Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 5.549
Filtrar
1.
Nat Commun ; 11(1): 5466, 2020 10 29.
Artigo em Inglês | MEDLINE | ID: mdl-33122719

RESUMO

Human Microrchidia 4 (MORC4) is associated with acute and chronic pancreatitis, inflammatory disorders and cancer but it remains largely uncharacterized. Here, we describe the structure-function relationship of MORC4 and define the molecular mechanism for MORC4 activation. Enzymatic and binding assays reveal that MORC4 has ATPase activity, which is dependent on DNA-binding functions of both the ATPase domain and CW domain of MORC4. The crystal structure of the ATPaseCW cassette of MORC4 and mutagenesis studies show that the DNA-binding site and the histone/ATPase binding site of CW are located on the opposite sides of the domain. The ATPase and CW domains cooperate in binding of MORC4 to the nucleosome core particle (NCP), enhancing the DNA wrapping around the histone core and impeding binding of DNA-associated proteins, such as transcription factors, to the NCP. In cells, MORC4 mediates formation of nuclear bodies in the nucleus and has a role in the progression of S-phase of the cell cycle, and both these functions require CW and catalytic activity of MORC4. Our findings highlight the mechanism for MORC4 activation, which is distinctly different from the mechanisms of action observed in other MORC family members.


Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas Nucleares , Sítios de Ligação , Ciclo Celular , Cristalografia por Raios X , DNA/metabolismo , Células HEK293 , Histonas/metabolismo , Humanos , Corpos de Inclusão Intranuclear/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Nucleossomos/metabolismo , Ligação Proteica , Domínios Proteicos/fisiologia , Pontos de Checagem da Fase S do Ciclo Celular , Espectrometria de Fluorescência , Fatores de Transcrição/metabolismo , Nucleases de Dedos de Zinco/química , Nucleases de Dedos de Zinco/metabolismo
2.
Nat Commun ; 11(1): 5095, 2020 10 09.
Artigo em Inglês | MEDLINE | ID: mdl-33037201

RESUMO

Nucleosome turnover concomitant with incorporation of the replication-independent histone variant H3.3 is a hallmark of regulatory regions in the animal genome. Nucleosome turnover is known to be universally linked to DNA accessibility and histone acetylation. In mouse embryonic stem cells, H3.3 is also highly enriched at interstitial heterochromatin, most prominently at intracisternal A-particle endogenous retroviral elements. Interstitial heterochromatin is established over confined domains by the TRIM28-KAP1/SETDB1 corepressor complex and has stereotypical features of repressive chromatin, such as H3K9me3 and recruitment of all HP1 isoforms. Here, we demonstrate that fast histone turnover and H3.3 incorporation is compatible with these hallmarks of heterochromatin. Further, we find that Smarcad1 chromatin remodeler evicts nucleosomes generating accessible DNA. Free DNA is repackaged via DAXX-mediated nucleosome assembly with histone variant H3.3 in this dynamic heterochromatin state. Loss of H3.3 in mouse embryonic stem cells elicits a highly specific opening of interstitial heterochromatin with minimal effects on other silent or active regions of the genome.


Assuntos
Células-Tronco Embrionárias/fisiologia , Heterocromatina/metabolismo , Histonas/metabolismo , Animais , Células Cultivadas , Imunoprecipitação da Cromatina , DNA/metabolismo , DNA Helicases/metabolismo , Heterocromatina/genética , Histonas/genética , Camundongos Knockout , Nucleossomos/genética , Nucleossomos/metabolismo , Células-Tronco Pluripotentes/fisiologia , Retroelementos/genética
3.
Nucleic Acids Res ; 48(19): 10877-10889, 2020 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-33010153

