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1.
Ann Lab Med ; 42(1): 79-88, 2022 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-34374352

RESUMO

Background: Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are genomic imprinting disorders that are mainly caused by a deletion on 15q11-q13, the uniparental disomy of chromosome 15, or an imprinting defect. We evaluated the utility of methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) as a diagnostic tool and for demonstrating the relationship between molecular mechanisms and clinical presentation. Methods: We performed MS-MLPA using DNA samples from 93 subjects (45 PWS, 24 AS, and 24 non-PWS/AS controls) who had previously undergone MS-PCR for the diagnosis of PWS/AS. We compared the results of both assays, and patients' clinical phenotypes were reviewed retrospectively. Results: MS-MLPA showed a 100% concordance rate with MS-PCR. Among the 45 PWS patients, 26 (57.8%) had a deletion of 15q11-q13, and the others (42.2%) had uniparental disomy 15 or an imprinting defect. Among the 24 AS patients, 16 (66.7%) had a deletion of 15q11-q13, 7 AS patients (29.2%) had uniparental disomy 15 or an imprinting defect, and one AS patient (4.2%) showed an imprinting center deletion. Conclusions: MS-MLPA has clinical utility for the diagnosis of PWS/AS, and it is superior to MS-PCR in that it can identify the molecular mechanism underlying the disease.


Assuntos
Síndrome de Angelman , Síndrome de Prader-Willi , Síndrome de Angelman/diagnóstico , Síndrome de Angelman/genética , Cromossomos Humanos Par 15/genética , Metilação de DNA , Humanos , Metilação , Reação em Cadeia da Polimerase Multiplex , Síndrome de Prader-Willi/diagnóstico , Síndrome de Prader-Willi/genética , Estudos Retrospectivos
2.
Enzyme Microb Technol ; 150: 109862, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34489021

RESUMO

Glycosylation and methylation of flavonoids are the main types of structural modifications and can endow flavonoids with greater stability, bioactivity, and bioavailability. In this study, five types of O-methyltransferases were screened for producing O-methylated luteolin, and the biosynthesis strategy of 3'-O-methylisoorientin from luteolin was determined. To improve the production of 3'-O-methylluteolin, the S-adenosyl-l-methionine synthesis pathway was reconstructed in the recombinant strain by introducing S-adenosyl-l-methionine synthetase genes. After optimizing the conversion conditions, maximal 3'-O-methylluteolin production reached 641 ± 25 mg/L with a corresponding molar conversion of 76.5 %, which was the highest titer of methylated flavonoids reported to date in Escherichia coli. 3'-O-Methylluteolin (127 mg) was prepared from 250 mL of the broth by silica gel column chromatography and preparative HPLC with a yield of 79.4 %. Subsequently, we used the biocatalytic cascade of Gentiana triflora C-glycosyltransferase (Gt6CGT) and Glycine max sucrose synthase (GmSUS) to biosynthesize 3'-O-methylisoorientin from 3'-O-methylluteolin in vitro. By optimizing the coupled reaction conditions and using the fed-batch operation, maximal 3'-O-methylisoorientin production reached 226 ± 8 mg/L with a corresponding molar conversion of 98 %. Therefore, this study provides an efficient method for the production of novel 3'-O-methylisoorientin and the biosynthesis strategy for methylated C-glycosylation flavonoids by selective O-methylation/C-glycosylation motif on flavonoids.


Assuntos
Flavonoides , Luteolina , Glicosilação , Metilação , Metiltransferases/metabolismo
3.
Int J Mol Sci ; 22(16)2021 Aug 09.
Artigo em Inglês | MEDLINE | ID: mdl-34445278

RESUMO

Epigenetics play a vital role in early embryo development. Offspring conceived via assisted reproductive technologies (ARTs) have a three times higher risk of epigenetic diseases than naturally conceived children. However, investigations into ART-associated placental histone modifications or sex-stratified analyses of ART-associated histone modifications remain limited. In the current study, we carried out immunohistochemistry, chip-sequence analysis, and a series of in vitro experiments. Our results demonstrated that placentas from intra-cytoplasmic sperm injection (ICSI), but not in vitro fertilization (IVF), showed global tri-methylated-histone-H3-lysine-4 (H3K4me3) alteration compared to those from natural conception. However, for acetylated-histone-H3-lysine-9 (H3K9ac) and acetylated-histone-H3-lysine-27 (H3K27ac), no significant differences between groups could be found. Further, sex -stratified analysis found that, compared with the same-gender newborn cord blood mononuclear cell (CBMC) from natural conceptions, CBMC from ICSI-boys presented more genes with differentially enriched H3K4me3 (n = 198) than those from ICSI-girls (n = 79), IVF-girls (n = 5), and IVF-boys (n = 2). We also found that varying oxygen conditions, RNA polymerase II subunit A (Polr2A), and lysine demethylase 5A (KDM5A) regulated H3K4me3. These findings revealed a difference between IVF and ICSI and a difference between boys and girls in H3K4me3 modification, providing greater insight into ART-associated epigenetic alteration.


Assuntos
RNA Polimerases Dirigidas por DNA/metabolismo , Epigênese Genética , Histonas/metabolismo , Proteína 2 de Ligação ao Retinoblastoma/metabolismo , Caracteres Sexuais , Injeções de Esperma Intracitoplásmicas , Adulto , Feminino , Humanos , Recém-Nascido , Masculino , Metilação , Gravidez
4.
Mol Cell ; 81(16): 3323-3338.e14, 2021 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-34352207

RESUMO

The emerging "epitranscriptomics" field is providing insights into the biological and pathological roles of different RNA modifications. The RNA methyltransferase METTL1 catalyzes N7-methylguanosine (m7G) modification of tRNAs. Here we find METTL1 is frequently amplified and overexpressed in cancers and is associated with poor patient survival. METTL1 depletion causes decreased abundance of m7G-modified tRNAs and altered cell cycle and inhibits oncogenicity. Conversely, METTL1 overexpression induces oncogenic cell transformation and cancer. Mechanistically, we find increased abundance of m7G-modified tRNAs, in particular Arg-TCT-4-1, and increased translation of mRNAs, including cell cycle regulators that are enriched in the corresponding AGA codon. Accordingly, Arg-TCT expression is elevated in many tumor types and is associated with patient survival, and strikingly, overexpression of this individual tRNA induces oncogenic transformation. Thus, METTL1-mediated tRNA modification drives oncogenic transformation through a remodeling of the mRNA "translatome" to increase expression of growth-promoting proteins and represents a promising anti-cancer target.


Assuntos
Carcinogênese/genética , Metiltransferases/genética , Neoplasias/genética , tRNA Metiltransferases/genética , Guanosina/análogos & derivados , Guanosina/genética , Humanos , Metilação , Neoplasias/patologia , Oncogenes/genética , Processamento Pós-Transcricional do RNA/genética , RNA Mensageiro/genética , RNA de Transferência/genética
5.
Mol Cell ; 81(16): 3368-3385.e9, 2021 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-34375583

RESUMO

The mechanistic understanding of nascent RNAs in transcriptional control remains limited. Here, by a high sensitivity method methylation-inscribed nascent transcripts sequencing (MINT-seq), we characterized the landscapes of N6-methyladenosine (m6A) on nascent RNAs. We uncover heavy but selective m6A deposition on nascent RNAs produced by transcription regulatory elements, including promoter upstream antisense RNAs and enhancer RNAs (eRNAs), which positively correlates with their length, inclusion of m6A motif, and RNA abundances. m6A-eRNAs mark highly active enhancers, where they recruit nuclear m6A reader YTHDC1 to phase separate into liquid-like condensates, in a manner dependent on its C terminus intrinsically disordered region and arginine residues. The m6A-eRNA/YTHDC1 condensate co-mixes with and facilitates the formation of BRD4 coactivator condensate. Consequently, YTHDC1 depletion diminished BRD4 condensate and its recruitment to enhancers, resulting in inhibited enhancer and gene activation. We propose that chemical modifications of eRNAs together with reader proteins play broad roles in enhancer activation and gene transcriptional control.


Assuntos
Adenosina/análogos & derivados , Proteínas de Ciclo Celular/genética , Proteínas do Tecido Nervoso/genética , Fatores de Processamento de RNA/genética , RNA/genética , Fatores de Transcrição/genética , Adenosina/genética , Elementos Facilitadores Genéticos/genética , Regulação da Expressão Gênica/genética , Humanos , Metilação , Elementos Reguladores de Transcrição/genética , Ativação Transcricional/genética
6.
Biol Res ; 54(1): 25, 2021 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-34362460

RESUMO

BACKGROUND: Peroxisome proliferator-activated receptor alpha (PPARα) is associated with diabetic retinopathy (DR), and the underlying mechanism is still unclear. Aim of this work was to investigate the mechanism of PPARα in DR. METHODS: Human retinal capillary pericytes (HRCPs) were treated with high glucose (HG) to induce DR cell model. DR mouse model was established by streptozotocin injection, and then received 5-Aza-2-deoxycytidine (DAC; DNA methyltransferase inhibitor) treatment. Hematoxylin-eosin staining was performed to assess retinal tissue damage. PPARα methylation was examined by Methylation-Specific PCR. Flow cytometry and DCFH-DA fluorescent probe was used to estimate apoptosis and reactive oxygen species (ROS). The interaction between DNA methyltransferase-1 (DNMT1) and PPARα promoter was examined by Chromatin Immunoprecipitation. Quantitative real-time PCR and western blot were performed to assess gene and protein expression. RESULTS: HG treatment enhanced the methylation levels of PPARα, and repressed PPARα expression in HRCPs. The levels of apoptotic cells and ROS were significantly increased in HRCPs in the presence of HG. Moreover, DNMT1 was highly expressed in HG-treated HRCPs, and DNMT1 interacted with PPARα promoter. PPARα overexpression suppressed apoptosis and ROS levels of HRCPs, which was rescued by DNMT1 up-regulation. In DR mice, DAC treatment inhibited PPARα methylation and reduced damage of retinal tissues. CONCLUSION: DNMT1-mediated PPARα methylation promotes apoptosis and ROS levels of HRCPs and aggravates damage of retinal tissues in DR mice. Thus, this study may highlight novel insights into DR pathogenesis.


Assuntos
DNA (Citosina-5-)-Metiltransferase 1/metabolismo , Retinopatia Diabética , PPAR alfa/genética , Retina/patologia , Animais , Apoptose , Células Cultivadas , Metilação de DNA , Diabetes Mellitus , Modelos Animais de Doenças , Humanos , Metilação , Camundongos , Regiões Promotoras Genéticas , Retina/citologia
7.
Int J Mol Sci ; 22(15)2021 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-34360892

RESUMO

The explosive development of next-generation sequencing-based technologies has allowed us to take an unprecedented look at many molecular signatures of the non-coding genome. In particular, the ChIP-seq (Chromatin ImmunoPrecipitation followed by sequencing) technique is now very commonly used to assess the proteins associated with different non-coding DNA regions genome-wide. While the analysis of such data related to transcription factor binding is relatively straightforward, many modified histone variants, such as H3K27me3, are very important for the process of gene regulation but are very difficult to interpret. We propose a novel method, called HERON (HiddEn MaRkov mOdel based peak calliNg), for genome-wide data analysis that is able to detect DNA regions enriched for a certain feature, even in difficult settings of weakly enriched long DNA domains. We demonstrate the performance of our method both on simulated and experimental data.


Assuntos
Sequenciamento de Cromatina por Imunoprecipitação/métodos , DNA/genética , DNA/metabolismo , Genoma Humano , Histonas/genética , Histonas/metabolismo , Adulto , Algoritmos , Expressão Gênica , Regulação da Expressão Gênica , Hipocampo/embriologia , Hipocampo/metabolismo , Código das Histonas/genética , Humanos , Fígado/metabolismo , Metilação , Distribuição Normal , Ligação Proteica
8.
Theriogenology ; 174: 85-93, 2021 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-34425304

RESUMO

The present study aims to investigate the expression and function of lysine-specific demethylase 4B (KDM4B) in yak cumulus cells (CCs) in order to reveal the mechanisms by which KDM4B regulates biological characteristics and function of CCs. The cellular location of KDM4B and the methylation pattern of H3K9 were detected using immunofluorescence (IF) staining in CCs. The mRNA expression levels of apoptosis-related genes (BCL-2, HAX1 and BAX) and genes related to the estrogen pathway (ESR2, CYP17 and 3B-HSD) were estimated by qRT-PCR after knockdown of KDM4B expression by siRNA in yak CCs. Then, a proliferation assay, Annexin V-FITC staining, and ELISA were utilized to explore the effects of KDM4B silencing on CCs proliferation, apoptosis, and estrogen (E2) secretion, respectively. The results showed that KDM4B is located in the nuclei of yak CCs and is distributed in a dotted pattern. Knockdown KDM4B induced a decrease in cell proliferation, an increase in apoptotic rate and a reduction in the levels of E2 secretion of CCs. Additionally, the methylation patterns of H3K9me2 and H3K9me3 were significantly increased in CCs transfected with KDM4B siRNA-1 (P < 0.05). The mRNA expression level of apoptosis promoting BAX genes was significantly upregulated, but 3B-HSD, ESR2 and anti-apoptotic HAX1 genes were significantly downregulated in transfected CCs (P < 0.05). Furthermore, the rate of embryos developing from the 2-cell stage to blastocysts was lower in the siRNA-1 transfection group than that of the control group (28.6 ± 2.9% vs 40.4 ± 2.4%, P < 0.05). In conclusion, our study indicates that KDM4B regulates the biological characteristics and physiological function of yak CCs mainly through changing the methylation patterns of H3K9 and related gene expression levels.


Assuntos
Células do Cúmulo , Histona Desmetilases com o Domínio Jumonji , Animais , Blastocisto/metabolismo , Bovinos , Células do Cúmulo/metabolismo , Feminino , Histona Desmetilases com o Domínio Jumonji/genética , Histona Desmetilases com o Domínio Jumonji/metabolismo , Metilação , Processamento de Proteína Pós-Traducional
9.
Medicine (Baltimore) ; 100(29): e26648, 2021 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-34398021

RESUMO

BACKGROUND: Endometrial carcinoma (EC) has become a common gynecologic malignancy with a high mortality. The m6A regulators have been identified to be closely associated with multiple human cancers including EC. However, the CpG methylation signature related to m6A regulators in EC remains unclear. METHOD: The methylation profiles of EC patients including cancer samples and adjacent normal samples were obtained from The Cancer Genome Atlas (TCGA) database. The CpG sites in 20 m6A regulators were identified. Univariate Cox regression and LASSO Cox regression analysis were used to screen key CpG sites which were located at m6A regulators and significantly related to the prognosis of EC. The predictive model for EC prognosis was constructed, and multivariate Cox regression analysis was applied to explore whether the risk score derived from the model could function as an independent signature for EC prognosis. Meanwhile, a nomogram model was constructed by combing the independent prognostic signatures for prediction of the long-term survival in EC patients. RESULTS: A total of 396 CpG sites located at 20 m6A regulators were identified. A specific predictive model for EC prognosis based on 7 optimal CpG sites was constructed, which presented good performance in prognosis prediction of EC patients. Moreover, risk score was determined to be an independent signature both in the training set and validation set. By bringing in three independent prognostic factors (age, risk score, and TNM stage), the nomogram was constructed and could effectively predict the 3- and 5-year survival rates of EC patients. CONCLUSION: Our study suggested that the CpG sites located at m6A regulators might be considered as potential prognostic signatures for EC patients.


Assuntos
Adenosina/análogos & derivados , Neoplasias do Endométrio/mortalidade , Adenosina/genética , Adenosina/metabolismo , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , China , Neoplasias do Endométrio/genética , Neoplasias do Endométrio/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Metilação , Pessoa de Meia-Idade , Nomogramas , Valor Preditivo dos Testes , Prognóstico , Modelos de Riscos Proporcionais , RNA/genética , Análise de Sobrevida
10.
World J Surg Oncol ; 19(1): 241, 2021 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-34389000

RESUMO

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most common malignancies in the world, and due to its complex pathogenic factors, its prognosis is poor. N6-methyladenosine (m6A) RNA methylation plays an important role in the tumorigenesis, progression, and prognosis of many tumors. The m6A RNA methylation regulator small nuclear ribonucleoprotein polypeptide C (SNRPC), which encodes one of the specific protein components of the U1 small nuclear ribonucleoprotein (snRNP) particle, has been proven to be related to the prognosis of patients with HCC. However, the effect of SNRPC on the tumor microenvironment and immunotherapy in HCC remains unclear. CASE PRESENTATION: The HCC RNA-seq profiles in The Cancer Genome Atlas (TCGA) and International Cancer Genome Consortium (ICGC) databases, including 421 LIHC and 440 LIRI-JP samples, respectively, were used in this study. Both the expression of SNRPC in HCC was upregulated in the TCGA and ICGC databases compared to normal tissues. Next, the expression of SNRPC was validated as a risk factor for prognosis by Kaplan-Meier analysis and employed to establish a nomogram with T pathologic stage. By gene set variation (GSVA) analysis and gene set enrichment (GSEA) analysis, we found that SNRPC was mainly related to protein metabolism and the immune process. Furthermore, the estimation of stromal and immune cells in malignant tumor tissues using expression (ESTIMATE), microenvironment cell population counter (MCP-counter), and single sample GSEA (ssGSEA) algorithms revealed that the high-SNRPC group had a lower stromal score, lower abundance of endothelial cells and fibroblasts, and lower immune infiltration. Ultimately, a tumor immune dysfunction and exclusion (TIDE) analysis revealed that patients in the low-SNRPC group may be more sensitive to immune checkpoint inhibitor therapy. CONCLUSION: SNRPC could serve as a promising prognostic and immunotherapeutic marker in HCC and might contribute to new directions and strategies for HCC treatment.


Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Adenosina/análogos & derivados , Biomarcadores Tumorais/genética , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/terapia , Células Endoteliais , Regulação Neoplásica da Expressão Gênica , Humanos , Imunoterapia , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/terapia , Metilação , Prognóstico , RNA/genética , Microambiente Tumoral
11.
Environ Pollut ; 285: 117509, 2021 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-34380217

RESUMO

Humans benefit from nuclear technologies but consequently experience nuclear disasters or side effects of iatrogenic radiation. Hematopoietic system injury first arises upon radiation exposure. As an intricate new layer of genetic control, the posttranscriptional m6A modification of RNA has recently come under investigation and has been demonstrated to play pivotal roles in multiple physiological and pathological processes. However, how the m6A methylome functions in the hematopoietic system after irradiation remains ambiguous. Here, we uncovered the time-varying epitranscriptome-wide m6A methylome and transcriptome alterations in γ-ray-exposed mouse bone marrow. 4 Gy γ-irradiation rapidly (5 min and 2 h) and severely impaired the mouse hematopoietic system, including spleen and thymus weight, blood components, tissue inflammation and malondialdehyde (MDA) levels. The m6A content and expression of m6A related enzymes were altered. Gamma-irradiation triggered dynamic and reversible m6A modification profiles and altered mRNA expression, where both m6A fold-enrichment and mRNA expression most followed the (5 min_up/2 h_down) pattern. The CDS enrichment region preferentially upregulated m6A peaks at 5 min. Moreover, the main GO and KEGG pathways were closely related to metabolism and the classical radiation response. Finally, m6A modifications correlated with transcriptional regulation of genes in multiple aspects. Blocking the expression of m6A demethylases FTO and ALKBH5 mitigated radiation hematopoietic toxicity. Together, our findings present the comprehensive landscape of mRNA m6A methylation in the mouse hematopoietic system in response to γ-irradiation, shedding light on the significance of m6A modifications in mammalian radiobiology. Regulation of the epitranscriptome may be exploited as a strategy against radiation damage.


Assuntos
Medula Óssea , Sistema Hematopoético , Adenosina/análogos & derivados , Animais , Sistema Hematopoético/metabolismo , Metilação , Camundongos , RNA Mensageiro/metabolismo
12.
Molecules ; 26(15)2021 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-34361784

RESUMO

The field of gas chromatography-mass spectrometry (GC-MS) in the analysis of chemical warfare agents (CWAs), specifically those involving the organophosphorus-based nerve agents (OPNAs), is a continually evolving and dynamic area of research. The ever-present interest in this field within analytical chemistry is driven by the constant threat posed by these lethal CWAs, highlighted by their use during the Tokyo subway attack in 1995, their deliberate use on civilians in Syria in 2013, and their use in the poisoning of Sergei and Yulia Skripal in Great Britain in 2018 and Alexei Navalny in 2020. These events coupled with their potential for mass destruction only serve to stress the importance of developing methods for their rapid and unambiguous detection. Although the direct detection of OPNAs is possible by GC-MS, in most instances, the analytical chemist must rely on the detection of the products arising from their degradation. To this end, derivatization reactions mainly in the form of silylations and alkylations employing a vast array of reagents have played a pivotal role in the efficient detection of these products that can be used retrospectively to identify the original OPNA.


Assuntos
Agentes Neurotóxicos/análise , Organofosfatos/análise , Compostos Organofosforados/análise , Compostos Organotiofosforados/análise , Sarina/análise , Soman/análise , Alquilação , Fluorbenzenos/química , Cromatografia Gasosa-Espectrometria de Massas/métodos , Humanos , Hidrólise , Metilação , Agentes Neurotóxicos/química , Organofosfatos/química , Compostos Organofosforados/química , Compostos Organotiofosforados/química , Sarina/química , Soman/química
13.
Front Cell Infect Microbiol ; 11: 676456, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34381738

RESUMO

Mycobacterium tuberculosis (Mtb) inhibits autophagy to promote its survival in host cells. However, the molecular mechanisms by which Mtb inhibits autophagy are poorly understood. Here, we report a previously unknown mechanism in which Mtb phosphoribosyltransferase (MtbPRT) inhibits autophagy in an mTOR, negative regulator of autophagy, independent manner by inducing histone hypermethylation (H3K9me2/3) at the Atg5 and Atg7 promoters by activating p38-MAPK- and EHMT2 methyltransferase-dependent signaling pathways. Additionally, we find that MtbPRT induces EZH2 methyltransferase-dependent H3K27me3 hypermethylation and reduces histone acetylation modifications (H3K9ac and H3K27ac) by upregulating histone deacetylase 3 to inhibit autophagy. In summary, this is the first demonstration that Mtb inhibits autophagy by inducing histone hypermethylation in autophagy-related genes to promote intracellular bacterial survival.


Assuntos
Histonas , Macrófagos/microbiologia , Mycobacterium tuberculosis , Pentosiltransferases/metabolismo , Autofagia , Histonas/metabolismo , Macrófagos/metabolismo , Metilação , Mycobacterium tuberculosis/enzimologia , Mycobacterium tuberculosis/genética , Transdução de Sinais
14.
Medicine (Baltimore) ; 100(31): e26747, 2021 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-34397817

RESUMO

OBJECTIVE: The incidence of non-suicidal self-injury (NSSI) behavior in adolescents is increasing year by year. Patients with a history of both depression and NSSI behavior tend to be at greater risk for suicide. At present, the mechanism of adolescent depressive disorder with NSSI behavior is not clear and still in research and exploration. The expression of the Silent Information Regulator 2 Related Enzyme 1 (SIRT1) gene is closely related to the level of serotonin in molecular mechanisms, and may be closely related to the occurrence and development of depressive disorder. This study aimed to explore the relationship between the SIRT1 gene and NSSI behaviors in adolescents with depressive disorder. METHODS: A total of 15 adolescent depressed patients with NSSI behavior and 15 healthy controls were enrolled in the study. Bisulfite Sequencing PCR (BSP) was used to test the methylation level of SIRT1 gene promoter region of the participants. The real-time fluorescent quantitative PCR was conducted to measure the mRNA expression level of SIRT1 gene. RESULTS: Our study found that the methylation level of SIRT1 gene promoter region at cytosine-guanine dinucleotide 5 (CpG5) site in depression group was higher than that of control group. Compared with that of control group, the plasma concentration of Sirt1 protein significantly decreased in depression group. CONCLUSION: Our study investigated the methylation level and the mRNA expression of SIRT1 gene in adolescent depressive patients with NSSI behavior. The study points towards finding an in vivo molecular marker for those adolescent patients.


Assuntos
Transtorno Depressivo/genética , Adolescente , Comportamento do Adolescente/psicologia , Distribuição de Qui-Quadrado , China/epidemiologia , Transtorno Depressivo/epidemiologia , Transtorno Depressivo/psicologia , Feminino , Voluntários Saudáveis , Humanos , Masculino , Metilação , RNA Mensageiro/genética , Fatores de Risco , Comportamento Autodestrutivo/epidemiologia , Comportamento Autodestrutivo/genética , Comportamento Autodestrutivo/psicologia , Sirtuína 1
15.
Nat Commun ; 12(1): 4778, 2021 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-34362929

RESUMO

N6,2'-O-dimethyladenosine (m6Am), a terminal modification adjacent to the mRNA cap, is a newly discovered reversible RNA modification. Yet, a specific and sensitive tool to directly map transcriptome-wide m6Am is lacking. Here, we report m6Am-seq, based on selective in vitro demethylation and RNA immunoprecipitation. m6Am-seq directly distinguishes m6Am and 5'-UTR N6-methyladenosine (m6A) and enables the identification of m6Am at single-base resolution and 5'-UTR m6A in the human transcriptome. Using m6Am-seq, we also find that m6Am and 5'-UTR m6A respond dynamically to stimuli, and identify key functional methylation sites that may facilitate cellular stress response. Collectively, m6Am-seq reveals the high-confidence m6Am and 5'-UTR m6A methylome and provides a robust tool for functional studies of the two epitranscriptomic marks.


Assuntos
Adenosina/análogos & derivados , Adenosina/metabolismo , Transcriptoma , Regiões 5' não Traduzidas , Fatores Ativadores da Transcrição , Adenosina/genética , Sequência de Bases , Células HEK293 , Humanos , Imunoprecipitação , Metilação , RNA Mensageiro/metabolismo
16.
Curr Protoc ; 1(8): e213, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34370893

RESUMO

Protein methyltransferases (PMTs) regulate many aspects of normal and disease processes through substrate methylation, with S-adenosyl-L-methionine (SAM) as a cofactor. It has been challenging to elucidate cellular protein lysine and arginine methylation because these modifications barely alter physical properties of target proteins and often are context dependent, transient, and substoichiometric. To reveal bona fide methylation events associated with specific PMT activities in native contexts, we developed the live-cell Bioorthogonal Profiling of Protein Methylation (lcBPPM) technology, in which the substrates of specific PMTs are labeled by engineered PMTs inside living cells, with in situ-synthesized SAM analogues as cofactors. The biorthogonality of this technology is achieved because these SAM analogue cofactors can only be processed by the engineered PMTs-and not native PMTs-to modify the substrates with distinct chemical groups. Here, we describe the latest lcBPPM protocol and its application to reveal proteome-wide methylation and validate specific methylation events. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Live-cell labeling of substrates of protein methyltransferases GLP1 and PRMT1 with lcBPPM-feasible enzymes and SAM analogue precursors Support Protocol: Gram-scale synthesis of Hey-Met Basic Protocol 2: Click labeling of lcBPPM cell lysates with a biotin-azide probe Alternate Protocol: Click labeling of small-scale lcBPPM cell lysates with a TAMRA-azide dye for in-gel fluorescence visualization Basic Protocol 3: Enrichment of biotinylated lcBPPM proteome with streptavidin beads Basic Protocol 4: Proteome-wide identification of lcBPPM targets with mass spectrometry Basic Protocol 5: Validation of individual lcBPPM targets by western blot.


Assuntos
Metionina , S-Adenosilmetionina , Humanos , Metilação , Proteínas Metiltransferases/metabolismo , Proteína-Arginina N-Metiltransferases/genética , Proteoma/metabolismo , Proteínas Repressoras , S-Adenosilmetionina/metabolismo
17.
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
18.
Zhonghua Yu Fang Yi Xue Za Zhi ; 55(8): 983-989, 2021 Aug 06.
Artigo em Chinês | MEDLINE | ID: mdl-34445837

RESUMO

Objective: To figure out the association between the expression of m6A RNA methylation regulators and the prognosis of children AML, and provide genetic markers for monitoring the progression and recurrence of AML. Methods: Twenty two m6A RNA methylation regulators were firstly analyzed using the data from Therapeutically Applicable Research To Generate Effective Treatments(TARGET) database and The Genotype-Tissue Expression(GTEx) database, Wilcoxon rank test was performed to analyze the differentially expression of m6A RNA methylation regulators between the AML and normal tissue, 296 AML children were divided into training cohort and validation cohort by simple random sampling method, Lasso regression was used to screen out the risk factors and the multivariate Cox regression was applied for establishing prognosis predicting model in training cohort. Kaplan-Meier survival curve, time-dependent ROC curve and multivariate Cox regression were used to estimate the efficiency of risk score calculated by predictive model in validation cohort. Results: Twenty one m6A genes were up regulated in AML compared to Normal patients. Five m6A RNA methylation regulators(ZC3H13, YTHDC2, HNRNPA2B1, METTL3, METTL5) were included in final predicting model. Risk score could independently predict the survival of AML patients in training cohort(HR:2.72, 95%CI: 1.54-4.81, P=0.000 6) and validation cohort(HR:2.01, 95%CI:1.14-3.50, P=0.016). Low-risk patients had better prognoses than high-risk patients both in training cohort(P=0.001 9) and validation cohort(P=0.023). Conclusion: This prognosis predicting model constructed by m6A RNA methylation regulators could independently predict the survival prognosis in AML children, and should be helpful for clinical therapy.


Assuntos
Leucemia Mieloide Aguda , Criança , Estudos de Coortes , Humanos , Leucemia Mieloide Aguda/genética , Metilação , RNA , RNA Helicases
19.
J Transl Med ; 19(1): 323, 2021 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-34330301

RESUMO

BACKGROUND: Pancreatic cancer is a fatal malignancy of the digestive system and the 5-year survival rate remains low. Therefore, new molecular therapeutic targets are required to improve treatments, prognosis, and the survival of patients. N6-methyladenosine (m6A) is the most prevalent reversible methylation in mammalian messenger RNA (mRNA) and has critical roles in the tumorigenesis and metastasis of various malignancies. However, the role of m6A in pancreatic cancer is still unclear. Exploring genetic alterations and functional networks of m6A regulators in pancreatic cancer may provide new strategies for its treatment. METHODS: In this study, we used data from the Cancer Genome Atlas (TCGA) database and other public databases through cBioPortal, LinkedOmics, UALCAN, GEPIA, STRING, and the database for annotation, visualization, and integrated discovery (DAVID) to systematically analyze the molecular alterations and functions of 20 main m6A regulators in pancreatic cancer. RESULTS: We found that m6A regulators had widespread genetic alterations, and that their expression levels were significantly correlated with pancreatic cancer malignancy. Moreover, m6A regulators were associated with the prognosis of pancreatic cancer patients. CONCLUSIONS: m6A regulators play a crucial part in the occurrence and development of pancreatic cancer. Our study will guide further studies of m6A RNA modification in pancreatic cancer and could potentially provide new strategies for pancreatic cancer treatment.


Assuntos
Regulação Neoplásica da Expressão Gênica , Neoplasias Pancreáticas , Animais , Mineração de Dados , Humanos , Metilação , Neoplasias Pancreáticas/genética , RNA Mensageiro/metabolismo
20.
mBio ; 12(4): e0106721, 2021 08 31.
Artigo em Inglês | MEDLINE | ID: mdl-34225491

RESUMO

The coronavirus disease 2019 pandemic caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is an ongoing global public crisis. Although viral RNA modification has been reported based on the transcriptome architecture, the types and functions of RNA modification are still unknown. In this study, we evaluated the roles of RNA N6-methyladenosine (m6A) modification in SARS-CoV-2. Our methylated RNA immunoprecipitation sequencing (MeRIP-Seq) and Nanopore direct RNA sequencing (DRS) analysis showed that SARS-CoV-2 RNA contained m6A modification. Moreover, SARS-CoV-2 infection not only increased the expression of methyltransferase-like 3 (METTL3) but also altered its distribution. Modification of METTL3 expression by short hairpin RNA or plasmid transfection for knockdown or overexpression, respectively, affected viral replication. Furthermore, the viral key protein RdRp interacted with METTL3, and METTL3 was distributed in both the nucleus and cytoplasm in the presence of RdRp. RdRp appeared to modulate the sumoylation and ubiquitination of METTL3 via an unknown mechanism. Taken together, our findings demonstrated that the host m6A modification complex interacted with viral proteins to modulate SARS-CoV-2 replication. IMPORTANCE Internal chemical modifications of viral RNA play key roles in the regulation of viral replication and gene expression. Although potential internal modifications have been reported in SARS-CoV-2 RNA, the function of the SARS-CoV-2 N6-methyladenosine (m6A) modification in the viral life cycle is unclear. In the current study, we demonstrated that SARS-CoV-2 RNA underwent m6A modification by host m6A machinery. SARS-CoV-2 infection altered the expression pattern of methyltransferases and demethylases, while the expression level of methyltransferase-like 3 (METTL3) and fat mass and obesity-associated protein (FTO) was linked to the viral replication. Further study showed that METTL3 interacted with viral RNA polymerase RNA-dependent RNA polymerase (RdRp), which influenced not only the distribution but also the posttranslational modification of METTL3. Our study provided evidence that host m6A components interacted with viral proteins to modulate viral replication.


Assuntos
Adenosina/análogos & derivados , Metiltransferases/genética , Metiltransferases/metabolismo , SARS-CoV-2/crescimento & desenvolvimento , Replicação Viral/genética , Adenosina/química , Adenosina/genética , Dioxigenase FTO Dependente de alfa-Cetoglutarato/metabolismo , COVID-19/patologia , Regulação da Expressão Gênica/genética , Humanos , Metilação , Processamento de Proteína Pós-Traducional/fisiologia , RNA Viral/química , RNA Viral/genética , RNA Polimerase Dependente de RNA/genética , SARS-CoV-2/genética
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