RESUMO
Chromatin adopts a diversity of regular and irregular fiber structures in vitro and in vivo. However, how an array of nucleosomes folds into and switches between different fiber conformations is poorly understood. We report the 9.7 Å resolution crystal structure of a 6-nucleosome array bound to linker histone H1 determined under ionic conditions that favor incomplete chromatin condensation. The structure reveals a flat two-start helix with uniform nucleosomal stacking interfaces and a nucleosome packing density that is only half that of a twisted 30-nm fiber. Hydroxyl radical footprinting indicates that H1 binds the array in an on-dyad configuration resembling that observed for mononucleosomes. Biophysical, cryo-EM, and crosslinking data validate the crystal structure and reveal that a minor change in ionic environment shifts the conformational landscape to a more compact, twisted form. These findings provide insights into the structural plasticity of chromatin and suggest a possible assembly pathway for a 30-nm fiber.
Assuntos
DNA/química , Histonas/química , Proteína 1 de Modelagem do Nucleossomo/química , Nucleossomos/ultraestrutura , Animais , Sítios de Ligação , Clonagem Molecular , Microscopia Crioeletrônica , Cristalografia por Raios X , DNA/genética , DNA/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Histonas/genética , Histonas/metabolismo , Humanos , Radical Hidroxila/química , Modelos Moleculares , Proteína 1 de Modelagem do Nucleossomo/genética , Proteína 1 de Modelagem do Nucleossomo/metabolismo , Nucleossomos/química , Nucleossomos/metabolismo , Concentração Osmolar , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Xenopus laevisRESUMO
Linker histones associate with nucleosomes to promote the formation of higher-order chromatin structure, but the underlying molecular details are unclear. We investigated the structure of a 197 bp nucleosome bearing symmetric 25 bp linker DNA arms in complex with vertebrate linker histone H1. We determined electron cryo-microscopy (cryo-EM) and crystal structures of unbound and H1-bound nucleosomes and validated these structures by site-directed protein cross-linking and hydroxyl radical footprinting experiments. Histone H1 shifts the conformational landscape of the nucleosome by drawing the two linkers together and reducing their flexibility. The H1 C-terminal domain (CTD) localizes primarily to a single linker, while the H1 globular domain contacts the nucleosome dyad and both linkers, associating more closely with the CTD-distal linker. These findings reveal that H1 imparts a strong degree of asymmetry to the nucleosome, which is likely to influence the assembly and architecture of higher-order structures.
Assuntos
Montagem e Desmontagem da Cromatina , Cromatina/metabolismo , DNA/metabolismo , Histonas/metabolismo , Nucleossomos/metabolismo , Animais , Pareamento de Bases , Sítios de Ligação , Cromatina/química , Cromatina/genética , Cromatina/ultraestrutura , Microscopia Crioeletrônica , DNA/química , DNA/genética , Histonas/química , Humanos , Modelos Moleculares , Nucleossomos/química , Nucleossomos/genética , Nucleossomos/ultraestrutura , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Relação Estrutura-Atividade , Fatores de Tempo , Xenopus laevis/genética , Xenopus laevis/metabolismoRESUMO
CENP-A is a histone variant, which replaces histone H3 at centromeres and confers unique properties to centromeric chromatin. The crystal structure of CENP-A nucleosome suggests flexible nucleosomal DNA ends, but their dynamics in solution remains elusive and their implication in centromere function is unknown. Using electron cryo-microscopy, we determined the dynamic solution properties of the CENP-A nucleosome. Our biochemical, proteomic, and genetic data reveal that higher flexibility of DNA ends impairs histone H1 binding to the CENP-A nucleosome. Substituting the 2-turn αN-helix of CENP-A with the 3-turn αN-helix of H3 results in compact particles with rigidified DNA ends, able to bind histone H1. In vivo replacement of CENP-A with H3-CENP-A hybrid nucleosomes leads to H1 recruitment, delocalization of kinetochore proteins, and significant mitotic and cytokinesis defects. Our data reveal that the evolutionarily conserved flexible ends of the CENP-A nucleosomes are essential to ensure the fidelity of the mitotic pathway.