RESUMO

The Saccharomyces cerevisiae HO gene is a model regulatory system with complex transcriptional regulation. Budding yeast divide asymmetrically and HO is expressed only in mother cells where a nucleosome eviction cascade along the promoter during the cell cycle enables activation. HO expression in daughter cells is inhibited by high concentration of Ash1 in daughters. To understand how Ash1 represses transcription, we used a myo4 mutation which boosts Ash1 accumulation in both mothers and daughters and show that Ash1 inhibits promoter recruitment of SWI/SNF and Gcn5. We show Ash1 is also required for the efficient nucleosome repopulation that occurs after eviction, and the strongest effects of Ash1 are seen when Ash1 has been degraded and at promoter locations distant from where Ash1 bound. Additionally, we defined a specific nucleosome/nucleosome-depleted region structure that restricts HO activation to one of two paralogous DNA-binding factors. We also show that nucleosome eviction occurs bidirectionally over a large distance. Significantly, eviction of the more distant nucleosomes is dependent upon the FACT histone chaperone, and FACT is recruited to these regions when eviction is beginning. These last observations, along with ChIP experiments involving the SBF factor, suggest a long-distance loop transiently forms at the HO promoter.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Desoxirribonucleases de Sítio Específico do Tipo II , Regulação Fúngica da Expressão Gênica , Proteínas de Grupo de Alta Mobilidade/metabolismo , Proteínas Repressoras/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Fatores de Elongação da Transcrição/metabolismo , Proteínas de Transporte/metabolismo , Desoxirribonucleases de Sítio Específico do Tipo II/genética , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Histona Acetiltransferases/metabolismo , Nucleossomos/metabolismo , Regiões Promotoras Genéticas , Proteínas de Saccharomyces cerevisiae/genética
4.
Nat Commun ; 11(1): 5244, 2020 10 16.
Artigo em Inglês | MEDLINE | ID: mdl-33067423

RESUMO

The protein deacetylase SIRT6 maintains cellular homeostasis through multiple pathways that include the deacetylation of histone H3 and repression of transcription. Prior work suggests that SIRT6 is associated with chromatin and can substantially reduce global levels of H3 acetylation, but how SIRT6 is able to accomplish this feat is unknown. Here, we describe an exquisitely tight interaction between SIRT6 and nucleosome core particles, in which a 2:1 enzyme:nucleosome complex assembles via asymmetric binding with distinct affinities. While both SIRT6 molecules associate with the acidic patch on the nucleosome, we find that the intrinsically disordered SIRT6 C-terminus promotes binding at the higher affinity site through recognition of nucleosomal DNA. Together, multivalent interactions couple productive binding to efficient deacetylation of histones on endogenous chromatin. Unique among histone deacetylases, SIRT6 possesses the intrinsic capacity to tightly interact with nucleosomes for efficient activity.


Assuntos
Cromatina/metabolismo , Nucleossomos/metabolismo , Sirtuínas/metabolismo , Acetilação , Cromatina/genética , Histonas/genética , Histonas/metabolismo , Humanos , Nucleossomos/genética , Ligação Proteica , Domínios Proteicos , Sirtuínas/química , Sirtuínas/genética
5.
Nat Commun ; 11(1): 5250, 2020 10 16.
Artigo em Inglês | MEDLINE | ID: mdl-33067435

RESUMO

Protein-DNA interactions are key to the functionality and stability of the genome. Identification and mapping of protein-DNA interaction interfaces and sites is crucial for understanding DNA-dependent processes. Here, we present a workflow that allows mass spectrometric (MS) identification of proteins in direct contact with DNA in reconstituted and native chromatin after cross-linking by ultraviolet (UV) light. Our approach enables the determination of contact interfaces at amino-acid level. With the example of chromatin-associated protein SCML2 we show that our technique allows differentiation of nucleosome-binding interfaces in distinct states. By UV cross-linking of isolated nuclei we determined the cross-linking sites of several factors including chromatin-modifying enzymes, demonstrating that our workflow is not restricted to reconstituted materials. As our approach can distinguish between protein-RNA and DNA interactions in one single experiment, we project that it will be possible to obtain insights into chromatin and its regulation in the future.


