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1.
Int J Mol Sci ; 22(19)2021 Sep 24.
Artigo em Inglês | MEDLINE | ID: mdl-34638614

RESUMO

Inflammation is the body's means of defense against harmful stimuli, with the ultimate aim being to restore homeostasis. Controlled acute inflammation transiently activates an immune response and can be beneficial as protection against infection or injury. However, dysregulated inflammatory responses, including chronic inflammation, disrupt the immune system's ability to maintain homeostatic balance, leading to increased susceptibility to infection, continuous tissue damage, and dysfunction. Aging is a risk factor for chronic inflammation; their coincidence is termed "inflammaging". Metabolic disorders including obesity, neurodegenerative diseases, and atherosclerosis are often encountered in old age. Therefore, it is important to understand the mechanistic relationship between aging, chronic inflammation, and metabolism. It has been established that the expression of inflammatory mediators is transcriptionally and translationally regulated. In addition, the post-translational modification of the mediators plays a crucial role in the response to inflammatory signaling. Chromatin regulation responds to metabolic status and controls homeostasis. However, chromatin structure is also changed by aging. In this review, we discuss the functional contributions of chromatin regulation to inflammaging.


Assuntos
Envelhecimento/imunologia , Envelhecimento/metabolismo , Cromatina/metabolismo , Macrófagos/imunologia , Macrófagos/metabolismo , Animais , Código das Histonas , Humanos , Inflamação/imunologia , Inflamação/metabolismo , Modelos Biológicos , Nucleossomos/imunologia , Nucleossomos/metabolismo
2.
Int J Mol Sci ; 22(19)2021 Sep 24.
Artigo em Inglês | MEDLINE | ID: mdl-34638632

RESUMO

Cellular identity is determined through complex patterns of gene expression. Chromatin, the dynamic structure containing genetic information, is regulated through epigenetic modulators, mainly by the histone code. One of the main challenges for the cell is maintaining functionality and identity, despite the accumulation of DNA damage throughout the aging process. Replicative cells can remain in a senescent state or develop a malign cancer phenotype. In contrast, post-mitotic cells such as pyramidal neurons maintain extraordinary functionality despite advanced age, but they lose their identity. This review focuses on tau, a protein that protects DNA, organizes chromatin, and plays a crucial role in genomic stability. In contrast, tau cytosolic aggregates are considered hallmarks of Alzheimer´s disease (AD) and other neurodegenerative disorders called tauopathies. Here, we explain AD as a phenomenon of chromatin dysregulation directly involving the epigenetic histone code and a progressive destabilization of the tau-chromatin interaction, leading to the consequent dysregulation of gene expression. Although this destabilization could be lethal for post-mitotic neurons, tau protein mediates profound cellular transformations that allow for their temporal survival.


Assuntos
Doença de Alzheimer/metabolismo , Cromatina/metabolismo , Proteínas tau/metabolismo , Doença de Alzheimer/etiologia , Doença de Alzheimer/genética , Cromatina/genética , DNA/química , DNA/genética , DNA/metabolismo , Dano ao DNA , Epigênese Genética , Instabilidade Genômica , Código das Histonas , Humanos , Nucleossomos/metabolismo , Fosforilação , Fatores de Tempo , Proteínas tau/química , Proteínas tau/genética
3.
Elife ; 102021 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-34486521

RESUMO

The chromatin remodeler ALC1 is recruited to and activated by DNA damage-induced poly(ADP-ribose) (PAR) chains deposited by PARP1/PARP2/HPF1 upon detection of DNA lesions. ALC1 has emerged as a candidate drug target for cancer therapy as its loss confers synthetic lethality in homologous recombination-deficient cells. However, structure-based drug design and molecular analysis of ALC1 have been hindered by the requirement for PARylation and the highly heterogeneous nature of this post-translational modification. Here, we reconstituted an ALC1 and PARylated nucleosome complex modified in vitro using PARP2 and HPF1. This complex was amenable to cryo-EM structure determination without cross-linking, which enabled visualization of several intermediate states of ALC1 from the recognition of the PARylated nucleosome to the tight binding and activation of the remodeler. Functional biochemical assays with PARylated nucleosomes highlight the importance of nucleosomal epitopes for productive remodeling and suggest that ALC1 preferentially slides nucleosomes away from DNA breaks.


Assuntos
Proteínas de Transporte/metabolismo , Montagem e Desmontagem da Cromatina , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas Nucleares/metabolismo , Nucleossomos/metabolismo , Poli(ADP-Ribose) Polimerase-1/metabolismo , Poli ADP Ribosilação , Poli(ADP-Ribose) Polimerases/metabolismo , Proteínas de Transporte/genética , Microscopia Crioeletrônica , DNA Helicases/genética , DNA Helicases/ultraestrutura , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/ultraestrutura , Humanos , Cinética , Modelos Moleculares , Proteínas Nucleares/genética , Nucleossomos/genética , Nucleossomos/ultraestrutura , Poli(ADP-Ribose) Polimerase-1/genética , Poli(ADP-Ribose) Polimerases/genética , Ligação Proteica , Conformação Proteica , Proteínas Recombinantes/metabolismo , Relação Estrutura-Atividade , Especificidade por Substrato
5.
Nat Commun ; 12(1): 5280, 2021 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-34489435

RESUMO

Little is known about the roles of histone tails in modulating nucleosomal DNA accessibility and its recognition by other macromolecules. Here we generate extensive atomic level conformational ensembles of histone tails in the context of the full nucleosome, totaling 65 microseconds of molecular dynamics simulations. We observe rapid conformational transitions between tail bound and unbound states, and characterize kinetic and thermodynamic properties of histone tail-DNA interactions. Different histone types exhibit distinct binding modes to specific DNA regions. Using a comprehensive set of experimental nucleosome complexes, we find that the majority of them target mutually exclusive regions with histone tails on nucleosomal/linker DNA around the super-helical locations ± 1, ± 2, and ± 7, and histone tails H3 and H4 contribute most to this process. These findings are explained within competitive binding and tail displacement models. Finally, we demonstrate the crosstalk between different histone tail post-translational modifications and mutations; those which change charge, suppress tail-DNA interactions and enhance histone tail dynamics and DNA accessibility.


Assuntos
DNA/química , Histonas/química , Nucleossomos/ultraestrutura , Processamento de Proteína Pós-Traducional , Proteínas Proto-Oncogênicas p21(ras)/química , Animais , Sítios de Ligação , DNA/genética , DNA/metabolismo , Genoma Humano , Histonas/genética , Histonas/metabolismo , Humanos , Simulação de Dinâmica Molecular , Conformação de Ácido Nucleico , Nucleossomos/genética , Nucleossomos/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Eletricidade Estática , Transcrição Genética , Xenopus laevis
6.
Cells ; 10(9)2021 08 29.
Artigo em Inglês | MEDLINE | ID: mdl-34571888

RESUMO

The compact nucleosomal structure limits DNA accessibility and regulates DNA-dependent cellular activities. Linker histones bind to nucleosomes and compact nucleosomal arrays into a higher-order chromatin structure. Recent developments in high throughput technologies and structural computational studies provide nucleosome positioning at a high resolution and contribute to the information of linker histone location within a chromatosome. However, the precise linker histone location within the chromatin fibre remains unclear. Using monomer extension, we mapped core particle and chromatosomal positions over a core histone-reconstituted, 1.5 kb stretch of DNA from the chicken adult ß-globin gene, after titration with linker histones and linker histone globular domains. Our results show that, although linker histone globular domains and linker histones display a wide variation in their binding affinity for different positioned nucleosomes, they do not alter nucleosome positions or generate new nucleosome positions. Furthermore, the extra ~20 bp of DNA protected in a chromatosome is usually symmetrically distributed at each end of the core particle, suggesting linker histones or linker histone globular domains are located close to the nucleosomal dyad axis.


Assuntos
Montagem e Desmontagem da Cromatina , DNA/metabolismo , Histonas/metabolismo , Nuclease do Micrococo/metabolismo , Nucleossomos/metabolismo , Globinas beta/metabolismo , Animais , Sítios de Ligação , Galinhas , DNA/genética , Histonas/genética , Hidrólise , Conformação de Ácido Nucleico , Nucleossomos/genética , Globinas beta/genética
7.
Science ; 373(6552): 306-315, 2021 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-34437148

RESUMO

Mammalian SWI/SNF (mSWI/SNF) adenosine triphosphate-dependent chromatin remodelers modulate genomic architecture and gene expression and are frequently mutated in disease. However, the specific chromatin features that govern their nucleosome binding and remodeling activities remain unknown. We subjected endogenously purified mSWI/SNF complexes and their constituent assembly modules to a diverse library of DNA-barcoded mononucleosomes, performing more than 25,000 binding and remodeling measurements. Here, we define histone modification-, variant-, and mutation-specific effects, alone and in combination, on mSWI/SNF activities and chromatin interactions. Further, we identify the combinatorial contributions of complex module components, reader domains, and nucleosome engagement properties to the localization of complexes to selectively permissive chromatin states. These findings uncover principles that shape the genomic binding and activity of a major chromatin remodeler complex family.


Assuntos
Montagem e Desmontagem da Cromatina , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Nucleossomos/metabolismo , Fatores de Transcrição/metabolismo , Adenosina Trifosfatases/química , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Proteínas Cromossômicas não Histona/química , Código das Histonas , Histonas/química , Histonas/metabolismo , Humanos , Modelos Moleculares , Complexos Multiproteicos/metabolismo , Mutação , Nucleossomos/química , Ligação Proteica , Domínios Proteicos , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Fatores de Transcrição/química
8.
Methods Mol Biol ; 2351: 93-104, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34382185

RESUMO

MNase-Seq is a genome-wide procedure that allows mapping of DNA associated to nucleosomes following micrococcal nuclease digestion. It is a rapid and robust technology useful for the analysis of chromatin properties genome-wide at the resolution of mono-nucleosomes. Here, we describe how to produce high-resolution nucleosome maps of cells grown in suspension or adherent mammalian cells. After only three steps: nuclei or cell preparation, native MNase digestion and DNA purification, libraries for high-throughput sequencing can be prepared. Genome-wide nucleosome maps allow analyzing chromatin opening at promoters or enhancers, nucleosome displacement, or labile nucleosome occupancy depending on the digestion condition used. As presented, MNase-Seq is a versatile tool for investigating chromatin dynamics, regulation, and to define open chromatin regions of regulatory elements in mammalian genomes.


Assuntos
Elementos Facilitadores Genéticos , Estudo de Associação Genômica Ampla/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Nucleossomos/metabolismo , Regiões Promotoras Genéticas , Animais , Células Cultivadas , Cromatina/genética , Cromatina/metabolismo , Mapeamento Cromossômico , Biologia Computacional/métodos , Biblioteca Gênica
9.
Methods Mol Biol ; 2351: 105-121, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34382186

RESUMO

Assay for Transposase-Accessible Chromatin using sequencing (ATAC-Seq) is a method to investigate the accessibility of chromatin in a genome-wide fashion. In this chapter, we provide a brief history of the chromatin accessibility field followed by a detailed protocol to perform ATAC-Seq assay.


Assuntos
Montagem e Desmontagem da Cromatina , Cromatina/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Biologia Computacional/métodos , Elementos de DNA Transponíveis , Análise de Dados , Regulação da Expressão Gênica , Biblioteca Gênica , Estudo de Associação Genômica Ampla , Humanos , Nucleossomos/metabolismo , Controle de Qualidade , Análise de Sequência de DNA , Transposases/metabolismo
10.
Methods Mol Biol ; 2351: 123-145, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34382187

RESUMO

The positioning of nucleosomes regulates the accessibility of genomic DNA and can impact the activities of functional elements. Nucleosome positioning is highly consistent at each genomic location in any particular cell-type, but can vary in an orchestrated fashion between different cell-types and between genomic loci according to their activities. Here, we describe a technique-"ChIP-MNase" (chromatin immunoprecipitation linked to micrococcal nuclease mapping)-to determine nucleosome positions at chosen sets of genomic features that can be defined by their molecular composition and recovered by chromatin immunoprecipitation. ChIP-MNase enables high-resolution analysis of nucleosome positioning at genomic regions-of-interest and can allow differential analysis of alleles undergoing distinct molecular processes.


Assuntos
Alelos , Sequenciamento de Cromatina por Imunoprecipitação/métodos , Imunoprecipitação da Cromatina/métodos , Mapeamento Cromossômico/métodos , Loci Gênicos , Nuclease do Micrococo/metabolismo , Nucleossomos/metabolismo , Sítios de Ligação , Células Cultivadas , Cromatina/genética , Cromatina/metabolismo , Montagem e Desmontagem da Cromatina , Ligação Proteica , Controle de Qualidade
11.
Methods Mol Biol ; 2351: 147-161, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34382188

RESUMO

Sequential ChIP (ChIP-reChIP) enables the characterization of the same nucleosome for two different types of modifications or histone subtypes. Here, we describe a ChIP-reChIP protocol to identify a heterotypic (asymmetric) H2A.Z-H2A-containing nucleosome. In this method, following MNase digestion of chromatin to mostly a mononucleosome fraction, H2A.Z-containing nucleosomes are first immunoprecipitated using affinity purified anti-H2A.Z antibodies. This H2A.Z-containing nucleosome fraction is then subsequently immunoprecipitated using anti-H2A affinity purified antibodies to yield an enriched population of heterotypic H2A.Z-H2A containing nucleosomes. This protocol can be adopted to investigate any pair-wise combination of any histone variant, histone posttranslational modification, or any other protein that binds to a modified nucleosome.


Assuntos
Montagem e Desmontagem da Cromatina , Imunoprecipitação da Cromatina/métodos , Cromatina/genética , Cromatina/metabolismo , Nucleossomos/metabolismo , Sítios de Ligação , Histonas/metabolismo , Ligação Proteica
12.
Methods Mol Biol ; 2351: 251-274, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34382194

RESUMO

In this chapter, we describe the proteomic approach named "Native Chromatin Proteomics" (N-ChroP) that couples a modified Chromatin ImmunoPrecipitation (ChIP) protocol with the mass spectrometry (MS) analysis of immunoprecipitated proteins to study the combinatorial enrichment or exclusion of histone post-translational modifications (PTMs) at specific genomic regions, such as promoters or enhancers. We describe the protocol steps from the digestion of chromatin and nucleosome immunoprecipitation to histone digestion and peptide enrichment prior to MS analysis, up to the MS raw data analysis. We also discuss current challenges and offer suggestions based on the direct hands-on experience acquired during the method setup.


Assuntos
Imunoprecipitação da Cromatina/métodos , Cromatina/genética , Cromatina/metabolismo , Genômica , Histonas/metabolismo , Processamento de Proteína Pós-Traducional , Proteômica , Cromatografia Líquida , Proteínas de Ligação a DNA/metabolismo , Análise de Dados , Genômica/métodos , Nucleossomos/metabolismo , Proteômica/métodos , Espectrometria de Massas em Tandem
13.
Nat Cell Biol ; 23(8): 834-845, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34354236

RESUMO

Pioneer transcription factors such as OCT4 can target silent genes embedded in nucleosome-dense regions. How nucleosome interaction enables transcription factors to target chromatin and determine cell identity remains elusive. Here, we systematically dissect OCT4 to show that nucleosome binding is encoded within the DNA-binding domain and yet can be uncoupled from free-DNA binding. Furthermore, accelerating the binding kinetics of OCT4 to DNA enhances nucleosome binding. In cells, uncoupling nucleosome binding diminishes the ability of OCT4 to individually access closed chromatin, while more dynamic nucleosome binding results in expansive genome scanning within closed chromatin. However, both uncoupling and enhancing nucleosome binding are detrimental to inducing pluripotency from differentiated cells. Remarkably, stable interactions between OCT4 and nucleosomes are continuously required for maintaining the accessibility of pluripotency enhancers in stem cells. Our findings reveal how the affinity and residence time of OCT4-nucleosome complexes modulate chromatin accessibility during cell fate changes and maintenance.


Assuntos
Nucleossomos/metabolismo , Fator 3 de Transcrição de Octâmero/fisiologia , Células-Tronco Pluripotentes/fisiologia , Animais , Sítios de Ligação/genética , Cromatina/metabolismo , Elementos Facilitadores Genéticos , Feminino , Fibroblastos , Biblioteca Gênica , Humanos , Camundongos , Modelos Moleculares , Mutação , Fator 3 de Transcrição de Octâmero/genética , Ligação Proteica , Fatores de Transcrição SOXB1/metabolismo
14.
DNA Repair (Amst) ; 106: 103193, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34339948

RESUMO

The nonhomologous DNA end joining pathway is required for repair of most double-strand breaks in the mammalian genome. Here we use a purified biochemical NHEJ system to compare the joining of free DNA with recombinant mononucleosomal and dinucleosomal substrates to investigate ligation and local DNA end resection. We find that the nucleosomal state permits ligation in a manner dependent on the presence of free DNA flanking the nucleosome core particle. Local resection at DNA ends by the Artemis:DNA-PKcs nuclease complex is completely suppressed in all mononucleosome substrates regardless of flanking DNA up to a length of 14 bp. Like mononucleosomes, dinucleosomes lacking flanking free DNA are not joined. Therefore, the nucleosomal state imposes severe constraints on NHEJ nuclease and ligase activities.


Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades , Proteína Quinase Ativada por DNA/metabolismo , Nucleossomos/metabolismo , Animais , Linhagem Celular , DNA/metabolismo , DNA Ligases/metabolismo , Células HeLa , Humanos , Spodoptera/metabolismo , Xenopus/metabolismo
15.
Int J Mol Sci ; 22(15)2021 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-34360997

RESUMO

Poly(dA:dT) tracts cause nucleosome depletion in many species, e.g., at promoters and replication origins. Their intrinsic biophysical sequence properties make them stiff and unfavorable for nucleosome assembly, as probed by in vitro nucleosome reconstitution. The mere correlation between nucleosome depletion over poly(dA:dT) tracts in in vitro reconstituted and in in vivo chromatin inspired an intrinsic nucleosome exclusion mechanism in vivo that is based only on DNA and histone properties. However, we compile here published and new evidence that this correlation does not reflect mechanistic causation. (1) Nucleosome depletion over poly(dA:dT) in vivo is not universal, e.g., very weak in S. pombe. (2) The energy penalty for incorporating poly(dA:dT) tracts into nucleosomes is modest (<10%) relative to ATP hydrolysis energy abundantly invested by chromatin remodelers. (3) Nucleosome depletion over poly(dA:dT) is much stronger in vivo than in vitro if monitored without MNase and (4) actively maintained in vivo. (5) S. cerevisiae promoters evolved a strand-biased poly(dA) versus poly(dT) distribution. (6) Nucleosome depletion over poly(dA) is directional in vivo. (7) The ATP dependent chromatin remodeler RSC preferentially and directionally displaces nucleosomes towards 5' of poly(dA). Especially distribution strand bias and displacement directionality would not be expected for an intrinsic mechanism. Together, this argues for an in vivo mechanism where active and species-specific read out of intrinsic sequence properties, e.g., by remodelers, shapes nucleosome organization.


Assuntos
Sequência Rica em At , Montagem e Desmontagem da Cromatina , Nucleossomos/genética , Regulação Fúngica da Expressão Gênica , Nucleossomos/química , Nucleossomos/metabolismo , Saccharomyces cerevisiae , Schizosaccharomyces
16.
Nat Commun ; 12(1): 4800, 2021 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-34417450

RESUMO

Histone lysine methylations have primarily been linked to selective recruitment of reader or effector proteins that subsequently modify chromatin regions and mediate genome functions. Here, we describe a divergent role for histone H4 lysine 20 mono-methylation (H4K20me1) and demonstrate that it directly facilitates chromatin openness and accessibility by disrupting chromatin folding. Thus, accumulation of H4K20me1 demarcates highly accessible chromatin at genes, and this is maintained throughout the cell cycle. In vitro, H4K20me1-containing nucleosomal arrays with nucleosome repeat lengths (NRL) of 187 and 197 are less compact than unmethylated (H4K20me0) or trimethylated (H4K20me3) arrays. Concordantly, and in contrast to trimethylated and unmethylated tails, solid-state NMR data shows that H4K20 mono-methylation changes the H4 conformational state and leads to more dynamic histone H4-tails. Notably, the increased chromatin accessibility mediated by H4K20me1 facilitates gene expression, particularly of housekeeping genes. Altogether, we show how the methylation state of a single histone H4 residue operates as a focal point in chromatin structure control. While H4K20me1 directly promotes chromatin openness at highly transcribed genes, it also serves as a stepping-stone for H4K20me3-dependent chromatin compaction.


Assuntos
Cromatina/metabolismo , Genes Essenciais , Histonas/metabolismo , Lisina/metabolismo , Transcrição Genética , Sequência de Aminoácidos , Animais , Ciclo Celular/genética , Linhagem Celular , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/química , Humanos , Espectroscopia de Ressonância Magnética , Metilação , Camundongos , Modelos Biológicos , Nucleossomos/metabolismo , Conformação Proteica
17.
Nat Commun ; 12(1): 5123, 2021 08 26.
Artigo em Inglês | MEDLINE | ID: mdl-34446700

RESUMO

Understanding the molecular underpinnings of pluripotency is a prerequisite for optimal maintenance and application of embryonic stem cells (ESCs). While the protein-protein interactions of core pluripotency factors have been identified in mouse ESCs, their interactome in human ESCs (hESCs) has not to date been explored. Here we mapped the OCT4 interactomes in naïve and primed hESCs, revealing extensive connections to mammalian ATP-dependent nucleosome remodeling complexes. In naïve hESCs, OCT4 is associated with both BRG1 and BRM, the two paralog ATPases of the BAF complex. Genome-wide location analyses and genetic studies reveal that these two enzymes cooperate in a functionally redundant manner in the transcriptional regulation of blastocyst-specific genes. In contrast, in primed hESCs, OCT4 cooperates with BRG1 and SOX2 to promote chromatin accessibility at ectodermal genes. This work reveals how a common transcription factor utilizes differential BAF complexes to control distinct transcriptional programs in naïve and primed hESCs.


Assuntos
Trifosfato de Adenosina/metabolismo , Cromatina/metabolismo , DNA Helicases/metabolismo , Células-Tronco Embrionárias/metabolismo , Proteínas Nucleares/metabolismo , Fator 3 de Transcrição de Octâmero/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Fatores de Transcrição/metabolismo , Cromatina/genética , Montagem e Desmontagem da Cromatina , DNA Helicases/genética , Regulação da Expressão Gênica , Humanos , Proteínas Nucleares/genética , Nucleossomos/genética , Nucleossomos/metabolismo , Fator 3 de Transcrição de Octâmero/genética , Ligação Proteica , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição/genética
18.
Nat Commun ; 12(1): 5136, 2021 08 26.
Artigo em Inglês | MEDLINE | ID: mdl-34446717

RESUMO

One fundamental yet unresolved question in biology remains how cells interpret the same signalling cues in a context-dependent manner resulting in lineage specification. A key step for decoding signalling cues is the establishment of a permissive chromatin environment at lineage-specific genes triggering transcriptional responses to inductive signals. For instance, bipotent neuromesodermal progenitors (NMPs) are equipped with a WNT-decoding module, which relies on TCFs/LEF activity to sustain both NMP expansion and paraxial mesoderm differentiation. However, how WNT signalling activates lineage specific genes in a temporal manner remains unclear. Here, we demonstrate that paraxial mesoderm induction relies on the TALE/HOX combinatorial activity that simultaneously represses NMP genes and activates the differentiation program. We identify the BRACHYURY-TALE/HOX code that destabilizes the nucleosomes at WNT-responsive regions and establishes the permissive chromatin landscape for de novo recruitment of the WNT-effector LEF1, unlocking the WNT-mediated transcriptional program that drives NMPs towards the paraxial mesodermal fate.


Assuntos
Proteínas Fetais/metabolismo , Mesoderma/metabolismo , Família Multigênica , Células-Tronco Neurais/metabolismo , Proteínas com Domínio T/metabolismo , Via de Sinalização Wnt , Animais , Diferenciação Celular , Linhagem da Célula , Proteínas Fetais/genética , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Mesoderma/embriologia , Camundongos , Camundongos Knockout , Células-Tronco Neurais/citologia , Nucleossomos/genética , Nucleossomos/metabolismo , Proteínas com Domínio T/genética
19.
Sci Rep ; 11(1): 16963, 2021 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-34417521

RESUMO

Suppressor of Hairy wing [Su(Hw)] is an insulator protein that participates in regulating chromatin architecture and gene repression in Drosophila. In previous studies we have shown that Su(Hw) is also required for pre-replication complex (pre-RC) recruitment on Su(Hw)-bound sites (SBSs) in Drosophila S2 cells and pupa. Here, we describe the effect of Su(Hw) on developmentally regulated amplification of 66D and 7F Drosophila amplicons in follicle cells (DAFCs), widely used as models in replication studies. We show Su(Hw) binding co-localizes with all known DAFCs in Drosophila ovaries, whereas disruption of Su(Hw) binding to 66D and 7F DAFCs causes a two-fold decrease in the amplification of these loci. The complete loss of Su(Hw) binding to chromatin impairs pre-RC recruitment to all amplification regulatory regions of 66D and 7F loci at early oogenesis (prior to DAFCs amplification). These changes coincide with a considerable Su(Hw)-dependent condensation of chromatin at 66D and 7F loci. Although we observed the Brm, ISWI, Mi-2, and CHD1 chromatin remodelers at SBSs genome wide, their remodeler activity does not appear to be responsible for chromatin decondensation at the 66D and 7F amplification regulatory regions. We have discovered that, in addition to the CBP/Nejire and Chameau histone acetyltransferases, the Gcn5 acetyltransferase binds to 66D and 7F DAFCs at SBSs and this binding is dependent on Su(Hw). We propose that the main function of Su(Hw) in developmental amplification of 66D and 7F DAFCs is to establish a chromatin structure that is permissive to pre-RC recruitment.


Assuntos
Córion/metabolismo , Cromatina/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Amplificação de Genes , Loci Gênicos , Proteínas Repressoras/genética , Animais , Montagem e Desmontagem da Cromatina/genética , Análise por Conglomerados , Replicação do DNA/genética , Proteínas de Drosophila/metabolismo , Feminino , Modelos Biológicos , Mutação/genética , Nucleossomos/metabolismo , Oogênese/genética , Folículo Ovariano/metabolismo , Ligação Proteica
20.
Nucleic Acids Res ; 49(16): 9053-9065, 2021 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-34352103

RESUMO

Centromeres are essential for chromosome movement. In independent taxa, species with holocentric chromosomes exist. In contrast to monocentric species, where no obvious dispersion of centromeres occurs during interphase, the organization of holocentromeres differs between condensed and decondensed chromosomes. During interphase, centromeres are dispersed into a large number of CENH3-positive nucleosome clusters in a number of holocentric species. With the onset of chromosome condensation, the centromeric nucleosomes join and form line-like holocentromeres. Using polymer simulations, we propose a mechanism relying on the interaction between centromeric nucleosomes and structural maintenance of chromosomes (SMC) proteins. Different sets of molecular dynamic simulations were evaluated by testing four parameters: (i) the concentration of Loop Extruders (LEs) corresponding to SMCs, (ii) the distribution and number of centromeric nucleosomes, (iii) the effect of centromeric nucleosomes on interacting LEs and (iv) the assembly of kinetochores bound to centromeric nucleosomes. We observed the formation of a line-like holocentromere, due to the aggregation of the centromeric nucleosomes when the chromosome was compacted into loops. A groove-like holocentromere structure formed after a kinetochore complex was simulated along the centromeric line. Similar mechanisms may also organize a monocentric chromosome constriction, and its regulation may cause different centromere types during evolution.


Assuntos
Ciclo Celular , Centrômero/metabolismo , Nucleossomos/química , Animais , Caenorhabditis elegans , Centrômero/química , Montagem e Desmontagem da Cromatina , Simulação por Computador , DNA/química , DNA/metabolismo , Histonas/química , Histonas/metabolismo , Nucleossomos/metabolismo
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