~80 nm to ~25 nm, whereas that of dynamin decreases from ~20 nm to ~10 nm. Author information * Abstract * Author information * Supplementary information Affiliations * Laboratory of Cell Biochemistry and Biology, National Institute of Diabetes, Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, USA. * Jason A Mears, * Shunming Fang & * Jenny E Hinshaw * Department of Molecular and Cellular Biology, Center for Genetics and Development, University of California, Davis, Davis, California, USA. * Laura L Lackner, * Elena Ingerman & * Jodi Nunnari * Current address: Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, California, USA. * Elena Ingerman Contributions J.A.M. prepared, imaged and processed the cryo-EM data. L.L.L., E.I. and J.N. made the protein. S.F. processed the data. L.L.L. and J.N. critiqued the manuscript. J.A.M. and J.E.H. analyzed the data and wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Jenny E Hinshaw Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (4M) Supplementary Figures 1–4 and Supplementary Methods Additional data - Transcription of functionally related constitutive genes is not coordinated
- Nat Struct Mol Biol 18(1):27-34 (2011)
Nature Structural & Molecular Biology | Article Transcription of functionally related constitutive genes is not coordinated * Saumil J Gandhi1 Search for this author in: * NPG journals * PubMed * Google Scholar * Daniel Zenklusen1 Search for this author in: * NPG journals * PubMed * Google Scholar * Timothée Lionnet1 Search for this author in: * NPG journals * PubMed * Google Scholar * Robert H Singer1 Contact Robert H Singer Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature Structural & Molecular BiologyVolume: 18,Pages:27–34Year published:(2011)DOI:doi:10.1038/nsmb.1934Received04 February 2010Accepted17 September 2010Published online05 December 2010Corrected online12 December 2010 Abstract * Abstract * Change history * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Expression of an individual gene can vary considerably among genetically identical cells because of stochastic fluctuations in transcription. However, proteins comprising essential complexes or pathways have similar abundances and lower variability. It is not known whether coordination in the expression of subunits of essential complexes occurs at the level of transcription, mRNA abundance or protein expression. To directly measure the level of coordination in the expression of genes, we used highly sensitive fluorescence in situ hybridization (FISH) to count individual mRNAs of functionally related and unrelated genes within single Saccharomyces cerevisiae cells. Our results revealed that transcript levels of temporally induced genes are highly correlated in individual cells. In contrast, transcription of constitutive genes encoding essential subunits of complexes is not coordinated because of stochastic fluctuations. The coordination of these functional complexes therefore! must occur post-transcriptionally, and likely post-translationally. View full text Figures at a glance * Figure 1: Highly coordinated transcription of genes in the galactose network. () Schematic diagram of the organization of three GAL genes and their promoters on chromosome II. () Nascent transcripts at the transcription site (TS) in the nucleus and individual transcripts in the cytoplasm detected with single-mRNA FISH. GAL7 mRNA (red) and GAL1 mRNA (green) were detected in the same cell with cyanine 3– and cyanine 3.5–labeled probes, respectively. DAPI (blue) was used to demarcate the nucleus. Differential interference contrast (DIC) images are shown in the last column. The scale bars are 1 μm. () Fraction of cells showing each of the four different modes of transcription shown in . Fraction of 196 cells with active transcription sites for only GAL7 (red), only GAL1 (green), both genes (yellow) and neither gene (black). () The same pairwise analysis of GAL10 and GAL1 transcription sites in 309 cells. Error bars indicate s.e.m. * Figure 2: Correlation between cytoplasmic mRNA abundance of GAL genes in individual cells. () Heat map of number of GAL7 and GAL1 mRNAs in 195 individual cells. The color of each point indicates the number of cells observed at that value as specified by the color bar at the bottom. The marginal histograms represent the frequency of GAL7 mRNAs per cell (top) and GAL1 mRNAs per cell (right) across the entire population. The expression of GAL7 (top) ranged between 0 and 40 mRNAs per cell with a mean (μGAL7) of 10.6 ± 0.7 and a s.d. (σGAL7) of 9.9 transcripts. The expression of GAL1 (right) ranged between 0 and 40 mRNAs per cell with μGAL1 = 9.0 ± 0.7 and σGAL1 = 11.1. The correlation (r) between transcripts of these two genes in the same cell was 0.69 ± 0.04. () Pairwise correlation between the number of GAL10 and GAL1 transcripts in 325 cells. Marginal histograms: μGAL10 = 7.6 ± 0.5, σGAL10 = 8.1 (top); μGAL1 = 9.2 ± 0.5, σGAL1 = 10.4 (right). Error bars indicate s.e.m. * Figure 3: Anti-correlation between cytoplasmic mRNA abundance of genes expressed during different cell cycle stages. () Cartoon of expression profile for NDD1 and its target genes SWI5 and CLB2 across different stages of the cell cycle. () Experimentally measured average mRNA abundance of NDD1, SWI5 and CLB2 across three different stages of the cell cycle. () Representative FISH images of mRNAs of the transcriptional activator NDD1 (red) and its target gene SWI5 (green) in an asynchronous population of cells. The nuclei are marked with DAPI (blue). The scale bar in the DIC image of cells is 1 μm. () NDD1 and SWI5 transcripts are anti-correlated in a subset of cells that excludes G1 cells. The distribution of mRNAs per cell for each gene across the population is depicted by the marginal histograms: μSWI5 = 2.9 ± 0.3, σSWI5 = 2.9 (top); μNDD1 = 3.6 ± 0.3, σNDD1 = 2.1 (right). () SWI5 and CLB2 mRNAs, expressed during the same cell cycle stage, are highly correlated in a subset that excludes G1 cells. Marginal histograms: μSWI5 = 4.1 ± 0.4, σSWI5 = 3.4 (top); μCLB2 = 4.7 ± 0.4, σN! DD1 = 3.4 (right). Error bars indicate s.e.m. * Figure 4: Correlation between cytoplasmic mRNA abundance of functionally unrelated constitutively active genes. () Representative FISH images of mRNAs of two functionally unrelated genes, PRP8 (green) and MDN1 (red), are shown along with the DIC image of cells. The nuclei are marked with DAPI (blue). The scale bar is 1 μm. () Heat map of number of MDN1 and PRP8 transcripts in 369 cells. The correlation (r) between transcripts of these two genes in the same cell was 0.26 ± 0.05. The distribution of mRNAs per cell for each gene across the population is depicted by the marginal histograms: μMDN1 = 4.3 ± 0.1, σMDN1 = 2.4 (top); μPRP8 = 2.5 ± 0.1, σPRP8 = 1.4 (right). () Pairwise correlation between PRP8 and KAP104 in 179 cells. Marginal histograms: μPRP8 = 3.1 ± 0.2, σPRP8 = 1.8 (top); μKAP104 = 3.3 ± 0.2, σKAP104 = 1.5 (right). () Correlation between MDN1 and KAP104 in 260 cells. Marginal histograms: μMDN1 = 4.4 ± 0.1, σMDN1 = 2.4 (top); μKAP104 = 3.1 ± 0.1, σKAP104 = 1.6 (right). Error bars indicate s.e.m. * Figure 5: Correlation between cytoplasmic mRNA abundance of essential genes encoding subunits of multi-protein complexes. () Mean abundance and pairwise correlation coefficients for transcripts of three genes encoding β-subunits of the proteasome 20S core particle. () Correlation coefficients for three genes encoding TATA binding protein associated factors involved in transcription initiation. () Correlation between three genes encoding subunits of RNA polymerase II. Errors indicate s.e.m. * Figure 6: Correlation between transcripts from two alleles of a constitutively active gene, MDN1, in diploid cells. () Schematic diagram of the PP7 array inserted in the 3′ untranslated region of one of the two endogenous MDN1 alleles. () Transcripts from both alleles were detected with cyanine 3–labeled probes hybridizing to the coding region of MDN1 (green). Transcripts from Allele 2 (yellow) were distinguished with colocalizing signals from cyanine 3.5–labeled probes against 11 binding sites in the 24 × PP7 array (red). The scale bar in the DIC image is 1 μm. () Heat map of number of transcripts from two MDN1 alleles in 217 diploid cells. The correlation coefficient (r) between transcripts from two alleles in the same cell was 0.33 ± 0.04. The distribution of mRNAs per cell for each allele across the population is depicted by the marginal histograms: μMDN1 = 6.6 ± 0.3, σMDN1 = 4.0 (top); μMDN1-PP7 = 4.8 ± 0.3, σMDN1-PP7 = 2.8 (right). Error bars indicate s.e.m. * Figure 7: Stochastic model predicts correlation coefficients from mean mRNA abundance and half-life times. () TAF6 and TAF12 mRNA distributions determined by FISH (blue bars) and analytical theory (black line). () Correlation coefficient as a function of mRNA half-life for various abundance levels. The analytical solution was obtained by solving the master equation. (,) Response to perturbation in the number of mRNAs as a result of cell division depends on the mRNA half-life. Gene 1 (red) and Gene 2 (blue) are simulated Monte Carlo time traces of transcript abundances for two genes in a single cell over three cell cycles. Analytical solution (black) is plotted along with the average of 100 simulations (green). (,) Experimentally measured average mRNA abundance of TAF6 and TAF12 across three different stages of the cell cycle. Error bars indicate s.e.m. Change history * Abstract * Change history * Author information * Supplementary informationErratum 12 December 2010In the version of this article initially published online, the color key in Figure 7c,d was incorrect. The error has been corrected for all versions of this article. Author information * Abstract * Change history * Author information * Supplementary information Affiliations * Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, New York, USA. * Saumil J Gandhi, * Daniel Zenklusen, * Timothée Lionnet & * Robert H Singer Contributions S.J.G. and D.Z. initiated the project. S.J.G. conducted the experiments and data analysis. S.J.G. and T.L. did the numerical simulations and T.L. derived the analytical solution. D.Z. and R.H.S. supervised the project. S.J.G. wrote the paper with editorial help from D.Z., T.L. and R.H.S. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Robert H Singer Supplementary information * Abstract * Change history * Author information * Supplementary information PDF files * Supplementary Text and Figures (2M) Supplementary Figures 1–11, Supplementary Table 1 and Supplementary Methods Additional data - Mapping the sequence of conformational changes underlying selectivity filter gating in the Kv11.1 potassium channel
- Nat Struct Mol Biol 18(1):35-41 (2011)
Nature Structural & Molecular Biology | Article Mapping the sequence of conformational changes underlying selectivity filter gating in the Kv11.1 potassium channel * David T Wang1, 2, 3 Search for this author in: * NPG journals * PubMed * Google Scholar * Adam P Hill1, 2, 3 Search for this author in: * NPG journals * PubMed * Google Scholar * Stefan A Mann1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * Peter S Tan1 Search for this author in: * NPG journals * PubMed * Google Scholar * Jamie I Vandenberg1, 2 Contact Jamie I Vandenberg Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Corresponding authorJournal name:Nature Structural & Molecular BiologyVolume: 18,Pages:35–41Year published:(2011)DOI:doi:10.1038/nsmb.1966Received09 May 2010Accepted18 October 2010Published online19 December 2010 Abstract * Abstract * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg The potassium channel selectivity filter both discriminates between K+ and sodium ions and contributes to gating of ion flow. Static structures of conducting (open) and nonconducting (inactivated) conformations of this filter are known; however, the sequence of protein rearrangements that connect these two states is not. We show that closure of the selectivity filter gate in the human Kv11.1 K+ channel (also known as hERG, for ether-a-go-go–related gene), a key regulator of the rhythm of the heartbeat, is initiated by K+ exit, followed in sequence by conformational rearrangements of the pore domain outer helix, extracellular turret region, voltage sensor domain, intracellular domains and pore domain inner helix. In contrast to the simple wave-like sequence of events proposed for opening of ligand-gated ion channels, a complex spatial and temporal sequence of widespread domain motions connect the open and inactivated states of the Kv11.1 K+ channel. View full text Figures at a glance * Figure 1: Schematic representation of topology and energetics of gating in Kv11.1 channels. () Topology of Kv11.1 channel showing two of the four subunits. The central ion conduction pore lined by the selectivity filter (red) with K+ ions shown as black spheres. Individual domains are numbered on the left hand subunit. () Reaction co-ordinate diagram for Kv11.1 channel gating. The transition from the closed (C) to open (O) state involves at least three kinetically distinct closed states, whereas the transition from the open to inactivated (I) state contains a single dominant transition state (red segment of trace)25. The dashed red line shows the effect of a perturbation that affects both the energetics of the transition state ΔΔG‡ as well as the energy difference between the open and inactivated states, ΔΔG0. * Figure 2: Measurement of inactivation in Kv11.1 channels. () Measurement of rates of recovery from inactivation using double-pulse protocol (left) and rates of inactivation using triple-pulse voltage protocol (right). Portions of the current traces in red indicate the sections used to measure rates of recovery from inactivation (left) and rates of inactivation (right). On right panel, arrows indicate peak currents and Iss indicates steady-state current levels that were used to calculate equilibrium values for inactivation (see panel ). () A plot of the logarithm of the observed rate constants of inactivation (red unfilled squares) and recovery from inactivation (red filled squares) versus voltage shows the typical chevron shape observed for reactions with a single dominant transition state between the two end states29. The solid line is a fit of equation (3) (Online Methods), kobs,V = kinact,V + krec,V, and the dashed lines are the derived unidirectional rate constants kinact,V and krec,V. The values for kinact,V and krec,V at 0 mV! , highlighted on the graph, are used to calculate the equilibrium constant at 0 mV: K0 = kinact,0 / krec,0. () Comparison of the equilibrium constant for inactivation, calculated using the ratio of kinact,V / krec,V (red line), with the ratio of steady-state current to peak current from the right hand graph in (black squares). * Figure 3: Mutations in the outer turret affect energetics of Kv11.1 inactivation. () Plots of log(kinact,0) versus log(K0) for mutations in the amphipathic α-helix in the S5P domain: (i) Asn588, (ii) Gln592 