Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 43
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
ACS Cent Sci ; 5(9): 1602-1613, 2019 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-31572787

RESUMO

Rhodamine dyes exist in equilibrium between a fluorescent zwitterion and a nonfluorescent lactone. Tuning this equilibrium toward the nonfluorescent lactone form can improve cell-permeability and allow creation of "fluorogenic" compounds-ligands that shift to the fluorescent zwitterion upon binding a biomolecular target. An archetype fluorogenic dye is the far-red tetramethyl-Si-rhodamine (SiR), which has been used to create exceptionally useful labels for advanced microscopy. Here, we develop a quantitative framework for the development of new fluorogenic dyes, determining that the lactone-zwitterion equilibrium constant (K L-Z) is sufficient to predict fluorogenicity. This rubric emerged from our analysis of known fluorophores and yielded new fluorescent and fluorogenic labels with improved performance in cellular imaging experiments. We then designed a novel fluorophore-Janelia Fluor 526 (JF526)-with SiR-like properties but shorter fluorescence excitation and emission wavelengths. JF526 is a versatile scaffold for fluorogenic probes including ligands for self-labeling tags, stains for endogenous structures, and spontaneously blinking labels for super-resolution immunofluorescence. JF526 constitutes a new label for advanced microscopy experiments, and our quantitative framework will enable the rational design of other fluorogenic probes for bioimaging.

2.
PLoS Biol ; 17(5): e3000277, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31107867

RESUMO

Chz1 is a specific chaperone for the histone variant H2A.Z in budding yeast. The ternary complex formed by Chz1 and H2A.Z-H2B dimer is the major in vivo substrate of Swi2/snif2-related 1 (SWR1), the ATP-dependent chromatin remodeling enzyme that deposits H2A.Z into chromatin. However, the structural basis for the binding preference of Chz1 for H2A.Z over H2A and the mechanism by which Chz1 modulates the histone replacement remain elusive. Here, we show that Chz1 utilizes 2 distinct structural domains to engage the H2A.Z-H2B dimer for optimal and specific recognition of H2A.Z. The middle region of Chz1 (Chz1-M) directly interacts with 2 highly conserved H2A.Z-specific residues (Gly98 and Ala57) and dictates a modest preference for H2A.Z-H2B. In addition, structural and biochemical analysis show that the C-terminal region of Chz1 (Chz1-C) harbors a conserved DEF/Y motif, which reflects the consecutive D/E residues followed by a single aromatic residue, to engage an arginine finger and a hydrophobic pocket in H2A.Z-H2B, enhancing the binding preference for H2A.Z-H2B. Furthermore, Chz1 facilitates SWR1-mediated H2A.Z deposition by alleviating inhibition caused by aggregation of excess free histones, providing insights into how Chz1 controls the bioavailability of H2A.Z to assist SWR1 in promoter-specific installation of a histone mark. Our study elucidates a novel H2A.Z-recognition mechanism and uncovers a molecular rationale for binding of free histone by specialized histone chaperones in vivo.


Assuntos
Chaperonas de Histonas/química , Chaperonas de Histonas/metabolismo , Histonas/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Cromatina/metabolismo , Ligação Proteica , Multimerização Proteica
3.
Nat Protoc ; 13(11): 2535-2556, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30341436

RESUMO

Hydroxyl-radical footprinting (HRF) is a powerful method for probing structures of nucleic acid-protein complexes with single-nucleotide resolution in solution. To tap the full quantitative potential of HRF, we describe a protocol, hydroxyl-radical footprinting interpretation for DNA (HYDROID), to quantify HRF data and integrate them with atomistic structural models. The stages of the HYDROID protocol are extraction of the lane profiles from gel images, quantification of the DNA cleavage frequency at each nucleotide and theoretical estimation of the DNA cleavage frequency from atomistic structural models, followed by comparison of experimental and theoretical results. Example scripts for each step of HRF data analysis and interpretation are provided for several nucleosome systems; they can be easily adapted to analyze user data. As input, HYDROID requires polyacrylamide gel electrophoresis (PAGE) images of HRF products and optionally can use a molecular model of the DNA-protein complex. The HYDROID protocol can be used to quantify HRF over DNA regions of up to 100 nucleotides per gel image. In addition, it can be applied to the analysis of RNA-protein complexes and free RNA or DNA molecules in solution. Compared with other methods reported to date, HYDROID is unique in its ability to simultaneously integrate HRF data with the analysis of atomistic structural models. HYDROID is freely available. The complete protocol takes ~3 h. Users should be familiar with the command-line interface, the Python scripting language and Protein Data Bank (PDB) file formats. A graphical user interface (GUI) with basic functionality (HYDROID_GUI) is also available.


Assuntos
Pegada de DNA/métodos , DNA/química , Radical Hidroxila/química , Pegadas de Proteínas/métodos , Proteínas/química , Software , DNA/metabolismo , Clivagem do DNA , Pegada de DNA/estatística & dados numéricos , Eletroforese em Gel de Poliacrilamida/estatística & dados numéricos , Humanos , Modelos Moleculares , Nucleossomos/química , Nucleossomos/metabolismo , Pegadas de Proteínas/estatística & dados numéricos , Proteínas/metabolismo , Soluções
4.
Genes Dev ; 31(19): 1958-1972, 2017 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-29074736

RESUMO

Histone CENP-A-containing nucleosomes play an important role in nucleating kinetochores at centromeres for chromosome segregation. However, the molecular mechanisms by which CENP-A nucleosomes engage with kinetochore proteins are not well understood. Here, we report the finding of a new function for the budding yeast Cse4/CENP-A histone-fold domain interacting with inner kinetochore protein Mif2/CENP-C. Strikingly, we also discovered that AT-rich centromere DNA has an important role for Mif2 recruitment. Mif2 contacts one side of the nucleosome dyad, engaging with both Cse4 residues and AT-rich nucleosomal DNA. Both interactions are directed by a contiguous DNA- and histone-binding domain (DHBD) harboring the conserved CENP-C motif, an AT hook, and RK clusters (clusters enriched for arginine-lysine residues). Human CENP-C has two related DHBDs that bind preferentially to DNA sequences of higher AT content. Our findings suggest that a DNA composition-based mechanism together with residues characteristic for the CENP-A histone variant contribute to the specification of centromere identity.


Assuntos
Proteína Centromérica A/metabolismo , Centrômero/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Modelos Moleculares , Nucleossomos/química , Nucleossomos/metabolismo , Saccharomyces cerevisiae , Sequência Rica em At , Centrômero/química , Proteína Centromérica A/química , Proteínas Cromossômicas não Histona/química , DNA Satélite/metabolismo , Proteínas de Ligação a DNA/metabolismo , Dimerização , Humanos , Ligação Proteica , Estrutura Terciária de Proteína , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo
5.
Nucleic Acids Res ; 45(16): 9229-9243, 2017 Sep 19.
Artigo em Inglês | MEDLINE | ID: mdl-28934480

RESUMO

Nucleosomes are the most abundant protein-DNA complexes in eukaryotes that provide compaction of genomic DNA and are implicated in regulation of transcription, DNA replication and repair. The details of DNA positioning on the nucleosome and the DNA conformation can provide key regulatory signals. Hydroxyl-radical footprinting (HRF) of protein-DNA complexes is a chemical technique that probes nucleosome organization in solution with a high precision unattainable by other methods. In this work we propose an integrative modeling method for constructing high-resolution atomistic models of nucleosomes based on HRF experiments. Our method precisely identifies DNA positioning on nucleosome by combining HRF data for both DNA strands with the pseudo-symmetry constraints. We performed high-resolution HRF for Saccharomyces cerevisiae centromeric nucleosome of unknown structure and characterized it using our integrative modeling approach. Our model provides the basis for further understanding the cooperative engagement and interplay between Cse4p protein and the A-tracts important for centromere function.


Assuntos
Pegada de DNA/métodos , DNA/química , Modelos Moleculares , Nucleossomos/química , Algoritmos , Centrômero/química , Proteínas Cromossômicas não Histona , Clivagem do DNA , Proteínas de Ligação a DNA , Radical Hidroxila , Conformação de Ácido Nucleico , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae
6.
Science ; 353(6297): 358, 2016 Jul 22.
Artigo em Inglês | MEDLINE | ID: mdl-27463665

RESUMO

Watanabe et al (Reports, 12 April 2013, p. 195) study the yeast SWR1/SWR-C complex responsible for depositing the histone variant H2A.Z by replacing nucleosomal H2A with H2A.Z. They report that reversal of H2A.Z replacement is mediated by SWR1 and related INO80 on an H2A.Z nucleosome carrying H3K56Q. Using multiple assays and reaction conditions, we find no evidence of such reversal of H2A.Z exchange.


Assuntos
Adenosina Trifosfatases/metabolismo , Montagem e Desmontagem da Cromatina , Histonas/metabolismo , Complexos Multienzimáticos/metabolismo , Nucleossomos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo
7.
Biomed Opt Express ; 7(3): 855-69, 2016 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-27231594

RESUMO

Multifocus microscopy (MFM) allows high-resolution instantaneous three-dimensional (3D) imaging and has been applied to study biological specimens ranging from single molecules inside cells nuclei to entire embryos. We here describe pattern designs and nanofabrication methods for diffractive optics that optimize the light-efficiency of the central optical component of MFM: the diffractive multifocus grating (MFG). We also implement a "precise color" MFM layout with MFGs tailored to individual fluorophores in separate optical arms. The reported advancements enable faster and brighter volumetric time-lapse imaging of biological samples. In live microscopy applications, photon budget is a critical parameter and light-efficiency must be optimized to obtain the fastest possible frame rate while minimizing photodamage. We provide comprehensive descriptions and code for designing diffractive optical devices, and a detailed methods description for nanofabrication of devices. Theoretical efficiencies of reported designs is ≈90% and we have obtained efficiencies of > 80% in MFGs of our own manufacture. We demonstrate the performance of a multi-phase MFG in 3D functional neuronal imaging in living C. elegans.

8.
Nat Struct Mol Biol ; 23(4): 317-23, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26974124

RESUMO

Histone variant H2A.Z, a universal mark of dynamic nucleosomes flanking gene promoters and enhancers, is incorporated into chromatin by SRCAP (SWR1), an ATP-dependent, multicomponent chromatin-remodeling complex. The YL1 (Swc2) subunit of SRCAP (SWR1) plays an essential role in H2A.Z recognition, but how it achieves this has been unclear. Here, we report the crystal structure of the H2A.Z-binding domain of Drosophila melanogaster YL1 (dYL1-Z) in complex with an H2A.Z-H2B dimer at 1.9-Å resolution. The dYL1-Z domain adopts a new whip-like structure that wraps over H2A.Z-H2B, and preferential recognition is largely conferred by three residues in loop 2, the hyperacidic patch and the extended αC helix of H2A.Z. Importantly, this domain is essential for deposition of budding yeast H2A.Z in vivo and SRCAP (SWR1)-catalyzed histone H2A.Z replacement in vitro. Our studies distinguish YL1-Z from known H2A.Z chaperones and suggest a hierarchical mechanism based on increasing binding affinity facilitating H2A.Z transfer from SRCAP (SWR1) to the nucleosome.


Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/química , Drosophila melanogaster/metabolismo , Chaperonas de Histonas/metabolismo , Histonas/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Montagem e Desmontagem da Cromatina , Cristalografia por Raios X , Proteínas de Drosophila/química , Chaperonas de Histonas/química , Histonas/química , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Multimerização Proteica , Estrutura Terciária de Proteína , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Alinhamento de Sequência
9.
Elife ; 4: e06845, 2015 Jun 27.
Artigo em Inglês | MEDLINE | ID: mdl-26116819

RESUMO

The histone variant H2A.Z is a universal mark of gene promoters, enhancers, and regulatory elements in eukaryotic chromatin. The chromatin remodeler SWR1 mediates site-specific incorporation of H2A.Z by a multi-step histone replacement reaction, evicting histone H2A-H2B from the canonical nucleosome and depositing the H2A.Z-H2B dimer. Binding of both substrates, the canonical nucleosome and the H2A.Z-H2B dimer, is essential for activation of SWR1. We found that SWR1 primarily recognizes key residues within the α2 helix in the histone-fold of nucleosomal histone H2A, a region not previously known to influence remodeler activity. Moreover, SWR1 interacts preferentially with nucleosomal DNA at superhelix location 2 on the nucleosome face distal to its linker-binding site. Our findings provide new molecular insights on recognition of the canonical nucleosome by a chromatin remodeler and have implications for ATP-driven mechanisms of histone eviction and deposition.


Assuntos
Adenosina Trifosfatases/metabolismo , Montagem e Desmontagem da Cromatina , DNA/metabolismo , Histonas/metabolismo , Nucleossomos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Cromatina/metabolismo , Ligação Proteica , Saccharomyces cerevisiae/genética
10.
Proc Natl Acad Sci U S A ; 111(49): 17480-5, 2014 Dec 09.
Artigo em Inglês | MEDLINE | ID: mdl-25422417

RESUMO

Single molecule-based superresolution imaging has become an essential tool in modern cell biology. Because of the limited depth of field of optical imaging systems, one of the major challenges in superresolution imaging resides in capturing the 3D nanoscale morphology of the whole cell. Despite many previous attempts to extend the application of photo-activated localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM) techniques into three dimensions, effective localization depths do not typically exceed 1.2 µm. Thus, 3D imaging of whole cells (or even large organelles) still demands sequential acquisition at different axial positions and, therefore, suffers from the combined effects of out-of-focus molecule activation (increased background) and bleaching (loss of detections). Here, we present the use of multifocus microscopy for volumetric multicolor superresolution imaging. By simultaneously imaging nine different focal planes, the multifocus microscope instantaneously captures the distribution of single molecules (either fluorescent proteins or synthetic dyes) throughout an ∼ 4-µm-deep volume, with lateral and axial localization precisions of ∼ 20 and 50 nm, respectively. The capabilities of multifocus microscopy to rapidly image the 3D organization of intracellular structures are illustrated by superresolution imaging of the mammalian mitochondrial network and yeast microtubules during cell division.


Assuntos
Microscopia de Fluorescência/instrumentação , Microscopia de Fluorescência/métodos , Mitocôndrias/metabolismo , Calibragem , Corantes Fluorescentes/química , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Processamento de Imagem Assistida por Computador , Imagem Tridimensional , Modelos Moleculares , Reprodutibilidade dos Testes , Saccharomyces cerevisiae/metabolismo
11.
Elife ; 3: e02203, 2014 May 20.
Artigo em Inglês | MEDLINE | ID: mdl-24844245

RESUMO

The budding yeast centromere contains Cse4, a specialized histone H3 variant. Fluorescence pulse-chase analysis of an internally tagged Cse4 reveals that it is replaced with newly synthesized molecules in S phase, remaining stably associated with centromeres thereafter. In contrast, C-terminally-tagged Cse4 is functionally impaired, showing slow cell growth, cell lethality at elevated temperatures, and extra-centromeric nuclear accumulation. Recent studies using such strains gave conflicting findings regarding the centromeric abundance and cell cycle dynamics of Cse4. Our findings indicate that internally tagged Cse4 is a better reporter of the biology of this histone variant. Furthermore, the size of centromeric Cse4 clusters was precisely mapped with a new 3D-PALM method, revealing substantial compaction during anaphase. Cse4-specific chaperone Scm3 displays steady-state, stoichiometric co-localization with Cse4 at centromeres throughout the cell cycle, while undergoing exchange with a nuclear pool. These findings suggest that a stable Cse4 nucleosome is maintained by dynamic chaperone-in-residence Scm3.DOI: http://dx.doi.org/10.7554/eLife.02203.001.


Assuntos
Centrômero , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Ligação a DNA/metabolismo , Fase S , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Anáfase , Replicação do DNA , DNA Fúngico/biossíntese , Fluorescência , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética
12.
Mol Cell ; 53(3): 498-505, 2014 Feb 06.
Artigo em Inglês | MEDLINE | ID: mdl-24507717

RESUMO

Histone variant H2A.Z-containing nucleosomes exist at most eukaryotic promoters and play important roles in gene transcription and genome stability. The multisubunit nucleosome-remodeling enzyme complex SWR1, conserved from yeast to mammals, catalyzes the ATP-dependent replacement of histone H2A in canonical nucleosomes with H2A.Z. How SWR1 catalyzes the replacement reaction is largely unknown. Here, we determined the crystal structure of the N-terminal region (599-627) of the catalytic subunit Swr1, termed Swr1-Z domain, in complex with the H2A.Z-H2B dimer at 1.78 Å resolution. The Swr1-Z domain forms a 310 helix and an irregular chain. A conserved LxxLF motif in the Swr1-Z 310 helix specifically recognizes the αC helix of H2A.Z. Our results show that the Swr1-Z domain can deliver the H2A.Z-H2B dimer to the DNA-(H3-H4)2 tetrasome to form the nucleosome by a histone chaperone mechanism.


Assuntos
Adenosina Trifosfatases/química , Histonas/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Adenosina Trifosfatases/fisiologia , Sequência de Aminoácidos , Montagem e Desmontagem da Cromatina/genética , Clonagem Molecular , Cristalografia por Raios X , Dimerização , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Subunidades Proteicas/química , Subunidades Proteicas/fisiologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/fisiologia , Difração de Raios X
13.
Cell ; 154(6): 1220-31, 2013 Sep 12.
Artigo em Inglês | MEDLINE | ID: mdl-24034246

RESUMO

The ATP-dependent chromatin-remodeling complex SWR1 exchanges a variant histone H2A.Z/H2B dimer for a canonical H2A/H2B dimer at nucleosomes flanking histone-depleted regions, such as promoters. This localization of H2A.Z is conserved throughout eukaryotes. SWR1 is a 1 megadalton complex containing 14 different polypeptides, including the AAA+ ATPases Rvb1 and Rvb2. Using electron microscopy, we obtained the three-dimensional structure of SWR1 and mapped its major functional components. Our data show that SWR1 contains a single heterohexameric Rvb1/Rvb2 ring that, together with the catalytic subunit Swr1, brackets two independently assembled multisubunit modules. We also show that SWR1 undergoes a large conformational change upon engaging a limited region of the nucleosome core particle. Our work suggests an important structural role for the Rvbs and a distinct substrate-handling mode by SWR1, thereby providing a structural framework for understanding the complex dimer-exchange reaction.


Assuntos
Adenosina Trifosfatases/química , Montagem e Desmontagem da Cromatina , DNA Helicases/química , Complexos Multiproteicos/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/química , Adenosina Trifosfatases/metabolismo , DNA Helicases/metabolismo , Dimerização , Complexos Multiproteicos/metabolismo , Complexos Multiproteicos/ultraestrutura , Nucleossomos/química , Nucleossomos/metabolismo , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/ultraestrutura , Fatores de Transcrição/metabolismo
14.
Cell ; 154(6): 1232-45, 2013 Sep 12.
Artigo em Inglês | MEDLINE | ID: mdl-24034247

RESUMO

The histone variant H2A.Z is a genome-wide signature of nucleosomes proximal to eukaryotic regulatory DNA. Whereas the multisubunit chromatin remodeler SWR1 is known to catalyze ATP-dependent deposition of H2A.Z, the mechanism of SWR1 recruitment to S. cerevisiae promoters has been unclear. A sensitive assay for competitive binding of dinucleosome substrates revealed that SWR1 preferentially binds long nucleosome-free DNA and the adjoining nucleosome core particle, allowing discrimination of gene promoters over gene bodies. Analysis of mutants indicates that the conserved Swc2/YL1 subunit and the adenosine triphosphatase domain of Swr1 are mainly responsible for binding to substrate. SWR1 binding is enhanced on nucleosomes acetylated by the NuA4 histone acetyltransferase, but recognition of nucleosome-free and nucleosomal DNA is dominant over interaction with acetylated histones. Such hierarchical cooperation between DNA and histone signals expands the dynamic range of genetic switches, unifying classical gene regulation by DNA-binding factors with ATP-dependent nucleosome remodeling and posttranslational histone modifications.


Assuntos
Montagem e Desmontagem da Cromatina , Histonas/metabolismo , Complexos Multiproteicos/metabolismo , Nucleossomos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Acetilação , Adenosina Trifosfatases/metabolismo , Sequência de Bases , Histona Acetiltransferases/metabolismo , Dados de Sequência Molecular , Processamento de Proteína Pós-Traducional , Saccharomyces cerevisiae/genética
15.
J Biol Chem ; 288(32): 23182-93, 2013 Aug 09.
Artigo em Inglês | MEDLINE | ID: mdl-23779104

RESUMO

The evolutionarily conserved ATP-dependent chromatin remodeling enzyme Fun30 has recently been shown to play important roles in heterochromatin silencing and DNA repair. However, how Fun30 remodels nucleosomes is not clear. Here we report a nucleosome sliding activity of Fun30 and its role in transcriptional repression. We observed that Fun30 repressed the expression of genes involved in amino acid and carbohydrate metabolism, the stress response, and meiosis. In addition, Fun30 was localized at the 5' and 3' ends of genes and within the open reading frames of its targets. Consistent with its role in gene repression, we observed that Fun30 target genes lacked histone modifications often associated with gene activation and showed an increased level of ubiquitinated histone H2B. Furthermore, a genome-wide nucleosome mapping analysis revealed that the length of the nucleosome-free region at the 5' end of a subset of genes was changed in Fun30-depleted cells. In addition, the positions of the -1, +2, and +3 nucleosomes at the 5' end of target genes were shifted significantly, whereas the position of the +1 nucleosome remained largely unchanged in the fun30Δ mutant. Finally, we demonstrated that affinity-purified, single-component Fun30 exhibited a nucleosome sliding activity in an ATP-dependent manner. These results define a role for Fun30 in the regulation of transcription and indicate that Fun30 remodels chromatin at the 5' end of genes by sliding promoter-proximal nucleosomes.


Assuntos
Trifosfato de Adenosina/metabolismo , Montagem e Desmontagem da Cromatina/fisiologia , Nucleossomos/metabolismo , Regiões Promotoras Genéticas/fisiologia , Proteínas Repressoras/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Genética/fisiologia , Trifosfato de Adenosina/genética , Histonas/genética , Histonas/metabolismo , Nucleossomos/genética , Proteínas Repressoras/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Transcrição/genética , Ubiquitinação/fisiologia
16.
Nat Methods ; 10(1): 60-3, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23223154

RESUMO

Conventional acquisition of three-dimensional (3D) microscopy data requires sequential z scanning and is often too slow to capture biological events. We report an aberration-corrected multifocus microscopy method capable of producing an instant focal stack of nine 2D images. Appended to an epifluorescence microscope, the multifocus system enables high-resolution 3D imaging in multiple colors with single-molecule sensitivity, at speeds limited by the camera readout time of a single image.