Assuntos
Cromatina/metabolismo , DNA/metabolismo , DNA/efeitos da radiação , Proteínas/metabolismo , Cromatina/química , Cromatina/genética , DNA/química , DNA/genética , Humanos , Espectrometria de Massas , Nucleossomos/química , Nucleossomos/genética , Nucleossomos/metabolismo , Proteínas do Grupo Polycomb/química , Proteínas do Grupo Polycomb/genética , Proteínas do Grupo Polycomb/metabolismo , Proteínas do Grupo Polycomb/efeitos da radiação , Ligação Proteica/efeitos da radiação , Proteínas/química , Proteínas/genética , Proteínas/efeitos da radiação , Raios Ultravioleta
6.
Nat Commun ; 11(1): 5063, 2020 10 08.
Artigo em Inglês | MEDLINE | ID: mdl-33033242

RESUMO

Genome-wide chromatin state underlies gene expression potential and cellular function. Epigenetic features and nucleosome positioning contribute to the accessibility of DNA, but widespread regulators of chromatin state are largely unknown. Our study investigates how coordination of ANP32E and H2A.Z contributes to genome-wide chromatin state in mouse fibroblasts. We define H2A.Z as a universal chromatin accessibility factor, and demonstrate that ANP32E antagonizes H2A.Z accumulation to restrict chromatin accessibility genome-wide. In the absence of ANP32E, H2A.Z accumulates at promoters in a hierarchical manner. H2A.Z initially localizes downstream of the transcription start site, and if H2A.Z is already present downstream, additional H2A.Z accumulates upstream. This hierarchical H2A.Z accumulation coincides with improved nucleosome positioning, heightened transcription factor binding, and increased expression of neighboring genes. Thus, ANP32E dramatically influences genome-wide chromatin accessibility through subtle refinement of H2A.Z patterns, providing a means to reprogram chromatin state and to hone gene expression levels.


Assuntos
Cromatina/metabolismo , Genoma , Chaperonas Moleculares/metabolismo , Animais , Diferenciação Celular/genética , DNA Helicases/metabolismo , Embrião de Mamíferos/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Regulação da Expressão Gênica , Histonas/metabolismo , Camundongos , Proteínas Nucleares/metabolismo , Nucleossomos/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , Fatores de Transcrição/metabolismo
7.
Nat Commun ; 11(1): 4747, 2020 09 21.
Artigo em Inglês | MEDLINE | ID: mdl-32958761

RESUMO

Chromosome structure at the multi-nucleosomal level has remained ambiguous in spite of its central role in epigenetic regulation and genome dynamics. Recent investigations of chromatin architecture portray diverse modes of interaction within and between nucleosome chains, but how this is realized at the atomic level is unclear. Here we present near-atomic resolution crystal structures of nucleosome fibres that assemble from cohesive-ended dinucleosomes with and without linker histone. As opposed to adopting folded helical '30 nm' structures, the fibres instead assume open zigzag conformations that are interdigitated with one another. Zigzag conformations obviate extreme bending of the linker DNA, while linker DNA size (nucleosome repeat length) dictates fibre configuration and thus fibre-fibre packing, which is supported by variable linker histone binding. This suggests that nucleosome chains have a predisposition to interdigitate with specific characteristics under condensing conditions, which rationalizes observations of local chromosome architecture and the general heterogeneity of chromatin structure.


Assuntos
Nucleossomos/química , Nucleossomos/metabolismo , Sequência de Bases , Cromatina/química , Cromatina/metabolismo , Cristalografia por Raios X , DNA/química , DNA/metabolismo , Histonas/química , Histonas/genética , Histonas/metabolismo , Humanos , Modelos Moleculares , Conformação Molecular , Ligação Proteica
8.
BMC Bioinformatics ; 21(Suppl 8): 326, 2020 Sep 16.
Artigo em Inglês | MEDLINE | ID: mdl-32938377

RESUMO

BACKGROUND: Nucleosomes wrap the DNA into the nucleus of the Eukaryote cell and regulate its transcription phase. Several studies indicate that nucleosomes are determined by the combined effects of several factors, including DNA sequence organization. Interestingly, the identification of nucleosomes on a genomic scale has been successfully performed by computational methods using DNA sequence as input data. RESULTS: In this work, we propose CORENup, a deep learning model for nucleosome identification. CORENup processes a DNA sequence as input using one-hot representation and combines in a parallel fashion a fully convolutional neural network and a recurrent layer. These two parallel levels are devoted to catching both non periodic and periodic DNA string features. A dense layer is devoted to their combination to give a final classification. CONCLUSIONS: Results computed on public data sets of different organisms show that CORENup is a state of the art methodology for nucleosome positioning identification based on a Deep Neural Network architecture. The comparisons have been carried out using two groups of datasets, currently adopted by the best performing methods, and CORENup has shown top performance both in terms of classification metrics and elapsed computation time.


