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1.
Nature ; 586(7827): 151-155, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32968275

RESUMO

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


Assuntos
Microscopia Crioeletrônica , DNA (Citosina-5-)-Metiltransferases/química , DNA (Citosina-5-)-Metiltransferases/metabolismo , Nucleossomos/metabolismo , Animais , Biocatálise , Montagem e Desmontagem da Cromatina , DNA/química , DNA/metabolismo , Metilação de DNA , Histonas/química , Histonas/genética , Histonas/metabolismo , Humanos , Modelos Moleculares , Nucleossomos/química , Ligação Proteica , Domínios Proteicos , Xenopus/genética
2.
Nature ; 585(7824): 277-282, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32879489

RESUMO

Abnormal epigenetic patterns correlate with effector T cell malfunction in tumours1-4, but the cause of this link is unknown. Here we show that tumour cells disrupt methionine metabolism in CD8+ T cells, thereby lowering intracellular levels of methionine and the methyl donor S-adenosylmethionine (SAM) and resulting in loss of dimethylation at lysine 79 of histone H3 (H3K79me2). Loss of H3K79me2 led to low expression of STAT5 and impaired T cell immunity. Mechanistically, tumour cells avidly consumed methionine and outcompeted T cells for methionine by expressing high levels of the methionine transporter SLC43A2. Genetic and biochemical inhibition of tumour SLC43A2 restored H3K79me2 in T cells, thereby boosting spontaneous and checkpoint-induced tumour immunity. Moreover, methionine supplementation improved the expression of H3K79me2 and STAT5 in T cells, and this was accompanied by increased T cell immunity in tumour-bearing mice and patients with colon cancer. Clinically, tumour SLC43A2 correlated negatively with T cell histone methylation and functional gene signatures. Our results identify a mechanistic connection between methionine metabolism, histone patterns, and T cell immunity in the tumour microenvironment. Thus, cancer methionine consumption is an immune evasion mechanism, and targeting cancer methionine signalling may provide an immunotherapeutic approach.


Assuntos
Sistema L de Transporte de Aminoácidos/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Histonas/metabolismo , Metionina/metabolismo , Metilação , Neoplasias/metabolismo , Sistema L de Transporte de Aminoácidos/deficiência , Animais , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/imunologia , Linhagem Celular Tumoral , Epigênese Genética , Feminino , Histonas/química , Humanos , Camundongos , Neoplasias/genética , Neoplasias/imunologia , Neoplasias/patologia , Receptores de Antígenos de Linfócitos T/metabolismo , Fator de Transcrição STAT5/metabolismo
3.
Nat Commun ; 11(1): 4747, 2020 09 21.
Artigo em Inglês | MEDLINE | ID: mdl-32958761

RESUMO

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


Assuntos
Nucleossomos/química , Nucleossomos/metabolismo , Sequência de Bases , Cromatina/química , Cromatina/metabolismo , Cristalografia por Raios X , DNA/química , DNA/metabolismo , Histonas/química , Histonas/genética , Histonas/metabolismo , Humanos , Modelos Moleculares , Conformação Molecular , Ligação Proteica
4.
Adv Exp Med Biol ; 1267: 117-133, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32894480

RESUMO

Antibiotic resistance is a global epidemic, becoming increasingly pressing due to its rapid spread. There is thus a critical need to develop new therapeutic approaches. In addition to searching for new antibiotics, looking into existing mechanisms of natural host defense may enable researchers to improve existing defense mechanisms, and to develop effective, synthetic drugs guided by natural principles. Histones, primarily known for their role in condensing mammalian DNA, are antimicrobial and share biochemical similarities with antimicrobial peptides (AMPs); however, the mechanism by which histones kill bacteria is largely unknown. Both AMPs and histones are similar in size, cationic, contain a high proportion of hydrophobic amino acids, and possess the ability to form alpha helices. AMPs, which mostly kill bacteria through permeabilization or disruption of the biological membrane, have recently garnered significant attention for playing a key role in host defenses. This chapter outlines the structure and function of histone proteins as they compare to AMPs and provides an overview of their role in innate immune responses, especially regarding the action of specific histones against microorganisms and their potential mechanism of action against microbial pathogens.


Assuntos
Antibacterianos/química , Antibacterianos/imunologia , Bactérias/imunologia , Histonas/química , Histonas/imunologia , Animais , Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/farmacologia , Bactérias/efeitos dos fármacos , Histonas/farmacologia , Imunidade Inata
5.
Proc Natl Acad Sci U S A ; 117(33): 19661-19663, 2020 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-32747537

RESUMO

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


Assuntos
Histonas/química , Histonas/metabolismo , Nucleossomos/metabolismo , Acetilação , Motivos de Aminoácidos , DNA/genética , DNA/metabolismo , Histonas/genética , Humanos , Conformação de Ácido Nucleico , Nucleossomos/genética
6.
Nat Commun ; 11(1): 3391, 2020 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-32636369

RESUMO

Neurodegeneration is a common hallmark of individuals with hereditary defects in DNA single-strand break repair; a process regulated by poly(ADP-ribose) metabolism. Recently, mutations in the ARH3 (ADPRHL2) hydrolase that removes ADP-ribose from proteins have been associated with neurodegenerative disease. Here, we show that ARH3-mutated patient cells accumulate mono(ADP-ribose) scars on core histones that are a molecular memory of recently repaired DNA single-strand breaks. We demonstrate that the ADP-ribose chromatin scars result in reduced endogenous levels of important chromatin modifications such as H3K9 acetylation, and that ARH3 patient cells exhibit measurable levels of deregulated transcription. Moreover, we show that the mono(ADP-ribose) scars are lost from the chromatin of ARH3-defective cells in the prolonged presence of PARP inhibition, and concomitantly that chromatin acetylation is restored to normal. Collectively, these data indicate that ARH3 can act as an eraser of ADP-ribose chromatin scars at sites of PARP activity during DNA single-strand break repair.


Assuntos
Adenosina Difosfato Ribose/química , Cromatina/química , Quebras de DNA de Cadeia Simples , Reparo do DNA , Glicosídeo Hidrolases/genética , Mutação , Linhagem Celular Tumoral , Sobrevivência Celular , Fibroblastos , Regulação da Expressão Gênica , Técnicas de Inativação de Genes , Células HEK293 , Histonas/química , Humanos , Doenças Neurodegenerativas/genética , Proteína 1 Complementadora Cruzada de Reparo de Raio-X/genética
7.
Science ; 369(6499): 59-64, 2020 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-32631887

RESUMO

Eukaryotic histone H3-H4 tetramers contain a putative copper (Cu2+) binding site at the H3-H3' dimerization interface with unknown function. The coincident emergence of eukaryotes with global oxygenation, which challenged cellular copper utilization, raised the possibility that histones may function in cellular copper homeostasis. We report that the recombinant Xenopus laevis H3-H4 tetramer is an oxidoreductase enzyme that binds Cu2+ and catalyzes its reduction to Cu1+ in vitro. Loss- and gain-of-function mutations of the putative active site residues correspondingly altered copper binding and the enzymatic activity, as well as intracellular Cu1+ abundance and copper-dependent mitochondrial respiration and Sod1 function in the yeast Saccharomyces cerevisiae The histone H3-H4 tetramer, therefore, has a role other than chromatin compaction or epigenetic regulation and generates biousable Cu1+ ions in eukaryotes.


Assuntos
Cobre/metabolismo , Histonas/química , Oxirredutases/química , Multimerização Proteica , Animais , Biocatálise , Domínio Catalítico/genética , Mutação com Ganho de Função , Histonas/genética , Histonas/metabolismo , Mitocôndrias/metabolismo , Proteínas Nucleares/metabolismo , Oxirredutases/genética , Oxirredutases/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Superóxido Dismutase-1/química , Fatores de Transcrição/metabolismo , Xenopus laevis
8.
BMC Bioinformatics ; 21(1): 317, 2020 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-32689977

RESUMO

BACKGROUND: The binding sites of transcription factors (TFs) and the localisation of histone modifications in the human genome can be quantified by the chromatin immunoprecipitation assay coupled with next-generation sequencing (ChIP-seq). The resulting chromatin feature data has been successfully adopted for genome-wide enhancer identification by several unsupervised and supervised machine learning methods. However, the current methods predict different numbers and different sets of enhancers for the same cell type and do not utilise the pattern of the ChIP-seq coverage profiles efficiently. RESULTS: In this work, we propose a PRobabilistic Enhancer PRedictIoN Tool (PREPRINT) that assumes characteristic coverage patterns of chromatin features at enhancers and employs a statistical model to account for their variability. PREPRINT defines probabilistic distance measures to quantify the similarity of the genomic query regions and the characteristic coverage patterns. The probabilistic scores of the enhancer and non-enhancer samples are utilised to train a kernel-based classifier. The performance of the method is demonstrated on ENCODE data for two cell lines. The predicted enhancers are computationally validated based on the transcriptional regulatory protein binding sites and compared to the predictions obtained by state-of-the-art methods. CONCLUSION: PREPRINT performs favorably to the state-of-the-art methods, especially when requiring the methods to predict a larger set of enhancers. PREPRINT generalises successfully to data from cell type not utilised for training, and often the PREPRINT performs better than the previous methods. The PREPRINT enhancers are less sensitive to the choice of prediction threshold. PREPRINT identifies biologically validated enhancers not predicted by the competing methods. The enhancers predicted by PREPRINT can aid the genome interpretation in functional genomics and clinical studies.


Assuntos
Cromatina/genética , Elementos Facilitadores Genéticos , Genoma Humano , Genômica/métodos , Histonas/genética , Modelos Estatísticos , Fatores de Transcrição/metabolismo , Cromatina/química , Cromatina/metabolismo , Código das Histonas , Histonas/química , Histonas/metabolismo , Humanos , Processamento de Proteína Pós-Traducional
9.
Nucleic Acids Res ; 48(15): e90, 2020 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-32609809

RESUMO

Specific genomic functions are dictated by macromolecular complexes (MCs) containing multiple proteins. Affinity purification of these complexes, often using antibodies, followed by mass spectrometry (MS) has revolutionized our ability to identify the composition of MCs. However, conventional immunoprecipitations suffer from contaminating antibody/serum-derived peptides that limit the sensitivity of detection for low-abundant interacting partners using MS. Here, we present AptA-MS (aptamer affinity-mass spectrometry), a robust strategy primarily using a specific, high-affinity RNA aptamer against Green Fluorescent Protein (GFP) to identify interactors of a GFP-tagged protein of interest by high-resolution MS. Utilizing this approach, we have identified the known molecular chaperones that interact with human Heat Shock Factor 1 (HSF1), and observed an increased association with several proteins upon heat shock, including translation elongation factors and histones. HSF1 is known to be regulated by multiple post-translational modifications (PTMs), and we observe both known and new sites of modifications on HSF1. We show that AptA-MS provides a dramatic target enrichment and detection sensitivity in evolutionarily diverse organisms and allows identification of PTMs without the need for modification-specific enrichments. In combination with the expanding libraries of GFP-tagged cell lines, this strategy offers a general, inexpensive, and high-resolution alternative to conventional approaches for studying MCs.


Assuntos
Aptâmeros de Nucleotídeos/química , Fatores de Transcrição de Choque Térmico/química , Substâncias Macromoleculares/isolamento & purificação , Espectrometria de Massas , Aptâmeros de Nucleotídeos/genética , Proteínas de Fluorescência Verde/genética , Fatores de Transcrição de Choque Térmico/genética , Histonas/química , Humanos , Imunoprecipitação , Substâncias Macromoleculares/química , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Peptídeos/química , Ligação Proteica , Processamento de Proteína Pós-Traducional
10.
Proc Natl Acad Sci U S A ; 117(31): 18439-18447, 2020 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-32675241

RESUMO

In mammals, repressive histone modifications such as trimethylation of histone H3 Lys9 (H3K9me3), frequently coexist with DNA methylation, producing a more stable and silenced chromatin state. However, it remains elusive how these epigenetic modifications crosstalk. Here, through structural and biochemical characterizations, we identified the replication foci targeting sequence (RFTS) domain of maintenance DNA methyltransferase DNMT1, a module known to bind the ubiquitylated H3 (H3Ub), as a specific reader for H3K9me3/H3Ub, with the recognition mode distinct from the typical trimethyl-lysine reader. Disruption of the interaction between RFTS and the H3K9me3Ub affects the localization of DNMT1 in stem cells and profoundly impairs the global DNA methylation and genomic stability. Together, this study reveals a previously unappreciated pathway through which H3K9me3 directly reinforces DNMT1-mediated maintenance DNA methylation.


Assuntos
DNA (Citosina-5-)-Metiltransferase 1/metabolismo , Metilação de DNA , Heterocromatina/metabolismo , Histonas/metabolismo , DNA (Citosina-5-)-Metiltransferase 1/genética , Heterocromatina/genética , Histonas/química , Histonas/genética , Humanos , Lisina/genética , Lisina/metabolismo , Metilação , Processamento de Proteína Pós-Traducional
11.
Nat Commun ; 11(1): 2818, 2020 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-32499524

RESUMO

In eukaryotes, trimethylation of lysine 9 on histone H3 (H3K9) is associated with transcriptional silencing of transposable elements (TEs). In drosophila ovaries, this heterochromatic repressive mark is thought to be deposited by SetDB1 on TE genomic loci after the initial recognition of nascent transcripts by PIWI-interacting RNAs (piRNAs) loaded on the Piwi protein. Here, we show that the nucleosome remodeler Mi-2, in complex with its partner MEP-1, forms a subunit that is transiently associated, in a MEP-1 C-terminus-dependent manner, with known Piwi interactors, including a recently reported SUMO ligase, Su(var)2-10. Together with the histone deacetylase Rpd3, this module is involved in the piRNA-dependent TE silencing, correlated with H3K9 deacetylation and trimethylation. Therefore, drosophila piRNA-mediated transcriptional silencing involves three epigenetic effectors, a remodeler, Mi-2, an eraser, Rpd3 and a writer, SetDB1, in addition to the Su(var)2-10 SUMO ligase.


Assuntos
Adenosina Trifosfatases/metabolismo , Autoantígenos/metabolismo , Proteínas de Drosophila/metabolismo , Heterocromatina/química , Histona Desacetilase 1/metabolismo , Nucleossomos/metabolismo , RNA Interferente Pequeno/metabolismo , Animais , Proteínas Argonauta/metabolismo , Drosophila melanogaster , Epigênese Genética , Feminino , Regulação da Expressão Gênica , Inativação Gênica , Histonas/química , Ovário/metabolismo , Proteínas Inibidoras de STAT Ativados
12.
PLoS One ; 15(6): e0235343, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32584896

RESUMO

Triple Negative Breast Cancer (TNBC) is a heterogeneous disease lacking known molecular drivers and effective targeted therapies. Cytotoxic chemotherapy remains the mainstay of treatment for TNBCs, which have significantly poorer survival rates compared to other breast cancer subtypes. In addition to changes within the coding genome, aberrant enhancer activity is a well-established contributor to tumorigenesis. Here we use H3K27Ac chromatin immunoprecipitation followed by sequencing (ChIP-Seq) to map the active cis-regulatory landscape in TNBC. We identify distinct disease subtypes associated with specific enhancer activity, and over 2,500 unique superenhancers acquired by tumor cells but absent from normal breast tissue. To identify potential actionable disease drivers, we probed the dependency on genes that associate with tumor-specific enhancers by CRISPR screening. In this way we identify a number of tumor-specific dependencies, including a previously uncharacterized dependency on the TGFß pseudo-receptor BAMBI.


Assuntos
Elementos Facilitadores Genéticos/genética , Oncogenes/genética , Neoplasias de Mama Triplo Negativas/patologia , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Feminino , Edição de Genes , Regulação Neoplásica da Expressão Gênica , Histonas/química , Histonas/genética , Histonas/metabolismo , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , RNA Guia/metabolismo , Neoplasias de Mama Triplo Negativas/genética
13.
Nat Protoc ; 15(8): 2503-2518, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32591768

RESUMO

Fixed-tissue ChIP-seq for H3K27 acetylation (H3K27ac) profiling (FiTAc-seq) is an epigenetic method for profiling active enhancers and promoters in formalin-fixed, paraffin-embedded (FFPE) tissues. We previously developed a modified ChIP-seq protocol (FiT-seq) for chromatin profiling in FFPE. FiT-seq produces high-quality chromatin profiles particularly for methylated histone marks but is not optimized for H3K27ac profiling. FiTAc-seq is a modified protocol that replaces the proteinase K digestion applied in FiT-seq with extended heating at 65 °C in a higher concentration of detergent and a minimized sonication step, to produce robust genome-wide H3K27ac maps from clinical samples. FiTAc-seq generates high-quality enhancer landscapes and super-enhancer (SE) annotation in numerous archived FFPE samples from distinct tumor types. This approach will be of great interest for both basic and clinical researchers. The entire protocol from FFPE blocks to sequence-ready library can be accomplished within 4 d.


Assuntos
Sequenciamento de Cromatina por Imunoprecipitação/métodos , Histonas/química , Histonas/metabolismo , Lisina/metabolismo , Inclusão em Parafina , Fixação de Tecidos , Acetilação , Animais , Fígado/citologia , Camundongos
14.
Proc Natl Acad Sci U S A ; 117(24): 13828-13838, 2020 06 16.
Artigo em Inglês | MEDLINE | ID: mdl-32461370

RESUMO

Despite its popularity, chromatin immunoprecipitation followed by sequencing (ChIP-seq) remains a tedious (>2 d), manually intensive, low-sensitivity and low-throughput approach. Here, we combine principles of microengineering, surface chemistry, and molecular biology to address the major limitations of standard ChIP-seq. The resulting technology, FloChIP, automates and miniaturizes ChIP in a beadless fashion while facilitating the downstream library preparation process through on-chip chromatin tagmentation. FloChIP is fast (<2 h), has a wide dynamic range (from 106 to 500 cells), is scalable and parallelized, and supports antibody- or sample-multiplexed ChIP on both histone marks and transcription factors. In addition, FloChIP's interconnected design allows for straightforward chromatin reimmunoprecipitation, which allows this technology to also act as a microfluidic sequential ChIP-seq system. Finally, we ran FloChIP for the transcription factor MEF2A in 32 distinct human lymphoblastoid cell lines, providing insights into the main factors driving collaborative DNA binding of MEF2A and into its role in B cell-specific gene regulation. Together, our results validate FloChIP as a flexible and reproducible automated solution for individual or sequential ChIP-seq.


Assuntos
Automação/métodos , Sequenciamento de Cromatina por Imunoprecipitação/métodos , Histonas/metabolismo , Fatores de Transcrição MEF2/metabolismo , Automação/instrumentação , Linfócitos B/química , Linfócitos B/metabolismo , Linhagem Celular Tumoral , Sequenciamento de Cromatina por Imunoprecipitação/instrumentação , Histonas/química , Histonas/genética , Humanos , Fatores de Transcrição MEF2/química , Fatores de Transcrição MEF2/genética , Ligação Proteica
15.
Nat Commun ; 11(1): 2462, 2020 05 18.
Artigo em Inglês | MEDLINE | ID: mdl-32424115

RESUMO

Histone ubiquitination plays an important role in the DNA damage response (DDR) pathway. RNF168 catalyzes H2A and H2AX ubiquitination on lysine 13/15 (K13/K15) upon DNA damage and promotes the accrual of downstream repair factors at damaged chromatin. Here, we report that RNF168 ubiquitinates the non-canonical H2A variants H2AZ and macroH2A1/2 at the divergent N-terminal tail lysine residue. In addition to their evolutionarily conserved nucleosome acidic patch, we identify the positively charged alpha1-extension helix as essential for RNF168-mediated ubiquitination of H2A variants. Moreover, mutation of the RNF168 UMI (UIM- and MIU-related UBD) hydrophilic acidic residues abolishes RNF168-mediated ubiquitination as well as 53BP1 and BRCA1 ionizing radiation-induced foci formation. Our results reveal a juxtaposed bipartite electrostatic interaction utilized by the nucleosome to direct RNF168 orientation towards the target lysine residues in proximity to the H2A alpha1-extension helix, which plays an important role in the DDR pathway.


Assuntos
Histonas/química , Histonas/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Sequência de Aminoácidos , Evolução Molecular , Células HEK293 , Histonas/genética , Humanos , Lisina/metabolismo , Estrutura Secundária de Proteína , Especificidade por Substrato , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo
16.
Nucleic Acids Res ; 48(10): 5318-5331, 2020 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-32356891

RESUMO

Intrinsically disordered proteins are crucial elements of chromatin heterogenous organization. While disorder in the histone tails enables a large variation of inter-nucleosome arrangements, disorder within the chromatin-binding proteins facilitates promiscuous binding to a wide range of different molecular targets, consistent with structural heterogeneity. Among the partially disordered chromatin-binding proteins, the H1 linker histone influences a myriad of chromatin characteristics including compaction, nucleosome spacing, transcription regulation, and the recruitment of other chromatin regulating proteins. Although it is now established that the long C-terminal domain (CTD) of H1 remains disordered upon nucleosome binding and that such disorder favours chromatin fluidity, the structural behaviour and thereby the role/function of the N-terminal domain (NTD) within chromatin is yet unresolved. On the basis of microsecond-long parallel-tempering metadynamics and temperature-replica exchange atomistic molecular dynamics simulations of different H1 NTD subtypes, we demonstrate that the NTD is completely unstructured in solution but undergoes an important disorder-to-order transition upon nucleosome binding: it forms a helix that enhances its DNA binding ability. Further, we show that the helical propensity of the H1 NTD is subtype-dependent and correlates with the experimentally observed binding affinity of H1 subtypes, suggesting an important functional implication of this disorder-to-order transition.


Assuntos
Histonas/química , Histonas/metabolismo , Nucleossomos/química , Nucleossomos/metabolismo , DNA/química , DNA/metabolismo , Humanos , Proteínas Intrinsicamente Desordenadas/química , Proteínas Intrinsicamente Desordenadas/metabolismo , Simulação de Dinâmica Molecular , Conformação de Ácido Nucleico , Peptídeos/química , Ligação Proteica , Domínios Proteicos
17.
Proc Natl Acad Sci U S A ; 117(20): 11000-11009, 2020 05 19.
Artigo em Inglês | MEDLINE | ID: mdl-32358196

RESUMO

African swine fever virus (ASFV) is a highly contagious nucleocytoplasmic large DNA virus (NCLDV) that causes nearly 100% mortality in swine. The development of effective vaccines and drugs against this virus is urgently needed. pA104R, an ASFV-derived histone-like protein, shares sequence and functional similarity with bacterial HU/IHF family members and is essential for viral replication. Herein, we solved the crystal structures of pA104R in its apo state as well as in complex with DNA. Apo-pA104R forms a homodimer and folds into an architecture conserved in bacterial heat-unstable nucleoid proteins/integration host factors (HUs/IHFs). The pA104R-DNA complex structure, however, uncovers that pA104R has a DNA binding pattern distinct from its bacterial homologs, that is, the ß-ribbon arms of pA104R stabilize DNA binding by contacting the major groove instead of the minor groove. Mutations of the basic residues at the base region of the ß-strand DNA binding region (BDR), rather than those in the ß-ribbon arms, completely abolished DNA binding, highlighting the major role of the BDR base in DNA binding. An overall DNA bending angle of 93.8° is observed in crystal packing of the pA104R-DNA complex structure, which is close to the DNA bending angle in the HU-DNA complex. Stilbene derivatives SD1 and SD4 were shown to disrupt the binding between pA104R and DNA and inhibit the replication of ASFV in primary porcine alveolar macrophages. Collectively, these results reveal the structural basis of pA104R binding to DNA highlighting the importance of the pA104R-DNA interaction in the ASFV replication cycle and provide inhibitor leads for ASFV chemotherapy.


Assuntos
Vírus da Febre Suína Africana/efeitos dos fármacos , Vírus da Febre Suína Africana/fisiologia , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/efeitos dos fármacos , DNA/química , Estilbenos/farmacologia , Febre Suína Africana/prevenção & controle , Vírus da Febre Suína Africana/genética , Animais , Sequência de Bases , Proteínas de Ligação a DNA/metabolismo , Escherichia coli , Histonas/química , Modelos Moleculares , Conformação Proteica , Suínos , Replicação Viral/efeitos dos fármacos
18.
Nucleic Acids Res ; 48(11): 6019-6031, 2020 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-32379321

RESUMO

ALT tumor cells often contain abundant DNA damage foci at telomeres and rely on the alternative lengthening of telomeres (ALT) mechanism to maintain their telomeres. How the telomere chromatin is regulated and maintained in these cells remains largely unknown. In this study, we present evidence that heterochromatin protein 1 binding protein 3 (HP1BP3) can localize to telomeres and is particularly enriched on telomeres in ALT cells. HP1BP3 inhibition led to preferential growth inhibition of ALT cells, which was accompanied by telomere chromatin decompaction, increased presence of C-circles, more pronounced ALT-associated phenotypes and elongated telomeres. Furthermore, HP1BP3 appeared to participate in regulating telomere histone H3K9me3 epigenetic marks. Taken together, our data suggest that HP1BP3 functions on telomeres to maintain telomere chromatin and represents a novel target for inhibiting ALT cancer cells.


Assuntos
Proliferação de Células , Montagem e Desmontagem da Cromatina , Heterocromatina/metabolismo , Histonas/metabolismo , Telômero/metabolismo , Linhagem Celular Tumoral , Dano ao DNA , Eucromatina/genética , Eucromatina/metabolismo , Técnicas de Silenciamento de Genes , Heterocromatina/genética , Código das Histonas , Histonas/química , Humanos , Metilação , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/deficiência , Proteínas Nucleares/metabolismo , Multimerização Proteica , Homeostase do Telômero
19.
Nucleic Acids Res ; 48(11): 5953-5966, 2020 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-32396165

RESUMO

The modification of histones by acetyl groups has a key role in the regulation of chromatin structure and transcription. The Arabidopsis thaliana histone acetyltransferase GCN5 regulates histone modifications as part of the Spt-Ada-Gcn5 Acetyltransferase (SAGA) transcriptional coactivator complex. GCN5 was previously shown to acetylate lysine 14 of histone 3 (H3K14ac) in the promoter regions of its target genes even though GCN5 binding did not systematically correlate with gene activation. Here, we explored the mechanism through which GCN5 controls transcription. First, we fine-mapped its GCN5 binding sites genome-wide and then used several global methodologies (ATAC-seq, ChIP-seq and RNA-seq) to assess the effect of GCN5 loss-of-function on the expression and epigenetic regulation of its target genes. These analyses provided evidence that GCN5 has a dual role in the regulation of H3K14ac levels in their 5' and 3' ends of its target genes. While the gcn5 mutation led to a genome-wide decrease of H3K14ac in the 5' end of the GCN5 down-regulated targets, it also led to an increase of H3K14ac in the 3' ends of GCN5 up-regulated targets. Furthermore, genome-wide changes in H3K14ac levels in the gcn5 mutant correlated with changes in H3K9ac at both 5' and 3' ends, providing evidence for a molecular link between the depositions of these two histone modifications. To understand the biological relevance of these regulations, we showed that GCN5 participates in the responses to biotic stress by repressing salicylic acid (SA) accumulation and SA-mediated immunity, highlighting the role of this protein in the regulation of the crosstalk between diverse developmental and stress-responsive physiological programs. Hence, our results demonstrate that GCN5, through the modulation of H3K14ac levels on its targets, controls the balance between biotic and abiotic stress responses and is a master regulator of plant-environmental interactions.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Regulação da Expressão Gênica de Plantas , Genes de Plantas/genética , Histona Acetiltransferases/metabolismo , Histonas/metabolismo , Homeostase , Lisina/metabolismo , Ácido Salicílico/metabolismo , Regiões 5' não Traduzidas/genética , Acetilação , Arabidopsis/imunologia , Histonas/química , Lisina/química , Imunidade Vegetal/genética , Regiões Promotoras Genéticas/genética , Transcrição Genética
20.
Nat Commun ; 11(1): 2219, 2020 05 06.
Artigo em Inglês | MEDLINE | ID: mdl-32376827

RESUMO

Heterochromatin in the eukaryotic genome is rigorously controlled by the concerted action of protein factors and RNAs. Here, we investigate the RNA binding function of ATRX, a chromatin remodeler with roles in silencing of repetitive regions of the genome and in recruitment of the polycomb repressive complex 2 (PRC2). We identify ATRX RNA binding regions (RBRs) and discover that the major ATRX RBR lies within the N-terminal region of the protein, distinct from its PHD and helicase domains. Deletion of this ATRX RBR (ATRXΔRBR) compromises ATRX interactions with RNAs in vitro and in vivo and alters its chromatin binding properties. Genome-wide studies reveal that loss of RNA interactions results in a redistribution of ATRX on chromatin. Finally, our studies identify a role for ATRX-RNA interactions in regulating PRC2 localization to a subset of polycomb target genes.


Assuntos
Cromatina/metabolismo , Complexo Repressor Polycomb 2/metabolismo , RNA/metabolismo , Proteína Nuclear Ligada ao X/genética , Animais , Montagem e Desmontagem da Cromatina/genética , Feminino , Fibroblastos/enzimologia , Fibroblastos/metabolismo , Heterocromatina/metabolismo , Histonas/química , Histonas/metabolismo , Metilação , Camundongos , Ligação Proteica , Domínios Proteicos/genética , Proteína Nuclear Ligada ao X/metabolismo
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