and (iii) Asp591. Color coding refers to location of mutated residues on topology diagram. Individual mutations are indicated by their single-letter amino acid codes. In each panel, the unfilled square shows WT Kv11.1 and error bars are ± s.e.m. for n = 3–8 oocytes. In some instances, error bars are within the symbols. () Summary plot showing all mutations for all residues in the S5P linker (see also Supplementary Table 1). () Plot of Φ value versus |Δlog(K0)| for each pair of mutants in the S5P domain. Colored squares depict pairs of mutants that both occur in Asn588 (red), Gln592 (blue) or Asp591 (green). The dashed line indicates the Φ value from . () Plot of mean Φ values for pairs of mutants where the absolute value of Δlog(K0) lies in the ranges indicated. The dashed line indicates the Φ value from . When Δlog(K0) is small, even small ! errors in the measurement of Δlog(K0) can lead to large errors in the calculated Φ value. Δlog(K0) needs to be > 0.5 to obtain an accurate estimate of Φ values. * Figure 4: Mutations adjacent to the selectivity filter affect a late step in Kv11.1 inactivation. () Plots of log(kinact,0) versus log(K0) for residues in the linker between the selectivity filter and S6. Left, plots for individual residues where max Δlog(K0) is > 0.5; right, summary plot for all mutants in the domain. () Plots of log(kinact,0) versus log(K0) for the linker between the pore-helix (p-helix) and selectivity filter (SF). Left and middle, plots for individual residues (mutations are indicated by their single-letter amino acid codes); right, summary plot for all residues. In each panel, the unfilled squares show WT Kv11.1, and error bars are ± s.e.m. for n = 2–8 oocytes. In some instances error bars are within the symbols. White dots in the topology diagrams at far left indicate the approximate location of residues highlighted in left panels of each row. For purposes of clarity, individual mutations are not identified in the summary plots on right of each row, but the properties of all mutants examined are summarized in Supplementary Table 1. * Figure 5: Mutations in transmembrane and cytoplasmic domains affect dynamics of Kv11.1 inactivation. Plots of log(kinact,0) versus log(K0) for individual residues, where max Δlog(K0) is > 0.5, and summary plots for all mutants in a given domain. () S6. () S5. () S4-S5. () S4. In plots to the left of each panel, individual mutations are indicated by their single-letter amino acid codes. In each panel, the unfilled square shows WT Kv11.1 and error bars are ± s.e.m. for n = 4–13 oocytes. In some instances, error bars are within the symbols. White dots in the topology diagrams at far left indicate the approximate location of residues highlighted in the left panels of each row. White '+' symbols indicate approximate location of charged residues in S4 that were mutated in this study. For purposes of clarity, individual mutations are not identified in the summary panels shown at the right of each row, but the properties of all mutants examined are summarized in Supplementary Table 1. * Figure 6: External K+ concentration affects energetics of K+ channel inactivation. () Current decay due to inactivation measured at 0 mV slows as external K+ is increased from 2 mM K+ (black) to 10 mM K+ (red) and 25 mM K+ (orange) (top). The initial increase in current due to recovery from inactivation at −120 mV (highlighted by gray shading) is relatively insensitive to changes in external K+ (bottom). Insets show voltage protocols used to record currents. Dashed horizontal lines indicate zero current level. () Plot of the logarithm of the measured rate constants versus voltage for 2, 10 and 25 mM K+. Dashed lines show the unilateral forward and reverse rate constants and arrows indicate kinact,0. () Plot of log(kinact,0) versus log(K0) for different external K+ concentrations (black: 2 mM; green: 5 mM; red: 10 mM; orange: 25 mM; blue: 98 mM). Each point represents an individual experiment. The slope of the fitted line gave a Φ value of 0.96. * Scheme 1: * Scheme 2: * Figure 7: Japanese puzzle box model of allosteric control of selectivity filter gating. A schematic diagram showing two of the four subunits of the Kv11.1 channel, color coded according to whether the domain moves early (black) or late (orange) during the open to inactivated state transition as determined by Φ values derived in Figures 3,4,5. The six steps for opening the puzzle are (1) exit of K+ ions from the external side of the selectivity filter (Φ ~ 1) followed by movements of (2) the outer helix of the pore domain (Φ ~ 0.75), (3) the S5P linker in the extracellular turret (Φ ~ 0.6), (4) the S4 domain, (5) the cytoplasmic S4-S5 linker (Φ ~ 0.45) and (6) the inner helix of the pore domain, the pore helix-selectivity filter linker and the PS6 linker (all with Φ ~ 0.25). The final putative step is collapse of the selectivity filter. This final step remains to be verified experimentally. The arrows indicate the sequence of allosteric communication rather than any specific movement. The exact nature of each of the depicted domain movements remains to be ! determined. Author information * Abstract * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * David T Wang & * Adam P Hill Affiliations * Mark Cowley Lidwill Research Program in Cardiac Electrophysiology, Molecular Cardiology and Biophysics Division, Victor Chang Cardiac Research Institute, New South Wales, Australia. * David T Wang, * Adam P Hill, * Stefan A Mann, * Peter S Tan & * Jamie I Vandenberg * St. Vincent's Clinical School, Faculty of Medicine, University of New South Wales, New South Wales, Australia. * David T Wang, * Adam P Hill, * Stefan A Mann & * Jamie I Vandenberg Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Jamie I Vandenberg Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (1M) Supplementary Figures 1–4 and Supplementary Table 1 Additional data - A methylation and phosphorylation switch between an adjacent lysine and serine determines human DNMT1 stability
- Nat Struct Mol Biol 18(1):42-48 (2011)
Nature Structural & Molecular Biology | Article A methylation and phosphorylation switch between an adjacent lysine and serine determines human DNMT1 stability * Pierre-Olivier Estève1 Search for this author in: * NPG journals * PubMed * Google Scholar * Yanqi Chang2 Search for this author in: * NPG journals * PubMed * Google Scholar * Mala Samaranayake1 Search for this author in: * NPG journals * PubMed * Google Scholar * Anup K Upadhyay2 Search for this author in: * NPG journals * PubMed * Google Scholar * John R Horton2 Search for this author in: * NPG journals * PubMed * Google Scholar * George R Feehery1 Search for this author in: * NPG journals * PubMed * Google Scholar * Xiaodong Cheng2 Search for this author in: * NPG journals * PubMed * Google Scholar * Sriharsa Pradhan1 Contact Sriharsa Pradhan Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature Structural & Molecular BiologyVolume: 18,Pages:42–48Year published:(2011)DOI:doi:10.1038/nsmb.1939Received24 February 2010Accepted29 September 2010Published online12 December 2010 Abstract * Abstract * Accession codes * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg The protein lysine methyltransferase SET7 regulates DNA methyltransferase-1 (DNMT1) activity in mammalian cells by promoting degradation of DNMT1 and thus allows epigenetic changes via DNA demethylation. Here we reveal an interplay between monomethylation of DNMT1 Lys142 by SET7 and phosphorylation of DNMT1 Ser143 by AKT1 kinase. These two modifications are mutually exclusive, and structural analysis suggests that Ser143 phosphorylation interferes with Lys142 monomethylation. AKT1 kinase colocalizes and directly interacts with DNMT1 and phosphorylates Ser143. Phosphorylated DNMT1 peaks during DNA synthesis, before DNMT1 methylation. Depletion of AKT1 or overexpression of dominant-negative AKT1 increases methylated DNMT1, resulting in a decrease in DNMT1 abundance. In mammalian cells, phosphorylated DNMT1 is more stable than methylated DNMT1. These results reveal cross-talk on DNMT1, through modifications mediated by AKT1 and SET7, that affects cellular DNMT1 levels. View full text Figures at a glance * Figure 1: Lysine methylation and serine phosphorylation on DNMT1. () The lysines targeted for methylation by SET7 in various known substrates are shown in red. The serine in the consensus AKT1 kinase sequence is aligned with histone H3 Ser10 and DNMT1 Ser143. () SET7 methylation reaction in the presence of various concentrations of DNMT1 peptides representing either native or phosphorylated Ser143. () MS of the products of in vitro methylation assays (0 min, before assay; 40 min, after assay) on DNMT1 peptides with either native (left) or phosphorylated Ser143 (right). The mass shift of 14 Da represents addition of one methyl group. () AKT1 kinase reaction in the presence of fixed concentrations of wild-type DNMT1 peptides or S143A mutant peptides. () Western blot assay showing AKT1 kinase activity on dephosphorylated recombinant full-length DNMT1 on Ser143. () Western blot showing Ser143 phosphorylation of full-length DNMT1 in the presence (+) or absence (−) of constitutively active AKT1 kinase. Antibodies used for probe are listed; act! in is shown as a loading control. MDM2 pSer166 is a positive control. () S143A mutation in full-length DNMT1 facilitates SET7-mediated Lys142 methylation. Western blot demonstrating effect of Ser143 phosphorylation and/or Lys142 methylation in either wild-type (WT) DsRed-DNMT1 or the mutant DsRed-DNMT1(S143A), as indicated by plus and minus signs, when coexpressed with GFP alone (lanes 1 and 2) or GFP-SET7 (lanes 3 and 4). * Figure 2: Structure of the SET7–DNMT1 complex. () The reaction occurred during crystallization. Omit electron densities, Fo – Fc contoured at 4σ above the mean, are shown for the monomethylated Lys142 (black mesh) and the methyl group (green mesh). () The substrate peptide and the reaction product AdoHcy occupy binding sites in the opposite ends of a narrow target-lysine channel. () Electrostatic interactions, hydrogen bonds and van der Waals interactions define SET7–DNMT1 peptide interactions (dashed lines). The network of interactions includes the following: (i) DNMT1 Arg139 forms a hydrogen bond with the main chain carbonyl oxygen atom of SET7 Gly336 (not shown) and an intramolecular interaction with the main chain carbonyl oxygen of DNMT1 Ser141; (ii) DNMT1 Arg140 forms an electrostatic salt bridge to SET7 Glu348 and a hydrogen bond with the main chain carbonyl oxygen atom of SET7 Arg258; (iii) DNMT1 Ser141 forms a serine-serine interaction with SET7 Ser268 and a water-mediated network with DNMT1 His252 (not sho! wn) and SET7 Asp256; (iv) the target nitrogen atom of DNMT1 Lys142me1 forms two hydrogen bonds with the side chain hydroxyl oxygen atoms of SET7 Tyr305 and Tyr245; and (v) DNMT1 Ser143 is involved in a polar interaction with SET7 Lys317 and a van der Waals contact with SET7 Leu267. In addition, two main chain atoms of DNMT1, the carbonyl oxygen of Arg140 and the amide nitrogen of Lys142me1, link through SET7 Thr266. * Figure 3: DNMT1 association with AKT1 kinase. () Colocalization of DNMT1 and AKT1 in COS-7 cells, as shown by transiently expressed DsRed-DNMT1 (red) and endogenous AKT1 kinase (green; stained with fluorescent anti-AKT1). Nuclei are Hoechst-stained (blue). Cells were released from G1/S arrest and followed through S and G2 phase for the time indicated at left. Percentages of cells showing similar pattern of colocalization are shown in parentheses. () Immunoprecipitation (IP) of AKT1, DNMT1 or GFP (control) from HEK293 nuclear extracts followed by western blot with indicated antibodies. () Coimmunoprecipitation of DNMT1–AKT1 complex at different time points after cell synchronization. () Expression different post-translationally modified (or total) DNMT1 species during the cell cycle. Cells were released from G1/S arrest and followed for the time shown at top. Extracts were western blotted and probed with the antibodies indicated. Anti-DNMT1 shows total enzyme. Cyclin A was used as the cell cycle marker. A representativ! e western blot for actin (which was used as a loading control) is shown. () Quantification of cell cycle–dependent expression of different DNMT1 species, normalized to actin levels. phDNMT1, DNMT1-pSer143; meDNMT1, DNMT1-Lys142me1. Data shown represent two independent western blots as in . Error bars show s.d. * Figure 4: AKT1 kinase–mediated phosphorylation stabilizes DNMT1. () Western blotting and RNA expression analysis of extracts from HeLa cells with AKT1 knockdown (si-AKT1) or control knockdown (si-Control). Left, western blot with indicated antibodies. Marker, biotinylated protein ladder showing relative molecular mass of DNMT1 as ~200 kDa. Right, RNA expression measured from quantitative PCR; error bars represent s.d. of three independent experiments. DNMT1-pSer143 is reduced in AKT1-knockdown cells, resulting in moderately higher DNMT1-Lys142me1 levels but no change in DNMT1 RNA levels. () Overexpression of the AKT1 dominant-negative mutant (AKT1DN) reduces DNMT1-pSer143 and increases DNMT1-Lys142me1 levels, as shown by western blotting with the indicated antibodies. () The AKT kinase inhibitor LY294002 reduces DNMT1-pSer143 levels and increases DNMT1-Lys142me1 levels in a time-dependent manner, as shown by western blotting with the indicated antibodies. () Effect of SET7 on DNMT1-Lys142me1 in the absence (−) or presence (+) of calycul! in A, a serine/threonine phosphatase inhibitor, as shown by western blotting with indicated antibodies. Cells were co-transfected with constructs expressing GFP alone with DsRed-DNMT1 (lanes 1 and 2) or GFP-SET7 with DsRed-DNMT1 (lanes 3 and 4). () Degradation of DNMT1 modified and unmodified species after cells were treated with cycloheximide (CHX). At indicated times, cells were lysed and western blotted with indicated antibodies. () Ubiquitinylation of total DNMT1 and DNMT1-Lys142me1. Wild-type DNMT1 or the DNMT1(S143A) mutant was overexpressed in cells, along with HA-ubiquitin and GFP-SET7, in the presence (+) or absence (−) of the proteasome inhibitor MG132. Total DNMT1 or DNMT1-Lys142me1 was immunoprecipitated (IP) from the nuclear extract and western blotted with antibody to HA. () A proposed model of cross-talk between DNMT1 (black), SET7 and AKT1 in mammalian cells. The other interactors that can reverse the post-translational modifications are an unknown phospha! tase and an unknown lysine-specific demethylase (KDMT). One su! ch KDMT may be LSD1 (ref. 17). Accession codes * Abstract * Accession codes * Author information * Supplementary information Primary accessions Protein Data Bank * 3OS5 * 3OS5 Referenced accessions Protein Data Bank * 3CBM * 3CBM Author information * Abstract * Accession codes * Author information * Supplementary information Affiliations * New England Biolabs, Ipswich, Massachusetts, USA. * Pierre-Olivier Estève, * Mala Samaranayake, * George R Feehery & * Sriharsa Pradhan * Department of Biochemistry, Emory University School of Medicine, Atlanta, Georgia, USA. * Yanqi Chang, * Anup K Upadhyay, * John R Horton & * Xiaodong Cheng Contributions P.-O.E. performed cell biology and biochemistry experiments. M.S. made constructs, tested kinetics and performed the pull-down assay. G.R.F. performed quantitative PCR. Y.C. performed SET7 purifications, MS-based methylation assays on DNMT1 peptide, mutagenesis of K142R and crystallization of SET7–DNMT1 peptide. A.K.U. purified the DNMT1 N-terminal domain and performed MS-based methylation assays on this fragment. J.R.H. performed crystallographic experiments. X.C. and S.P. organized and analyzed data and wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Sriharsa Pradhan Supplementary information * Abstract * Accession codes * Author information * Supplementary information PDF files * Supplementary Text and Figures (5M) Supplementary Figures 1–7 Additional data - β2-microglobulin forms three-dimensional domain-swapped amyloid fibrils with disulfide linkages