Assuntos
Genômica/métodos , Redes Neurais de Computação , Nucleossomos/metabolismo , Humanos
9.
Nature ; 586(7827): 151-155, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32968275

RESUMO

CpG methylation by de novo DNA methyltransferases (DNMTs) 3A and 3B is essential for mammalian development and differentiation and is frequently dysregulated in cancer1. These two DNMTs preferentially bind to nucleosomes, yet cannot methylate the DNA wrapped around the nucleosome core2, and they favour the methylation of linker DNA at positioned nucleosomes3,4. Here we present the cryo-electron microscopy structure of a ternary complex of catalytically competent DNMT3A2, the catalytically inactive accessory subunit DNMT3B3 and a nucleosome core particle flanked by linker DNA. The catalytic-like domain of the accessory DNMT3B3 binds to the acidic patch of the nucleosome core, which orients the binding of DNMT3A2 to the linker DNA. The steric constraints of this arrangement suggest that nucleosomal DNA must be moved relative to the nucleosome core for de novo methylation to occur.


Assuntos
Microscopia Crioeletrônica , DNA (Citosina-5-)-Metiltransferases/química , DNA (Citosina-5-)-Metiltransferases/metabolismo , Nucleossomos/metabolismo , Animais , Biocatálise , Montagem e Desmontagem da Cromatina , DNA/química , DNA/metabolismo , Metilação de DNA , Histonas/química , Histonas/genética , Histonas/metabolismo , Humanos , Modelos Moleculares , Nucleossomos/química , Ligação Proteica , Domínios Proteicos , Xenopus/genética
10.
Mol Cell ; 80(2): 311-326.e4, 2020 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-32970994

RESUMO

To determine whether double-strand break (DSB) mobility enhances the physical search for an ectopic template during homology-directed repair (HDR), we tested the effects of factors that control chromatin dynamics, including cohesin loading and kinetochore anchoring. The former but not the latter is altered in response to DSBs. Loss of the nonhistone high-mobility group protein Nhp6 reduces histone occupancy and increases chromatin movement, decompaction, and ectopic HDR. The loss of nucleosome remodeler INO80-C did the opposite. To see whether enhanced HDR depends on DSB mobility or the global chromatin response, we tested the ubiquitin ligase mutant uls1Δ, which selectively impairs local but not global movement in response to a DSB. Strand invasion occurs in uls1Δ cells with wild-type kinetics, arguing that global histone depletion rather than DSB movement is rate limiting for HDR. Impaired break movement in uls1Δ correlates with elevated MRX and cohesin loading, despite normal resection and checkpoint activation.


Assuntos
Quebras de DNA de Cadeia Dupla , Nucleossomos/metabolismo , Saccharomyces cerevisiae/metabolismo , Bleomicina/farmacologia , Ciclo Celular , Proteínas de Ciclo Celular/metabolismo , Centrômero/metabolismo , Cromatina/metabolismo , Montagem e Desmontagem da Cromatina , Proteínas Cromossômicas não Histona/metabolismo , DNA Fúngico/metabolismo , Histonas/metabolismo , Modelos Biológicos , Fosforilação , Saccharomyces cerevisiae/citologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Corpos Polares do Fuso/metabolismo
11.
Nature ; 585(7826): 609-613, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32939087