Assuntos
Autoantígenos/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , DNA/metabolismo , Histonas/metabolismo , Cinetocoros/metabolismo , Mitose/fisiologia , Nucleossomos/metabolismo , Animais , Autoantígenos/genética , Autoantígenos/ultraestrutura , Sítios de Ligação , Proteína Centromérica A , Proteínas Cromossômicas não Histona/deficiência , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/ultraestrutura , Microscopia Crioeletrônica , Citocinese , DNA/química , Genótipo , Células HeLa , Humanos , Cinetocoros/ultraestrutura , Camundongos , Camundongos Knockout , Modelos Moleculares , Mutação , Conformação de Ácido Nucleico , Nucleossomos/ultraestrutura , Fenótipo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Relação Estrutura-Atividade , TransfecçãoRESUMO
RNA polymerase V (Pol V) long noncoding RNAs (lncRNAs) have been proposed to guide ARGONAUTE4 (AGO4) to chromatin in RNA-directed DNA methylation (RdDM) in plants. Here, we provide evidence, based on laser UV-assisted zero-length cross-linking, for functionally relevant AGO4-DNA interaction at RdDM targets. We further demonstrate that Pol V lncRNAs or the act of their transcription are required to lock Pol V holoenzyme into a stable DNA-bound state that allows AGO4 recruitment via redundant glycine-tryptophan/tryptophan-glycine AGO hook motifs present on both Pol V and its associated factor, SPT5L. We propose a model in which AGO4-DNA interaction could be responsible for the unique specificities of RdDM.
Assuntos
Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Metilação de DNA , DNA de Plantas/metabolismo , RNA de Plantas/metabolismo , Motivos de Aminoácidos/genética , Proteínas de Arabidopsis/química , Proteínas Argonautas/química , Cromatina/metabolismo , Metilação de DNA/genética , RNA Polimerases Dirigidas por DNA/química , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Inativação Gênica , Modelos Biológicos , Interferência de RNA , Transcrição Gênica/genéticaRESUMO
Pioneer transcription factors (PTFs) have the remarkable ability to directly bind to chromatin to stimulate vital cellular processes. In this work, we dissect the universal binding mode of Sox PTF by combining extensive molecular simulations and physiochemistry approaches, along with DNA footprinting techniques. As a result, we show that when Sox consensus DNA is located at the solvent-facing DNA strand, Sox binds to the compact nucleosome without imposing any significant conformational changes. We also reveal that the base-specific Sox:DNA interactions (base reading) and Sox-induced DNA changes (shape reading) are concurrently required for sequence-specific nucleosomal DNA recognition. Among three different nucleosome positions located on the positive DNA arm, a sequence-specific reading mechanism is solely satisfied at the superhelical location 2 (SHL2). While SHL2 acts transparently for solvent-facing Sox binding, among the other two positions, SHL4 permits only shape reading. The final position, SHL0 (dyad), on the other hand, allows no reading mechanism. These findings demonstrate that Sox-based nucleosome recognition is essentially guided by intrinsic nucleosome properties, permitting varying degrees of DNA recognition.
Assuntos
Nucleossomos , Fatores de Transcrição , Fatores de Transcrição/química , DNA/química , Regulação da Expressão GênicaRESUMO
The histone H3 variant CENP-A marks centromeres epigenetically and is essential for mitotic fidelity. Previous crystallographic studies of the CENP-A nucleosome core particle (NCP) reconstituted with a human α-satellite DNA derivative revealed both DNA ends to be highly flexible, a feature important for CENP-A mitotic functions. However, recent cryo-EM studies of CENP-A NCP complexes comprising primarily Widom 601 DNA reported well-ordered DNA ends. Here, we report the cryo-EM structure of the CENP-A 601 NCP determined by Volta phase-plate imaging. The data reveal that one ('left') 601 DNA end is well ordered whereas the other ('right') end is flexible and partly detached from the histone core, suggesting sequence-dependent dynamics of the DNA termini. Indeed, a molecular dynamics simulation of the CENP-A 601 NCP confirmed the distinct dynamics of the two DNA extremities. Reprocessing the image data using two-fold symmetry yielded a cryo-EM map in which both DNA ends appeared well ordered, indicating that such an artefact may inadvertently arise if NCP asymmetry is lost during image processing. These findings enhance our understanding of the dynamic features that discriminate CENP-A from H3 nucleosomes by revealing that DNA end flexibility can be fine-tuned in a sequence-dependent manner.
Assuntos
Proteína Centromérica A/química , DNA/química , Nucleossomos/química , Microscopia Crioeletrônica , Humanos , Simulação de Dinâmica Molecular , Nucleossomos/ultraestruturaRESUMO
FACT, in addition to its role in transcription, is likely implicated in both transcription-coupled nucleotide excision repair and DNA double strand break repair. Here, we present evidence that FACT could be directly involved in Base Excision Repair and elucidate the chromatin remodeling mechanisms of FACT during BER. We found that, upon oxidative stress, FACT is released from transcription related protein complexes to get associated with repair proteins and chromatin remodelers from the SWI/SNF family. We also showed the rapid recruitment of FACT to the site of damage, coincident with the glycosylase OGG1, upon the local generation of oxidized DNA. Interestingly, FACT facilitates uracil-DNA glycosylase in the removal of uracil from nucleosomal DNA thanks to an enhancement in the remodeling activity of RSC. This discloses a novel property of FACT wherein it has a co-remodeling activity and strongly enhances the remodeling capacity of the chromatin remodelers. Altogether, our data suggest that FACT may acts in concert with RSC to facilitate excision of DNA lesions during the initial step of BER.
Assuntos
Reparo do DNA/genética , Proteínas de Ligação a DNA/genética , Proteínas de Grupo de Alta Mobilidade/genética , Histonas/genética , Transcrição Gênica , Fatores de Elongação da Transcrição/genética , Animais , Cromatina/genética , Montagem e Desmontagem da Cromatina/genética , Proteínas Cromossômicas não Histona/genética , Dano ao DNA/genética , Proteínas de Ligação a DNA/biossíntese , Células HeLa , Proteínas de Grupo de Alta Mobilidade/biossíntese , Histonas/metabolismo , Humanos , Nucleossomos/genética , Estresse Oxidativo/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Transcrição/genética , Fatores de Elongação da Transcrição/biossíntese , Uracila/metabolismo , Xenopus laevisRESUMO
Thyroid hormones control various aspects of gut development and homeostasis. The best-known example is in gastrointestinal tract remodeling during amphibian metamorphosis. It is well documented that these hormones act via the TR nuclear receptors, which are hormone-modulated transcription factors. Several studies have shown that thyroid hormones regulate the expression of several genes in the Notch signaling pathway, indicating a possible means by which they participate in the control of gut physiology. However, the mechanisms and biological significance of this control have remained unexplored. Using multiple in vivo and in vitro approaches, we show that thyroid hormones positively regulate Notch activity through the TRα1 receptor. From a molecular point of view, TRα1 indirectly controls Notch1, Dll1, Dll4 and Hes1 expression but acts as a direct transcriptional regulator of the Jag1 gene by binding to a responsive element in the Jag1 promoter. Our findings show that the TRα1 nuclear receptor plays a key role in intestinal crypt progenitor/stem cell biology by controlling the Notch pathway and hence the balance between cell proliferation and cell differentiation.
Assuntos
Linhagem da Célula/fisiologia , Hipertireoidismo/metabolismo , Intestinos/citologia , Receptor Notch1/metabolismo , Transdução de Sinais/fisiologia , Receptores alfa dos Hormônios Tireóideos/genética , Animais , Western Blotting , Imunoprecipitação da Cromatina , Ensaio de Desvio de Mobilidade Eletroforética , Células Epiteliais/fisiologia , Imuno-Histoquímica , Intestinos/fisiologia , Camundongos , Microscopia ConfocalRESUMO
NF-κB is a key transcription factor regulating the expression of inflammatory responsive genes. How NF-κB binds to naked DNA templates is well documented, but how it interacts with chromatin is far from being clear. Here we used a combination of UV laser footprinting, hydroxyl footprinting and electrophoretic mobility shift assay to investigate the binding of NF-κB to nucleosomal templates. We show that NF-κB p50 homodimer is able to bind to its recognition sequence, when it is localized at the edge of the core particle, but not when the recognition sequence is at the interior of the nucleosome. Remodeling of the nucleosome by the chromatin remodeling machine RSC was not sufficient to allow binding of NF-κB to its recognition sequence located in the vicinity of the nucleosome dyad, but RSC-induced histone octamer sliding allowed clearly detectable binding of NF-κB with the slid particle. Importantly, nucleosome dilution-driven removal of H2A-H2B dimer led to complete accessibility of the site located close to the dyad to NF-κB. Finally, we found that NF-κB was able to displace histone H1 and prevent its binding to nucleosome. These data provide important insight on the role of chromatin structure in the regulation of transcription of NF-κB dependent genes.