- Nat Struct Mol Biol 18(1):49-55 (2011)
Nature Structural & Molecular Biology | Article β2-microglobulin forms three-dimensional domain-swapped amyloid fibrils with disulfide linkages * Cong Liu1, 2, 3 Search for this author in: * NPG journals * PubMed * Google Scholar * Michael R Sawaya1, 2, 3 Search for this author in: * NPG journals * PubMed * Google Scholar * David Eisenberg1, 2, 3 Contact David Eisenberg Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature Structural & Molecular BiologyVolume: 18,Pages:49–55Year published:(2011)DOI:doi:10.1038/nsmb.1948Received15 March 2010Accepted16 September 2010Published online05 December 2010 Abstract * Abstract * Accession codes * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg β2-microglobulin (β2m) is the light chain of the type I major histocompatibility complex. It deposits as amyloid fibrils within joints during long-term hemodialysis treatment. Despite the devastating effects of dialysis-related amyloidosis, full understanding of how fibrils form from soluble β2m remains elusive. Here we show that β2m can oligomerize and fibrillize via three-dimensional domain swapping. Isolating a covalently bound, domain-swapped dimer from β2m oligomers on the pathway to fibrils, we were able to determine its crystal structure. The hinge loop that connects the swapped domain to the core domain includes the fibrillizing segment LSFSKD, whose atomic structure we also determined. The LSFSKD structure reveals a class 5 steric zipper, akin to other amyloid spines. The structures of the dimer and the zipper spine fit well into an atomic model for this fibrillar form of β2m, which assembles slowly under physiological conditions. View full text Figures at a glance * Figure 1: Characterization of β2m oligomers. () SDS-PAGE of β2m oligomerization under non-reducing and reducing conditions. Oligomerization started from monomeric β2m with or without DTT agitated at 37 °C for various times. After 5 d, without DTT, oligomer ladders still formed (lane 1), but much slower than with DTT (lane 3). Over time, the disulfide-bridged oligomer ladders were observed (lane 6, 8, 10 and 12). After 10 d, even higher oligomers formed and remained at the boundary between the stacking gel and running gel (lanes 10 and 12, open arrows). After 30 d, protofilaments formed and were stuck in the loading well (lane 12, filled arrow). The oligomers were dissociated into monomers upon reduction in SDS loading buffer (+DTT) (lanes 2, 4, 5, 7, 9 and 11). Lane 13 is the BenchMark Protein Ladder (Invitrogen). The additional band above the dimer that exists in all β2m samples is DTT resistant, unlike other oligomeric species. It is composed of β2m, as found by amino acid sequencing, but the mechanism of its fo! rmation remains to be determined. () Electron micrograph showing the protofilaments formed in . The scale bar is 200 nm. () Experimental fibril X-ray diffraction pattern of protofilaments visualized in . * Figure 2: Refolding and purification of β2m dimer. () Analytical size-exclusion chromatography elution profiles of β2m after refolding. The dotted profiles show five molecular weight markers (Bio-Rad gel filtration standard). From right to left: vitamin B12 (Mr 1,350), equine myoglobin (Mr 17,000), chicken ovalbumin (Mr 44,000), bovine gamma globulin (Mr 158,000) and thyroglobulin (Mr 670,000). The solid profiles show β2m refolding from inclusion bodies with or without βME. AU, absorbance units. () Purified dimer on SDS-PAGE. Lane 1, BenchMark Protein Ladder; lanes 2, 3, β2m dimer in the absence or presence of DTT in SDS loading dye. * Figure 3: Structure of the domain-swapped β2m dimer. () Ribbon diagram of the crystal structure of monomeric β2m (PDB entry 1LDS34). () Topology diagrams of β2m monomer and dimer. Intra- and intermolecular disulfide bonds are highlighted in the same color as the backbones. () Ribbon diagram of the crystal structure of β2m domain-swapped dimer. * Figure 4: Systematic screening for amyloidogenic segments in the hinge loop (residues 52–65). Five segments were selected and synthesized. LSFSKD and KDWSFY in solid lines formed fibrils, and LSFSKD also formed microcrystals. The scale bars are 100 nm for electron microscopy (fibrils) and 50 μm for light microscopy (microcrystals). The experimental X-ray diffraction images show a typical cross-β fibril diffraction pattern. * Figure 5: Crystal structure of segment LSFSKD and schematics for β2m fibrillation. () Atomic structure of hinge loop segment LSFSKD. Yellow spheres represent water molecules. The backbone of one sheet is magenta, and the backbone of the other sheet is blue. The interdigitated side chains between adjacent β-sheets form a dry interface, as shown by the projection down the fibril axis on the left. The LSFSKD structure is a typical steric zipper structure, belonging to class 5 (ref. 5). () Schematic model for β2m fibrillation via domain swapping. Upon reduction of the intramolecular disulfide bond, the β2m monomer can assemble to 'closed-ended' oligomers, such as the dimer characterized in this work, or 'open-ended' runaway domain-swapped oligomers. Each subunit is colored in either blue or magenta. The formation of intermolecular disulfide bonds stabilizes the domain-swapped oligomers. The self-association of hinge loops into a zipper spine accomplishes the transformation from oligomers into fibrils. Accession codes * Abstract * Accession codes * Author information * Supplementary information Primary accessions Referenced accessions Protein Data Bank * 1DUZ * 1LDS * 3LOW * 3LOZ * 1DUZ * 1LDS * 3LOW * 3LOZ Author information * Abstract * Accession codes * Author information * Supplementary information Affiliations * University of California Los Angeles–United States Department of Energy Institute for Genomics and Proteomics, University of California, Los Angeles, Los Angeles, California, USA. * Cong Liu, * Michael R Sawaya & * David Eisenberg * Howard Hughes Medical Institute, University of California, Los Angeles, Los Angeles, California, USA. * Cong Liu, * Michael R Sawaya & * David Eisenberg * Molecular Biology Institute, University of California, Los Angeles, Los Angeles, California, USA. * Cong Liu, * Michael R Sawaya & * David Eisenberg Contributions C.L. designed the research, with advice from D.E., and carried out all the experiments; M.R.S. calculated and built the fibril models; C.L. wrote the paper; all authors discussed the results and revised the manuscript; D.E. supervised the work. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * David Eisenberg Supplementary information * Abstract * Accession codes * Author information * Supplementary information PDF files * Supplementary Text and Figures (928K) Supplementary Figures 1–7 and Supplementary Methods Additional data - The resistance of DMC1 D-loops to dissociation may account for the DMC1 requirement in meiosis
- Nat Struct Mol Biol 18(1):56-60 (2011)
Nature Structural & Molecular Biology | Article The resistance of DMC1 D-loops to dissociation may account for the DMC1 requirement in meiosis * Dmitry V Bugreev1, 5 Search for this author in: * NPG journals * PubMed * Google Scholar * Roberto J Pezza2, 4, 5 Search for this author in: * NPG journals * PubMed * Google Scholar * Olga M Mazina1 Search for this author in: * NPG journals * PubMed * Google Scholar * Oleg N Voloshin2 Search for this author in: * NPG journals * PubMed * Google Scholar * R Daniel Camerini-Otero2 Contact R Daniel Camerini-Otero Search for this author in: * NPG journals * PubMed * Google Scholar * Alexander V Mazin1, 3 Contact Alexander V Mazin Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorsJournal name:Nature Structural & Molecular BiologyVolume: 18,Pages:56–60Year published:(2011)DOI:doi:10.1038/nsmb.1946Received16 April 2010Accepted01 October 2010Published online12 December 2010 Abstract * Abstract * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg The ubiquitously expressed Rad51 recombinase and the meiosis-specific Dmc1 recombinase promote the formation of strand-invasion products (D-loops) between homologous molecules. Strand-invasion products are processed by either the double-strand break repair (DSBR) or synthesis-dependent strand annealing (SDSA) pathway. D-loops destined to be processed by SDSA need to dissociate, producing non-crossovers, and those destined for DSBR should resist dissociation to generate crossovers. The mechanism that channels recombination intermediates into different homologous-recombination pathways is unknown. Here we show that D-loops in a human DMC1-driven reaction are substantially more resistant to dissociation by branch-migration proteins such as RAD54 than those formed by RAD51. We propose that the intrinsic resistance to dissociation of DMC1 strand-invasion intermediates may account for why DMC1 is essential to ensure the proper segregation of chromosomes in meiosis. View full text Figures at a glance * Figure 1: Pathways of homologous recombination. Repair of a DSB proceeds either through the DSBR mechanism (left), which results in crossovers and is more common in meiosis than in mitosis; or through the SDSA mechanism (right), which results in non-crossovers. Red and blue represent DNA copies of two different homologous chromosomes. * Figure 2: RAD54 does not dissociate non-deproteinized joint molecules (D-loops) formed by DMC1. () The experimental approach. The asterisk indicates 32P-label; balls represent nucleoprotein complexes; tailed DNA, oligonucleotides 199 and 209 (Table 1); scDNA, supercoiled DNA. () Dissociation of DMC1-formed D-loops by RAD54, analyzed by gel electrophoresis. The reaction was initiated by mixing of non-deproteinized DMC1-generated D-loops (containing DMC1) with RAD54, either in the presence of Ca2+ or after Ca2+ depletion by EGTA, and was carried out for the indicated periods of time. () Graph of data from . () Dissociation of non-deproteinized RAD51-formed D-loop by RAD54, analyzed by gel electrophoresis, as in . () Graph of data from . Error bars indicate s.e.m. * Figure 3: RAD54 dissociates native D-loops formed by RAD51, but not DMC1, in the presence of Hop2–Mnd1. () Experimental scheme. Asterisk indicates 32P-label; balls represent nucleoprotein complexes; scDNA, supercoiled DNA. () The kinetics of D-loop dissociation was initiated by mixing the D-loops formed by DMC1 (1 μM) or RAD51 (1 μM) in the presence of Hop2–Mnd1 (0.2 μM) with RAD54 (0.32 μM). DNA products were analyzed by gel electrophoresis. () Graph of results from . () Effect of RAD54 concentration on D-loop dissociation. D-loops formed by DMC1 or RAD51 in the presence of Hop2–Mnd1 were mixed with RAD54 at the indicated concentrations. The results are shown as a graph. In and , a D-loop measurement of 100% represents the amount of D-loops formed in the absence of RAD54; error bars indicate s.e.m. Author information * Abstract * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Dmitry V Bugreev & * Roberto J Pezza Affiliations * Department of Biochemistry and Molecular Biology, Drexel University College of Medicine, Philadelphia, Pennsylvania, USA. * Dmitry V Bugreev, * Olga M Mazina & * Alexander V Mazin * Genetics and Biochemistry Branch, National Institute of Diabetes and Digestive and Kidney Diseases, US National Institutes of Health, Bethesda, Maryland, USA. * Roberto J Pezza, * Oleg N Voloshin & * R Daniel Camerini-Otero * Institute of Cytology and Genetics, Siberian Branch of the Russian Academy of Science, Novosibirsk, Russia. * Alexander V Mazin * Current address: Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma, USA. * Roberto J Pezza Contributions D.V.B., R.J.P., A.V.M. and R.D.C.-O. conceived the general ideas for this study. All authors planned experiments and interpreted data. D.V.B., R.J.P., O.N.V. and O.M.M. performed experiments. A.V.M. and R.D.C.-O. wrote the manuscript, and all authors provided editorial input. Competing financial interests The authors declare no competing financial interests. Corresponding authors Correspondence to: * R Daniel Camerini-Otero or * Alexander V Mazin Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (3M) Supplementary Methods, Supplementary Figures 1–5 and Supplementary Table 1 Additional data - Mixed Hsp90–cochaperone complexes are important for the progression of the reaction cycle
- Nat Struct Mol Biol 18(1):61-66 (2011)
