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1.
Nat Commun ; 11(1): 4940, 2020 10 02.
Artigo em Inglês | MEDLINE | ID: mdl-33009411

RESUMO

The HUSH complex represses retroviruses, transposons and genes to maintain the integrity of vertebrate genomes. HUSH regulates deposition of the epigenetic mark H3K9me3, but how its three core subunits - TASOR, MPP8 and Periphilin - contribute to assembly and targeting of the complex remains unknown. Here, we define the biochemical basis of HUSH assembly and find that its modular architecture resembles the yeast RNA-induced transcriptional silencing complex. TASOR, the central HUSH subunit, associates with RNA processing components. TASOR is required for H3K9me3 deposition over LINE-1 repeats and repetitive exons in transcribed genes. In the context of previous studies, this suggests that an RNA intermediate is important for HUSH activity. We dissect the TASOR and MPP8 domains necessary for transgene repression. Structure-function analyses reveal TASOR bears a catalytically-inactive PARP domain necessary for targeted H3K9me3 deposition. We conclude that TASOR is a multifunctional pseudo-PARP that directs HUSH assembly and epigenetic regulation of repetitive genomic targets.


Assuntos
Elementos de DNA Transponíveis/genética , Epigênese Genética , Complexos Multiproteicos/metabolismo , Proteínas Nucleares/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Sequência de Aminoácidos , Antígenos de Neoplasias/metabolismo , Sítios de Ligação , Éxons/genética , Genoma , Células HEK293 , Células HeLa , Histonas/metabolismo , Humanos , Lisina/metabolismo , Espectroscopia de Ressonância Magnética , Metilação , NAD/metabolismo , Proteínas Nucleares/química , Fosfoproteínas/metabolismo , Ligação Proteica , Domínios Proteicos , RNA/metabolismo , Processamento Pós-Transcricional do RNA , Transcrição Genética
2.
Nat Commun ; 11(1): 4914, 2020 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-33004788

RESUMO

Oxepinamides are derivatives of anthranilyl-containing tripeptides and share an oxepin ring and a fused pyrimidinone moiety. To the best of our knowledge, no studies have been reported on the elucidation of an oxepinamide biosynthetic pathway and conversion of a quinazolinone to a pyrimidinone-fused 1H-oxepin framework by a cytochrome P450 enzyme in fungal natural product biosynthesis. Here we report the isolation of oxepinamide F from Aspergillus ustus and identification of its biosynthetic pathway by gene deletion, heterologous expression, feeding experiments, and enzyme assays. The nonribosomal peptide synthase (NRPS) OpaA assembles the quinazolinone core with D-Phe incorporation. The cytochrome P450 enzyme OpaB catalyzes alone the oxepin ring formation. The flavoenzyme OpaC installs subsequently one hydroxyl group at the oxepin ring, accompanied by double bond migration. The epimerase OpaE changes the D-Phe residue back to L-form, which is essential for the final methylation by OpaF.


Assuntos
Amidas/metabolismo , Aspergillus/enzimologia , Proteínas Fúngicas/metabolismo , Oxepinas/metabolismo , Amidas/química , Amidas/isolamento & purificação , Aspergillus/genética , Vias Biossintéticas , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Ensaios Enzimáticos , Proteínas Fúngicas/genética , Hidroxilação , Isomerismo , Metilação , Oxepinas/química , Oxepinas/isolamento & purificação , Peptídeo Sintases/genética , Peptídeo Sintases/metabolismo , Fenilalanina/química , Fenilalanina/metabolismo , Proteína O-Metiltransferase/genética , Proteína O-Metiltransferase/metabolismo , Quinazolinonas/metabolismo , Racemases e Epimerases/genética , Racemases e Epimerases/metabolismo
3.
Nat Commun ; 11(1): 4501, 2020 09 09.
Artigo em Inglês | MEDLINE | ID: mdl-32908132

RESUMO

Streptovaricin C is a naphthalenic ansamycin antibiotic structurally similar to rifamycins with potential anti-MRSA bioactivities. However, the formation mechanism of the most fascinating and bioactivity-related methylenedioxy bridge (MDB) moiety in streptovaricins is unclear. Based on genetic and biochemical evidences, we herein clarify that the P450 enzyme StvP2 catalyzes the MDB formation in streptovaricins, with an atypical substrate inhibition kinetics. Furthermore, X-ray crystal structures in complex with substrate and structure-based mutagenesis reveal the intrinsic details of the enzymatic reaction. The mechanism of MDB formation is proposed to be an intramolecular nucleophilic substitution resulting from the hydroxylation by the heme core and the keto-enol tautomerization via a crucial catalytic triad (Asp89-His92-Arg72) in StvP2. In addition, in vitro reconstitution uncovers that C6-O-methylation and C4-O-acetylation of streptovaricins are necessary prerequisites for the MDB formation. This work provides insight for the MDB formation and adds evidence in support of the functional versatility of P450 enzymes.


Assuntos
Proteínas de Bactérias/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Streptomyces/metabolismo , Estreptovaricina/análogos & derivados , Acetilação , Proteínas de Bactérias/genética , Proteínas de Bactérias/ultraestrutura , Biocatálise , Cristalografia por Raios X , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/ultraestrutura , Ensaios Enzimáticos , Metilação , Mutagênese Sítio-Dirigida , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura , Estreptovaricina/biossíntese , Estreptovaricina/química , Estreptovaricina/metabolismo
5.
Zhongguo Dang Dai Er Ke Za Zhi ; 22(9): 1001-1006, 2020 Sep.
Artigo em Chinês | MEDLINE | ID: mdl-32933634

RESUMO

OBJECTIVE: To study the clinical screening and genetic diagnosis of children suspected of Prader-Willi syndrome (PWS), as well as the differences in the scores of clinical diagnostic criteria among the children with a confirmed diagnosis of PWS. METHODS: A total of 94 children suspected of PWS who were admitted from July 2016 to December 2018 were enrolled as subjects. Methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) was performed to confirm the diagnosis. For the children with a confirmed diagnosis of PWS, the scores of clinical diagnostic criteria were determined, and the perinatal characteristics were analyzed. RESULTS: A total of 11 children with PWS were confirmed by MS-MLPA, with a detection rate of 12%, among whom there were 7 boys and 4 girls, with a median age of 3 years and 4 months (range 25 days to 6 years and 8 months) at the time of confirmed diagnosis. Among the 11 children with PWS, only 5 children (45%) met the criteria for clinical diagnosis. The main perinatal characteristics of the children with PWS were decreased fetal movement (9 cases, 82%), cesarean section birth (11 cases, 100%), hypotonia (11 cases, 100%), feeding difficulties (11 cases, 100%), and weak crying (11 cases, 100%). CONCLUSIONS: Gene testing should be performed as early as possible for children suspected of PWS by clinical screening. PWS may be missed if only based on the scores of clinical diagnostic criteria.


Assuntos
Síndrome de Prader-Willi , Cesárea , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Metilação , Hipotonia Muscular , Gravidez
6.
Nat Commun ; 11(1): 4673, 2020 09 16.
Artigo em Inglês | MEDLINE | ID: mdl-32938917

RESUMO

RAS-MAPK signaling mediates processes critical to normal development including cell proliferation, survival, and differentiation. Germline mutation of RAS-MAPK genes lead to the Noonan-spectrum of syndromes. Here, we present a patient affected by a 6p-interstitial microdeletion with unknown underlying molecular etiology. Examination of 6p-interstitial microdeletion cases reveals shared clinical features consistent with Noonan-spectrum disorders including short stature, facial dysmorphia and cardiovascular abnormalities. We find the RAS-responsive element binding protein-1 (RREB1) is the common deleted gene in multiple 6p-interstitial microdeletion cases. Rreb1 hemizygous mice display orbital hypertelorism and cardiac hypertrophy phenocopying the human syndrome. Rreb1 haploinsufficiency leads to sensitization of MAPK signaling. Rreb1 recruits Sin3a and Kdm1a to control H3K4 methylation at MAPK pathway gene promoters. Haploinsufficiency of SIN3A and mutations in KDM1A cause syndromes similar to RREB1 haploinsufficiency suggesting genetic perturbation of the RREB1-SIN3A-KDM1A complex represents a new category of RASopathy-like syndromes arising through epigenetic reprogramming of MAPK pathway genes.


Assuntos
Proteínas de Ligação a DNA/genética , Haploinsuficiência , Sistema de Sinalização das MAP Quinases/genética , Síndrome de Noonan/etiologia , Fatores de Transcrição/genética , Proteínas ras/metabolismo , Anormalidades Múltiplas/genética , Animais , Deleção Cromossômica , Cromossomos Humanos Par 6 , Proteínas de Ligação a DNA/metabolismo , Epigênese Genética , Feminino , Fatores de Crescimento de Fibroblastos/genética , Fatores de Crescimento de Fibroblastos/metabolismo , Histona Desmetilases/genética , Histona Desmetilases/metabolismo , Histonas/metabolismo , Humanos , Masculino , Metilação , Camundongos Endogâmicos C57BL , Camundongos Knockout , Complexo Correpressor Histona Desacetilase e Sin3/genética , Complexo Correpressor Histona Desacetilase e Sin3/metabolismo , Fatores de Transcrição/metabolismo , Proteínas ras/genética
7.
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
8.
PLoS One ; 15(9): e0236081, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32960890

RESUMO

Type 2 diabetes mellitus (T2DM), one of the most common metabolic diseases, is characterized by insulin resistance and inadequate insulin secretion of ß cells. Glycogen phosphorylase (GP) is the key enzyme in glycogen breakdown, and contributes to hepatic glucose production during fasting or during insulin resistance. Pharmacological GP inhibitors are potential glucose lowering agents, which may be used in T2DM therapy. A natural product isolated from the cultured broth of the fungal strain No. 138354, called 2,3-bis(4-hydroxycinnamoyloxy)glutaric acid (FR258900), was discovered a decade ago. In vivo studies showed that FR258900 significantly reduced blood glucose levels in diabetic mice. We previously showed that GP inhibitors can potently enhance the function of ß cells. The purpose of this study was to assess whether an analogue of FR258900 can influence ß cell function. BF142 (Meso-Dimethyl 2,3-bis[(E)-3-(4-acetoxyphenyl)prop-2-enamido]butanedioate) treatment activated the glucose-stimulated insulin secretion pathway, as indicated by enhanced glycolysis, increased mitochondrial oxidation, significantly increased ATP production, and elevated calcium influx in MIN6 cells. Furthermore, BF142 induced mTORC1-specific phosphorylation of S6K, increased levels of PDX1 and insulin protein, and increased insulin secretion. Our data suggest that BF142 can influence ß cell function and can support the insulin producing ability of ß cells.


Assuntos
Cinamatos/farmacologia , Inibidores Enzimáticos/farmacologia , Glutaratos/farmacologia , Glicogênio Fosforilase/antagonistas & inibidores , Secreção de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/efeitos dos fármacos , Ácido Succínico/farmacologia , Animais , Linhagem Celular Tumoral , Cinamatos/química , Inibidores Enzimáticos/química , Glucose/metabolismo , Glutaratos/química , Glicogênio Fosforilase/metabolismo , Glicólise/efeitos dos fármacos , Insulina/metabolismo , Células Secretoras de Insulina/metabolismo , Metilação , Camundongos , Ácido Succínico/química
9.
Sci Signal ; 13(651)2020 09 29.
Artigo em Inglês | MEDLINE | ID: mdl-32994211

RESUMO

There are currently no antiviral therapies specific for SARS-CoV-2, the virus responsible for the global pandemic disease COVID-19. To facilitate structure-based drug design, we conducted an x-ray crystallographic study of the SARS-CoV-2 nsp16-nsp10 2'-O-methyltransferase complex, which methylates Cap-0 viral mRNAs to improve viral protein translation and to avoid host immune detection. We determined the structures for nsp16-nsp10 heterodimers bound to the methyl donor S-adenosylmethionine (SAM), the reaction product S-adenosylhomocysteine (SAH), or the SAH analog sinefungin (SFG). We also solved structures for nsp16-nsp10 in complex with the methylated Cap-0 analog m7GpppA and either SAM or SAH. Comparative analyses between these structures and published structures for nsp16 from other betacoronaviruses revealed flexible loops in open and closed conformations at the m7GpppA-binding pocket. Bound sulfates in several of the structures suggested the location of the ribonucleic acid backbone phosphates in the ribonucleotide-binding groove. Additional nucleotide-binding sites were found on the face of the protein opposite the active site. These various sites and the conserved dimer interface could be exploited for the development of antiviral inhibitors.


Assuntos
Betacoronavirus/enzimologia , Infecções por Coronavirus/tratamento farmacológico , Metiltransferases/química , Pneumonia Viral/tratamento farmacológico , Proteínas não Estruturais Virais/química , Adenosina/análogos & derivados , Adenosina/metabolismo , Adenosina/farmacologia , Betacoronavirus/efeitos dos fármacos , Sítios de Ligação , Domínio Catalítico , Cristalografia por Raios X , Dimerização , Genes Virais/genética , Humanos , Metilação , Metiltransferases/antagonistas & inibidores , Modelos Moleculares , Fases de Leitura Aberta/genética , Pandemias , Ligação Proteica , Conformação Proteica , Análogos de Capuz de RNA/metabolismo , Processamento Pós-Transcricional do RNA , RNA Viral/metabolismo , S-Adenosil-Homocisteína/metabolismo , S-Adenosilmetionina/metabolismo , Relação Estrutura-Atividade , Proteínas não Estruturais Virais/antagonistas & inibidores , Proteínas não Estruturais Virais/metabolismo
10.
Virology ; 548: 59-72, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32838947

RESUMO

Methylation of the N6 position of adenosine (m6A) is a widespread RNA modification that is critical for various physiological and pathological processes. Although this modification was also found in the RNA of several viruses almost 40 years ago, its biological functions during viral infection have been elucidated recently. Here, we investigated the effects of viral and host RNA methylation during porcine epidemic diarrhea virus (PEDV) infection. The results demonstrated that the m6A modification was abundant in the PEDV genome and the host methyltransferases METTL3 and METTL14 and demethylase FTO were involved in the regulation of viral replication. The knockdown of the methyltransferases increased PEDV replication while silencing the demethylase decreased PEDV output. Moreover, the proteins of the YTHDF family regulated the PEDV replication by affecting the stability of m6A-modified viral RNA. In particular, PEDV infection could trigger an increasement of m6A in host RNA and decrease the expression of FTO. The m6A modification sites in mRNAs and target genes were also altered during PEDV infection. Additionally, part of the host responses to PEDV infection was controlled by m6A modification, which could be reversed by the expression of FTO. Taken together, our results identified the role of m6A modification in PEDV replication and interactions with the host.


Assuntos
Adenosina/análogos & derivados , Infecções por Coronavirus/veterinária , Regulação da Expressão Gênica , Interações Hospedeiro-Patógeno/genética , Vírus da Diarreia Epidêmica Suína/fisiologia , Doenças dos Suínos/genética , Doenças dos Suínos/virologia , Replicação Viral , Adenosina/metabolismo , Animais , Linhagem Celular , Chlorocebus aethiops , Genoma Viral , Metilação , Vírus da Diarreia Epidêmica Suína/ultraestrutura , Ligação Proteica , RNA Viral , Proteínas de Ligação a RNA/metabolismo , Suínos , Células Vero
11.
Mol Cell ; 79(3): 361-362, 2020 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-32763222

RESUMO

In this issue of Molecular Cell, Zhang et al. (2020) reveal that ATM triggers RNA methylation of DNA-RNA hybrids formed at double-strand breaks (DSBs) to modulate repair, adding a new layer of complexity to RNA's role in the DNA damage response.


Assuntos
Quebras de DNA de Cadeia Dupla , RNA , Adenosina/análogos & derivados , Proteínas Mutadas de Ataxia Telangiectasia , DNA , Reparo do DNA , Metilação
12.
Ecotoxicol Environ Saf ; 205: 111089, 2020 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-32810645

RESUMO

Early molecular events after the exposure of heavy metals, such as aberrant DNA methylation, suggest that DNA methylation was important in regulating physiological processes for animals and accordingly could be used as environmental biomarkers. In the present study, we found that copper (Cu) exposure increased lipid content and induced the DNA hypermethylation at the whole genome level. Especially, Cu induced hypermethylation of glucose-regulated protein 78 (grp78) and peroxisome proliferator-activated receptor gamma coactivator-1α (pgc1α). CCAAT/enhancer binding protein α (C/EBPα) could bind to the methylated sequence of grp78, whereas C/EBPß could not bind to the methylated sequence of grp78. These synergistically influenced grp78 expression and increased lipogenesis. In contrast, DNA methylation of PGC1α blocked the specific protein 1 (SP1) binding and interfered mitochondrial function. Moreover, Cu increased reactive oxygen species (ROS) production, activated endoplasmic reticulum (ER) stress and damaged mitochondrial function, and accordingly increased lipid deposition. Notably, we found a new toxicological mechanism for Cu-induced lipid deposition at DNA methylation level. The measurement of DNA methylation facilitated the use of these epigenetic biomarkers for the evaluation of environmental risk.


Assuntos
Carpas/fisiologia , Cobre/toxicidade , Poluentes Químicos da Água/toxicidade , Animais , Carpas/metabolismo , Cobre/metabolismo , Estresse do Retículo Endoplasmático , Glucose/metabolismo , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Lipídeos , Metilação , Mitocôndrias/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Ativação Transcricional , Regulação para Cima
13.
Nat Commun ; 11(1): 4092, 2020 08 14.
Artigo em Inglês | MEDLINE | ID: mdl-32796837

RESUMO

Single nucleotide polymorphisms (SNPs) in the gene encoding kinesin family member 3A, KIF3A, have been associated with atopic dermatitis (AD), a chronic inflammatory skin disorder. We find that KIF3A SNP rs11740584 and rs2299007 risk alleles create cytosine-phosphate-guanine sites, which are highly methylated and result in lower KIF3A expression, and this methylation is associated with increased transepidermal water loss (TEWL) in risk allele carriers. Kif3aK14∆/∆ mice have increased TEWL, disrupted junctional proteins, and increased susceptibility to develop AD. Thus, KIF3A is required for skin barrier homeostasis whereby decreased KIF3A skin expression causes disrupted skin barrier function and promotes development of AD.


Assuntos
Dermatite Atópica/metabolismo , Cinesina/metabolismo , Pele/metabolismo , Adolescente , Adulto , Alelos , Animais , Criança , Dermatite Atópica/genética , Dermatite Atópica/patologia , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Cinesina/genética , Masculino , Metilação , Camundongos , Reação em Cadeia da Polimerase em Tempo Real , Pele/patologia , Adulto Jovem
14.
Life Sci ; 259: 118169, 2020 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-32738360

RESUMO

AIMS: The recent outbreak of pandemic severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has led the world towards a global health emergency. Currently, no proper medicine or effective treatment strategies are available; therefore, repurposing of FDA approved drugs may play an important role in overcoming the situation. MATERIALS AND METHODS: The SARS-CoV-2 genome encodes for 2-O-methyltransferase (2'OMTase), which plays a key role in methylation of viral RNA for evading host immune system. In the present study, the protein sequence of 2'OMTase of SARS-CoV-2 was analyzed, and its structure was modeled by a comparative modeling approach and validated. The library of 3000 drugs was screened against the active site of 2'OMTase followed by re-docking analysis. The apo and ligand-bound 2'OMTase were further validated and analyzed by using molecular dynamics simulation. KEY FINDINGS: The modeled structure displayed the conserved characteristic fold of class I MTase family. The quality assessment analysis by SAVES server reveals that the modeled structure follows protein folding rules and of excellent quality. The docking analysis displayed that the active site of 2'OMTase accommodates an array of drugs, which includes alkaloids, antivirals, cardiac glycosides, anticancer, steroids, and other drugs. The redocking and MD simulation analysis of the best 5 FDA approved drugs reveals that these drugs form a stable conformation with the 2'OMTase. SIGNIFICANCE: The results suggested that these drugs may be used as potential inhibitors for 2'OMTase for combating the SARS-CoV-2 infection.


Assuntos
Betacoronavirus/efeitos dos fármacos , Betacoronavirus/enzimologia , Infecções por Coronavirus/tratamento farmacológico , Metiltransferases/antagonistas & inibidores , Pneumonia Viral/tratamento farmacológico , Antivirais/química , Antivirais/farmacologia , Biologia Computacional/métodos , Infecções por Coronavirus/virologia , Reposicionamento de Medicamentos/métodos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Humanos , Metilação/efeitos dos fármacos , Metiltransferases/química , Metiltransferases/metabolismo , Metiltransferases/ultraestrutura , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Terapia de Alvo Molecular , Pandemias , Pneumonia Viral/virologia , Homologia de Sequência de Aminoácidos
15.
Free Radic Res ; 54(7): 540-555, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32842802

RESUMO

Radiotherapy is an important treatment regime for lung cancer, worldwide. However, radiation-induced pneumonitis and fibrosis are the treatment-limiting toxicities among patients who have undergone radiotherapy. The epithelial cells via epithelial to mesenchymal transition [EMT] acquires mesenchymal phenotype, which ultimately leads to fibrosis. Many investigations are focussed on understanding the signalling pathways mediating in EMT, however, the role of histone methylation is less understood in radiation-induced lung EMT. In the present study, we analysed the effect of vanillin, an antioxidant, on histone methylation during radiation-induced EMT. The thoracic region of Wistar rats was irradiated with a fractionated dose of X-ray (3 Gy/day) for two weeks (total of 30 Gy). The irradiated animals were sacrificed at the 8th and 16th weeks and tissues were used for analyses. Our data showed that radiation decreased the level of antioxidant enzymes such as SOD, catalase and reduced glutathione that would ultimately enhance oxidative stress in the tissues. Histopathological analysis revealed that radiation increased the infiltration of inflammatory cells to the tissue injury site. Total global histone methylation was increased upon irradiation, which was effectively prevented by vanillin administration. Vanillin enhanced E-cadherin expression and decreased the mesenchymal markers N-cadherin and vimentin in the irradiated lung tissue. The ChIP-qPCR analysis suggested that snail expression in the nucleus might involve in the enrichment of suppressive marker H3K9me3 on the E-cadherin promoter. Finally, we suggested that vanillin administration decreased radiation-induced oxidative stress and EMT expression. Additionally, irradiation increased the H3K9 methylation status with nuclear translocation of snail during lung EMT.


Assuntos
Antígenos CD/metabolismo , Benzaldeídos/metabolismo , Caderinas/metabolismo , Histonas/metabolismo , Pulmão/efeitos da radiação , Células A549 , Animais , Antígenos CD/genética , Caderinas/genética , Transição Epitelial-Mesenquimal , Feminino , Humanos , Pulmão/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/radioterapia , Metilação/efeitos da radiação , Estresse Oxidativo/efeitos da radiação , Regiões Promotoras Genéticas , Ratos , Ratos Wistar
16.
Nat Cell Biol ; 22(7): 767-778, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32601371

RESUMO

Following fertilization in mammals, the gametes are reprogrammed to create a totipotent zygote, a process that involves de novo establishment of chromatin domains. A major feature occurring during preimplantation development is the dramatic remodelling of constitutive heterochromatin, although the functional relevance of this is unknown. Here, we show that heterochromatin establishment relies on the stepwise expression and regulated activity of SUV39H enzymes. Enforcing precocious acquisition of constitutive heterochromatin results in compromised development and epigenetic reprogramming, which demonstrates that heterochromatin remodelling is essential for natural reprogramming at fertilization. We find that de novo H3K9 trimethylation (H3K9me3) in the paternal pronucleus after fertilization is catalysed by SUV39H2 and that pericentromeric RNAs inhibit SUV39H2 activity and reduce H3K9me3. De novo H3K9me3 is initially non-repressive for gene expression, but instead bookmarks promoters for compaction. Overall, we uncover the functional importance for the restricted transmission of constitutive heterochromatin during reprogramming and a non-repressive role for H3K9me3.


Assuntos
Centrômero/genética , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Desenvolvimento Embrionário , Heterocromatina/metabolismo , Histonas/metabolismo , RNA/metabolismo , Animais , Núcleo Celular/genética , Núcleo Celular/metabolismo , Epigênese Genética , Feminino , Heterocromatina/genética , Histonas/genética , Masculino , Metilação , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , RNA/genética
17.
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
18.
Nat Commun ; 11(1): 3326, 2020 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-32620791

RESUMO

Tumour cells adapt to nutrient deprivation in vivo, yet strategies targeting the nutrient poor microenvironment remain unexplored. In melanoma, tumour cells often experience low glutamine levels, which promote cell dedifferentiation. Here, we show that dietary glutamine supplementation significantly inhibits melanoma tumour growth, prolongs survival in a transgenic melanoma mouse model, and increases sensitivity to a BRAF inhibitor. Metabolomic analysis reveals that dietary uptake of glutamine effectively increases the concentration of glutamine in tumours and its downstream metabolite, αKG, without increasing biosynthetic intermediates necessary for cell proliferation. Mechanistically, we find that glutamine supplementation uniformly alters the transcriptome in tumours. Our data further demonstrate that increase in intra-tumoural αKG concentration drives hypomethylation of H3K4me3, thereby suppressing epigenetically-activated oncogenic pathways in melanoma. Therefore, our findings provide evidence that glutamine supplementation can serve as a potential dietary intervention to block melanoma tumour growth and sensitize tumours to targeted therapy via epigenetic reprogramming.


Assuntos
Proliferação de Células/efeitos dos fármacos , Suplementos Nutricionais , Epigênese Genética/efeitos dos fármacos , Glutamina/farmacologia , Melanoma/prevenção & controle , Transdução de Sinais/efeitos dos fármacos , Animais , Linhagem Celular Tumoral , Proliferação de Células/genética , Epigênese Genética/genética , Glutamina/administração & dosagem , Histonas/metabolismo , Humanos , Lisina/metabolismo , Masculino , Melanoma/genética , Melanoma/patologia , Metilação/efeitos dos fármacos , Camundongos Nus , Transdução de Sinais/genética , Transcriptoma/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto/métodos
19.
Food Chem ; 332: 127366, 2020 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-32619940

RESUMO

High methyl-esterified citrus pectin (HMCP) molecules could be self-assembled into micelles in water. The morphology of HMCP micelles in water was irregular spheres, long rods, and arc-shaped. Most of HMCP molecules cross-linked with HMCP micelles in the presence of calcium chloride and increased the range of size distribution of HMCP micelles. A little number of HMCP molecules cross-linked with each other to form 80 nn ~ 200 nm microgel particles. Calcium chloride could improve HMCP emulsification when its concentration was more than 70 mmol/L. HMCP micelles could be adsorbed on the surface of emulsion droplets. The emulsion prepared with HMCP and calcium chloride was similar to the Pickering emulsion.


Assuntos
Cloreto de Cálcio/química , Pectinas/química , Emulsões , Esterificação , Metilação , Micelas , Água/química
20.
PLoS Biol ; 18(7): e3000782, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32692742

RESUMO

Tight regulation of gene transcription and mRNA splicing is essential for plant growth and development. Here we demonstrate that a plant-specific protein, EMBRYO DEFECTIVE 1579 (EMB1579), controls multiple growth and developmental processes in Arabidopsis. We demonstrate that EMB1579 forms liquid-like condensates both in vitro and in vivo, and the formation of normal-sized EMB1579 condensates is crucial for its cellular functions. We found that some chromosomal and RNA-related proteins interact with EMB1579 compartments, and loss of function of EMB1579 affects global gene transcription and mRNA splicing. Using floral transition as a physiological process, we demonstrate that EMB1579 is involved in FLOWERING LOCUS C (FLC)-mediated repression of flowering. Interestingly, we found that EMB1579 physically interacts with a homologue of Drosophila nucleosome remodeling factor 55-kDa (p55) called MULTIPLE SUPPRESSOR OF IRA 4 (MSI4), which has been implicated in repressing the expression of FLC by forming a complex with DNA Damage Binding Protein 1 (DDB1) and Cullin 4 (CUL4). This complex, named CUL4-DDB1MSI4, physically associates with a CURLY LEAF (CLF)-containing Polycomb Repressive Complex 2 (CLF-PRC2). We further demonstrate that EMB1579 interacts with CUL4 and DDB1, and EMB1579 condensates can recruit and condense MSI4 and DDB1. Furthermore, emb1579 phenocopies msi4 in terms of the level of H3K27 trimethylation on FLC. This allows us to propose that EMB1579 condensates recruit and condense CUL4-DDB1MSI4 complex, which facilitates the interaction of CUL4-DDB1MSI4 with CLF-PRC2 and promotes the role of CLF-PRC2 in establishing and/or maintaining the level of H3K27 trimethylation on FLC. Thus, we report a new mechanism for regulating plant gene transcription, mRNA splicing, and growth and development.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/genética , Proteínas de Ligação ao Cálcio/metabolismo , Desenvolvimento Vegetal/genética , Processamento de RNA/genética , Transcrição Genética , Proteínas de Arabidopsis/genética , Proteínas de Ligação ao Cálcio/genética , Núcleo Celular/metabolismo , Flores/fisiologia , Histonas/metabolismo , Mutação com Perda de Função , Lisina/metabolismo , Metilação , Proteínas Nucleares/metabolismo , Fenótipo , Raízes de Plantas/citologia , Ligação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Sequências Repetitivas de Aminoácidos
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