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1.
Nucleic Acids Res ; 49(15): 8961-8973, 2021 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-34365506

RESUMO

Histone recognition constitutes a key epigenetic mechanism in gene regulation and cell fate decision. PHF14 is a conserved multi-PHD finger protein that has been implicated in organ development, tissue homeostasis, and tumorigenesis. Here we show that PHF14 reads unmodified histone H3(1-34) through an integrated PHD1-ZnK-PHD2 cassette (PHF14PZP). Our binding, structural and HDX-MS analyses revealed a feature of bipartite recognition, in which PHF14PZP utilizes two distinct surfaces for concurrent yet separable engagement of segments H3-Nter (e.g. 1-15) and H3-middle (e.g. 14-34) of H3(1-34). Structural studies revealed a novel histone H3 binding mode by PHD1 of PHF14PZP, in which a PHF14-unique insertion loop but not the core ß-strands of a PHD finger dominates H3K4 readout. Binding studies showed that H3-PHF14PZP engagement is sensitive to modifications occurring to H3 R2, T3, K4, R8 and K23 but not K9 and K27, suggesting multiple layers of modification switch. Collectively, our work calls attention to PHF14 as a 'ground' state (unmodified) H3(1-34) reader that can be negatively regulated by active marks, thus providing molecular insights into a repressive function of PHF14 and its derepression.


Assuntos
Histonas/química , Histonas/metabolismo , Proteínas de Peixe-Zebra/química , Proteínas de Peixe-Zebra/metabolismo , Regulação Alostérica , Animais , Cristalografia por Raios X , Humanos , Modelos Moleculares , Mutagênese , Proteínas Nucleares/química , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Fatores de Transcrição/química , Proteínas de Peixe-Zebra/genética
2.
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
3.
Int J Mol Sci ; 22(16)2021 Aug 11.
Artigo em Inglês | MEDLINE | ID: mdl-34445348

RESUMO

The periodontal ligament is a soft connective tissue embedded between the alveolar bone and cementum, the surface hard tissue of teeth. Periodontal ligament fibroblasts (PDLF) actively express osteo/cementogenic genes, which contribute to periodontal tissue homeostasis. However, the key factors maintaining the osteo/cementogenic abilities of PDLF remain unclear. We herein demonstrated that PPARγ was expressed by in vivo periodontal ligament tissue and its distribution pattern correlated with alkaline phosphate enzyme activity. The knockdown of PPARγ markedly reduced the osteo/cementogenic abilities of PDLF in vitro, whereas PPARγ agonists exerted the opposite effects. PPARγ was required to maintain the acetylation status of H3K9 and H3K27, active chromatin markers, and the supplementation of acetyl-CoA, a donor of histone acetylation, restored PPARγ knockdown-induced decreases in the osteo/cementogenic abilities of PDLF. An RNA-seq/ChIP-seq combined analysis identified four osteogenic transcripts, RUNX2, SULF2, RCAN2, and RGMA, in the PPARγ-dependent active chromatin region marked by H3K27ac. Furthermore, RUNX2-binding sites were selectively enriched in the PPARγ-dependent active chromatin region. Collectively, these results identified PPARγ as the key transcriptional factor maintaining the osteo/cementogenic abilities of PDLF and revealed that global H3K27ac modifications play a role in the comprehensive osteo/cementogenic transcriptional alterations mediated by PPARγ.


Assuntos
Fibroblastos/fisiologia , Histonas/metabolismo , PPAR gama/fisiologia , Ligamento Periodontal/fisiologia , Acetilação , Diferenciação Celular/genética , Células Cultivadas , Cementogênese/genética , Cementogênese/fisiologia , Regulação da Expressão Gênica , Histona Acetiltransferases/metabolismo , Histonas/química , Humanos , Osteogênese/genética , Osteogênese/fisiologia , Ligamento Periodontal/citologia , Processamento de Proteína Pós-Traducional/genética
4.
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
5.
Nat Commun ; 12(1): 3497, 2021 06 09.
Artigo em Inglês | MEDLINE | ID: mdl-34108453

RESUMO

Decoding the role of histone posttranslational modifications (PTMs) is key to understand the fundamental process of epigenetic regulation. This is well studied for PTMs of core histones but not for linker histone H1 in general and its ubiquitylation in particular due to a lack of proper tools. Here, we report on the chemical synthesis of site-specifically mono-ubiquitylated H1.2 and identify its ubiquitin-dependent interactome on a proteome-wide scale. We show that site-specific ubiquitylation of H1 at position K64 modulates interactions with deubiquitylating enzymes and the deacetylase SIRT1. Moreover, it affects H1-dependent chromatosome assembly and phase separation resulting in a more open chromatosome conformation generally associated with a transcriptionally active chromatin state. In summary, we propose that site-specific ubiquitylation plays a general regulatory role for linker histone H1.


Assuntos
Histonas/metabolismo , Ubiquitinação/fisiologia , Cromatina/química , Cromatina/metabolismo , Enzimas Desubiquitinantes/metabolismo , Epigênese Genética , Histonas/química , Humanos , Nucleossomos/química , Nucleossomos/metabolismo , Ligação Proteica , Mapas de Interação de Proteínas , Sirtuína 1/metabolismo , Ubiquitina/química , Ubiquitina/metabolismo
6.
J Phys Chem Lett ; 12(25): 6014-6019, 2021 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-34165307

RESUMO

Apurinic/apyrimidinic sites are the most common forms of DNA damage under physiological conditions, yet their structural and dynamical behavior within nucleosome core particles has just begun to be investigated and is dramatically different from that of abasic sites in B-DNA. Clusters of two or more abasic sites are repaired even less efficiently and hence constitute hot spots of high mutagenicity notably due to enhanced double-strand break formation. On the basis of an X-ray structure of a 146 bp DNA wrapped onto a histone core, we investigate the structural behavior of two bistranded abasic sites positioned at mutational hot spots during microsecond-range molecular dynamics simulations. Our simulations allow us to probe interactions of histone tails at clustered abasic site locations, with a definitive assignment of the key residues involved in the NCP-catalyzed formation of DNA-protein cross-linking in line with recent experimental findings, and pave the way for a systematic assessment of the response of histone tails to DNA lesions.


Assuntos
Histonas/química , Nucleossomos/metabolismo , Histonas/genética , Histonas/metabolismo , Modelos Moleculares , Mutagênese , Mutação , Nucleossomos/genética , Conformação Proteica
7.
J Phys Chem Lett ; 12(26): 6174-6181, 2021 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-34184895

RESUMO

Chromatin is a supramolecular DNA-protein complex that compacts eukaryotic genomes and regulates their accessibility and functions. Dynamically disordered histone H3 N-terminal tails are among key chromatin regulatory components. Here, we used high-resolution-magic-angle-spinning NMR measurements of backbone amide 15N spin relaxation rates to investigate, with residue-specific detail, the dynamics and interactions of H3 tails in recombinant 13C,15N-enriched nucleosome arrays containing 15, 30, or 60 bp linker DNA between the nucleosome repeats. These measurements were compared to analogous data available for mononucleosomes devoid of linker DNA or containing two 20 bp DNA overhangs. The H3 tail dynamics in nucleosome arrays were found to be considerably attenuated compared with nucleosomes with or without linker DNA due to transient electrostatic interactions with the linker DNA segments and the structured chromatin environment. Remarkably, however, the H3 tail dynamics were not modulated by the specific linker DNA length within the 15-60 bp range investigated here.


Assuntos
Cromatina/química , Histonas/química , Cromatina/metabolismo , DNA/metabolismo , Histonas/metabolismo , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Conformação Proteica , Eletricidade Estática
8.
Nucleic Acids Res ; 49(13): 7347-7360, 2021 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-34165567

RESUMO

Lysine 2-hydroxyisobutyrylation (Khib) is a novel type of histone acylation whose prevalence and function in plants remain unclear. Here, we identified 41 Khib sites on histones in Arabidopsis thaliana, which did not overlap with frequently modified N-tail lysines (e.g. H3K4, H3K9 and H4K8). Chromatin immunoprecipitation-sequencing (ChIP-seq) assays revealed histone Khib in 35% of protein-coding genes. Most Khib peaks were located in genic regions, and they were highly enriched at the transcription start sites. Histone Khib is highly correlated with acetylation (ac), particularly H3K23ac, which it largely resembles in its genomic and genic distribution. Notably, co-enrichment of histone Khib and H3K23ac correlates with high gene expression levels. Metabolic profiling, transcriptome analyses, and ChIP-qPCR revealed that histone Khib and H3K23ac are co-enriched on genes involved in starch and sucrose metabolism, pentose and glucuronate interconversions, and phenylpropanoid biosynthesis, and help fine-tune plant response to dark-induced starvation. These findings suggest that Khib and H3K23ac may act in concert to promote high levels of gene transcription and regulate cellular metabolism to facilitate plant adaption to stress. Finally, HDA6 and HDA9 are involved in removing histone Khib. Our findings reveal Khib as a conserved yet unique plant histone mark acting with lysine acetylation in transcription-associated epigenomic processes.


Assuntos
Arabidopsis/genética , Arabidopsis/metabolismo , Epigênese Genética , Código das Histonas , Histonas/metabolismo , Lisina/metabolismo , Acetilação , Proteínas de Arabidopsis/fisiologia , Escuridão , Regulação da Expressão Gênica de Plantas , Histona Desacetilases/fisiologia , Histonas/química , Redes e Vias Metabólicas/genética
9.
Methods Mol Biol ; 2329: 71-80, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34085216

RESUMO

This chapter describes a method used to analyze the behavior of histone modifications in S phase in Arabidopsis using a whole-mount immunostaining technique. Previous studies have demonstrated that dramatic changes in local chromatin structure are required for the initiation and progression of DNA replication, and that histone modifications play an essential role in the determination of chromatin structure in S phase. Since euchromatic and heterochromatic regions are replicated in distinct S-phase stages, it is important to identify histone modifications at each stage. Here, we introduce a protocol for whole-mount immunostaining combined with 5-ethynyl-2'-deoxyuridine (EdU) staining, which enables the visualization of spatial patterns in histone modifications in the early and late S-phase nuclei of Arabidopsis roots.


Assuntos
Arabidopsis/fisiologia , Cromatina/metabolismo , Desoxiuridina/análogos & derivados , Histonas/metabolismo , Proteínas de Arabidopsis/metabolismo , Desoxiuridina/química , Epigênese Genética , Código das Histonas , Histonas/química , Imuno-Histoquímica , Microscopia Confocal , Raízes de Plantas/fisiologia , Fase S
10.
Methods Mol Biol ; 2329: 237-247, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34085227

RESUMO

Posttranslational histone modifications are critical for the regulation of genome function. The levels of histone modifications oscillate during the cell cycle. Most modifications are diluted after DNA replication and then their levels are restored during the rest of the cell cycle with different kinetics depending on the modification. Some modifications, like histone H4 Lys20 monomethylation (H4K20me1), exhibit cell cycle-dependent dynamic changes. To track histone modifications in living cells, we have developed genetically encoded probes termed modification specific intracellular antibodies, or "mintbodies." As mintbodies shuttle between the cytoplasm and nucleus by diffusion, their nuclear concentration depends on the target modification level. By measuring the nuclear to cytoplasmic intensity ratio of H4K20me1-specific mintbody, we have monitored the increase of H4K20me1 in the G2 phase. Here we describe how the mintbody-based methods can be applied to track a specific chromosome, such as the inactive X chromosome (Xi), on which genes are repressed through histone H3 Lys27 trimethylation (H3K27me3). When H3K27me3-specific mintbodies are expressed in cells that harbor Xi, the mintbodies are concentrated on Xi and the dynamic behavior of Xi can be tracked using a confocal microscope. After acquiring 3D time-lapse images, an image analysis allows measuring the volume, shape and H3K27me3 level of Xi during the cell cycle.


Assuntos
Corantes Fluorescentes/química , Histonas/metabolismo , Inativação do Cromossomo X , Cromossomo X/genética , Animais , Ciclo Celular , Linhagem Celular Tumoral , Células HeLa , Código das Histonas , Histonas/química , Humanos , Metilação , Camundongos , Microscopia Confocal , Imagem com Lapso de Tempo , Cromossomo X/química
11.
Phys Rev Lett ; 126(22): 228101, 2021 Jun 04.
Artigo em Inglês | MEDLINE | ID: mdl-34152157

RESUMO

Cell differentiation, the process by which stem cells become specialized cells, is associated with chromatin reorganization inside the cell nucleus. Here, we measure the chromatin distribution and dynamics in embryonic stem cells in vivo before and after differentiation. We find that undifferentiated chromatin is less compact, more homogeneous, and more dynamic than differentiated chromatin. Furthermore, we present a noninvasive rheological analysis using intrinsic chromatin dynamics, which reveals that undifferentiated chromatin behaves like a Maxwell fluid, while differentiated chromatin shows a coexistence of fluidlike (sol) and solidlike (gel) phases. Our data suggest that chromatin undergoes a local sol-gel transition upon cell differentiation, corresponding to the formation of the more dense and transcriptionally inactive heterochromatin.


Assuntos
Células-Tronco Embrionárias/química , Células-Tronco Embrionárias/citologia , Modelos Biológicos , Diferenciação Celular/fisiologia , Cromatina/química , Cromatina/metabolismo , Células-Tronco Embrionárias/metabolismo , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/metabolismo , Histonas/química , Histonas/metabolismo , Humanos , Interfase , Transição de Fase , Reologia
12.
J Mol Biol ; 433(15): 167110, 2021 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-34153285

RESUMO

The nucleosome comprises two histone dimers of H2A-H2B and one histone tetramer of (H3-H4)2, wrapped around by ~145 bp of DNA. Detailed core structures of nucleosomes have been established by X-ray and cryo-EM, however, histone tails have not been visualized. Here, we have examined the dynamic structures of the H2A and H2B tails in 145-bp and 193-bp nucleosomes using NMR, and have compared them with those of the H2A and H2B tail peptides unbound and bound to DNA. Whereas the H2A C-tail adopts a single but different conformation in both nucleosomes, the N-tails of H2A and H2B adopt two distinct conformations in each nucleosome. To clarify these conformations, we conducted molecular dynamics (MD) simulations, which suggest that the H2A N-tail can locate stably in either the major or minor grooves of nucleosomal DNA. While the H2B N-tail, which sticks out between two DNA gyres in the nucleosome, was considered to adopt two different orientations, one toward the entry/exit side and one on the opposite side. Then, the H2A N-tail minor groove conformation was obtained in the H2B opposite side and the H2B N-tail interacts with DNA similarly in both sides, though more varied conformations are obtained in the entry/exit side. Collectively, the NMR findings and MD simulations suggest that the minor groove conformer of the H2A N-tail is likely to contact DNA more strongly than the major groove conformer, and the H2A N-tail reduces contact with DNA in the major groove when the H2B N-tail is located in the entry/exit side.


Assuntos
DNA/metabolismo , Histonas/química , Histonas/metabolismo , Microscopia Crioeletrônica , Cristalografia por Raios X , DNA/genética , Humanos , Simulação de Dinâmica Molecular , Nucleossomos/metabolismo , Conformação Proteica
13.
ACS Appl Mater Interfaces ; 13(23): 26712-26720, 2021 Jun 16.
Artigo em Inglês | MEDLINE | ID: mdl-34082523

RESUMO

Delivering hydrophobic molecules through the intestine can be challenging due to limited cargo solubility and the harsh biochemical environment of the stomach. Here, we show that a protein-based nanocarrier system based on the abundant protein histone and the natural cross-linker genipin can deliver hydrophobic cargos, such as dyes and therapeutic molecules, through the gastrointestinal tract. Using hydrophobic near-infrared dyes as model cargos, a panel of potential protein carriers was screened, and histone was identified as the one with the best loading capability. The resulting nanoparticles had a positive ζ potential and were mucoadhesive. Cross-linking of the amine-rich nanocarrier with genipin was particularly effective relative to other proteins and increased the stability of the system during incubation with pepsin. Cross-linking was required for successful delivery of a hydrophobic dye to the colon of mice after oral gavage. To assess the platform for therapeutic delivery, another hydrophobic model compound, curcumin, was delivered using cross-linked histone nanoparticles in a murine colitis model and significantly alleviated the disease. Taken together, these results demonstrate that histone is a cationic, mucoadhesive, and cross-linkable protein nanocarrier that can be considered for oral delivery.


Assuntos
Colite/tratamento farmacológico , Curcumina/farmacologia , Portadores de Fármacos/química , Histonas/química , Iridoides/química , Nanopartículas/administração & dosagem , Animais , Anti-Inflamatórios não Esteroides/farmacologia , Colite/patologia , Reagentes para Ligações Cruzadas/química , Feminino , Trato Gastrointestinal/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Camundongos , Camundongos Endogâmicos ICR , Nanopartículas/química
14.
Mol Cell ; 81(13): 2765-2777.e6, 2021 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-34102105

RESUMO

The BRCA1-BARD1 complex directs the DNA double-strand break (DSB) repair pathway choice to error-free homologous recombination (HR) during the S-G2 stages. Targeting BRCA1-BARD1 to DSB-proximal sites requires BARD1-mediated nucleosome interaction and histone mark recognition. Here, we report the cryo-EM structure of BARD1 bound to a ubiquitinated nucleosome core particle (NCPUb) at 3.1 Å resolution and illustrate how BARD1 simultaneously recognizes the DNA damage-induced mark H2AK15ub and DNA replication-associated mark H4K20me0 on the nucleosome. In vitro and in vivo analyses reveal that the BARD1-NCPUb complex is stabilized by BARD1-nucleosome interaction, BARD1-ubiquitin interaction, and BARD1 ARD domain-BARD1 BRCT domain interaction, and abrogating these interactions is detrimental to HR activity. We further identify multiple disease-causing BARD1 mutations that disrupt BARD1-NCPUb interactions and hence impair HR. Together, this study elucidates the mechanism of BRCA1-BARD1 complex recruitment and retention by DSB-flanking nucleosomes and sheds important light on cancer therapeutic avenues.


Assuntos
Proteína BRCA1/química , Histonas/química , Complexos Multiproteicos/química , Nucleossomos/química , Proteínas Supressoras de Tumor/química , Ubiquitina-Proteína Ligases/química , Proteínas de Xenopus/química , Animais , Proteína BRCA1/genética , Histonas/genética , Humanos , Modelos Moleculares , Complexos Multiproteicos/genética , Mutação , Nucleossomos/genética , Proteínas Supressoras de Tumor/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitinação , Proteínas de Xenopus/genética , Xenopus laevis
15.
Commun Biol ; 4(1): 571, 2021 05 13.
Artigo em Inglês | MEDLINE | ID: mdl-33986449

RESUMO

Histone H3 lysine 9 dimethylation (H3K9me2) is a highly conserved silencing epigenetic mark. Chromatin marked with H3K9me2 forms large domains in mammalian cells and overlaps well with lamina-associated domains and the B compartment defined by Hi-C. However, the role of H3K9me2 in 3-dimensional (3D) genome organization remains unclear. Here, we investigated genome-wide H3K9me2 distribution, transcriptome, and 3D genome organization in mouse embryonic stem cells following the inhibition or depletion of H3K9 methyltransferases (MTases): G9a, GLP, SETDB1, SUV39H1, and SUV39H2. We show that H3K9me2 is regulated by all five MTases; however, H3K9me2 and transcription in the A and B compartments are regulated by different MTases. H3K9me2 in the A compartments is primarily regulated by G9a/GLP and SETDB1, while H3K9me2 in the B compartments is regulated by all five MTases. Furthermore, decreased H3K9me2 correlates with changes to more active compartmental state that accompanied transcriptional activation. Thus, H3K9me2 contributes to inactive compartment setting.


Assuntos
Cromatina/metabolismo , Metilação de DNA , Fibroblastos/metabolismo , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/metabolismo , Lisina/metabolismo , Células-Tronco Embrionárias Murinas/metabolismo , Animais , Cromatina/química , Cromatina/genética , Fibroblastos/citologia , Genoma , Histona-Lisina N-Metiltransferase/genética , Histonas/química , Histonas/genética , Lisina/química , Lisina/genética , Camundongos , Células-Tronco Embrionárias Murinas/citologia
16.
J Biol Chem ; 297(1): 100844, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34058198

RESUMO

RAD51-associated protein 1 (RAD51AP1) is a key protein in the homologous recombination (HR) DNA repair pathway. Loss of RAD51AP1 leads to defective HR, genome instability, and telomere erosion. RAD51AP1 physically interacts with the RAD51 recombinase and promotes RAD51-mediated capture of donor DNA, synaptic complex assembly, and displacement-loop formation when tested with nucleosome-free DNA substrates. In cells, however, DNA is packaged into chromatin, posing an additional barrier to the complexities of the HR reaction. In this study, we show that RAD51AP1 binds to nucleosome core particles (NCPs), the minimum basic unit of chromatin in which approximately two superhelical turns of 147 bp double-stranded DNA are wrapped around one histone octamer with no free DNA ends remaining. We identified a C-terminal region in RAD51AP1, including its previously mapped DNA-binding domain, as critical for mediating the association between RAD51AP1 and both the NCP and the histone octamer. Using in vitro surrogate assays of HR activity, we show that RAD51AP1 is capable of promoting duplex DNA capture and initiating joint-molecule formation with the NCP and chromatinized template DNA, respectively. Together, our results suggest that RAD51AP1 directly assists in the RAD51-mediated search for donor DNA in chromatin. We present a model, in which RAD51AP1 anchors the DNA template through affinity for its nucleosomes to the RAD51-ssDNA nucleoprotein filament.


Assuntos
Cromatina/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a RNA/genética , Rad51 Recombinase/genética , Reparo de DNA por Recombinação/genética , Cromatina/química , Pareamento Cromossômico/genética , DNA/química , DNA/genética , Proteínas de Ligação a DNA/química , Instabilidade Genômica/genética , Histonas/química , Histonas/genética , Humanos , Nucleossomos/genética , Domínios Proteicos/genética , Proteínas de Ligação a RNA/química , Rad51 Recombinase/química , Telômero/genética
17.
Nat Commun ; 12(1): 2883, 2021 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-34001913

RESUMO

Liquid-liquid phase separation (LLPS) is an important mechanism that helps explain the membraneless compartmentalization of the nucleus. Because chromatin compaction and LLPS are collective phenomena, linking their modulation to the physicochemical features of nucleosomes is challenging. Here, we develop an advanced multiscale chromatin model-integrating atomistic representations, a chemically-specific coarse-grained model, and a minimal model-to resolve individual nucleosomes within sub-Mb chromatin domains and phase-separated systems. To overcome the difficulty of sampling chromatin at high resolution, we devise a transferable enhanced-sampling Debye-length replica-exchange molecular dynamics approach. We find that nucleosome thermal fluctuations become significant at physiological salt concentrations and destabilize the 30-nm fiber. Our simulations show that nucleosome breathing favors stochastic folding of chromatin and promotes LLPS by simultaneously boosting the transient nature and heterogeneity of nucleosome-nucleosome contacts, and the effective nucleosome valency. Our work puts forward the intrinsic plasticity of nucleosomes as a key element in the liquid-like behavior of nucleosomes within chromatin, and the regulation of chromatin LLPS.


Assuntos
Cromatina/metabolismo , DNA/metabolismo , Histonas/metabolismo , Conformação de Ácido Nucleico , Nucleossomos/metabolismo , Algoritmos , Cromatina/química , Cromatina/genética , Simulação por Computador , DNA/química , DNA/genética , Histonas/química , Modelos Genéticos , Simulação de Dinâmica Molecular , Nucleossomos/química , Nucleossomos/genética
18.
Annu Rev Biophys ; 50: 95-116, 2021 05 06.
Artigo em Inglês | MEDLINE | ID: mdl-33957053

RESUMO

In eukaryotes, genomic DNA is packaged into chromatin in the nucleus. The accessibility of DNA is dependent on the chromatin structure and dynamics, which essentially control DNA-related processes, including transcription, DNA replication, and repair. All of the factors that affect the structure and dynamics of nucleosomes, the nucleosome-nucleosome interaction interfaces, and the binding of linker histones or other chromatin-binding proteins need to be considered to understand the organization and function of chromatin fibers. In this review, we provide a summary of recent progress on the structure of chromatin fibers in vitro and in the nucleus, highlight studies on the dynamic regulation of chromatin fibers, and discuss their related biological functions and abnormal organization in diseases.


Assuntos
Cromatina/química , Cromatina/metabolismo , Animais , Replicação do DNA , Histonas/química , Histonas/genética , Histonas/metabolismo , Humanos , Nucleossomos/metabolismo
19.
Nucleic Acids Res ; 49(8): 4750-4767, 2021 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-33856458

RESUMO

Hexasomes and tetrasomes are intermediates in nucleosome assembly and disassembly. Their formation is promoted by histone chaperones, ATP-dependent remodelers, and RNA polymerase II. In addition, hexasomes are maintained in transcribed genes and could be an important regulatory factor. While nucleosome composition has been shown to affect the structure and accessibility of DNA, its influence on histone tails is largely unknown. Here, we investigate the conformational dynamics of the H3 tail in the hexasome and tetrasome. Using a combination of NMR spectroscopy, MD simulations, and trypsin proteolysis, we find that the conformational ensemble of the H3 tail is regulated by nucleosome composition. As has been found for the nucleosome, the H3 tails bind robustly to DNA within the hexasome and tetrasome, but upon loss of the H2A/H2B dimer, we determined that the adjacent H3 tail has an altered conformational ensemble, increase in dynamics, and increase in accessibility. Similar to observations of DNA dynamics, this is seen to be asymmetric in the hexasome. Our results indicate that nucleosome composition has the potential to regulate chromatin signaling and ultimately help shape the chromatin landscape.


Assuntos
Montagem e Desmontagem da Cromatina , Cromatina/metabolismo , DNA/química , Histonas/química , Histonas/metabolismo , Conformação de Ácido Nucleico , Nucleossomos/química , Nucleossomos/metabolismo , Dimerização , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Simulação de Dinâmica Molecular , Análise de Componente Principal , Conformação Proteica , Proteólise , Tripsina/química
20.
J Biol Chem ; 296: 100635, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33823156

RESUMO

The methyltransferases MLL3 and MLL4 primarily catalyze the monomethylation of histone H3 lysine 4 (H3K4) on enhancers to regulate cell-type-specific gene expression and cell fate transition. MLL3 and MLL4 share almost identical binding partners and biochemical activities, but perform specific and nonredundant functions. The features and functions that distinguish MLL3 and MLL4 remain elusive. Here, we characterize the kinetic mechanisms of MLL3 and MLL4 ternary complexes containing the catalytic SET domain from MLL3 or MLL4 (MLL3SET or MLL4SET), the SPRY domain of ASH2L (ASH2LSPRY), and a short fragment of RBBP5 (RBBP5AS-ABM) to search for possible explanations. Steady-state kinetic analyses and inhibition studies reveal that the MLL3 complex catalyzes methylation in a random sequential bi-bi mechanism. In contrast, the MLL4 complex adopts an ordered sequential bi-bi mechanism, in which the cofactor S-adenosylmethionine (AdoMet) binds to the enzyme prior to the H3 peptide, and the methylated H3 peptide dissociates from the enzyme before S-adenosylhomocysteine (AdoHcy) detaches after methylation. Substrate-binding assays using fluorescence polarization (FP) confirm that AdoMet binding is a prerequisite for H3 binding for the MLL4 complex but not for the MLL3 complex. Molecular dynamic simulations reveal that the binding of AdoMet exclusively induces conformational constraints on the AdoMet-binding groove and the H3 substrate-binding pocket of MLL4, therefore stabilizing a specific active conformation to ease entry of the substrate H3. The distinct kinetic mechanisms and conformational plasticities provide important insights into the differential functions of MLL3 and MLL4 and may also guide the development of selective inhibitors targeting MLL3 or MLL4.


Assuntos
Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Histona-Lisina N-Metiltransferase/química , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/metabolismo , Processamento de Proteína Pós-Traducional , Catálise , Proteínas de Ligação a DNA/genética , Histona-Lisina N-Metiltransferase/genética , Histonas/química , Humanos , Cinética , Metilação , Ligação Proteica
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