Assuntos
Cromatina/genética , Histonas/genética , NF-kappa B/metabolismo , Nucleossomos/genética , Biossíntese de Proteínas , Animais , Sítios de Ligação , Cromatina/ultraestrutura , Proteínas de Ligação a DNA/genética , Dimerização , Histonas/metabolismo , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase , NF-kappa B/genética , Nucleossomos/metabolismo , Ligação Proteica , Raios Ultravioleta , Xenopus/genéticaRESUMO
In this work we have studied the effect of chromatin structure on the base excision repair (BER) efficiency of 8-oxoG. As a model system we have used precisely positioned dinucleosomes assembled with linker histone H1. A single 8-oxoG was inserted either in the linker or the core particle DNA within the dinucleosomal template. We found that in the absence of histone H1 the glycosylase OGG1 removed 8-oxoG from the linker DNA and cleaved DNA with identical efficiency as in the naked DNA. In contrast, the presence of histone H1 resulted in close to 10-fold decrease in the efficiency of 8-oxoG initiation of repair in linker DNA independently of linker DNA length. The repair of 8-oxoG in nucleosomal DNA was very highly impeded in both absence and presence of histone H1. Chaperone-induced uptake of H1 restored the efficiency of the glycosylase induced removal of 8-oxoG from linker DNA, but not from the nucleosomal DNA. We show, however, that removal of histone H1 and nucleosome remodelling are both necessary and sufficient for an efficient removal of 8-oxoG in nucleosomal DNA. Finally, a model for BER of 8-oxoG in chromatin templates is suggested.
Assuntos
Reparo do DNA , Guanina/análogos & derivados , Nucleossomos/metabolismo , Animais , DNA/química , DNA/metabolismo , DNA Glicosilases/metabolismo , Guanina/metabolismo , Histonas/metabolismo , Camundongos , Modelos Genéticos , Proteína 1 de Modelagem do Nucleossomo/metabolismoRESUMO
This review article is aimed at providing updated information on the contribution of immediate and delayed oxidative reactions to the photo-induced damage to cellular DNA/skin under exposure to UVB/UVA radiations and visible light. Low-intensity UVC and UVB radiations that operate predominantly through direct excitation of the nucleobases are very poor oxidizing agents giving rise to very low amounts of 8-oxo-7,8-dihydroguanine and DNA strand breaks with respect to the overwhelming bipyrimidine dimeric photoproducts. The importance of these two classes of oxidatively generated damage to DNA significantly increases together with a smaller contribution of oxidized pyrimidine bases upon UVA irradiation. This is rationalized in terms of sensitized photooxidation reactions predominantly mediated by singlet oxygen together with a small contribution of hydroxyl radical that appear to also be implicated in the photodynamic effects of the blue light component of visible light. Chemiexcitation-mediated formation of "dark" cyclobutane pyrimidine dimers in UVA-irradiated melanocytes is a recent major discovery that implicates in the initial stage, a delayed generation of reactive oxygen and nitrogen species giving rise to triplet excited carbonyl intermediate and possibly singlet oxygen. High-intensity UVC nanosecond laser radiation constitutes a suitable source of light to generate pyrimidine and purine radical cations in cellular DNA via efficient biphotonic ionization.
RESUMO
The linker histone H1 C-terminal domain (CTD) plays a pivotal role in chromatin condensation. De novo frameshift mutations within the CTD coding region of H1.4 have recently been reported to be associated with Rahman syndrome, a neurological disease that causes intellectual disability and overgrowth. To investigate the mechanisms and pathogenesis of Rahman syndrome, we developed a cellular model using murine embryonic stem cells (mESCs) and CRISPR/Cas9 genome engineering. Our engineered mES cells facilitate detailed investigations, such as H1-4 dynamics, immunoprecipitation, and nuclear localization; in addition, we tagged the mutant H1-4 with a photoactivatable GFP (PA-GFP) and an HA tag to facilitate pulldown assays. We anticipate that these engineered cells could also be used for the development of a mouse model to study the in vivo role of the H1-4 protein.
Assuntos
Histonas , Células-Tronco Embrionárias Murinas , Animais , Camundongos , Cromatina , Histonas/metabolismo , Células-Tronco Embrionárias Murinas/metabolismoRESUMO
DNA is not the static entity that structural pictures suggest. It has been longly known that it 'breathes' and fluctuates by local opening of the bases. Here we show that the effect of structural fluctuations, exhibited by AT-rich low stability regions present in some common transcription initiation regions, influences the properties of DNA in a distant range of at least 10 bp. This observation is confirmed by experiments on genuine gene promoter regions of DNA. The spatial correlations revealed by these experiments throw a new light on the physics of DNA and could have biological implications, for instance by contributing to the cooperative effects needed to assemble the molecular machinery that forms the transcription complex.
Assuntos
Sequência Rica em At , DNA/química , Guanina/química , Regiões Promotoras Genéticas , Modelos Moleculares , TemperaturaRESUMO
The 'remodels structure of chromatin' (RSC) complex is an essential chromatin remodeling factor that is required for the control of several processes including transcription, repair and replication. The ability of RSC to relocate centrally positioned mononucleosomes at the end of nucleosomal DNA is firmly established, but the data on RSC action on oligo-nucleosomal templates remains still scarce. By using atomic force microscopy (AFM) imaging, we have quantitatively studied the RSC-induced mobilization of positioned di- and trinucleosomes as well as the directionality of mobilization on mononucleosomal template labeled at one end with streptavidin. AFM imaging showed only a limited set of distinct configurational states for the remodeling products. No stepwise or preferred directionality of the nucleosome motion was observed. Analysis of the corresponding reaction pathways allows deciphering the mechanistic features of RSC-induced nucleosome relocation. The final outcome of RSC remodeling of oligosome templates is the packing of the nucleosomes at the edge of the template, providing large stretches of DNA depleted of nucleosomes. This feature of RSC may be used by the cell to overcome the barrier imposed by the presence of nucleosomes.
Assuntos
Montagem e Desmontagem da Cromatina , Proteínas de Ligação a DNA/metabolismo , Nucleossomos/ultraestrutura , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo , DNA/química , DNA/metabolismo , Microscopia de Força Atômica , Nucleossomos/química , Nucleossomos/metabolismoRESUMO
Histone variants within the H2A family show high divergences in their C-terminal regions. In this work, we have studied how these divergences and in particular, how a part of the H2A COOH-terminus, the docking domain, is implicated in both structural and functional properties of the nucleosome. Using biochemical methods in combination with Atomic Force Microscopy and Electron Cryo-Microscopy, we show that the H2A-docking domain is a key structural feature within the nucleosome. Deletion of this domain or replacement with the incomplete docking domain from the variant H2A.Bbd results in significant structural alterations in the nucleosome, including an increase in overall accessibility to nucleases, un-wrapping of â¼10 bp of DNA from each end of the nucleosome and associated changes in the entry/exit angle of DNA ends. These structural alterations are associated with a reduced ability of the chromatin remodeler RSC to both remodel and mobilize the nucleosomes. Linker histone H1 binding is also abrogated in nucleosomes containing the incomplete docking domain of H2A.Bbd. Our data illustrate the unique role of the H2A-docking domain in coordinating the structural-functional aspects of the nucleosome properties. Moreover, our data suggest that incorporation of a 'defective' docking domain may be a primary structural role of H2A.Bbd in chromatin.
Assuntos
Montagem e Desmontagem da Cromatina , Proteínas de Ligação a DNA/metabolismo , Histonas/química , Nucleossomos/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo , DNA/química , DNA/metabolismo , Histonas/genética , Histonas/metabolismo , Nucleossomos/metabolismo , Nucleossomos/ultraestrutura , Ligação Proteica , Estrutura Terciária de Proteína , Deleção de SequênciaRESUMO
The interaction of histone H1 with linker DNA results in the formation of the nucleosomal stem structure, with considerable influence on chromatin organization. In a recent paper [Syed,S.H., Goutte-Gattat,D., Becker,N., Meyer,S., Shukla,M.S., Hayes,J.J., Everaers,R., Angelov,D., Bednar,J. and Dimitrov,S. (2010) Single-base resolution mapping of H1-nucleosome interactions and 3D organization of the nucleosome. Proc. Natl Acad. Sci. USA, 107, 9620-9625], we published results of biochemical footprinting and cryo-electron-micrographs of reconstituted mono-, di- and tri-nucleosomes, for H1 variants with different lengths of the cationic C-terminus. Here, we present a detailed account of the analysis of the experimental data and we include thermal fluctuations into our nano-scale model of the stem structure. By combining (i) crystal and NMR structures of the nucleosome core particle and H1, (ii) the known nano-scale structure and elasticity of DNA, (iii) footprinting information on the location of protected sites on the DNA backbone and (iv) cryo-electron micrographs of reconstituted tri-nucleosomes, we arrive at a description of a polymorphic, hierarchically organized stem with a typical length of 20 ± 2 base pairs. A comparison to linker conformations inferred for poly-601 fibers with different linker lengths suggests, that intra-stem interactions stabilize and facilitate the formation of dense chromatin fibers.
Assuntos
Modelos Moleculares , Nucleossomos/química , Fenômenos Biomecânicos , Microscopia Crioeletrônica , Cristalografia por Raios X , DNA/química , Histonas/química , Ressonância Magnética Nuclear Biomolecular , Pegadas de ProteínasRESUMO
Chromatin remodelers are sophisticated nano-machines that are able to alter histone-DNA interactions and to mobilize nucleosomes. Neither the mechanism of their action nor the conformation of the remodeled nucleosomes are, however, yet well understood. We have studied the mechanism of Remodels Structure of Chromatin (RSC)-nucleosome mobilization by using high-resolution microscopy and biochemical techniques. Atomic force microscopy and electron cryomicroscopy (EC-M) analyses show that two types of products are generated during the RSC remodeling: (i) stable non-mobilized particles, termed remosomes that contain about 180 bp of DNA associated with the histone octamer and, (ii) mobilized particles located at the end of DNA. EC-M reveals that individual remosomes exhibit a distinct, variable, highly-irregular DNA trajectory. The use of the unique "one pot assays" for studying the accessibility of nucleosomal DNA towards restriction enzymes, DNase I footprinting and ExoIII mapping demonstrate that the histone-DNA interactions within the remosomes are strongly perturbed, particularly in the vicinity of the nucleosome dyad. The data suggest a two-step mechanism of RSC-nucleosome remodeling consisting of an initial formation of a remosome followed by mobilization. In agreement with this model, we show experimentally that the remosomes are intermediate products generated during the first step of the remodeling reaction that are further efficiently mobilized by RSC.
Assuntos
Montagem e Desmontagem da Cromatina , DNA/química , Histonas/química , Nucleossomos/química , Animais , Microscopia Crioeletrônica , DNA/ultraestrutura , Histonas/ultraestrutura , Técnicas In Vitro , Microscopia de Força Atômica , Conformação de Ácido Nucleico , Nucleossomos/ultraestrutura , Estrutura Quaternária de Proteína , Xenopus laevisRESUMO
Despite the key role of the linker histone H1 in chromatin structure and dynamics, its location and interactions with nucleosomal DNA have not been elucidated. In this work we have used a combination of electron cryomicroscopy, hydroxyl radical footprinting, and nanoscale modeling to analyze the structure of precisely positioned mono-, di-, and trinucleosomes containing physiologically assembled full-length histone H1 or truncated mutants of this protein. Single-base resolution *OH footprinting shows that the globular domain of histone H1 (GH1) interacts with the DNA minor groove located at the center of the nucleosome and contacts a 10-bp region of DNA localized symmetrically with respect to the nucleosomal dyad. In addition, GH1 interacts with and organizes about one helical turn of DNA in each linker region of the nucleosome. We also find that a seven amino acid residue region (121-127) in the COOH terminus of histone H1 was required for the formation of the stem structure of the linker DNA. A molecular model on the basis of these data and coarse-grain DNA mechanics provides novel insights on how the different domains of H1 interact with the nucleosome and predicts a specific H1-mediated stem structure within linker DNA.
Assuntos
Histonas/química , Histonas/metabolismo , Nucleossomos/química , Nucleossomos/metabolismo , Sequência de Aminoácidos , Histonas/genética , Histonas/ultraestrutura , Radical Hidroxila/metabolismo , Microscopia Eletrônica , Modelos Moleculares , Conformação Molecular , Dados de Sequência Molecular , Mutação , Nucleossomos/ultraestrutura , Ligação ProteicaRESUMO
Interactions of DNA with structural proteins such as histones, regulatory proteins and enzymes play a crucial role in major cellular processes such as transcription, replication and repair. The in vivo mapping and characterization of the binding sites of the involved biomolecules are of primary importance for a better understanding of genomic deployment that is implicated in tissue and developmental stage-specific gene expression regulation. The most powerful and commonly used approach to date is immunoprecipitation of chemically cross-linked chromatin (XChIP) coupled with sequencing analysis (ChIP-seq). While the resolution and the sensitivity of the high-throughput sequencing techniques have been constantly improved, little progress has been achieved in the cross-linking step. Because of its low efficiency, the use of the conventional UVC lamps remains very limited while the formaldehyde method was established as the "gold standard" cross-linking agent. Efficient biphotonic cross-linking of directly interacting nucleic acid-protein complexes by a single short UV laser pulse has been introduced as an innovative technique for overcoming limitations of conventionally used chemical and photochemical approaches. In this survey, the main available methods including the laser approach are critically reviewed for their ability to generate DNA-protein cross-links in vitro model systems and cells.