RESUMO

Breaks in DNA strands recruit the protein PARP1 and its paralogue PARP2 to modify histones and other substrates through the addition of mono- and poly(ADP-ribose) (PAR)1-5. In the DNA damage responses, this post-translational modification occurs predominantly on serine residues6-8 and requires HPF1, an accessory factor that switches the amino acid specificity of PARP1 and PARP2 from aspartate or glutamate to serine9,10. Poly(ADP) ribosylation (PARylation) is important for subsequent chromatin decompaction and provides an anchor for the recruitment of downstream signalling and repair factors to the sites of DNA breaks2,11. Here, to understand the molecular mechanism by which PARP enzymes recognize DNA breaks within chromatin, we determined the cryo-electron-microscopic structure of human PARP2-HPF1 bound to a nucleosome. This showed that PARP2-HPF1 bridges two nucleosomes, with the broken DNA aligned in a position suitable for ligation, revealing the initial step in the repair of double-strand DNA breaks. The bridging induces structural changes in PARP2 that signal the recognition of a DNA break to the catalytic domain, which licenses HPF1 binding and PARP2 activation. Our data suggest that active PARP2 cycles through different conformational states to exchange NAD+ and substrate, which may enable PARP enzymes to act processively while bound to chromatin. The processes of PARP activation and the PARP catalytic cycle we describe can explain mechanisms of resistance to PARP inhibitors and will aid the development of better inhibitors as cancer treatments12-16.


Assuntos
Proteínas de Transporte/metabolismo , Quebras de DNA de Cadeia Dupla , Proteínas Nucleares/metabolismo , Nucleossomos/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Biocatálise , Proteínas de Transporte/química , Proteínas de Transporte/ultraestrutura , Microscopia Crioeletrônica , DNA/metabolismo , Reparo do DNA , Ativação Enzimática , Humanos , Modelos Moleculares , NAD/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/ultraestrutura , Nucleossomos/química , Nucleossomos/ultraestrutura , Poli(ADP-Ribose) Polimerases/química , Poli(ADP-Ribose) Polimerases/ultraestrutura , Domínios Proteicos
12.
Nat Struct Mol Biol ; 27(9): 836-845, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32747783

RESUMO

Interactions between chromatin-associated proteins and the histone landscape play major roles in dictating genome topology and gene expression. Cancer-specific fusion oncoproteins, which display unique chromatin localization patterns, often lack classical DNA-binding domains, presenting challenges in identifying mechanisms governing their site-specific chromatin targeting and function. Here we identify a minimal region of the human SS18-SSX fusion oncoprotein (the hallmark driver of synovial sarcoma) that mediates a direct interaction between the mSWI/SNF complex and the nucleosome acidic patch. This binding results in altered mSWI/SNF composition and nucleosome engagement, driving cancer-specific mSWI/SNF complex targeting and gene expression. Furthermore, the C-terminal region of SSX confers preferential affinity to repressed, H2AK119Ub-marked nucleosomes, underlying the selective targeting to polycomb-marked genomic regions and synovial sarcoma-specific dependency on PRC1 function. Together, our results describe a functional interplay between a key nucleosome binding hub and a histone modification that underlies the disease-specific recruitment of a major chromatin remodeling complex.


Assuntos
Proteínas Cromossômicas não Histona/metabolismo , Histonas/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas de Fusão Oncogênica/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Repressoras/metabolismo , Sarcoma Sinovial/metabolismo , Fatores de Transcrição/metabolismo , Ubiquitinas/metabolismo , Linhagem Celular Tumoral , Proteínas Cromossômicas não Histona/química , Células HEK293 , Humanos , Modelos Moleculares , Proteínas de Neoplasias/química , Nucleossomos/metabolismo , Nucleossomos/patologia , Proteínas de Fusão Oncogênica/química , Conformação Proteica , Proteínas Proto-Oncogênicas/química , Proteínas Repressoras/química , Sarcoma Sinovial/patologia , Fatores de Transcrição/química , Ubiquitinação
13.
Proc Natl Acad Sci U S A ; 117(33): 19888-19895, 2020 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-32747552

RESUMO

More than 30% of genes in higher eukaryotes are regulated by RNA polymerase II (Pol II) promoter proximal pausing. Pausing is released by the positive transcription elongation factor complex (P-TEFb). However, the exact mechanism by which this occurs and whether phosphorylation of the carboxyl-terminal domain of Pol II is involved in the process remains unknown. We previously reported that JMJD5 could generate tailless nucleosomes at position +1 from transcription start sites (TSS), thus perhaps enable progression of Pol II. Here we find that knockout of JMJD5 leads to accumulation of nucleosomes at position +1. Absence of JMJD5 also results in loss of or lowered transcription of a large number of genes. Interestingly, we found that phosphorylation, by CDK9, of Ser2 within two neighboring heptad repeats in the carboxyl-terminal domain of Pol II, together with phosphorylation of Ser5 within the second repeat, HR-Ser2p (1, 2)-Ser5p (2) for short, allows Pol II to bind JMJD5 via engagement of the N-terminal domain of JMJD5. We suggest that these events bring JMJD5 near the nucleosome at position +1, thus allowing JMJD5 to clip histones on this nucleosome, a phenomenon that may contribute to release of Pol II pausing.


Assuntos
Quinase 9 Dependente de Ciclina/metabolismo , Histona Desmetilases/metabolismo , RNA Polimerase II/metabolismo , Transcrição Genética , Linhagem Celular Tumoral , Quinase 9 Dependente de Ciclina/genética , Histona Desmetilases/química , Histona Desmetilases/genética , Humanos , Nucleossomos/genética , Nucleossomos/metabolismo , Fosforilação , Fator B de Elongação Transcricional Positiva/genética , Fator B de Elongação Transcricional Positiva/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , Domínios Proteicos , RNA Polimerase II/genética
14.
Nucleic Acids Res ; 48(17): 9538-9549, 2020 09 25.
Artigo em Inglês | MEDLINE | ID: mdl-32766790

RESUMO

Posttranslational modifications (PTMs) of histones represent a crucial regulatory mechanism of nucleosome and chromatin dynamics in various of DNA-based cellular processes, such as replication, transcription and DNA damage repair. Lysine succinylation (Ksucc) is a newly identified histone PTM, but its regulation and function in chromatin remain poorly understood. Here, we utilized an expressed protein ligation (EPL) strategy to synthesize histone H4 with site-specific succinylation at K77 residue (H4K77succ), an evolutionarily conserved succinylation site at the nucleosomal DNA-histone interface. We then assembled mononucleosomes with the semisynthetic H4K77succ in vitro. We demonstrated that this succinylation impacts nucleosome dynamics and promotes DNA unwrapping from the histone surface, which allows proteins such as transcription factors to rapidly access buried regions of the nucleosomal DNA. In budding yeast, a lysine-to-glutamic acid mutation, which mimics Ksucc, at the H4K77 site reduced nucleosome stability and led to defects in DNA damage repair and telomere silencing in vivo. Our findings revealed this uncharacterized histone modification has important roles in nucleosome and chromatin dynamics.


Assuntos
DNA/metabolismo , Histonas/síntese química , Histonas/metabolismo , Lisina/metabolismo , Nucleossomos/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Cromatina/química , Cromatina/metabolismo , Transferência Ressonante de Energia de Fluorescência , Histonas/genética , Lisina/química , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes/síntese química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo
15.
Mol Cell ; 79(3): 371-375, 2020 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-32763226

RESUMO

Whereas the core nucleosome is thought to serve as a packaging device for the coiling and contraction in length of genomic DNA, we suggest that it serves primarily in the regulation of transcription. A nucleosome on a promoter prevents the initiation of transcription. The association of nucleosomes with most genomic DNA prevents initiation from cryptic promoters. The nucleosome thus serves not only as a general gene repressor, but also as a repressor of all transcription (genic, intragenic, and intergenic). The core nucleosome performs a fundamental regulatory role, apart from the histone "tails," which modulate gene activity.


Assuntos
Proteínas Cromossômicas não Histona/genética , Nucleossomos/metabolismo , RNA Polimerase II/genética , Saccharomyces cerevisiae/genética , Fatores de Transcrição/genética , Transcrição Genética , Animais , Sítios de Ligação , Montagem e Desmontagem da Cromatina , Proteínas Cromossômicas não Histona/metabolismo , Evolução Molecular , Regulação da Expressão Gênica , Histonas/genética , Histonas/metabolismo , Humanos , Nucleossomos/ultraestrutura , Regiões Promotoras Genéticas , RNA Polimerase II/metabolismo , Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo
16.
Nat Commun ; 11(1): 4136, 2020 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-32811816

RESUMO

During cellular reprogramming, the pioneer transcription factor GATA3 binds chromatin, and in a context-dependent manner directs local chromatin remodeling and enhancer formation. Here, we use high-resolution nucleosome mapping in human cells to explore the impact of the position of GATA motifs on the surface of nucleosomes on productive enhancer formation, finding productivity correlates with binding sites located near the nucleosomal dyad axis. Biochemical experiments with model nucleosomes demonstrate sufficiently stable transcription factor-nucleosome interaction to empower cryo-electron microscopy structure determination of the complex at 3.15 Å resolution. The GATA3 zinc fingers efficiently bind their target 5'-GAT-3' sequences in the nucleosome when they are located in solvent accessible, consecutive major grooves without significant changes in nucleosome structure. Analysis of genomic loci bound by GATA3 during reprogramming suggests a correlation of recognition motif sequence and spacing that may distinguish productivity of new enhancer formation.


Assuntos
Montagem e Desmontagem da Cromatina/genética , Fator de Transcrição GATA3/química , Nucleossomos/química , Nucleossomos/genética , Motivos de Aminoácidos/genética , Sítios de Ligação , Sequenciamento de Cromatina por Imunoprecipitação , Microscopia Crioeletrônica , Elementos Facilitadores Genéticos , Fator de Transcrição GATA3/genética , Fator de Transcrição GATA3/metabolismo , Fator de Transcrição GATA3/ultraestrutura , Histonas/metabolismo , Humanos , Nucleossomos/metabolismo , Nucleossomos/ultraestrutura , Ligação Proteica , Dedos de Zinco/genética
17.
Proc Natl Acad Sci U S A ; 117(34): 20586-20596, 2020 08 25.
Artigo em Inglês | MEDLINE | ID: mdl-32778600

RESUMO

While recent experiments revealed that some pioneer transcription factors (TFs) can bind to their target DNA sequences inside a nucleosome, the binding dynamics of their target recognitions are poorly understood. Here we used the latest coarse-grained models and molecular dynamics simulations to study the nucleosome-binding procedure of the two pioneer TFs, Sox2 and Oct4. In the simulations for a strongly positioning nucleosome, Sox2 selected its target DNA sequence only when the target was exposed. Otherwise, Sox2 entropically bound to the dyad region nonspecifically. In contrast, Oct4 plastically bound on the nucleosome mainly in two ways. First, the two POU domains of Oct4 separately bound to the two parallel gyres of the nucleosomal DNA, supporting the previous experimental results of the partial motif recognition. Second, the POUS domain of Oct4 favored binding on the acidic patch of histones. Then, simulating the TFs binding to a genomic nucleosome, the LIN28B nucleosome, we found that the recognition of a pseudo motif by Sox2 induced the local DNA bending and shifted the population of the rotational position of the nucleosomal DNA. The redistributed DNA phase, in turn, changed the accessibility of a distant TF binding site, which consequently affected the binding probability of a second Sox2 or Oct4. These results revealed a nucleosomal DNA-mediated allosteric mechanism, through which one TF binding event can change the global conformation, and effectively regulate the binding of another TF at distant sites. Our simulations provide insights into the binding mechanism of single and multiple TFs on the nucleosome.


Assuntos
Nucleossomos/metabolismo , Fator 3 de Transcrição de Octâmero/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Regulação Alostérica , Humanos , Simulação de Dinâmica Molecular , Proteínas de Ligação a RNA/genética
18.
Virology ; 548: 124-131, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32838933

RESUMO

SP1 binding in SV40 chromatin in vitro and in vivo was characterized in order to better understand its role during the initiation of early transcription. We observed that chromatin from disrupted virions, but not minichromosomes, was efficiently bound by HIS-tagged SP1 in vitro, while the opposite was true for the presence of endogenous SP1 introduced in vivo. Using ChIP-Seq to compare the location of SP1 to nucleosomes carrying modified histones, we found that SP1 could occupy its whole binding site in virion chromatin but only the early side of its binding site in most of the minichromosomes carrying modified histones due to the presence of overlapping nucleosomes. The results suggest that during the initiation of an SV40 infection, SP1 binds to an open region in SV40 virion chromatin but quickly triggers chromatin reorganization and its own removal.