Assuntos
Caenorhabditis elegans/citologia , Rastreamento de Células , Imagem Tridimensional/métodos , Microscopia de Fluorescência , Neurônios/citologia , Saccharomyces cerevisiae/citologia , Animais , Neoplasias Ósseas/enzimologia , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Ligação a DNA/metabolismo , Humanos , Osteossarcoma/enzimologia , RNA Polimerase II/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo
19.
Mol Cell ; 43(3): 369-80, 2011 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-21816344

RESUMO

The molecular architecture of centromere-specific nucleosomes containing histone variant CenH3 is controversial. We have biochemically reconstituted two distinct populations of nucleosomes containing Saccharomyces cerevisiae CenH3 (Cse4). Reconstitution of octameric nucleosomes containing histones Cse4/H4/H2A/H2B is robust on noncentromere DNA, but inefficient on AT-rich centromere DNA. However, nonhistone Scm3, which is required for Cse4 deposition in vivo, facilitates in vitro reconstitution of Cse4/H4/Scm3 complexes on AT-rich centromere sequences. Scm3 has a nonspecific DNA binding domain that shows preference for AT-rich DNA and a histone chaperone domain that promotes specific loading of Cse4/H4. In live cells, Scm3-GFP is enriched at centromeres in all cell cycle phases. Chromatin immunoprecipitation confirms that Scm3 occupies centromere DNA throughout the cell cycle, even when Cse4 and H4 are temporarily dislodged in S phase. These findings suggest a model in which centromere-bound Scm3 aids recruitment of Cse4/H4 to assemble and maintain an H2A/H2B-deficient centromeric nucleosome.


Assuntos
Centrômero/química , Proteínas Cromossômicas não Histona/química , Proteínas Cromossômicas não Histona/fisiologia , Proteínas de Ligação a DNA/química , Histonas/química , Nucleossomos/química , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/genética , Sequência Rica em At , Sítios de Ligação , Ciclo Celular/genética , Centrômero/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Cromossomos Fúngicos/metabolismo , DNA Fúngico/química , Proteínas de Ligação a DNA/metabolismo , Chaperonas de Histonas/química , Chaperonas de Histonas/metabolismo , Chaperonas de Histonas/fisiologia , Histonas/metabolismo , Modelos Moleculares , Nucleossomos/metabolismo , Estrutura Terciária de Proteína , Proteínas de Saccharomyces cerevisiae/metabolismo
20.
Blood ; 118(5): 1386-94, 2011 Aug 04.
Artigo em Inglês | MEDLINE | ID: mdl-21653943

RESUMO

Chromatin insulators protect erythroid genes from being silenced during erythropoiesis, and the disruption of barrier insulator function in erythroid membrane gene loci results in mild or severe anemia. We showed previously that the USF1/2-bound 5'HS4 insulator mediates chromatin barrier activity in the erythroid-specific chicken ß-globin locus. It is currently not known how insulators establish such a barrier. To understand the function of USF1, we purified USF1-associated protein complexes and found that USF1 forms a multiprotein complex with hSET1 and NURF, thus exhibiting histone H3K4 methyltransferase- and ATP-dependent nucleosome remodeling activities, respectively. Both SET1 and NURF are recruited to the 5'HS4 insulator by USF1 to retain the active chromatin structure in erythrocytes. Knock-down of NURF resulted in a rapid loss of barrier activity accompanied by an alteration of nucleosome positioning, increased occupancy of the nucleosome-free linker region at the insulator site, and increased repressive H3K27me3 levels in the vicinity of the HS4 insulator. Furthermore, suppression of SET1 reduced barrier activity, decreased H3K4me2 and acH3K9/K14, and diminished the recruitment of BPTF at several erythroid-specific barrier insulator sites. Therefore, our data reveal a synergistic role of hSET1 and NURF in regulating the USF-bound barrier insulator to prevent erythroid genes from encroachment of heterochromatin.


Assuntos
Montagem e Desmontagem da Cromatina/genética , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/fisiologia , Histona-Lisina N-Metiltransferase/fisiologia , Animais , Galinhas , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Células HeLa , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Elementos Isolantes/fisiologia , Células K562 , Modelos Biológicos , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Complexos Multiproteicos/fisiologia , Ligação Proteica/genética , Ligação Proteica/fisiologia , Células Tumorais Cultivadas , Fatores Estimuladores Upstream/metabolismo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA