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1.
Phys Rev Lett ; 123(20): 208103, 2019 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-31809067

RESUMO

Within a living cell, the myriad of proteins that bind DNA introduce heterogeneously spaced kinks into an otherwise semiflexible DNA double helix. To investigate the effects of heterogeneous nucleosome binding on chromatin organization, we extend the wormlike chain model to include statistically spaced, rigid kinks. On timescales where nucleosome positions are fixed, we find that the probability of chromatin loop formation can vary by up to six orders of magnitude between two sets of nucleosome positions drawn from the same distribution. On longer timescales, we show that continuous rerandomization due to nucleosome turnover results in chromatin tracing out an effective WLC with a dramatically smaller Kuhn length than bare DNA. Together, these observations demonstrate that nucleosome spacing acts as the primary source of the structural heterogeneity that dominates local and global chromatin organization.


Assuntos
Cromatina/química , Cromatina/metabolismo , Modelos Biológicos , Cromatina/genética , DNA/química , DNA/genética , DNA/metabolismo , Calefação , Humanos , Modelos Químicos , Modelos Genéticos , Modelos Moleculares , Nucleossomos/química , Nucleossomos/genética , Nucleossomos/metabolismo
2.
Phys Rev Lett ; 123(20): 208102, 2019 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-31809105

RESUMO

Nucleosome positioning controls the accessible regions of chromatin and plays essential roles in DNA-templated processes. ATP driven remodeling enzymes are known to be crucial for its establishment in vivo, but their nonequilibrium nature has hindered the development of a unified theoretical framework for nucleosome positioning. Using a perturbation theory, we show that the effect of these enzymes can be well approximated by effective equilibrium models with rescaled temperatures and interactions. Numerical simulations support the accuracy of the theory in predicting both kinetic and steady-state quantities, including the effective temperature and the radial distribution function, in biologically relevant regimes. The energy landscape view emerging from our study provides an intuitive understanding for the impact of remodeling enzymes in either reinforcing or overwriting intrinsic signals for nucleosome positioning, and may help improve the accuracy of computational models for its prediction in silico.


Assuntos
Cromatina/metabolismo , Modelos Biológicos , Trifosfato de Adenosina/metabolismo , Cromatina/enzimologia , Cromatina/genética , DNA/genética , DNA/metabolismo , Humanos , Nucleossomos/enzimologia , Nucleossomos/genética , Nucleossomos/metabolismo
3.
Mol Biol (Mosk) ; 53(6): 933-953, 2019.
Artigo em Russo | MEDLINE | ID: mdl-31876274

RESUMO

Chromatin packing in eukaryotic chromosomes has been traditionally viewed as a hierarchical process, in which nucleosome chains fold into helical chromatin fibers. These fibers would then fold into more complex regular structures. However, recent chromatin imaging studies and analyses of chromosomal DNA contacts within the 3D space of the cell nucleus have necessitated a radical revision of the hierarchical chromatin packing model. According to the new studies, the nucleosome chain has a free spatial configuration without regular helical fibers in most cell types. The overall 3D organization of DNA in the cell nucleus includes chromatin loops and contact domains of up to several million base pairs in size. During cell differentiation, individual structure-functional chromatin domains marked by similar types of histone modifications and functional states can merge together and form chromosomal subcompartments suited for local gene activation or repression. This "attraction of likenesses" may be mediated by direct self-association of nucleosome chains as well as by architectural chromatin proteins making oligomeric protein "bridges" between nucleosomes as well as larger dynamic condensates leading to liquid-liquid phase separation inside the cell nucleus. Future studies of mechanisms of chromatin self-association and compartmentalization will require a combination of molecular, imaging, and computational approaches capable of revealing the 3D organization of the eukaryotic genome with nucleosomal resolution.


Assuntos
Cromatina/química , Cromatina/metabolismo , Posicionamento Cromossômico , Eucariotos/genética , DNA/química , DNA/metabolismo , Eucariotos/citologia , Histonas/química , Histonas/metabolismo , Nucleossomos/química , Nucleossomos/metabolismo
4.
Nature ; 574(7777): 278-282, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31578520

RESUMO

In eukaryotes, accurate chromosome segregation in mitosis and meiosis maintains genome stability and prevents aneuploidy. Kinetochores are large protein complexes that, by assembling onto specialized Cenp-A nucleosomes1,2, function to connect centromeric chromatin to microtubules of the mitotic spindle3,4. Whereas the centromeres of vertebrate chromosomes comprise millions of DNA base pairs and attach to multiple microtubules, the simple point centromeres of budding yeast are connected to individual microtubules5,6. All 16 budding yeast chromosomes assemble complete kinetochores using a single Cenp-A nucleosome (Cenp-ANuc), each of which is perfectly centred on its cognate centromere7-9. The inner and outer kinetochore modules are responsible for interacting with centromeric chromatin and microtubules, respectively. Here we describe the cryo-electron microscopy structure of the Saccharomyces cerevisiae inner kinetochore module, the constitutive centromere associated network (CCAN) complex, assembled onto a Cenp-A nucleosome (CCAN-Cenp-ANuc). The structure explains the interdependency of the constituent subcomplexes of CCAN and shows how the Y-shaped opening of CCAN accommodates Cenp-ANuc to enable specific CCAN subunits to contact the nucleosomal DNA and histone subunits. Interactions with the unwrapped DNA duplex at the two termini of Cenp-ANuc are mediated predominantly by a DNA-binding groove in the Cenp-L-Cenp-N subcomplex. Disruption of these interactions impairs assembly of CCAN onto Cenp-ANuc. Our data indicate a mechanism of Cenp-A nucleosome recognition by CCAN and how CCAN acts as a platform for assembly of the outer kinetochore to link centromeres to the mitotic spindle for chromosome segregation.


Assuntos
Proteína Centromérica A/metabolismo , Cinetocoros/química , Cinetocoros/metabolismo , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Nucleossomos/química , Nucleossomos/metabolismo , Proteína Centromérica A/química , Proteína Centromérica A/ultraestrutura , Microscopia Crioeletrônica , DNA/química , DNA/metabolismo , DNA/ultraestrutura , Cinetocoros/ultraestrutura , Modelos Moleculares , Complexos Multiproteicos/ultraestrutura , Nucleossomos/ultraestrutura , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/ultraestrutura
5.
Nat Commun ; 10(1): 4436, 2019 09 30.
Artigo em Inglês | MEDLINE | ID: mdl-31570711

RESUMO

CENP-A is an essential histone H3 variant that epigenetically marks the centromeric region of chromosomes. Here we show that CENP-A nucleosomes form characteristic clusters during the G1 phase of the cell cycle. 2D and 3D super-resolution microscopy and segmentation analysis reveal that these clusters encompass a globular rosette-like structure, which evolves into a more compact structure in late G1. The rosette-like clusters contain numerous CENP-A molecules and form a large cellular structure of ∼250-300 nm diameter with remarkably similar shapes for each centromere. Co-localization analysis shows that HJURP, the CENP-A chaperone, is located in the center of the rosette and serves as a nucleation point. The discovery of an HJURP-mediated CENP-A nucleation in human cells and its structural description provide important insights into the mechanism of CENP-A deposition and the organization of CENP-A chromatin in the centromeric region.


Assuntos
Proteína Centromérica A/metabolismo , Proteína Centromérica A/ultraestrutura , Proteínas de Ligação a DNA/metabolismo , Fase G1/fisiologia , Nucleossomos/metabolismo , Animais , Ciclo Celular/fisiologia , Linhagem Celular , Centrômero/metabolismo , Centrômero/ultraestrutura , Cromatina , Montagem e Desmontagem da Cromatina/fisiologia , Proteínas de Ligação a DNA/química , Epigenômica , Células HeLa , Humanos , Imagem Tridimensional , Camundongos , Camundongos Endogâmicos C57BL/embriologia , Chaperonas Moleculares/química , Nucleossomos/ultraestrutura , Imagem Óptica
6.
Nat Commun ; 10(1): 4189, 2019 09 13.
Artigo em Inglês | MEDLINE | ID: mdl-31519882

RESUMO

Retroviral integrase can efficiently utilise nucleosomes for insertion of the reverse-transcribed viral DNA. In face of the structural constraints imposed by the nucleosomal structure, integrase gains access to the scissile phosphodiester bonds by lifting DNA off the histone octamer at the site of integration. To clarify the mechanism of DNA looping by integrase, we determined a 3.9 Å resolution structure of the prototype foamy virus intasome engaged with a nucleosome core particle. The structural data along with complementary single-molecule Förster resonance energy transfer measurements reveal twisting and sliding of the nucleosomal DNA arm proximal to the integration site. Sliding the nucleosomal DNA by approximately two base pairs along the histone octamer accommodates the necessary DNA lifting from the histone H2A-H2B subunits to allow engagement with the intasome. Thus, retroviral integration into nucleosomes involves the looping-and-sliding mechanism for nucleosomal DNA repositioning, bearing unexpected similarities to chromatin remodelers.


Assuntos
Microscopia Crioeletrônica/métodos , DNA/química , Histonas/química , Nucleossomos/metabolismo , DNA/ultraestrutura , Transferência Ressonante de Energia de Fluorescência , Histonas/ultraestrutura , Humanos , Nucleoproteínas/química , Nucleoproteínas/ultraestrutura , Nucleossomos/ultraestrutura , Estrutura Quaternária de Proteína
7.
Nat Commun ; 10(1): 4372, 2019 09 26.
Artigo em Inglês | MEDLINE | ID: mdl-31558720

RESUMO

Dynamic disruption and reassembly of promoter-proximal nucleosomes is a conserved hallmark of transcriptionally active chromatin. Histone H3-K56 acetylation (H3K56Ac) enhances these turnover events and promotes nucleosome assembly during S phase. Here we sequence nascent transcripts to investigate the impact of H3K56Ac on transcription throughout the yeast cell cycle. We find that H3K56Ac is a genome-wide activator of transcription. While H3K56Ac has a major impact on transcription initiation, it also appears to promote elongation and/or termination. In contrast, H3K56Ac represses promiscuous transcription that occurs immediately following replication fork passage, in this case by promoting efficient nucleosome assembly. We also detect a stepwise increase in transcription as cells transit S phase and enter G2, but this response to increased gene dosage does not require H3K56Ac. Thus, a single histone mark can exert both positive and negative impacts on transcription that are coupled to different cell cycle events.


Assuntos
Ciclo Celular/genética , Histonas/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Ativação Transcricional , Acetilação , Montagem e Desmontagem da Cromatina/genética , Código das Histonas/genética , Histonas/metabolismo , Lisina/metabolismo , Nucleossomos/genética , Nucleossomos/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo
8.
Genes Dev ; 33(17-18): 1159-1174, 2019 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-31371436

RESUMO

Accessibility of the genomic regulatory information is largely controlled by the nucleosome-organizing activity of transcription factors (TFs). While stimulus-induced TFs bind to genomic regions that are maintained accessible by lineage-determining TFs, they also increase accessibility of thousands of cis-regulatory elements. Nucleosome remodeling events underlying such changes and their interplay with basal positioning are unknown. Here, we devised a novel quantitative framework discriminating different types of nucleosome remodeling events in micrococcal nuclease ChIP-seq (chromatin immunoprecipitation [ChIP] combined with high-throughput sequencing) data sets and used it to analyze nucleosome dynamics at stimulus-regulated cis-regulatory elements. At enhancers, remodeling preferentially affected poorly positioned nucleosomes while sparing well-positioned nucleosomes flanking the enhancer core, indicating that inducible TFs do not suffice to overrule basal nucleosomal organization maintained by lineage-determining TFs. Remodeling events appeared to be combinatorially driven by multiple TFs, with distinct TFs showing, however, different remodeling efficiencies. Overall, these data provide a systematic view of the impact of stimulation on nucleosome organization and genome accessibility in mammalian cells.


Assuntos
Nucleossomos/metabolismo , Elementos Reguladores de Transcrição/fisiologia , Fatores de Transcrição/metabolismo , Animais , Células Cultivadas , Imunoprecipitação da Cromatina , Sequenciamento de Nucleotídeos em Larga Escala , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Nuclease do Micrococo/metabolismo
9.
Nat Commun ; 10(1): 3795, 2019 08 22.
Artigo em Inglês | MEDLINE | ID: mdl-31439846

RESUMO

Histone H3 lysine 36 methylation (H3K36me) is a conserved histone modification deposited by the Set2 methyltransferases. Recent findings show that over-expression or mutation of Set2 enzymes promotes cancer progression, however, mechanisms of H3K36me are poorly understood. Set2 enzymes show spurious activity on histones and histone tails, and it is unknown how they obtain specificity to methylate H3K36 on the nucleosome. In this study, we present 3.8 Å cryo-EM structure of Set2 bound to the mimic of H2B ubiquitinated nucleosome. Our structure shows that Set2 makes extensive interactions with the H3 αN, the H3 tail, the H2A C-terminal tail and stabilizes DNA in the unwrapped conformation, which positions Set2 to specifically methylate H3K36. Moreover, we show that ubiquitin contributes to Set2 positioning on the nucleosome and stimulates the methyltransferase activity. Notably, our structure uncovers interfaces that can be targeted by small molecules for development of future cancer therapies.


Assuntos
Proteínas Fúngicas/metabolismo , Histonas/metabolismo , Metiltransferases/metabolismo , Nucleossomos/metabolismo , Ubiquitina/metabolismo , Chaetomium , Microscopia Crioeletrônica , Metilação de DNA , Proteínas Fúngicas/isolamento & purificação , Proteínas Fúngicas/ultraestrutura , Código das Histonas , Histonas/isolamento & purificação , Histonas/ultraestrutura , Metiltransferases/isolamento & purificação , Metiltransferases/ultraestrutura , Modelos Moleculares , Nucleossomos/ultraestrutura , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura , Ubiquitina/ultraestrutura
10.
Mol Cell ; 75(4): 700-710.e6, 2019 08 22.
Artigo em Inglês | MEDLINE | ID: mdl-31442422

RESUMO

Microrchidia (MORC) ATPases are critical for gene silencing and chromatin compaction in multiple eukaryotic systems, but the mechanisms by which MORC proteins act are poorly understood. Here, we apply a series of biochemical, single-molecule, and cell-based imaging approaches to better understand the function of the Caenorhabditis elegans MORC-1 protein. We find that MORC-1 binds to DNA in a length-dependent but sequence non-specific manner and compacts DNA by forming DNA loops. MORC-1 molecules diffuse along DNA but become static as they grow into foci that are topologically entrapped on DNA. Consistent with the observed MORC-1 multimeric assemblies, MORC-1 forms nuclear puncta in cells and can also form phase-separated droplets in vitro. We also demonstrate that MORC-1 compacts nucleosome templates. These results suggest that MORCs affect genome structure and gene silencing by forming multimeric assemblages to topologically entrap and progressively loop and compact chromatin.


Assuntos
Proteínas de Caenorhabditis elegans/química , Caenorhabditis elegans/química , DNA de Helmintos/química , Proteínas Nucleares/química , Conformação de Ácido Nucleico , Nucleossomos/química , Multimerização Proteica , Animais , Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/ultraestrutura , DNA de Helmintos/metabolismo , Nucleossomos/metabolismo , Nucleossomos/ultraestrutura
11.
Elife ; 82019 07 31.
Artigo em Inglês | MEDLINE | ID: mdl-31364986

RESUMO

Nucleosomes represent mechanical and energetic barriers that RNA Polymerase II (Pol II) must overcome during transcription. A high-resolution description of the barrier topography, its modulation by epigenetic modifications, and their effects on Pol II nucleosome crossing dynamics, is still missing. Here, we obtain topographic and transcriptional (Pol II residence time) maps of canonical, H2A.Z, and monoubiquitinated H2B (uH2B) nucleosomes at near base-pair resolution and accuracy. Pol II crossing dynamics are complex, displaying pauses at specific loci, backtracking, and nucleosome hopping between wrapped states. While H2A.Z widens the barrier, uH2B heightens it, and both modifications greatly lengthen Pol II crossing time. Using the dwell times of Pol II at each nucleosomal position we extract the energetics of the barrier. The orthogonal barrier modifications of H2A.Z and uH2B, and their effects on Pol II dynamics rationalize their observed enrichment in +1 nucleosomes and suggest a mechanism for selective control of gene expression.


Assuntos
Epigênese Genética , Nucleossomos/metabolismo , RNA Polimerase II/metabolismo , Transcrição Genética , Animais , Histonas/metabolismo , Xenopus
12.
PLoS Genet ; 15(8): e1008326, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31381567

RESUMO

The SWR1 chromatin remodeling complex, which deposits the histone variant H2A.Z into nucleosomes, has been well characterized in yeast and animals, but its composition in plants has remained uncertain. We used the conserved SWR1 subunit ACTIN RELATED PROTEIN 6 (ARP6) as bait in tandem affinity purification experiments to isolate associated proteins from Arabidopsis thaliana. We identified all 11 subunits found in yeast SWR1 and the homologous mammalian SRCAP complexes, demonstrating that this complex is conserved in plants. We also identified several additional proteins not previously associated with SWR1, including Methyl-CpG-BINDING DOMAIN 9 (MBD9) and three members of the Alfin1-like protein family, all of which have been shown to bind modified histone tails. Since mbd9 mutant plants were phenotypically similar to arp6 mutants, we explored a potential role for MBD9 in H2A.Z deposition. We found that MBD9 is required for proper H2A.Z incorporation at thousands of discrete sites, which represent a subset of the genomic regions normally enriched with H2A.Z. We also discovered that MBD9 preferentially interacts with acetylated histone H4 peptides, as well as those carrying mono- or dimethylated H3 lysine 4, or dimethylated H3 arginine 2 or 8. Considering that MBD9-dependent H2A.Z sites show a distinct histone modification profile, we propose that MBD9 recognizes particular nucleosome modifications via its PHD- and Bromo-domains and thereby guides SWR1 to these sites for H2A.Z deposition. Our data establish the SWR1 complex as being conserved across eukaryotes and suggest that MBD9 may be involved in targeting the complex to specific genomic sites through nucleosomal interactions. The finding that MBD9 does not appear to be a core subunit of the Arabidopsis SWR1 complex, along with the synergistic phenotype of arp6;mbd9 double mutants, suggests that MBD9 also has important roles beyond H2A.Z deposition.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Montagem e Desmontagem da Cromatina , Genoma de Planta/genética , Histonas/metabolismo , Proteínas de Arabidopsis/genética , Cromatina/genética , Cromatina/metabolismo , Mutação , Nucleossomos/genética , Nucleossomos/metabolismo , Plantas Geneticamente Modificadas
13.
Nat Commun ; 10(1): 3072, 2019 07 11.
Artigo em Inglês | MEDLINE | ID: mdl-31296853

RESUMO

Faithful transcription initiation is critical for accurate gene expression, yet the mechanisms underlying specific transcription start site (TSS) selection in mammals remain unclear. Here, we show that the histone-fold domain protein NF-Y, a ubiquitously expressed transcription factor, controls the fidelity of transcription initiation at gene promoters in mouse embryonic stem cells. We report that NF-Y maintains the region upstream of TSSs in a nucleosome-depleted state while simultaneously protecting this accessible region against aberrant and/or ectopic transcription initiation. We find that loss of NF-Y binding in mammalian cells disrupts the promoter chromatin landscape, leading to nucleosomal encroachment over the canonical TSS. Importantly, this chromatin rearrangement is accompanied by upstream relocation of the transcription pre-initiation complex and ectopic transcription initiation. Further, this phenomenon generates aberrant extended transcripts that undergo translation, disrupting gene expression profiles. These results suggest NF-Y is a central player in TSS selection in metazoans and highlight the deleterious consequences of inaccurate transcription initiation.


Assuntos
Fator de Ligação a CCAAT/metabolismo , Nucleossomos/metabolismo , Sítio de Iniciação de Transcrição , Iniciação da Transcrição Genética , Animais , Fator de Ligação a CCAAT/genética , Linhagem Celular , Cromatina/genética , Cromatina/metabolismo , Células-Tronco Embrionárias , Técnicas de Silenciamento de Genes , Camundongos , Nucleossomos/genética , Regiões Promotoras Genéticas/genética , RNA Interferente Pequeno/metabolismo
14.
Nat Commun ; 10(1): 3221, 2019 07 19.
Artigo em Inglês | MEDLINE | ID: mdl-31324780

RESUMO

The Satb1 genome organizer regulates multiple cellular and developmental processes. It is not yet clear how Satb1 selects different sets of targets throughout the genome. Here we have used live-cell single molecule imaging and deep sequencing to assess determinants of Satb1 binding-site selectivity. We have found that Satb1 preferentially targets nucleosome-dense regions and can directly bind consensus motifs within nucleosomes. Some genomic regions harbor multiple, regularly spaced Satb1 binding motifs (typical separation ~1 turn of the DNA helix) characterized by highly cooperative binding. The Satb1 homeodomain is dispensable for high affinity binding but is essential for specificity. Finally, we find that Satb1-DNA interactions are mechanosensitive. Increasing negative torsional stress in DNA enhances Satb1 binding and Satb1 stabilizes base unpairing regions against melting by molecular machines. The ability of Satb1 to control diverse biological programs may reflect its ability to combinatorially use multiple site selection criteria.


Assuntos
Sítios de Ligação , Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Proteínas de Ligação à Região de Interação com a Matriz/metabolismo , Nucleossomos/metabolismo , Sequência de Bases , Linhagem Celular , Cromatina , Proteínas de Ligação a DNA/genética , Técnicas de Inativação de Genes , Genoma , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Proteínas de Ligação à Região de Interação com a Matriz/genética , Ligação Proteica , Domínios Proteicos
15.
Phys Rev E ; 99(6-1): 060401, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31330635

RESUMO

Chromatin remodelers are multidomain enzymatic motor complexes that displace nucleosomes along DNA and hence "remodel chromatin structure," i.e., they dynamically reorganize nucleosome positions in both gene activation and gene repression. Recently, experimental insights from structural biology methods and remodeling assays have substantially advanced the understanding of these key chromatin components. Here we confront the kinetic proofreading scenario of chromatin remodeling, which proposes a mechanical link between histone residue modifications and the ATP-dependent action of remodelers, with recent experiments. We show that recent high-throughput data on nucleosome libraries assayed with remodelers from the Imitation Switch family are in accord with our earlier predictions of the kinetic proofreading scenario. We make suggestions for experimentally verifiable predictions of the kinetic proofreading scenarios for remodelers from other families.


Assuntos
Código das Histonas , Modelos Biológicos , Nucleossomos/metabolismo , Transporte Biológico , Montagem e Desmontagem da Cromatina , Cinética
16.
Mol Cell ; 75(5): 921-932.e6, 2019 09 05.
Artigo em Inglês | MEDLINE | ID: mdl-31303471

RESUMO

Fate-changing transcription factors (TFs) scan chromatin to initiate new genetic programs during cell differentiation and reprogramming. Yet the protein structure domains that allow TFs to target nucleosomal DNA remain unexplored. We screened diverse TFs for binding to nucleosomes containing motif-enriched sequences targeted by pioneer factors in vivo. FOXA1, OCT4, ASCL1/E12α, PU1, CEBPα, and ZELDA display a range of nucleosome binding affinities that correlate with their cell reprogramming potential. We further screened 593 full-length human TFs on protein microarrays against different nucleosome sequences, followed by confirmation in solution, to distinguish among factors that bound nucleosomes, such as the neuronal AP-2α/ß/γ, versus factors that only bound free DNA. Structural comparisons of DNA binding domains revealed that efficient nucleosome binders use short anchoring α helices to bind DNA, whereas weak nucleosome binders use unstructured regions and/or ß sheets. Thus, specific modes of DNA interaction allow nucleosome scanning that confers pioneer activity to transcription factors.


Assuntos
DNA/química , Nucleossomos/química , Fatores de Transcrição/química , Animais , DNA/metabolismo , Humanos , Camundongos , Nucleossomos/metabolismo , Ligação Proteica , Domínios Proteicos , Fatores de Transcrição/metabolismo
17.
Artif Cells Nanomed Biotechnol ; 47(1): 2891-2899, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31307234

RESUMO

JHDM1A participates in cancer development via demethylate dimethyl histone H3 lysine 36 (H3K36me2). p300 is an intrinsic acetyltransferase. This study explored the acetyltransferase activity of p300 on JHDM1A and analyzed the JHDM1A acetylation on H3K36me2 demethylation in osteosarcoma. Co-immunoprecipitation (CoIP) and immunoblotting assay found that p300 directly acetylated JHDM1A at K409 residue in osteosarcoma MG-63 and HOS cells. Nucleosomes and mononucleosomes were prepared and found that acetylation of JHDMIA disrupted its association with nucleosomes and thereby impaired its capability to induce H3K36me2 demethylation. Moreover, chromatin immunoprecipitation (ChIP) assay discovered that the input levels of H3K36me2 in the promoter regions of p21 and puma were increased after acetylation of JHDM1A, which raised the p21 and puma mRNA levels in the cells. Finally, the analysis of JHDM1A acetylation on osteosarcoma cell proliferation and invasion, along with tumor growth pointed out that acetylation of JHDMIA inhibited the proliferation and invasion of osteosarcoma HOS cells, as well as suppressed the tumor growth of osteosarcoma. In conclusion, the outcomes of our research verified that p300 could directly acetylate JHDM1A at K409 site, which reduces the demethylation of H3K36me2, enhanced the transcription of p21 and puma, and thereby inhibited the growth and metastasis of osteosarcoma.


Assuntos
Carcinogênese , Proteína p300 Associada a E1A/metabolismo , Proteínas F-Box/metabolismo , Histona Desmetilases com o Domínio Jumonji/metabolismo , Osteossarcoma/patologia , Acetilação , Animais , Proteínas Reguladoras de Apoptose/genética , Inibidor de Quinase Dependente de Ciclina p21/genética , Proteínas F-Box/química , Histonas/metabolismo , Humanos , Histona Desmetilases com o Domínio Jumonji/química , Lisina/metabolismo , Metilação , Camundongos , Nucleossomos/metabolismo , Proteínas Proto-Oncogênicas/genética , Transcrição Genética
18.
Nucleic Acids Res ; 47(16): 8563-8580, 2019 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-31291457

RESUMO

Creating access to DNA double-strand break (DSB) sites in the chromatin context is an essential step during the repair process, but much remains to be determined about its regulatory mechanisms. Here, using a novel reporter cassette for simultaneous detection of homologous recombination (HR) and nonhomologous end joining (NHEJ) at the same chromosomal site, we report that the efficiency of HR but not NHEJ negatively correlates with nucleosome density. We demonstrate that PARP1 is required for HR by modulating nucleosome density at damage sites. Mechanistic studies indicate that the ATPase domain of BRG1 and the ZnF domain of SIRT1 interact with poly-ADP ribose (PAR) in response to DNA damage, and are responsible for bringing the two factors to broken DNA ends. At DNA damage sites, BRG1 and SIRT1 physically interact, whereupon SIRT1 deacetylates BRG1 at lysine residues 1029 and 1033, stimulating its ATPase activity to remodel chromatin and promote HR.


Assuntos
DNA Helicases/genética , DNA/genética , Proteínas Nucleares/genética , Nucleossomos/metabolismo , Poli(ADP-Ribose) Polimerase-1/genética , Reparo de DNA por Recombinação , Sirtuína 1/genética , Fatores de Transcrição/genética , Sítios de Ligação , Linhagem Celular , Linhagem Celular Tumoral , Cloroquina/farmacologia , DNA/metabolismo , Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades , DNA Helicases/metabolismo , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Regulação da Expressão Gênica , Genes Reporter , Células HEK293 , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Proteínas Nucleares/metabolismo , Nucleossomos/química , Nucleossomos/efeitos dos fármacos , Fenantrenos/farmacologia , Ftalazinas/farmacologia , Piperazinas/farmacologia , Poli(ADP-Ribose) Polimerase-1/antagonistas & inibidores , Poli(ADP-Ribose) Polimerase-1/metabolismo , Poli Adenosina Difosfato Ribose/metabolismo , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Sirtuína 1/metabolismo , Fatores de Transcrição/metabolismo
19.
Nucleic Acids Res ; 47(16): 8470-8484, 2019 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-31287868

RESUMO

Chromatin organization is crucial for regulating gene expression. Previously, we showed that nucleosomes form groups, termed clutches. Clutch size correlated with the pluripotency grade of mouse embryonic stem cells and human induced pluripotent stem cells. Recently, it was also shown that regions of the chromatin containing activating epigenetic marks were composed of small and dispersed chromatin nanodomains with lower DNA density compared to the larger silenced domains. Overall, these results suggest that clutch size may regulate DNA packing density and gene activity. To directly test this model, we carried out 3D, two-color super-resolution microscopy of histones and DNA with and without increased histone tail acetylation. Our results showed that lower percentage of DNA was associated with nucleosome clutches in hyperacetylated cells. We further showed that the radius and compaction level of clutch-associated DNA decreased in hyperacetylated cells, especially in regions containing several neighboring clutches. Importantly, this change was independent of clutch size but dependent on the acetylation state of the clutch. Our results directly link the epigenetic state of nucleosome clutches to their DNA packing density. Our results further provide in vivo support to previous in vitro models that showed a disruption of nucleosome-DNA interactions upon hyperacetylation.


Assuntos
DNA/química , Epigênese Genética , Heterocromatina/metabolismo , Histonas/metabolismo , Nucleossomos/metabolismo , Processamento de Proteína Pós-Traducional , Acetilação , Ciclo Celular/genética , Linhagem Celular , DNA/genética , DNA/metabolismo , Fibroblastos/metabolismo , Fibroblastos/ultraestrutura , Heterocromatina/ultraestrutura , Histonas/genética , Humanos , Microscopia/métodos , Nucleossomos/ultraestrutura
20.
Nat Commun ; 10(1): 2894, 2019 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-31263106

RESUMO

The Origin Recognition Complex (ORC) is essential for replication, heterochromatin formation, telomere maintenance and genome stability in eukaryotes. Here we present the structure of the yeast Orc1 BAH domain bound to the nucleosome core particle. Our data reveal that Orc1, unlike its close homolog Sir3 involved in gene silencing, does not appear to discriminate between acetylated and non-acetylated lysine 16, modification states of the histone H4 tail that specify open and closed chromatin respectively. We elucidate the mechanism for this unique feature of Orc1 and hypothesize that its ability to interact with nucleosomes regardless of K16 modification state enables it to perform critical functions in both hetero- and euchromatin. We also show that direct interactions with nucleosomes are essential for Orc1 to maintain the integrity of rDNA borders during meiosis, a process distinct and independent from its known roles in silencing and replication.


Assuntos
Nucleossomos/metabolismo , Complexo de Reconhecimento de Origem/química , Complexo de Reconhecimento de Origem/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Ciclo Celular , Montagem e Desmontagem da Cromatina , Eucromatina/genética , Eucromatina/metabolismo , Heterocromatina/genética , Heterocromatina/metabolismo , Histonas/genética , Histonas/metabolismo , Nucleossomos/genética , Complexo de Reconhecimento de Origem/genética , Ligação Proteica , Domínios Proteicos , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas Reguladoras de Informação Silenciosa de Saccharomyces cerevisiae/genética , Proteínas Reguladoras de Informação Silenciosa de Saccharomyces cerevisiae/metabolismo
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