Nature Structural & Molecular Biology | Article Mixed Hsp90–cochaperone complexes are important for the progression of the reaction cycle * Jing Li1 Search for this author in: * NPG journals * PubMed * Google Scholar * Klaus Richter1 Contact Klaus Richter Search for this author in: * NPG journals * PubMed * Google Scholar * Johannes Buchner1 Contact Johannes Buchner Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorsJournal name:Nature Structural & Molecular BiologyVolume: 18,Pages:61–66Year published:(2011)DOI:doi:10.1038/nsmb.1965Received11 June 2010Accepted08 October 2010Published online19 December 2010 Abstract * Abstract * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg The chaperone cycle of heat shock protein-90 (Hsp90) involves progression through defined complexes with different cochaperones. It is still enigmatic how the exchange of cochaperones is regulated. The first cochaperone entering the cycle is the Hsp90 ATPase inhibitor Sti1 (Hop in human), which later is replaced by a prolyl isomerase (PPIase) and p23. We found, unexpectedly, that one Sti1 molecule is sufficient to completely inhibit the ATPase of the Hsp90 dimer. Upon addition of a PPIase cochaperone to the Hsp90–Sti1 complex, an asymmetric ternary complex is preferentially formed. This PPIase–Hsp90–Sti1 intermediate is important for the progression of the cycle. To expel the bound Sti1, the concerted action of ATP and p23 is required. This mechanism, which is strictly conserved between the yeast and human Hsp90 systems, presents an example of how, in a cyclic process, directionality of assembly and disassembly of protein complexes can be achieved. View full text Figures at a glance * Figure 1: Interaction of Sti1 and Cpr6 with Hsp90. () Inhibition of the ATPase activity of yeast Hsp90 by Sti1. Different concentrations of Sti1 were added to 4 μM Hsp90, and the resulting ATPase activities were measured (black). Identical experiments were performed in the presence of Cpr6 (red, 2 μM Cpr6; green, 12 μM Cpr6). Error bars represent s.d. () Affinity isolation of Sti1-containing and Cpr6-containing complexes from yeast cell lysates. Ni-NTA beads complexed with His-tagged Sti1 or Cpr6 were pulled down, and the proteins indicated were detected by western blotting. First column (Lysate) shows the presence of the proteins in the lysate. Control pull-down experiments were performed with beads to which a His-tagged Escherichia coli protein (YjiE) was coupled. The stoichiometries (Stoi.) of the pulled-down proteins were calculated from a dilution standard of purified proteins. Left gels, pull-down of His-tagged Sti1 from lysates of a STI1-deletion strain. Right gels, pull-down of His-tagged Cpr6 from lysates of a CP! R6-deletion strain. () Titration of ATTO550-tagged Hsp90 (*Hsp90, acceptor) to Alexa Fluor 488–tagged Sti1 (*Sti1, donor). Different concentrations of *Hsp90 were added (0 nM, 25 nM, 50 nM, 75 nM, 100 nM, 150 nM, 200 nM, 300 nM, 400 nM, 600 nM and 800 nM, represented by different colors in plots) to 300 nM *Sti1, and the emission spectra were recorded at 25 °C. The excitation wavelength was set to 494 nm. AU, arbitrary units. () Release of Sti1 from the Hsp90 complex. We added 300 nM *Hsp90 to 300 nM *Sti1, and the binding kinetics were monitored (black plot) at 25 °C in standard assay buffer (see Online Methods). Excess amounts of unlabeled Sti1 (blue) or Cpr6 (red) were added to compete with the donor fluorophore (*Sti1) for the formation of Hsp90 complexes. The orange arrow indicates the addition of proteins. * Figure 2: aUC analysis of the Sti1-Hsp90-Cpr6 interaction. () Binding of Hsp90 to Sti1, investigated using aUC. Δc/Δt profiles (Δc/Δt represents the change in fluorescence signal intensity over time) are shown for 0.5 μM labeled *Sti1 alone (black), *Sti1 mixed with 1 μM Hsp90 (red) and *Sti1 mixed with 1 μM Hsp90 and 1 μM Cpr6 (blue). The yellow symbol indicates that *Sti1 was detected. () Binding of Hsp90 to Cpr6, investigated using aUC. Δc/Δt profiles are shown for 0.5 μM labeled *Cpr6 alone (black), *Cpr6 mixed with 1 μM Hsp90 (red) and *Cpr6 mixed with 1 μM Hsp90 and 1 μM Sti1 (blue). The yellow symbol indicates that *Cpr6 was detected. * Figure 3: Regulation of the asymmetric complex by nucleotides and p23. () Regulation of the asymmetric complex by AMP-PNP, investigated using aUC. Δc/Δt profiles are shown for the *Sti1–Hsp90–Cpr6 asymmetric complex in the absence (black) or presence (blue) of AMP-PNP, and for the asymmetric complex with 2 μM p23 and 2 mM AMP-PNP (olive green). No effect of p23 was observed in the absence of AMP-PNP (red). () The release of Sti1 from the Hsp90 complex, investigated by FRET. We added 300 nM *Hsp90 (acceptor) to 300 nM *Sti1 (donor), and the binding kinetics (black) were monitored at 25 °C in standard reaction buffer (see Online Methods). We added Cpr6 alone (red) Cpr6 and AMP-PNP (light blue), Cpr6, AMP-PNP and p23 (turquoise), or AMP-PNP and p23 (dark blue) to the *Sti1–*Hsp90 complex to trace the kinetics of *Sti1 release. As controls, AMP-PNP (pink) and p23 (olive green) were added alone. The orange arrow indicates the addition of nucleotide or protein. AU, arbitrary units. * Figure 4: Asymmetric complex formation in the human HSP90 system. () Generation of the Fkbp51–Hsp90–Hop asymmetric complex. Binding of the human Hsp90β to Fkbp51 was investigated using aUC. Δc/Δt profiles are shown for Fkbp51 with Hsp90 (black) and for Fkbp51 with both Hsp90 and Hop (red). () Generation of the Xap2–Hsp90–Hop asymmetric complex. Binding of the human Hsp90β to Xap2 was investigated using aUC. Δc/Δt profiles are shown for Xap2 with Hsp90 alone (black) and with both Hsp90 and Hop (red). () Quantitative evaluation of Hop–Xap2–Hsp90 complex formation. Raw sedimentation-velocity runs were evaluated by UltraScan to obtain the complexed and free concentrations of Xap2 in the presence of Hsp90 at different concentrations of Hop, and the complexed fraction of Xap2 was plotted. Separately, we simulated random binding using the binding constant for Hop that best matched the initial decrease in the complexed Xap2 fraction (red line). () The regulation of the asymmetric Xap2-containing complex, investigated using aUC. �! �c/Δt profiles are shown for the Hop–Hsp90–Xap2 asymmetric complex (black), and for the complex with the addition of p23 (2 μM) in the absence (red) or presence of 2 mM AMP-PNP (blue). Results for the complex plus AMP-PNP alone are depicted in green. * Figure 5: Model of the Hsp90 cochaperone cycle. Hop/Sti1 binds to the open conformation of Hsp90. One Sti1 molecule bound is sufficient to inhibit the Hsp90 ATPase activity. For simplicity, Hsc70 is depicted to enter the cycle together with client protein after Sti1 is bound to Hsp90. It is reasonable to assume that, alternatively, Hsc70 might already be bound to Sti1 when Sti1 enters the complex. The other TPR-acceptor site is then preferentially occupied by a PPIase, leading to an asymmetric Hsp90 complex. Hsp90 converts to the closed conformation after binding of ATP and binding of p23. This reaction weakens the binding of Sti1 and therefore promotes its exit from the complex. Another PPIase (dashed outline) can potentially bind to form the final complex together with Hsp90 and p23. After ATP hydrolysis, p23, PPIase and the folded client are released from Hsp90. Author information * Abstract * Author information * Supplementary information Affiliations * Center for Integrated Protein Science, Department Chemie, Technische Universität München, Munich, Germany. * Jing Li, * Klaus Richter & * Johannes Buchner Contributions J.L. designed, performed and analyzed experiments and wrote the first draft of the paper. K.R. was responsible for aUC and data analysis and contributed to writing the manuscript. J.B. designed and supervised experiments and wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding authors Correspondence to: * Johannes Buchner or * Klaus Richter Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (1M) Supplementary Figures 1–7 and Supplementary Methods Additional data - On the structural basis of modal gating behavior in K+ channels
- Nat Struct Mol Biol 18(1):67-74 (2011)
Nature Structural & Molecular Biology | Article On the structural basis of modal gating behavior in K+ channels * Sudha Chakrapani1, 2, 3 Search for this author in: * NPG journals * PubMed * Google Scholar * Julio F Cordero-Morales1, 2, 3 Search for this author in: * NPG journals * PubMed * Google Scholar * Vishwanath Jogini1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * Albert C Pan1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * D Marien Cortes1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * Benoît Roux1 Search for this author in: * NPG journals * PubMed * Google Scholar * Eduardo Perozo1 Contact Eduardo Perozo Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature Structural & Molecular BiologyVolume: 18,Pages:67–74Year published:(2011)DOI:doi:10.1038/nsmb.1968Received20 March 2010Accepted18 October 2010Published online26 December 2010 Abstract * Abstract * Accession codes * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Modal-gating shifts represent an effective regulatory mechanism by which ion channels control the extent and time course of ionic fluxes. Under steady-state conditions, the K+ channel KcsA shows three distinct gating modes, high-Po, low-Po and a high-frequency flicker mode, each with about an order of magnitude difference in their mean open times. Here we show that in the absence of C-type inactivation, mutations at the pore-helix position Glu71 unmask a series of kinetically distinct modes of gating in a side chain–specific way. These gating modes mirror those seen in wild-type channels and suggest that specific interactions in the side chain network surrounding the selectivity filter, in concert with ion occupancy, alter the relative stability of pre-existing conformational states of the pore. The present results highlight the key role of the selectivity filter in regulating modal gating behavior in K+ channels. View full text Figures at a glance * Figure 1: Modal gating behavior of WT KcsA. () A continuous recording of KcsA single-channel currents measured under steady-state conditions at pH 3.0 and +150 mV in 200 mM symmetric K+ solutions. () Left, KcsA shows a highly variable kinetic behavior that arises from a combination of three distinct modes of channel activity, the high-Po, low-Po and flicker modes. Right, histograms show a distribution of open times within bursts for each of the three modes of channel activity with corresponding values indicated above. () Channels occasionally switch between modes within a burst of activity, suggesting that modes arise from a homogenous population of channels. * Figure 2: Glu71 mutants stabilize individual gating modes in a side chain–specific way. () Macroscopic responses of WT and various Glu71 mutants elicited by pH jumps from 8.0 to 4.0 using a rapid solution exchanger in the presence of 200 mM KCl and with the membrane potential held at +150 mV. The current trace for the E71G mutant is shown at a relative amplitude compared with the other traces; the inset shows the same trace expanded in the current axis. () A plot of Isteady/Ipeak for various Glu71 mutants (n > 5). () Single-channel currents were recorded under steady-state conditions at pH 4.0 and +150 mV in 200 mM symmetric K+ solutions. Gray box highlights mutants that are focused on in this study. () Selectivity versus Na+ estimated from single-channel current-voltage (I-V) ramps under bi-ionic conditions. No detectable Na+ currents were seen in any of the mutants. Eapparent is the voltage value at which outward K+ currents can last be resolved. Error bars, s.d (n > 5). * Figure 3: Kinetic behavior of Glu71 mutants. Left, representative single-channel activity for Glu71 mutants. Middle, histograms show a distribution of closed- and open-channel lifetimes for the entire recordings. Single-channel current recordings were best fit by three closed and one open state for Glu71 mutants. Right, the closed states were defined as F, Ii and Is on the basis of their lifetimes. Rate constants of recovery from Is for WT and E71H are overestimated as a result of low Po and uncertainty in the number of channels in the patch. * Figure 4: Crystal structure of E71I. () Single-subunit line representation of the P-loop of E71I overlaid onto the WT structure5 (PDB entry 1K4C) highlights the conductive conformation of the selectivity filter backbone. () One-dimensional electron density profile along the central symmetry (z) axis is shown. S1–S4 denote the K+ binding sites. Gray peaks in the background correspond to one-dimensional electron density profile of the WT structure. () Electron density map of residues 60–84 from two diagonally symmetric subunits. Sticks, polypeptide chain; blue mesh, 2σ contour of the 2Fo − Fc electron density map for the protein; magenta mesh, 6σ contour of the Fo − Fc omit map for the ions; red mesh, 4σ contour of the Fo − Fc omit map for the waters. () A single-subunit P-loop is shown, with side chains at Glu71 and Asp80 in stick representation. The H-bond interaction between the three crystallographic water molecules within the cavity behind the filter and the rest of the protein are represented b! y black dotted lines. * Figure 5: Crystal structure of E71Q. () Single-subunit line representation of the P-loop of E71Q overlaid onto the WT structure5 (PDB entry 1K4C) highlights the conductive conformation of the selectivity filter backbone. () One-dimensional electron density profile along the central symmetry (z) axis is shown. S1–S4 denote the K+ binding sites. Gray peaks in the background correspond to one-dimensional electron density profile of the WT structure. () Electron density map of residues 60–84 from two diagonally symmetric subunits. Sticks, polypeptide chain; blue mesh, 2.5σ contour of the 2Fo − Fc electron density map for the protein; magenta mesh, (4–6)σ contour of the Fo − Fc omit map for the ions; red mesh, 5σ contour of the Fo − Fc omit map for the waters. () A single-subunit P-loop is shown, with side chains at Glu71 and Asp80 in stick representation. At 2.7-Å resolution, we observe no crystallographic waters within the cavity behind the filter. * Figure 6: Underlying conformational dynamics of the selectivity filter and the fast gating kinetics. () Structural snapshots of outward-facing carbonyl conformations. () Dynamics of carbonyl reorientation in KcsA. Time traces of the Val76 carbonyl dihedral angle (N-CA-C-O) during 20-ns molecular dynamics trajectories. Different colored lines correspond to different subunits. Potassium ions were initially placed in the cavity and sites S1 and S3. () Distribution of Glu71-Asp80 Cα-Cα distances. The green and magenta fits correspond to populations with Asp80 facing down (centered at ~ 10.3 Å) and 'flipped' outward (centered at ~ 11.1 Å), respectively. Accession codes * Abstract * Accession codes * Author information * Supplementary information Primary accessions Protein Data Bank * 3OR7 * 3OR6 * 3OR7 * 3OR6 Author information * Abstract * Accession codes * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Sudha Chakrapani & * Julio F Cordero-Morales Affiliations * Department of Biochemistry and Molecular Biology, University of Chicago, Center for Integrative Science, Chicago, Illinois, USA. * Sudha Chakrapani, * Julio F Cordero-Morales, * Vishwanath Jogini, * Albert C Pan, * D Marien Cortes, * Benoît Roux & * Eduardo Perozo * Present addresses: Department of Physiology and Biophysics, Case Western Reserve University, Cleveland, Ohio, USA (S.C.); Department of Physiology, University of California San Francisco, San Francisco, California, USA (J.F.C.); D.E. Shaw Research, Hyderabad, India (V.J); D.E. Shaw Research, New York, New York, USA (A.C.P.); Department of Cell Physiology and Molecular Biophysics, Texas Tech University, Lubbock, Texas, USA (D.M.C.). * Sudha Chakrapani, * Julio F Cordero-Morales, * Vishwanath Jogini, * Albert C Pan & * D Marien Cortes Contributions S.C., J.F.C.-M. and E.P. designed the research. S.C. and J.F.C.-M. carried out electrophysiology measurements and kinetic analysis. D.M.C. made Fab preparations. J.F.C.-M. and D.M.C. crystallized the mutant proteins. V.J. determined and analyzed the structures. A.C.P. and B.R. did the computation analysis. S.C., J.F.C.-M. and E.P. analyzed the data and wrote the paper. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Eduardo Perozo Supplementary information * Abstract * Accession codes * Author information * Supplementary information PDF files * Supplementary Text and Figures (3M) Supplementary Figures 1–6 Additional data - CtIP promotes microhomology-mediated alternative end joining during class-switch recombination
- Nat Struct Mol Biol 18(1):75-79 (2011)
Nature Structural & Molecular Biology | Article CtIP promotes microhomology-mediated alternative end joining during class-switch recombination * Mieun Lee-Theilen1 Search for this author in: * NPG journals * PubMed * Google Scholar * Allysia J Matthews2 Search for this author in: * NPG journals * PubMed * Google Scholar * Dierdre Kelly3 Search for this author in: * NPG journals * PubMed * Google Scholar * Simin Zheng2 Search for this author in: * NPG journals * PubMed * Google Scholar * Jayanta Chaudhuri1, 2, 3 Contact Jayanta Chaudhuri Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature Structural & Molecular BiologyVolume: 18,Pages:75–79Year published:(2011)DOI:doi:10.1038/nsmb.1942Received29 June 2010Accepted10 September 2010Published online05 December 2010 Abstract * Abstract * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Immunoglobulin heavy chain (Igh locus) class-switch recombination (CSR) requires targeted introduction of DNA double strand breaks (DSBs) into repetitive 'switch'-region DNA elements in the Igh locus and subsequent ligation between distal DSBs. Both canonical nonhomologous end joining (C-NHEJ) that seals DNA ends with little or no homology and a poorly defined alternative end joining (A-NHEJ, also known as alt-NHEJ) process that requires microhomology ends for ligation have been implicated in CSR. Here, we show that the DNA end-processing factor CtIP is required for microhomology-directed A-NHEJ during CSR. Additionally, we demonstrate that microhomology joins that are enriched upon depletion of the C-NHEJ component Ku70 require CtIP. Finally, we show that CtIP binds to switch-region DNA in a fashion dependent on activation-induced cytidine deaminase. Our results establish CtIP as a bona fide component of microhomology-dependent A-NHEJ and unmask a hitherto unrecognized phys! iological role of microhomology-mediated end joining in a C-NHEJ–proficient environment. View full text Figures at a glance * Figure 1: CtIP knockdown alters CSR in CH12 cells. () Extracts derived from unstimulated (−) or CIT-stimulated (+) CH12 cells expressing two different shRNAs (CtIP-1 or CtIP-2) against CtIP or control scrambled shRNA were analyzed by western blotting using antibodies against CtIP or GAPDH (loading control). The arrow indicates the polypeptide corresponding to CtIP. () CSR to IgA was measured by flow cytometry. CSR frequency in scrambled shRNA infected cells was assigned an arbitrary unit (AU) of 100. The data are the means of 14 independent experiments, with error bars depicting s.d. () Levels of AID transcripts of scrambled or CtIP knockdown cells were measured 48 h after CIT stimulation by quantitative real-time PCR. The values are the means of three independent experiments and the error bars represent s.d. from the mean. () An empty lentiviral vector or one harboring human CtIP (hsCtIP) cDNA was introduced into control or CtIP knockdown cells, and western blotting was done to determine expression of CtIP. () ChIP analys! is was done by using CIT-stimulated CH12 cells with anti-AID, and the amount of AID bound to Sμ was determined by real-time quantitative PCR. The values represent the average of three independent experiments, with error bars representing s.d. from the mean. () CSR was measured by flow cytometry in CtIP knockdown cells transduced with either hsCtIP or empty expression vector. * Figure 2: CtIP knockdown alters end joining during CSR. () Sμ-Sα junctions from CIT-stimulated CH12 cells infected with control or CtIP-1 shRNA were analyzed. Sequence data were compiled from six independent experiments. () The spectrum of junctions in control versus CtIP knockdown cells was tabulated. The difference in the percentage of junctions with microhomology of four nucleotides or more was statistically significant (P = 0.02). () Sμ-Sα junctions in CtIP knockdown cells transduced with empty vector or vector encoding hsCtIP were analyzed. * Figure 3: CtIP knockdown alters end joining in Ku70-deficient cells. () CIT-stimulated CH12 cells transduced with scrambled or shRNAs (Ku70-1, Ku70-2) against Ku70 were analyzed by western blotting using antibodies to Ku70 or to GAPDH (loading control). () CSR to IgA in cells transduced with Ku70-1 or Ku70-2 shRNA cells was measured by flow cytometry. CSR frequency in cells transduced with scrambled shRNA was assigned an arbitrary unit (AU) of 100. The data are the means of at least three independent experiments, with error bars indicating s.d. from the mean. () Ku70 knockdown or control cells were transduced with scrambled or CtIP-1 shRNA, and expression of Ku70, CtIP and GAPDH was determined by western blot analysis. () CH12 cells with the indicated shRNAs were stimulated with CIT for 72 h, and CSR to IgA was measured by flow cytometry. CSR frequency in cells transduced with scrambled shRNA was assigned an AU of 100. The data are the means of nine independent experiments, with error bars representing s.d. from the mean. () Sμ-Sα junctions! from Ku70-1+scrambled– and Ku70-1+CtIP-1–transduced cells were cloned and sequenced, and the percentage of cells with the indicated lengths of microhomology was plotted. () The distribution of microhomology at the Sμ-Sα junctions is tabulated. The difference in the number of junctions with microhomology of four nucleotides or more between the Ku70-1+scrambled and Ku70-1+CtIP-1 knockdown cells was statistically significant (P = 0.04). * Figure 4: CtIP binds to S-region DNA. () ChIP was carried out in CIT-stimulated CH12 cells with antibodies to AID or CtIP or control nonspecific IgG antibodies. DNA from the ChIP samples was diluted by a factor of three, amplified by PCR and the presence of Sμ, μ promoter (Iμ promoter) or p53 was determined by analyzing the PCR products on agarose gels. () The amount of CtIP bound to Sμ in the indicated cells was measured by real-time quantitative PCR. Data are the means of three independent experiments, with error bars representing s.d. from the mean. () CtIP binding to Sμ in unstimulated or CIT-stimulated CH12 cells was assayed by ChIP and quantified by real-time PCR. The values represent the mean of three independent experiments, with error bars representing s.d. from the mean. () Splenic B cells from wild-type or Aid−/− mice were stimulated with α-CD40 and IL-4 for 48 h, and ChIP analysis was done with antibodies to CtIP, AID and H3 and control nonspecific IgG antibodies. Immunoprecipitated DNA was! diluted by a factor of four and amplified by PCR for the presence of Sμ, Sγ1, Iμ promoter or p53. * Figure 5: A model for the role of CtIP in CSR. AID activity induces formation of staggered DSBs in two distinct S regions. The DSBs are initially processed by the Mre11–Rad50–Nbs1 (MRN) complex, possibly to generate blunt ends, and then channeled into either the C-NHEJ or the A-NHEJ pathway. For C-NHEJ, blunt DSBs are bound by Ku (and other C-NHEJ proteins) and subsequently ligated by DNA ligase IV (Lig. IV) and XRCC4, which do not require microhomology at the DNA ends. For A-NHEJ, CtIP, alone or in conjunction with MRN, further processes the DNA ends to reveal stretches of microhomology before ligation. Because CtIP binding to S regions is enhanced when Ku protein is depleted (Fig. 4), Ku could possibly compete with or suppress the A-NHEJ pathway. Additional components of A-NHEJ that participate in CSR, including the ligase that seals the DNA ends, are yet to be elucidated. Author information * Abstract * Author information * Supplementary information Affiliations * Immunology Program, Memorial Sloan-Kettering Cancer Center, New York, New York, USA. * Mieun Lee-Theilen & * Jayanta Chaudhuri * Immunology and Microbial Pathogenesis Program, Weill-Cornell Medical School, New York, New York, USA. * Allysia J Matthews, * Simin Zheng & * Jayanta Chaudhuri * Gerstner Sloan-Kettering Graduate School of Biomedical Sciences, Memorial Sloan-Kettering Cancer Center, New York, New York, USA. * Dierdre Kelly & * Jayanta Chaudhuri Contributions M.L.-T., A.J.M. and D.K. conducted experiments; A.J.M. and S.Z. devised protocols for shRNA-mediated knockdown in CH12 cell lines; M.L.-T. and J.C. devised experiments, analyzed data and wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Jayanta Chaudhuri Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (1012K) Supplementary Figures 1–6 and Supplementary Tables 1–2 Additional data - An essential role for CtIP in chromosomal translocation formation through an alternative end-joining pathway
- Nat Struct Mol Biol 18(1):80-84 (2011)
Nature Structural & Molecular Biology | Article An essential role for CtIP in chromosomal translocation formation through an alternative end-joining pathway * Yu Zhang1 Search for this author in: * NPG journals * PubMed * Google Scholar * Maria Jasin1 Contact Maria Jasin Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature Structural & Molecular BiologyVolume: 18,Pages:80–84Year published:(2011)DOI:doi:10.1038/nsmb.1940Received29 June 2010Accepted10 September 2010Published online05 December 2010 Abstract * Abstract * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Chromosomal translocations arise from the misjoining of DNA breaks, but the identity of the DNA repair factors and activities involved in their formation has been elusive. Here we show that depletion of CtIP, a DNA end-resection factor, results in a substantial decrease in chromosomal translocation frequency in mouse cells. Moreover, microhomology usage, a signature of the alternative nonhomologous end-joining pathway (alt-NHEJ), is significantly lower in translocation breakpoint junctions recovered from CtIP-depleted cells than in those from wild-type cells. Thus, we directly demonstrate that CtIP-mediated alt-NHEJ has a primary role in translocation formation. CtIP depletion in Ku70−/− cells reduces translocation frequency without affecting microhomology, indicating that Ku70-dependent NHEJ generates a fraction of translocations in wild-type cells. Translocations from both wild-type and Ku70−/− cells have smaller deletions on the participating chromosomes when CtIP! is depleted, implicating the end-resection activity of CtIP in translocation formation. View full text Figures at a glance * Figure 1: CtIP is essential for efficient chromosomal translocation formation by microhomology-prone alt-NHEJ. () Diagram of the pCr15 translocation reporter. NHEJ of DSBs induced on chromosomes 17 and 14 by the I-SceI endonuclease generates a chromosomal translocation with a neo+ gene on der(17) in mouse cells. neoSD, neo splice donor; SAneo, splice acceptor neo. () Flow chart of shRNA-mediated depletion of CtIP to quantify translocation efficiency. In rescue experiments, an expression vector bearing human CtIP (hCtIP) is included. At the time of the second shRNA transfection, the I-SceI expression vector is introduced to generate DSBs. () Representative western blots showing CtIP knockdown with shRNAs CtIP-1 and CtIP-2 or control shRNA to the luciferase gene (Luc). The times relative to the first shRNA transfection are indicated. Black line indicates lanes merged from separate gels. () Translocation frequencies of wild-type pCr15 cells treated with the indicated shRNAs alone (blue bars), with an empty expression vector (green bars) and with an hCtIP expression vector (orange bars),! as compared to that of mock-treated cells (black bars). All translocation frequencies are normalized for colony survival after shRNA transfection (Supplementary Fig. 1c). Data represent the mean ± 1 s.d. from three or more independent experiments. () Representative western blots showing mouse CtIP knockdown rescued by hCtIP expression. Black line indicates lanes merged from separate gels. () Distribution of microhomology lengths at der(17) breakpoint junctions from pCr15 cells treated with the control Luc shRNA (black bars), CtIP-2 shRNA (blue bars) or CtIP-2 shRNA rescued by an hCtIP expression vector (orange bars) or an empty vector (green bars). () Distribution of deletion lengths for der(17) breakpoint junctions from pCr15 cells treated as in . P values were calculated with a two-tailed Mann-Whitney test. * Figure 2: In Ku70−/− cells, CtIP is essential for efficient chromosomal translocation formation but does not affect microhomology at breakpoint junctions. (,) Representative western blots showing CtIP knockdown with shRNAs CtIP-1 and CtIP-2 or control Luc shRNA () and complementation by expressing human CtIP (hCtIP; ). The times relative to the first shRNA transfection are indicated. Black line indicates lanes merged from separate gels. () Translocation frequencies of Ku70−/− pCr15 cells treated with the indicated shRNAs alone (blue bars), with an empty expression vector (green bars) and with an hCtIP expression vector (orange bars) as compared to that of mock-treated Ku70−/− cells (black bars). All translocation frequencies are normalized for colony survival after shRNA transfection (Supplementary Fig. 1c). Data represent the mean ± 1 s.d. from three or more independent experiments. () Distribution of microhomology lengths at der(17) breakpoint junctions from Ku70−/− pCr15 cells treated with the control Luc shRNA (black bars), CtIP-2 shRNA (blue bars) or CtIP-2 shRNA rescued by an hCtIP expression vector (orange ! bars) or an empty vector (green bars). () Distribution of deletion lengths for der(17) breakpoint junctions from Ku70−/− pCr15 cells treated as in . P values were calculated by a two-tailed Mann-Whitney test. * Figure 3: Model for pathways involved in chromosomal translocations. Translocations primarily arise from an alt-NHEJ pathway that is largely dependent on CtIP. CtIP promotes the resection of DNA ends to uncover microhomologies that anneal for end joining. In the absence of Ku, resection factors such as CtIP (and possibly other unknown ones, represented by the question mark) have greater access to DNA ends, such that translocations increase. In wild-type cells, a minor portion of translocations may arise through the canonical NHEJ pathway, which can efficiently join ends without microhomology. Author information * Abstract * Author information * Supplementary information Affiliations * Developmental Biology Program, Memorial Sloan-Kettering Cancer Center, New York, New York, USA. * Yu Zhang & * Maria Jasin Contributions Y.Z. performed the experiments. Y.Z. and M.J. designed the research and wrote the paper. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Maria Jasin Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (704K) Supplementary Figures 1 and 2 and Supplementary Table 1 Additional data - Selective silencing of mutated mRNAs in DM1 by using modified hU7-snRNAs
- Nat Struct Mol Biol 18(1):85-87 (2011)
Nature Structural & Molecular Biology | Brief Communication Selective silencing of mutated mRNAs in DM1 by using modified hU7-snRNAs * Virginie François1, 2, 3, 4, 5 Search for this author in: * NPG journals * PubMed * Google Scholar * Arnaud F Klein1, 2, 3, 4, 5 Search for this author in: * NPG journals * PubMed * Google Scholar * Cyriaque Beley1, 2, 3, 4 Search for this author in: * NPG journals * PubMed * Google Scholar * Arnaud Jollet1, 2, 3, 4 Search for this author in: * NPG journals * PubMed * Google Scholar * Camille Lemercier1, 2, 3, 4 Search for this author in: * NPG journals * PubMed * Google Scholar * Luis Garcia1, 2, 3, 4, 6 Search for this author in: * NPG journals * PubMed * Google Scholar * Denis Furling1, 2, 3, 4, 6 Contact Denis Furling Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature Structural & Molecular BiologyVolume: 18,Pages:85–87Year published:(2011)DOI:doi:10.1038/nsmb.1958Received18 May 2010Accepted19 October 2010Published online26 December 2010 Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg We describe a function for modified human U7 small nuclear RNAs (hU7-snRNAs) distinct from modification of pre-mRNA splicing events. Engineered hU7-snRNAs containing a poly-CAG antisense sequence targeting the expanded CUG repeats of mutant DMPK transcripts in myotonic dystrophy caused specific degradation of pathogenic DMPK mRNAs without affecting the products of wild-type DMPK alleles. Abolition of the RNA gain-of-function toxicity that is responsible for pathogenesis supports the use of hU7-snRNAs for gene silencing in RNA-dominant disorders in which expanded repeats are expressed. View full text Figures at a glance * Figure 1: Silencing of CUGexp RNAs by hU7-(CAG)15. () Structure of the hU7-snRNA-(CAG)15 indicating the loop, Sm-Opt and CAG antisense sequences. () Representative northern blot and analysis (n = 5) of DMPK mRNA expression in DM1 muscle cells (800CTG) transduced with hU7-(CAG)15 lentiviral vector (4–8 × 106 vector genomes (vg) per ml). () FISH analysis (n = 4) of the number of CUGexp-mRNA foci (red spots) in the nuclei (blue) of DM1 cells (800CTG) transduced with hU7-(CAG)15 (8 × 106 vg per ml). () RT-PCR assay of normal and CUGexp-DMPK mRNA, GAPDH mRNA, U6 snRNA and DMPK pre-RNA in nuclear and cytoplasmic fractions of DM1-converted muscle cells (1300CTG). BpmI restriction site polymorphism in exon 10 of expanded DMPK allele identifies normal and CUGexp allele products. Error bars, s.e.m.; **P < 0.01, ***P < 0.001. * Figure 2: Consequences of hU7-(CAG)n expression in DM1 muscle cells. () Expression of normal and CUGexp-DMPK mRNAs in DM1 cells (800CTG) transduced with hU7-(CAG)n vectors (4 × 106 vg per ml) containing antisense sequences of 7, 11 or 15 CAGs (northern blot, n = 3). () Localization of the splicing regulator MBNL1 in DM1 cells. () Correction of alternative splicing misregulation of BIN1, DMD and LDB3 transcripts in differentiated DM1 muscle cells (2000CTG) transduced with hU7-(CAG)15 vectors (8 × 106 vg per ml; RT-PCR, n = 3). () Effect of hU7-(CAG)15 expression on myogenic differentiation of DM1 muscle cells (2000CTG) quantified as fusion index (n = 6). Error bars, s.e.m.; *P < 0.05, **P < 0.01, ***P < 0.001. Author information * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Virginie François & * Arnaud F Klein Affiliations * Université Pierre et Marie Curie-Paris6, Um76, Paris, France. * Virginie François, * Arnaud F Klein, * Cyriaque Beley, * Arnaud Jollet, * Camille Lemercier, * Luis Garcia & * Denis Furling * Centre Nationale de la Recherche Scientifique, Unité Mixte de Recherche 7215, Paris, France. * Virginie François, * Arnaud F Klein, * Cyriaque Beley, * Arnaud Jollet, * Camille Lemercier, * Luis Garcia & * Denis Furling * INSERM, U974, Paris, France. * Virginie François, * Arnaud F Klein, * Cyriaque Beley, * Arnaud Jollet, * Camille Lemercier, * Luis Garcia & * Denis Furling * Institut de Myologie, Paris, France. * Virginie François, * Arnaud F Klein, * Cyriaque Beley, * Arnaud Jollet, * Camille Lemercier, * Luis Garcia & * Denis Furling * These authors jointly directed this work. * Luis Garcia & * Denis Furling Contributions V.F. and A.F.K. conducted most of the experiments. C.B. made the constructs. A.J. produced the lentiviral vectors. C.L. supported some experiments. L.G. engineered the modified hU7-snRNA. D.F. supervised the entire project. L.G. and D.F. wrote the manuscript. Competing financial interests L.G., D.F., C.B. and T.V. are named as inventors on a patent application to the US Patent Office covering the method described in this paper. Corresponding author Correspondence to: * Denis Furling Supplementary information * Author information * Supplementary information PDF files * Supplementary Text and Figures (1M) Supplementary Figures 1–9 and Supplementary Methods Additional data - Cryo-EM structure of the E. coli translating ribosome in complex with SRP and its receptor
- Nat Struct Mol Biol 18(1):88-90 (2011)
Nature Structural & Molecular Biology | Brief Communication Cryo-EM structure of the E. coli translating ribosome in complex with SRP and its receptor * Leandro F Estrozi1, 2, 5 Search for this author in: * NPG journals * PubMed * Google Scholar * Daniel Boehringer3, 5 Search for this author in: * NPG journals * PubMed * Google Scholar * Shu-ou Shan4 Search for this author in: * NPG journals * PubMed * Google Scholar * Nenad Ban3 Contact Nenad Ban Search for this author in: * NPG journals * PubMed * Google Scholar * Christiane Schaffitzel1, 2 Contact Christiane Schaffitzel Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorsJournal name: Nature Structural & Molecular BiologyVolume: 18,Pages:88–90Year published:(2011)DOI:doi:10.1038/nsmb.1952Received25 April 2010Accepted08 October 2010Published online12 December 2010 Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg We report the 'early' conformation of the Escherichia coli signal recognition particle (SRP) and its receptor FtsY bound to the translating ribosome, as determined by cryo-EM. FtsY binds to the tetraloop of the SRP RNA, whereas the NG domains of the SRP protein and FtsY interact weakly in this conformation. Our results suggest that optimal positioning of the SRP RNA tetraloop and the Ffh NG domain leads to FtsY recruitment. View full text Figures at a glance * Figure 1: Generation and characterization of RNC–SRP–FtsY. () Schematic of the single-chain SRP–FtsY construct (scSRP). () Cocrystal structure of the Ffh (green)–FtsY (magenta) NG domain complex8, 9. The bound GTP analogs are represented as spheres. () Coomassie-stained SDS-PAGE gel showing binding of the SRP (Ffh), FtsY and scSRP to RNC analyzed by ribosomal pelleting. scSRP binds in presence and absence of non-hydrolyzable GTP (GMPPNP). () GTPase activity of the scSRP construct is within a factor of 2 of that of unlinked SRP and FtsY and is inhibited by RNCs. Error bars, s.d. from three different experiments. () Cryo-EM structure of RNC-scSRP. 30S, yellow; 50S, blue; scSRP, red; star, tunnel exit. All figures were produced with the programs Adobe Illustrator and PyMOL (http://www.pymol.org/). * Figure 2: Atomic model of the early conformation of scSRP. () View on the tunnel exit. () Fitting of the NG domains of Ffh and FtsY. () Contact between FtsY G domain and the RNA tetraloop (green) via Lys399, Arg402 and Lys406 (blue). View from the tunnel exit. The side chain placement is based on the Ffh–FtsY NG-domain complex structure8. () Ribosomal connection formed by M-domain helices 2 and 3 and rRNA helix 24 (h24). Experimental density, light gray; 4.5S RNA, orange; Ffh M domain (helices 2 and 3), yellow; Ffh NG domain, lime green; FtsY, magenta; ribosomal RNA helix 24, blue; rRNA, dark gray. * Figure 3: Cartoon model of co-translational targeting. The Ffh N domain binds L23 (left). Upon recognition of a signal sequence, the SRP binds with high affinity to the RNC (middle) and is pre-positioned to bind FtsY. In the early conformation (right), the FtsY NG domain contacts the 4.5S RNA tetraloop, initiating the rearrangement of the GTPase domains and release of the RNC. The colored outlines are based on EM reconstructions. Accession codes * Accession codes * Author information * Supplementary information Primary accessions Protein Data Bank * 2XKV * EMD-1762 * 2XKV * EMD-1762 Author information * Accession codes * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Leandro F Estrozi & * Daniel Boehringer Affiliations * European Molecular Biology Laboratory, Grenoble Outstation, Grenoble, France. * Leandro F Estrozi & * Christiane Schaffitzel * Unit of Virus Host-Cell Interactions, Unité Mixte Internationale 3265, Grenoble, France. * Leandro F Estrozi & * Christiane Schaffitzel * ETH Zurich (Swiss Federal Institute of Technology), Institute of Molecular Biology and Biophysics, Zurich, Switzerland. * Daniel Boehringer & * Nenad Ban * Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California, USA. * Shu-ou Shan Contributions C.S. conceived the scSRP construct and performed sample preparations; S.-o.S. performed activity assays; D.B. did the electron microscopy; D.B., L.F.E. and C.S. performed the image analysis and model building; C.S., N.B., D.B. and S.-o.S. prepared the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding authors Correspondence to: * Nenad Ban or * Christiane Schaffitzel Supplementary information * Accession codes * Author information * Supplementary information PDF files * Supplementary Text and Figures (740K) Supplementary Figures 1–5, Supplementary Methods and Supplementary Discussion Additional data - An assessment of histone-modification antibody quality
- Nat Struct Mol Biol 18(1):91-93 (2011)
Nature Structural & Molecular Biology | Resource An assessment of histone-modification antibody quality * Thea A Egelhofer1, 16 Search for this author in: * NPG journals * PubMed * Google Scholar * Aki Minoda2, 3, 16 Search for this author in: * NPG journals * PubMed * Google Scholar * Sarit Klugman4, 5, 16 Search for this author in: * NPG journals * PubMed * Google Scholar * Kyungjoon Lee6 Search for this author in: * NPG journals * PubMed * Google Scholar * Paulina Kolasinska-Zwierz7, 8 Search for this author in: * NPG journals * PubMed * Google Scholar * Artyom A Alekseyenko9, 10 Search for this author in: * NPG journals * PubMed * Google Scholar * Ming-Sin Cheung7, 8 Search for this author in: * NPG journals * PubMed * Google Scholar * Daniel S Day6 Search for this author in: * NPG journals * PubMed * Google Scholar * Sarah Gadel11 Search for this author in: * NPG journals * PubMed * Google Scholar * Andrey A Gorchakov9, 10 Search for this author in: * NPG journals * PubMed * Google Scholar * Tingting Gu11 Search for this author in: * NPG journals * PubMed * Google Scholar * Peter V Kharchenko6 Search for this author in: * NPG journals * PubMed * Google Scholar * Samantha Kuan4, 5 Search for this author in: * NPG journals * PubMed * Google Scholar * Isabel Latorre7, 8 Search for this author in: * NPG journals * PubMed * Google Scholar * Daniela Linder-Basso12 Search for this author in: * NPG journals * PubMed * Google Scholar * Ying Luu4, 5 Search for this author in: * NPG journals * PubMed * Google Scholar * Queminh Ngo4, 5 Search for this author in: * NPG journals * PubMed * Google Scholar * Marc Perry13 Search for this author in: * NPG journals * PubMed * Google Scholar * Andreas Rechtsteiner1 Search for this author in: * NPG journals * PubMed * Google Scholar * Nicole C Riddle11 Search for this author in: * NPG journals * PubMed * Google Scholar * Yuri B Schwartz12 Search for this author in: * NPG journals * PubMed * Google Scholar * Gregory A Shanower12 Search for this author in: * NPG journals * PubMed * Google Scholar * Anne Vielle7, 8 Search for this author in: * NPG journals * PubMed * Google Scholar * Julie Ahringer7, 8 Search for this author in: * NPG journals * PubMed * Google Scholar * Sarah C R Elgin11 Search for this author in: * NPG journals * PubMed * Google Scholar * Mitzi I Kuroda9, 10 Search for this author in: * NPG journals * PubMed * Google Scholar * Vincenzo Pirrotta12 Search for this author in: * NPG journals * PubMed * Google Scholar * Bing Ren4, 5 Search for this author in: * NPG journals * PubMed * Google Scholar * Susan Strome1 Search for this author in: * NPG journals * PubMed * Google Scholar * Peter J Park6 Contact Peter J Park Search for this author in: * NPG journals * PubMed * Google Scholar * Gary H Karpen2, 3 Contact Gary H Karpen Search for this author in: * NPG journals * PubMed * Google Scholar * R David Hawkins4, 5 Contact R David Hawkins Search for this author in: * NPG journals * PubMed * Google Scholar * Jason D Lieb14, 15 Contact Jason D Lieb Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorsJournal name:Nature Structural & Molecular BiologyVolume: 18,Pages:91–93Year published:(2011)DOI:doi:10.1038/nsmb.1972Received07 June 2010Accepted09 November 2010Published online05 December 2010 Abstract * Abstract * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg We have tested the specificity and utility of more than 200 antibodies raised against 57 different histone modifications in Drosophila melanogaster, Caenorhabditis elegans and human cells. Although most antibodies performed well, more than 25% failed specificity tests by dot blot or western blot. Among specific antibodies, more than 20% failed in chromatin immunoprecipitation experiments. We advise rigorous testing of histone-modification antibodies before use, and we provide a website for posting new test results (http://compbio.med.harvard.edu/antibodies/). View full text Figures at a glance * Figure 1: Representative western assays and results. () Western blot of anti-H3K4me2 (Millipore, 07-030, lot DAM1543701), anti-H3S10ph (Wako, 303-35199) and anti-H4K20me3 (Diagenode, CS-057, lot A9-002). Left, Coomassie blue–stained gel of worm nuclear extract (Nuc. ext.) and recombinant H3 (Recomb. H3) (Active Motif, 31207), showing the amount of protein loaded in each lane and approximately equal levels of histone H3 in the nuclear extract and recombinant H3 sets of lanes. Arrowhead, histone H3; asterisk, histone H4. Anti-H3K4me2 passed, because it recognized only H3 in the nuclear extract and not unmodified H3. Anti-H3S10ph failed, because it recognized unmodified H3 with equal intensity to H3 in the nuclear extract. Anti-H4K20me3 failed, because it recognized nonhistone proteins and perhaps H3 instead of H4 in nuclear extract. All western blot images are available at http://compbio.med.harvard.edu/antibodies/. Images are also available at http://www.modencode.org/docs/hmav.html (worm and fly) and http://epigenome.ucsd.ed! u/antibodies.html (human). () Summary of results of fly and worm western blots. Antibodies to core histones are not included, as they are expected to detect recombinant histones. For three antibodies, test results differed among groups (pass versus no signal, or fail versus no signal), and these three were included in the pass or fail categories, respectively. () Performance of antibodies tested in fly and worm nuclear extracts. Antibody results were binned into five mutually exclusive groups; the percentage is plotted, with the number of antibodies shown above each bar. The same exceptions were applied as in . () Performance of antibodies tested in human whole-cell extracts (WCE). Many antibodies classified as 'Histone + other bands' passed ChIP tests. * Figure 2: Representative dot blot assays and results. () Dot blot characterization of anti-H3K4me2 (Abcam, ab32356, lot 577702) and anti-H3K27ac (Abcam, ab4729, lot 726657). Top, positions of histone tail peptides spotted on membranes. Anti-H3K4me2 passed. Anti-H3K27ac failed owing to detection of multiple peptides. Human, fly and worm dot blot images are available at the websites listed in the legend to Figure 1. () Summary of peptide blot results. We classified 149 antibodies as described in the text. Low signal indicates that only the highest peptide concentration was detected by the antibody. See Supplementary Figure 1 for a description of the peptide array used for each antibody and Supplementary Table 1 for enumeration of cross-reacting peptides. * Figure 3: ChIP-chip and ChIP-seq. () Representative ChIP-chip characterization with anti-H3K36me1 from two different sources (Abcam, ab9048 lot no. 18882, and H. Kimura, 1H1). A ~60-kb region of C. elegans chromosome IV is shown, with annotated genes (x axis) and ChIP-chip z scores (standardized log2 ratios of ChIP/input signals; y axis) plotted for biological replicates using both antibodies. The replicates were highly correlated using the Abcam antibody (passed), but not using the 1H1 antibody (failed). () Summary of results. Antibodies to core histones are not included in the summary. Author information * Abstract * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Thea A Egelhofer, * Aki Minoda & * Sarit Klugman Affiliations * Department of Molecular, Cell and Developmental Biology, University of California Santa Cruz, Santa Cruz, California, USA. * Thea A Egelhofer, * Andreas Rechtsteiner & * Susan Strome * Department of Molecular and Cell Biology, University of California at Berkeley, Berkeley, California, USA. * Aki Minoda & * Gary H Karpen * Department of Genome Dynamics, Lawrence Berkeley National Lab, Berkeley, California, USA. * Aki Minoda & * Gary H Karpen * Ludwig Institute for Cancer Research, La Jolla, California, USA. * Sarit Klugman, * Samantha Kuan, * Ying Luu, * Queminh Ngo, * Bing Ren & * R David Hawkins * Department of Cellular and Molecular Medicine, University of California, San Diego School of Medicine, La Jolla, California, USA. * Sarit Klugman, * Samantha Kuan, * Ying Luu, * Queminh Ngo, * Bing Ren & * R David Hawkins * Center for Biomedical Informatics, Harvard Medical School, Boston, Massachusetts, USA. * Kyungjoon Lee, * Daniel S Day, * Peter V Kharchenko & * Peter J Park * The Gurdon Institute, University of Cambridge, Cambridge, UK. * Paulina Kolasinska-Zwierz, * Ming-Sin Cheung, * Isabel Latorre, * Anne Vielle & * Julie Ahringer * Department of Genetics, University of Cambridge, Cambridge, UK. * Paulina Kolasinska-Zwierz, * Ming-Sin Cheung, * Isabel Latorre, * Anne Vielle & * Julie Ahringer * Division of Genetics, Department of Medicine, Brigham & Women's Hospital, Boston, Massachusetts, USA. * Artyom A Alekseyenko, * Andrey A Gorchakov & * Mitzi I Kuroda * Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA. * Artyom A Alekseyenko, * Andrey A Gorchakov & * Mitzi I Kuroda * Department of Biology, Washington University in St. Louis, St. Louis, Missouri, USA. * Sarah Gadel, * Tingting Gu, * Nicole C Riddle & * Sarah C R Elgin * Department of Molecular Biology & Biochemistry, Rutgers University, Piscataway, New Jersey, USA. * Daniela Linder-Basso, * Yuri B Schwartz, * Gregory A Shanower & * Vincenzo Pirrotta * Ontario Institute for Cancer Research, Toronto, Ontario, Canada. * Marc Perry * Department of Biology, Carolina Center for Genome Sciences, Chapel Hill, North Carolina, USA. * Jason D Lieb * Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA. * Jason D Lieb Contributions J.A., A.A.A., M.-S.C., D.S.D., T.A.E., S.C.R.E., S.G., A.A.G., T.G., R.D.H., G.H.K., P.V.K., S. Klugman, P.K.-Z., S. Kuan, M.I.K., I.L., K.L., J.D.L., D.L.-B., Y.L., A.M., Q.N., P.J.P., M.P., V.P., A.R., B.R., N.C.R., Y.B.S., G.A.S., S.S. and A.V. designed, executed, and analyzed the experiments. T.A.E., R.D.H., G.H.K., J.D.L., A.M. and S.S. wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding authors Correspondence to: * Jason D Lieb or * R David Hawkins or * Gary H Karpen or * Peter J Park Supplementary information * Abstract * Author information * Supplementary information Excel files * Supplementary Table 1 (132K) Tested antibodies and results. PDF files * Supplementary Text and Figures (252K) Supplementary Figure 1 Additional data - Dicer associates with chromatin to repress genome activity in Schizosaccharomyces pombe
- Nat Struct Mol Biol 18(1):94-99 (2011)
Nature Structural & Molecular Biology | Resource Dicer associates with chromatin to repress genome activity in Schizosaccharomyces pombe * Katrina J Woolcock1 Search for this author in: * NPG journals * PubMed * Google Scholar * Dimos Gaidatzis1 Search for this author in: * NPG journals * PubMed * Google Scholar * Tanel Punga1 Search for this author in: * NPG journals * PubMed * Google Scholar * Marc Bühler1 Contact Marc Bühler Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature Structural & Molecular BiologyVolume: 18,Pages:94–99Year published:(2011)DOI:doi:10.1038/nsmb.1935Received28 April 2010Accepted20 September 2010Published online12 December 2010 Abstract * Abstract * Accession codes * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg In the fission yeast S. pombe, the RNA interference (RNAi) pathway is required to generate small interfering RNAs (siRNAs) that mediate heterochromatic silencing of centromeric repeats. Here, we demonstrate that RNAi also functions to repress genomic elements other than constitutive heterochromatin. Using DNA adenine methyltransferase identification (DamID), we show that the RNAi proteins Dcr1 and Rdp1 physically associate with some euchromatic genes, noncoding RNA genes and retrotransposon long terminal repeats, and that this association is independent of the Clr4 histone methyltransferase. Physical association of RNAi with chromatin is sufficient to trigger a silencing response but not to assemble heterochromatin. The mode of silencing at the newly identified RNAi targets is consistent with a co-transcriptional gene silencing model, as proposed earlier, and functions with trace amounts of siRNAs. We anticipate that similar mechanisms could also be operational in other euka! ryotes. View full text Figures at a glance * Figure 1: Identification of previously known and novel Swi6 and Rdp1 binding sites by DamID. () In DamID, a fusion protein consisting of a protein of interest and DNA adenine methyltransferase (Dam) from Escherichia coli is expressed at very low levels. On interaction of the fusion protein with chromatin, Dam methylates the N6 position of adenine in the sequence context GATC. Thus, Dam leaves G6mATC marks close to the genomic binding sites, which can be mapped by a methylation-specific PCR protocol (Supplementary Fig. 1). (,) Swi6 and Rdp1 maps for chromosome II as determined by DamID or ChIP-on-chip. () DamID map of Swi6 interactions for chromosome II in wild-type and dcr1Δ cells. For the DamID maps, the signal was averaged over every 500 probes. y axes are on log2 scale; x axes indicate position on chromosome II. * Figure 2: Dcr1 physically associates with centromeric chromatin. Dcr1, Rdp1 and Swi6 DamID maps for the centromeric region of chromosome III with flanking internal repeat elements (IRC3L/R), outermost repeats (dg/dh), innermost repeats (imr) and central core domain (cnt). The signal was averaged over every 50 probes. y axes are on log2 scale; x axes indicate position on chromosome III. * Figure 3: Clr4 dependency of Dcr1 or Rdp1 association with chromatin. () Northern blot for centromeric siRNAs in wild-type and heterochromatin-defective (H3K9R) cells. snoR69 serves as a loading control. () Swi6, Rdp1 and Dcr1 enrichment at centromeric repeat elements in wild-type and heterochromatin-defective (clr4Δδ) cells. Error bars represent the s.e.m. (n = 3). P values were generated using the Student's t-test. (,) Comparisons of DamID signal quantified at annotated features across the genome in wild-type and heterochromatin-defective (clr4Δδ) cells. Each axis shows the average of three independent biological replicates. () Dcr1 association is Clr4-independent across the whole genome. () Rdp1 association depends on Clr4 at some loci but not at others. * Figure 4: Enrichment of Dam fusion proteins at different genomic features. (–) Dcr1, Rdp1 and Swi6 enrichments (log2) at the indicated genomic features in wild-type and heterochromatin-deficient (clr4Δ) cells. For all experiments, three biological replicates are shown. () ChIP experiment confirming physical association of Dcr1 with LTRs. The fold enrichment of Dcr1-TAP compared to a nontagged control, and normalized to actin and input, is shown. Error bars represent s.e.m. (n = 3). P value was generated using the Student's t-test; cendg, centromeric repeat element. * Figure 5: The S. pombe RNAi machinery contributes to LTR repression. () Schematic representation of LTRs and their position relative to protein coding genes (dark blue) or Tf2 retrotransposon ORFs (light blue). () Tf2 LTR and ORF transcript levels in the indicated mutant strains. () Dcr1, Ago1, Rdp1 and Clr4 are equally important for repression of centromeric repeats (cendg). () Genes within 5 kb of an LTR (measured from the middle of the gene to the middle of the LTR) were assessed for expression in Dcr1-deficient cells and for Dcr1 association with the nearby LTR. Genes whose expression was at least 1.5-fold increased in a dcr1Δ mutant and whose nearby LTR had at least 1.4-fold enrichment in the DamID data are highlighted in red and listed in Supplementary Table 5. () Tf2 LTR transcript levels in different dcr1 mutants. D937A and D1127A are mutated sites in the RNaseIII catalytic centers of Dcr1. (,,) RNA levels were normalized to actin and represented as fold increase compared to wild type. Error bars represent s.e.m., n = 6 biological re! plicates for dcr1Δ, n = 3 biological replicates for all other mutants. P values were generated using the Student's t-test. Accession codes * Abstract * Accession codes * Author information * Supplementary information Referenced accessions Gene Expression Omnibus * GSE24360 Author information * Abstract * Accession codes * Author information * Supplementary information Affiliations * Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland. * Katrina J Woolcock, * Dimos Gaidatzis, * Tanel Punga & * Marc Bühler Contributions K.J.W. and M.B. designed the research. K.J.W. designed and conducted experiments. D.G. wrote R scripts for data analysis. K.J.W. and D.G. analyzed the DamID data. T.P. conducted experiments. M.B. and K.J.W. analyzed the results and wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Marc Bühler Supplementary information * Abstract * Accession codes * Author information * Supplementary information PDF files * Supplementary Text and Figures (532K) Supplementary Figures 1–4 and Supplementary Tables 1–8 Additional data - A portable RNA sequence whose recognition by a synthetic antibody facilitates structural determination
- Nat Struct Mol Biol 18(1):100-106 (2011)
Nature Structural & Molecular Biology | Technical Report A portable RNA sequence whose recognition by a synthetic antibody facilitates structural determination * Yelena Koldobskaya1 Search for this author in: * NPG journals * PubMed * Google Scholar * Erica M Duguid2 Search for this author in: * NPG journals * PubMed * Google Scholar * David M Shechner3, 4, 7 Search for this author in: * NPG journals * PubMed * Google Scholar * Nikolai B Suslov2 Search for this author in: * NPG journals * PubMed * Google Scholar * Jingdong Ye5 Search for this author in: * NPG journals * PubMed * Google Scholar * Sachdev S Sidhu6 Search for this author in: * NPG journals * PubMed * Google Scholar * David P Bartel3, 4 Search for this author in: * NPG journals * PubMed * Google Scholar * Shohei Koide2 Search for this author in: * NPG journals * PubMed * Google Scholar * Anthony A Kossiakoff2 Contact Anthony A Kossiakoff Search for this author in: * NPG journals * PubMed * Google Scholar * Joseph A Piccirilli1, 2 Contact Joseph A Piccirilli Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorsJournal name:Nature Structural & Molecular BiologyVolume: 18,Pages:100–106Year published:(2011)DOI:doi:10.1038/nsmb.1945Received12 May 2010Accepted01 October 2010Published online12 December 2010 Abstract * Abstract * Accession codes * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg RNA crystallization and phasing represent major bottlenecks in RNA structure determination. Seeking to exploit antibody fragments as RNA crystallization chaperones, we have used an arginine-enriched synthetic Fab library displayed on phage to obtain Fabs against the class I ligase ribozyme. We solved the structure of a Fab–ligase complex at 3.1-Å resolution using molecular replacement with Fab coordinates, confirming the ribozyme architecture and revealing the chaperone's role in RNA recognition and crystal contacts. The epitope resides in the GAAACAC sequence that caps the P5 helix, and this sequence retains high-affinity Fab binding within the context of other structured RNAs. This portable epitope provides a new RNA crystallization chaperone system that easily can be screened in parallel to the U1A RNA-binding protein, with the advantages of a smaller loop and Fabs′ high molecular weight, large surface area and phasing power. View full text Figures at a glance * Figure 1: Selection of class I ligase–binding Fabs from the YSGR Fab superlibrary. () Secondary structure of the ligase ribozyme and its substrate36. The arrow indicates the self-ligation reaction, in which the substrate′s 3′-terminal hydroxyl (nucleotide A(–1), red) attacks the α-phosphate of the ribozyme′s 5′-terminal triphosphate (nucleotide G1, red). () CDR sequences of ligase-binding Fabs selected from the YSGR superlibrary. Red, serine; yellow, tyrosine; green, glycine; blue, arginine. Residues are numbered according to the Kabat system53. () Nitrocellulose binding assay reveals low-micromolar Kd values for Fab–class I ligase binding. BL1 Kd = 478 nM; BL2 Kd > 2,000 nM; BL3 Kd = 338 nM. Reactions were carried out as described in Online Methods. * Figure 2: Affinity maturation of class I ligase–binding Fab-BL3 by error-prone PCR. () CDR sequences of BL3-derived affinity-matured Fabs selected from error-prone PCR Fab libraries. Red, serine; yellow, tyrosine; green, glycine; blue, arginine or lysine; lavender, aspartate or glutamate; gray, leucine or isoleucine. () BL3-derived Fabs bind to the class I ligase product with nanomolar Kd values. Parent BL3 Fab, Kd = 338 nM; BL3-1, Kd = 270 nM; BL3-2, Kd = 66 nM; BL3-3, Kd = 127 nM; BL3-4, Kd = 138 nM; BL3-5, Kd = 44 nM; BL3-6, Kd = 35 nM. Nitrocellulose filter binding assay conditions were identical to those for Figure 1c. The variation of endpoint (fraction RNA bound) values observed for BL3 Fab binding to the ligase product, ranging from 0.4 to 0.75, probably reflects the faster dissociation of weaker-binding Fabs in the context of the nitrocellulose filter binding assay. Accordingly, we used this assay only as a convenient, qualitative method to assay Fab-RNA affinity. As an independent method to assess BL3 Fab Kd values, we used hydroxyl-radical footpr! inting (Supplementary Fig. 2b, BL3-6 Fab). () Hydroxyl-radical footprinting of self-labeled class I ligase in the presence of 5 nM to 1 mM BL3 affinity-matured Fabs BL3-2, BL3-5 and BL3-6. Increasing Fab concentrations are denoted by black triangles; red boxes show P7 and P5 Fab binding sites and the corresponding areas of protection. T1, samples treated with ribonuclease T1. Ligase ribozyme secondary structure (see Fig. 1a) is shown at right. * Figure 3: Crystal structure of the Fab BL3-6–ligase complex. () Crystal structure of the BL3-6 Fab–ligase complex at 3.1 Å resolution. () Overlay of the Fab–ligase structure (green) with the U1A–ligase structure41 (blue); red indicates the ligation junction. Nucleotides in the U1A-binding loop have been omitted. All-atom r.m.s. deviation is 1.26 Å, omitting the residues in L5. * Figure 4: Details of Fab-P5 loop interactions. () Contacts to P5 loop are formed by Fab CDRs L3, H1, H2 and H3. () CDR-H3 arginine residues form contacts to A62 and to the G59-C65 terminal base pair. () Phe95 (CDR-L3) stacks with Tyr62 (CDR-H2) and the A62 base. () C63 forms contacts to Ser58 and Ser60 (CDR-H2). () A61 and Tyr57 (CDR-H2) stack with G59 from the terminal base pair. * Figure 5: Fab–ligase crystal packing. () Crystal packing along the a-b plane. () Crystal packing along the b-c plane. Green, Fab; blue, ligase. * Figure 6: Analysis of the Fab–ligase P5 epitope. () Design of P5 and P7 hairpin oligos to test for Fab binding to isolated RNA hairpin loops. Red boxes indicate Fab–class I ligase binding sites. () BL3 Fabs bind to the P5 hairpin-loop RNA oligo but not to the P7 oligo. Binding curves show results for the P5 hairpin incubated with BL3-1 (Kd = 360 nM), BL3-2 (Kd = 105 nM), BL3-3 (Kd = 230 nM), BL3-4 (Kd = 270 nM), BL3-5 (Kd = 26 nM) and BL3-6 (Kd = 28 nM). Also plotted are results for the P7 hairpin incubated with BL3 Fabs. Nitrocellulose filter binding assay conditions were identical to those for Figure 1c. () P5 RNA oligonucleotides that bind the BL3-6 Fab. () P5 RNA and DNA oligonucleotides that do not bind the BL3-6 Fab. Accession codes * Abstract * Accession codes * Author information * Supplementary information Primary accessions Protein Data Bank * 3IVK * 3IVK Author information * Abstract * Accession codes * Author information * Supplementary information Affiliations * Department of Chemistry, University of Chicago, Chicago, Illinois, USA. * Yelena Koldobskaya & * Joseph A Piccirilli * Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, Illinois, USA. * Erica M Duguid, * Nikolai B Suslov, * Shohei Koide, * Anthony A Kossiakoff & * Joseph A Piccirilli * Whitehead Institute for Biomedical Research, Cambridge, Massachusetts, USA. * David M Shechner & * David P Bartel * Department of Biology and Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA. * David M Shechner & * David P Bartel * Department of Chemistry, University of Central Florida, Orlando, Florida, USA. * Jingdong Ye * Banting and Best Department of Medical Research, University of Toronto, Toronto, Ontario, Canada. * Sachdev S Sidhu * Current address: Broad Institute and Harvard University Department of Stem Cell and Regenerative Biology, Cambridge, Massachusetts, USA. * David M Shechner Contributions All authors designed research; Y.K., E.M.D., D.M.S. and N.B.S. performed experiments; Y.K., E.M.D., D.M.S., S.K., A.A.K. and J.A.P. analyzed data; Y.K., E.M.D., D.M.S., N.B.S., D.P.B., S.K., A.A.K. and J.A.P wrote the paper. Competing financial interests The authors declare no competing financial interests. Corresponding authors Correspondence to: * Joseph A Piccirilli or * Anthony A Kossiakoff Supplementary information * Abstract * Accession codes * Author information * Supplementary information PDF files * Supplementary Text and Figures (5M) Supplementary Methods, Supplementary Note and Supplementary Figures 1–6 Additional data - The three-dimensional folding of the α-globin gene domain reveals formation of chromatin globules
- Nat Struct Mol Biol 18(1):107-114 (2011)
Nature Structural & Molecular Biology | Technical Report The three-dimensional folding of the α-globin gene domain reveals formation of chromatin globules * Davide Baù1, 4 Search for this author in: * NPG journals * PubMed * Google Scholar * Amartya Sanyal2, 4 Search for this author in: * NPG journals * PubMed * Google Scholar * Bryan R Lajoie2, 4 Search for this author in: * NPG journals * PubMed * Google Scholar * Emidio Capriotti1 Search for this author in: * NPG journals * PubMed * Google Scholar * Meg Byron3 Search for this author in: * NPG journals * PubMed * Google Scholar * Jeanne B Lawrence3 Search for this author in: * NPG journals * PubMed * Google Scholar * Job Dekker2 Contact Job Dekker Search for this author in: * NPG journals * PubMed * Google Scholar * Marc A Marti-Renom1 Contact Marc A Marti-Renom Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorsJournal name:Nature Structural & Molecular BiologyVolume: 18,Pages:107–114Year published:(2011)DOI:doi:10.1038/nsmb.1936Received22 November 2009Accepted20 September 2010Published online05 December 2010 Abstract * Abstract * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg We developed a general approach that combines chromosome conformation capture carbon copy (5C) with the Integrated Modeling Platform (IMP) to generate high-resolution three-dimensional models of chromatin at the megabase scale. We applied this approach to the ENm008 domain on human chromosome 16, containing the α-globin locus, which is expressed in K562 cells and silenced in lymphoblastoid cells (GM12878). The models accurately reproduce the known looping interactions between the α-globin genes and their distal regulatory elements. Further, we find using our approach that the domain folds into a single globular conformation in GM12878 cells, whereas two globules are formed in K562 cells. The central cores of these globules are enriched for transcribed genes, whereas nontranscribed chromatin is more peripheral. We propose that globule formation represents a higher-order folding state related to clustering of transcribed genes around shared transcription machineries, as prev! iously observed by microscopy. View full text Figures at a glance * Figure 1: ENCODE region ENm008 on human chromosome 16. () Map of ENm008, including the ζ-, μ-, α2-, α1- and θ-globin genes. Genes are indicated by gray lines above the linear representation. Vertical black lines indicate HindIII restriction sites. Colored restriction fragments contain annotated genes. Red, orange and green circles mark the HS40 sites, other α-globin–related HSs and CTCF sites, respectively. () ENCODE annotations for the ENm008 region. RNA expression data, CTCF data, histone modification data (H3K4me3) and DNase I sensitivity data56, 57 are generated by the ENCODE project (http://genome.ucsc.edu/ENCODE/). Red and blue bands indicate the ENCODE track intensity for K562 and GM12878 cell lines, respectively. * Figure 2: 5C analysis of the 500-kb ENCODE region ENm008. () 5C experimental data for GM12878 cell lines. Upper plot shows 5C count matrix colored yellow to blue to indicate low to high counts. For easy inspection, the axis labels are replaced by the linear representation of the forward and reverse fragments of the ENm008 region. Lower plots show 5C interaction profiles for fragments containing HS48, HS46, HS40, HBM, HBA2, HBA1 and 3′ end of LUC7L, respectively. The plots show the 5C counts and their associated s.e.m. of interactions between the anchor fragment (indicated by vertical arrows) and the rest of the queried fragments in the ENm008 region; colored bars indicate the positions of HS elements (red), globin genes (green) and LUC7L gene (blue). Blue solid lines show the average and s.e.m. of the expected relationship between interaction frequency (5C counts) and genomic distance (kb), determined by LOESS smoothing of the complete data set (Supplementary Fig. 2). Red circles show the observed 5C counts for each of the querie! d fragments. () 5C experimental data for K562 cell lines. Data are represented as in . * Figure 3: Ensemble of solutions. () Cluster analysis for the selected 10,000 GM12878 models. Upper plot shows the number of models per cluster plotted against the cluster number. Points are colored on a grayscale proportional to the lowest IMP objective function in the cluster. IMP mirroring is illustrated by the superimposition of cluster 1 (red arrow) and cluster 2 (blue arrow) centroids (that is, the solution closest to the center of each cluster). Lower plot shows the structural relationship between the top cluster centroids. The tree was generated on the basis of the structural similarity between each of the centroids; branch thickness is proportional to the number of solutions at each branch point. A structure of each centroid, colored as in its linear representation (Fig. 1a), is placed on a vertical scale proportional to the lowest IMP objective function within the cluster it represents. () Cluster analysis for the selected 10,000 K562 models. Data are represented as in panel . () Model consistency ! for the ensemble of solutions in cluster 1 of GM12878 models (blue) and cluster 2 of K562 models (red). * Figure 4: 3D models of the ENm008 ENCODE region containing the α-globin locus. () 3D structure of the GM12878 models represented by the centroid of cluster 1. The 3D model is colored as in its linear representation (Fig. 1a). Regulatory elements are represented as spheres colored red (HS40), orange (other HSs) and green (CTCF). () 3D structure of the K562 models represented by the centroid of cluster 2. Data are represented as in panel . () Distances between the α-globin genes (restriction fragments 31 and 32) and other restriction fragments in ENm008. The plot shows the distribution and s.d. of the mean of distances for GM12878 models in cluster 1 (blue) and K562 models in cluster 2 (red). () Average distances (and their s.e.m.) between a pair of loci located on either end of the ENm008 domain, as determined by FISH with two fosmid probes (see Online Methods) and from a 2D representation of the IMP-generated models in both cell lines. () Example images obtained with FISH of GM12878 and K562 cell lines. The images show smaller distances between the pr! obes in GM12878 than in K562 cell lines. * Figure 5: Analysis of chromatin globules. () Frequency contact map differences between models in cluster 1 of GM12878 cells and cluster 2 of K562 cells. Differential expression levels are shown next to the 1D representation of the ENm008 on each axis of the plot. () Relative abundance of different ENm008 fragment types, plotted against the center of the chromatin globule, for GM12878 (upper plot) and K562 (lower plot). Plots show cumulative relative abundance of annotations versus radial position in the globule. Active genes and promoters are enriched in the center. () Observed loops in the centroids of selected clusters for GM12878 (top) and K562 (bottom) models. The loops are placed over the 1D representation of the ENm008 region (Fig. 1a). Loop height is proportional to the path length of the loop. Loops are colored according to the distance between the anchor points (dark, near; light, far). Loop sizes in kilobases (kb) are indicated at the tip of each loop. () Chromatin density for the ensemble of solutions in ! cluster 1 of GM12878 models (blue) and cluster 2 of K562 models (red). HSs are shown next to the 1D representation of the ENm008 on the x axis of the plot. * Figure 6: Diagram of the proposed chromatin-globule model for higher-order chromatin folding of actively transcribed genomic regions. Author information * Abstract * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Davide Baù, * Amartya Sanyal & * Bryan R Lajoie Affiliations * Structural Genomics Unit, Bioinformatics and Genomics Department, Centro de Investigación Príncipe Felipe, Valencia, Spain. * Davide Baù, * Emidio Capriotti & * Marc A Marti-Renom * Program in Gene Function and Expression, Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, Massachusetts, USA. * Amartya Sanyal, * Bryan R Lajoie & * Job Dekker * Department of Cell Biology, University of Massachusetts Medical School, Worcester, Massachusetts, USA. * Meg Byron & * Jeanne B Lawrence Contributions B.R.L. performed the bioinformatics design and analysis of the 5C experiments. A.S. performed the 5C experiments. D.B., E.C. and M.A.M.-R. carried out the IMP computational modeling. M.B., A.S. and J.B.L. performed the FISH experiments. D.B., B.R.L., A.S., J.D. and M.A.M.-R. wrote the manuscript. J.D. and M.A.M.-R. conceived the work. Competing financial interests The authors declare no competing financial interests. Corresponding authors Correspondence to: * Job Dekker or * Marc A Marti-Renom Supplementary information * Abstract * Author information * Supplementary information Text files * Supplementary Data 1 (16K) Zip file with 5C results and model analysis. * Supplementary Data 2 (5K) 5C frequency counts matrix for ENm008 in GM12878 cells in a tabulated text file. * Supplementary Data 3 (4K) 5C frequency counts matrix for ENm008 in K562 cells in a tabulated text file. * Supplementary Data 4 (21K) Contact map for ENm008 in GM12878 cells in a tabulated text file. * Supplementary Data 5 (18K) Contact map for ENm008 in K562 cells in a tabulated text file. * Supplementary Data 6 (403K) Contact map for ENm008 in GM12878 cells as BED formatted file for direct upload into the UCSC Genome Browser. * Supplementary Data 7 (247K) Contact map for ENm008 in K562 cells as BED formatted file for direct upload into the UCSC Genome Browser. Movies * Supplementary Video 1 (4M) Video of the spinning 3D structure for the ENm008 region in GM12878 cell lines. The region includes the α-globin locus, which contains, from telomere to centromere, the ζ, μ (also known as αD), α2, α1, and θ globin genes. Colored fragments contain annotated genes. Red (HS40), orange (other HSs) and green (CTCF-bound elements) spheres localize regulatory elements. * Supplementary Video 2 (4M) Video of the spinning 3D structure for the ENm008 region in K562 cell lines. The region includes the α-globin locus, which contains, from telomere to centromere, the ζ, μ (also known as αD), α2, α1, and θ globin genes. Colored fragments contain annotated genes. Red (HS40), orange (other HSs) and green (CTCF-bound elements) spheres localize regulatory elements. PDF files * Supplementary Text and Figures (3M) Supplementary Methods, Supplementary Table 1 and Supplementary Figures 1–6 Additional data
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