Assuntos
Cromatina/virologia , Infecções por Polyomavirus/metabolismo , Infecções por Polyomavirus/virologia , Vírus 40 dos Símios/metabolismo , Fator de Transcrição Sp1/metabolismo , Vírion/metabolismo , Cromatina/genética , Cromatina/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Nucleossomos/genética , Nucleossomos/metabolismo , Infecções por Polyomavirus/genética , Ligação Proteica , Vírus 40 dos Símios/genética , Fator de Transcrição Sp1/genética , Vírion/genética
19.
Proc Natl Acad Sci U S A ; 117(33): 19661-19663, 2020 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-32747537

RESUMO

The structural unit of eukaryotic chromatin is a nucleosome, comprising two histone H2A-H2B heterodimers and one histone (H3-H4)2 tetramer, wrapped around by ∼146 bp of DNA. The N-terminal flexible histone tails stick out from the histone core and have extensive posttranslational modifications, causing epigenetic changes of chromatin. Although crystal and cryogenic electron microscopy structures of nucleosomes are available, the flexible tail structures remain elusive. Using NMR, we have examined the dynamics of histone H3 tails in nucleosomes containing unmodified and tetra-acetylated H4 tails. In unmodified nucleosome, the H3 tail adopts a dynamic equilibrium structure between DNA-contact and reduced-contact states. In acetylated H4 nucleosome, however, the H3 tail equilibrium shifts to a mainly DNA-contact state with a minor reduced-contact state. The acetylated H4 tail is dynamically released from its own DNA-contact state to a reduced-contact state, while the H3 tail DNA-contact state becomes major. Notably, H3 K14 in the acetylated H4 nucleosome is much more accessible to acetyltransferase Gcn5 relative to unmodified nucleosome, possibly due to the formation of a favorable H3 tail conformation for Gcn5. In summary, each histone tail adopts a characteristic dynamic state but regulates one other, probably creating a histone tail network even on a nucleosome.


Assuntos
Histonas/química , Histonas/metabolismo , Nucleossomos/metabolismo , Acetilação , Motivos de Aminoácidos , DNA/genética , DNA/metabolismo , Histonas/genética , Humanos , Conformação de Ácido Nucleico , Nucleossomos/genética
20.
Nat Rev Mol Cell Biol ; 21(9): 522-541, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32665685

RESUMO

Histones serve to both package and organize DNA within the nucleus. In addition to histone post-translational modification and chromatin remodelling complexes, histone variants contribute to the complexity of epigenetic regulation of the genome. Histone variants are characterized by a distinct protein sequence and a selection of designated chaperone systems and chromatin remodelling complexes that regulate their localization in the genome. In addition, histone variants can be enriched with specific post-translational modifications, which in turn can provide a scaffold for recruitment of variant-specific interacting proteins to chromatin. Thus, through these properties, histone variants have the capacity to endow specific regions of chromatin with unique character and function in a regulated manner. In this Review, we provide an overview of recent advances in our understanding of the contribution of histone variants to chromatin function in mammalian systems. First, we discuss new molecular insights into chaperone-mediated histone variant deposition. Next, we discuss mechanisms by which histone variants influence chromatin properties such as nucleosome stability and the local chromatin environment both through histone variant sequence-specific effects and through their role in recruiting different chromatin-associated complexes. Finally, we focus on histone variant function in the context of both embryonic development and human disease, specifically developmental syndromes and cancer.


Assuntos
Cromatina/metabolismo , Histonas/genética , Histonas/metabolismo , Animais , DNA/metabolismo , Reparo do DNA/genética , Epigênese Genética/genética , Humanos , Chaperonas Moleculares/metabolismo , Nucleossomos/genética , Nucleossomos/metabolismo , Processamento de Proteína Pós-Traducional/genética , Fatores de Transcrição/metabolismo , Transcrição Genética/fisiologia
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA