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1.
Nat Genet ; 53(8): 1156-1165, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34211177

RESUMO

The most prevalent post-transcriptional mRNA modification, N6-methyladenosine (m6A), plays diverse RNA-regulatory roles, but its genetic control in human tissues remains uncharted. Here we report 129 transcriptome-wide m6A profiles, covering 91 individuals and 4 tissues (brain, lung, muscle and heart) from GTEx/eGTEx. We integrate these with interindividual genetic and expression variation, revealing 8,843 tissue-specific and 469 tissue-shared m6A quantitative trait loci (QTLs), which are modestly enriched in, but mostly orthogonal to, expression QTLs. We integrate m6A QTLs with disease genetics, identifying 184 GWAS-colocalized m6A QTL, including brain m6A QTLs underlying neuroticism, depression, schizophrenia and anxiety; lung m6A QTLs underlying expiratory flow and asthma; and muscle/heart m6A QTLs underlying coronary artery disease. Last, we predict novel m6A regulators that show preferential binding in m6A QTLs, protein interactions with known m6A regulators and expression correlation with the m6A levels of their targets. Our results provide important insights and resources for understanding both cis and trans regulation of epitranscriptomic modifications, their interindividual variation and their roles in human disease.


Assuntos
Adenosina/análogos & derivados , Encéfalo/fisiologia , Pulmão/fisiologia , Músculo Esquelético/fisiologia , Locos de Características Quantitativas , Adenosina/genética , Adenosina/metabolismo , Estudo de Associação Genômica Ampla , Coração/fisiologia , Humanos , Metilação , Especificidade de Órgãos , Polimorfismo de Nucleotídeo Único , Processamento Pós-Transcricional do RNA , Proteínas de Ligação a RNA/genética , Reprodutibilidade dos Testes
2.
Cell Rep ; 20(9): 2262-2276, 2017 Aug 29.
Artigo em Inglês | MEDLINE | ID: mdl-28854373

RESUMO

N6-methyladenosine (m6A) is the most abundant internal modification of mRNAs and is implicated in all aspects of post-transcriptional RNA metabolism. However, little is known about m6A modifications to circular (circ) RNAs. We developed a computational pipeline (AutoCirc) that, together with depletion of ribosomal RNA and m6A immunoprecipitation, defined thousands of m6A circRNAs with cell-type-specific expression. The presence of m6A circRNAs is corroborated by interaction between circRNAs and YTHDF1/YTHDF2, proteins that read m6A sites in mRNAs, and by reduced m6A levels upon depletion of METTL3, the m6A writer. Despite sharing m6A readers and writers, m6A circRNAs are frequently derived from exons that are not methylated in mRNAs, whereas mRNAs that are methylated on the same exons that compose m6A circRNAs exhibit less stability in a process regulated by YTHDF2. These results expand our understanding of the breadth of m6A modifications and uncover regulation of circRNAs through m6A modification.


Assuntos
Adenosina/análogos & derivados , Genoma Humano , RNA/metabolismo , Adenosina/metabolismo , Sequência de Bases , Biologia Computacional , Elementos de DNA Transponíveis/genética , Éxons/genética , Exorribonucleases/metabolismo , Regulação da Expressão Gênica , Meia-Vida , Células HeLa , Células-Tronco Embrionárias Humanas/metabolismo , Humanos , Metilação , Metiltransferases/metabolismo , RNA/genética , Estabilidade de RNA , RNA Circular , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo
3.
Methods Mol Biol ; 1562: 45-53, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28349453

RESUMO

N6-methyladenosine-sequencing (m6A-seq) is a critical tool to obtain an unbiased genome-wide picture of m6A sites of modification at high resolution. It allows the study of the impact of various perturbations on m6A modification distribution and the study of m6A functions. Herein, we describe the m6A-seq protocol, which entails RNA immunoprecipitation (RIP) performed on fragmented poly(A) RNA utilizing anti-m6A antibodies. The captured/enriched m6A positive RNA fragments are subsequently sequenced by RNA-seq in parallel with background control non-immunoprecipitated input RNA fragments. Analyses reveal peaks of m6A enrichment containing sites of modifications analogous to chromatin modification immunoprecipitation experiments.


Assuntos
Adenosina/análogos & derivados , Genoma , RNA , Análise de Sequência de RNA , Biologia Computacional/métodos , Perfilação da Expressão Gênica , Metilação , RNA/química , RNA/isolamento & purificação , RNA/metabolismo , RNA Mensageiro/química , RNA Mensageiro/isolamento & purificação , RNA Mensageiro/metabolismo , Transcriptoma , Fluxo de Trabalho
4.
Nat Methods ; 13(8): 692-8, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27376769

RESUMO

N(6)-Methyladenosine (m(6)A) is a widespread, reversible chemical modification of RNA molecules, implicated in many aspects of RNA metabolism. Little quantitative information exists as to either how many transcript copies of particular genes are m(6)A modified ('m(6)A levels') or the relationship of m(6)A modification(s) to alternative RNA isoforms. To deconvolute the m(6)A epitranscriptome, we developed m(6)A-level and isoform-characterization sequencing (m(6)A-LAIC-seq). We found that cells exhibit a broad range of nonstoichiometric m(6)A levels with cell-type specificity. At the level of isoform characterization, we discovered widespread differences in the use of tandem alternative polyadenylation (APA) sites by methylated and nonmethylated transcript isoforms of individual genes. Strikingly, there is a strong bias for methylated transcripts to be coupled with proximal APA sites, resulting in shortened 3' untranslated regions, while nonmethylated transcript isoforms tend to use distal APA sites. m(6)A-LAIC-seq yields a new perspective on transcriptome complexity and links APA usage to m(6)A modifications.


Assuntos
Adenosina/análogos & derivados , Epigênese Genética/genética , Epigenômica/métodos , Regulação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Transcriptoma , Regiões 3' não Traduzidas/genética , Adenosina/química , Adenosina/genética , Células Cultivadas , Humanos , Poliadenilação , Isoformas de Proteínas , RNA Mensageiro/genética , Células-Tronco/metabolismo
5.
Science ; 350(6263): 978-81, 2015 Nov 20.
Artigo em Inglês | MEDLINE | ID: mdl-26516199

RESUMO

Transcription factors (TFs) bind specific sequences in promoter-proximal and -distal DNA elements to regulate gene transcription. RNA is transcribed from both of these DNA elements, and some DNA binding TFs bind RNA. Hence, RNA transcribed from regulatory elements may contribute to stable TF occupancy at these sites. We show that the ubiquitously expressed TF Yin-Yang 1 (YY1) binds to both gene regulatory elements and their associated RNA species across the entire genome. Reduced transcription of regulatory elements diminishes YY1 occupancy, whereas artificial tethering of RNA enhances YY1 occupancy at these elements. We propose that RNA makes a modest but important contribution to the maintenance of certain TFs at gene regulatory elements and suggest that transcription of regulatory elements produces a positive-feedback loop that contributes to the stability of gene expression programs.


Assuntos
Elementos Facilitadores Genéticos , Regulação da Expressão Gênica , Regiões Promotoras Genéticas , RNA Mensageiro/metabolismo , Transcrição Genética , Fator de Transcrição YY1/metabolismo , Animais , Sequência de Bases , Sítios de Ligação , Linhagem Celular , Sequência Consenso , DNA/metabolismo , Células-Tronco Embrionárias/metabolismo , Camundongos
6.
Cell Stem Cell ; 15(6): 707-19, 2014 Dec 04.
Artigo em Inglês | MEDLINE | ID: mdl-25456834

RESUMO

N6-methyl-adenosine (m(6)A) is the most abundant modification on messenger RNAs and is linked to human diseases, but its functions in mammalian development are poorly understood. Here we reveal the evolutionary conservation and function of m(6)A by mapping the m(6)A methylome in mouse and human embryonic stem cells. Thousands of messenger and long noncoding RNAs show conserved m(6)A modification, including transcripts encoding core pluripotency transcription factors. m(6)A is enriched over 3' untranslated regions at defined sequence motifs and marks unstable transcripts, including transcripts turned over upon differentiation. Genetic inactivation or depletion of mouse and human Mettl3, one of the m(6)A methylases, led to m(6)A erasure on select target genes, prolonged Nanog expression upon differentiation, and impaired ESC exit from self-renewal toward differentiation into several lineages in vitro and in vivo. Thus, m(6)A is a mark of transcriptome flexibility required for stem cells to differentiate to specific lineages.


Assuntos
Adenina/análogos & derivados , Células-Tronco Embrionárias/fisiologia , Proteínas de Homeodomínio/metabolismo , Metiltransferases/metabolismo , Adenina/metabolismo , Animais , Sequência de Bases , Diferenciação Celular/genética , Linhagem Celular , Linhagem da Célula/genética , Proliferação de Células/genética , Sequência Conservada/genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/genética , Humanos , Metiltransferases/genética , Camundongos , Camundongos SCID , Dados de Sequência Molecular , Mutação/genética , Proteína Homeobox Nanog , Processamento Pós-Transcricional do RNA/genética , RNA Interferente Pequeno/genética , Transcriptoma
7.
J Immunol ; 190(11): 5578-87, 2013 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-23616578

RESUMO

Profiling studies of mRNA and microRNA, particularly microarray-based studies, have been extensively used to create compendia of genes that are preferentially expressed in the immune system. In some instances, functional studies have been subsequently pursued. Recent efforts such as the Encyclopedia of DNA Elements have demonstrated the benefit of coupling RNA sequencing analysis with information from expressed sequence tags (ESTs) for transcriptomic analysis. However, the full characterization and identification of transcripts that function as modulators of human immune responses remains incomplete. In this study, we demonstrate that an integrated analysis of human ESTs provides a robust platform to identify the immune transcriptome. Beyond recovering a reference set of immune-enriched genes and providing large-scale cross-validation of previous microarray studies, we discovered hundreds of novel genes preferentially expressed in the immune system, including noncoding RNAs. As a result, we have established the Immunogene database, representing an integrated EST road map of gene expression in human immune cells, which can be used to further investigate the function of coding and noncoding genes in the immune system. Using this approach, we have uncovered a unique metabolic gene signature of human macrophages and identified PRDM15 as a novel overexpressed gene in human lymphomas. Thus, we demonstrate the utility of EST profiling as a basis for further deconstruction of physiologic and pathologic immune processes.


Assuntos
Etiquetas de Sequências Expressas , Perfilação da Expressão Gênica , Estudo de Associação Genômica Ampla , Sistema Imunitário/metabolismo , Animais , Análise por Conglomerados , Biologia Computacional/métodos , Proteínas de Ligação a DNA/genética , Bases de Dados de Ácidos Nucleicos , Redes Reguladoras de Genes , Genômica , Humanos , Doenças do Sistema Imunitário/genética , Linfoma de Células B/genética , Camundongos , Anotação de Sequência Molecular , RNA Longo não Codificante/genética , Reprodutibilidade dos Testes , Fatores de Transcrição/genética , Transcriptoma
8.
Proc Natl Acad Sci U S A ; 110(8): 2876-81, 2013 Feb 19.
Artigo em Inglês | MEDLINE | ID: mdl-23382218

RESUMO

Many long noncoding RNA (lncRNA) species have been identified in mammalian cells, but the genomic origin and regulation of these molecules in individual cell types is poorly understood. We have generated catalogs of lncRNA species expressed in human and murine embryonic stem cells and mapped their genomic origin. A surprisingly large fraction of these transcripts (>60%) originate from divergent transcription at promoters of active protein-coding genes. The divergently transcribed lncRNA/mRNA gene pairs exhibit coordinated changes in transcription when embryonic stem cells are differentiated into endoderm. Our results reveal that transcription of most lncRNA genes is coordinated with transcription of protein-coding genes.


Assuntos
Células-Tronco Embrionárias/metabolismo , RNA Mensageiro/genética , RNA não Traduzido/genética , Transcrição Genética , Animais , Diferenciação Celular , Células-Tronco Embrionárias/citologia , Humanos , Camundongos
9.
Cell ; 147(1): 107-19, 2011 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-21962511

RESUMO

Whereas chromosomal translocations are common pathogenetic events in cancer, mechanisms that promote them are poorly understood. To elucidate translocation mechanisms in mammalian cells, we developed high-throughput, genome-wide translocation sequencing (HTGTS). We employed HTGTS to identify tens of thousands of independent translocation junctions involving fixed I-SceI meganuclease-generated DNA double-strand breaks (DSBs) within the c-myc oncogene or IgH locus of B lymphocytes induced for activation-induced cytidine deaminase (AID)-dependent IgH class switching. DSBs translocated widely across the genome but were preferentially targeted to transcribed chromosomal regions. Additionally, numerous AID-dependent and AID-independent hot spots were targeted, with the latter comprising mainly cryptic I-SceI targets. Comparison of translocation junctions with genome-wide nuclear run-ons revealed a marked association between transcription start sites and translocation targeting. The majority of translocation junctions were formed via end-joining with short microhomologies. Our findings have implications for diverse fields, including gene therapy and cancer genomics.


Assuntos
Linfócitos B/metabolismo , Quebra Cromossômica , Genoma , Mutagênese , Translocação Genética , Animais , Células Cultivadas , Quebras de DNA de Cadeia Dupla , Genes myc , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Cadeias Pesadas de Imunoglobulinas/genética , Camundongos , Neoplasias/genética , Baço/citologia
10.
Drug News Perspect ; 22(5): 247-54, 2009 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-19609462

RESUMO

The regulatory mechanisms governing the expression of specific genes associated with prostate cancer cannot all be explained by monitoring DNA sequences and potential mutations. This suggests the involvement of additional modes of regulating gene expression, such as epigenetics. The contribution of epigenetic events, although fully recognized, remains to be fully understood. There is increasing evidence for the regulatory roles of both DNA methylation and the composition and conformation of chromatin. These epigenetic changes can be modulated by molecules that are part of our daily diet. Genistein, an isoflavonoid present in soy products, has been shown to have anticancer effects. Epidemiological studies have indicated a link between a low occurrence of prostate cancer and a genistein-rich diet. This review summarizes the current knowledge about genistein-modulated genetic and epigenetic effects on both the gene expression and biological pathways associated with prostate cancer.


Assuntos
Anticarcinógenos/farmacologia , Genisteína/farmacologia , Neoplasias da Próstata/tratamento farmacológico , Animais , Sistemas de Liberação de Medicamentos , Epigênese Genética/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Masculino , Neoplasias da Próstata/genética , Inibidores de Proteínas Quinases/farmacologia
11.
Biophys J ; 96(5): 1944-51, 2009 Mar 04.
Artigo em Inglês | MEDLINE | ID: mdl-19254554

RESUMO

Studies on the stability of nucleosome core particles as a function of concentration have indicated a lower limit of approximately 5 ng/microL, below which the complexes start to spontaneously destabilize. Until recently little information was available on the effect of low concentration on chromatin. Using the well-characterized array of tandemly repeated 5S rDNA reconstituted into chromatin, we have investigated the effect of dilution. In this study, we demonstrate that the stability of saturated nucleosomal arrays and that of nucleosome core particles are within the same order of magnitude, and no significant loss of histones is monitored down to a concentration of 2.5 ng/microL. We observed that levels of subsaturation of the nucleosomal arrays were directly correlated with an increased sensitivity to histone loss, suggesting a shielding effect. The loss of histones from our linear nucleosomal arrays was shown not to be random, with a significant likelihood to occur at the end of the template than toward the center. This observation indicates that centrally located nucleosomes are more stable than those close to the end of the DNA templates. Itis important to take this information into account for the proper design of experiments pertaining to histone composition and the folding ability of chromatin samples.


Assuntos
Cromatina/química , DNA Ribossômico/química , Histonas/química , Animais , Galinhas , Eletroforese em Gel de Ágar , Lytechinus , Microscopia de Força Atômica , Nucleossomos/química , Nucleossomos/fisiologia , Multimerização Proteica , Estabilidade Proteica
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