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1.
Cell ; 185(26): 4937-4953.e23, 2022 12 22.
Artigo em Inglês | MEDLINE | ID: mdl-36563664

RESUMO

To define the multi-cellular epigenomic and transcriptional landscape of cardiac cellular development, we generated single-cell chromatin accessibility maps of human fetal heart tissues. We identified eight major differentiation trajectories involving primary cardiac cell types, each associated with dynamic transcription factor (TF) activity signatures. We contrasted regulatory landscapes of iPSC-derived cardiac cell types and their in vivo counterparts, which enabled optimization of in vitro differentiation of epicardial cells. Further, we interpreted sequence based deep learning models of cell-type-resolved chromatin accessibility profiles to decipher underlying TF motif lexicons. De novo mutations predicted to affect chromatin accessibility in arterial endothelium were enriched in congenital heart disease (CHD) cases vs. controls. In vitro studies in iPSCs validated the functional impact of identified variation on the predicted developmental cell types. This work thus defines the cell-type-resolved cis-regulatory sequence determinants of heart development and identifies disruption of cell type-specific regulatory elements in CHD.


Assuntos
Cromatina , Cardiopatias Congênitas , Humanos , Cromatina/genética , Cardiopatias Congênitas/genética , Coração , Mutação , Análise de Célula Única
2.
Cell ; 173(1): 90-103.e19, 2018 03 22.
Artigo em Inglês | MEDLINE | ID: mdl-29551269

RESUMO

Blood cell formation is classically thought to occur through a hierarchical differentiation process, although recent studies have shown that lineage commitment may occur earlier in hematopoietic stem and progenitor cells (HSPCs). The relevance to human blood diseases and the underlying regulation of these refined models remain poorly understood. By studying a genetic blood disorder, Diamond-Blackfan anemia (DBA), where the majority of mutations affect ribosomal proteins and the erythroid lineage is selectively perturbed, we are able to gain mechanistic insight into how lineage commitment is programmed normally and disrupted in disease. We show that in DBA, the pool of available ribosomes is limited, while ribosome composition remains constant. Surprisingly, this global reduction in ribosome levels more profoundly alters translation of a select subset of transcripts. We show how the reduced translation of select transcripts in HSPCs can impair erythroid lineage commitment, illuminating a regulatory role for ribosome levels in cellular differentiation.


Assuntos
Anemia de Diamond-Blackfan/patologia , Ribossomos/metabolismo , Regiões 5' não Traduzidas , Anemia de Diamond-Blackfan/genética , Proteínas Reguladoras de Apoptose/antagonistas & inibidores , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Células da Medula Óssea/metabolismo , Células Cultivadas , Feminino , Fator de Transcrição GATA1/genética , Fator de Transcrição GATA1/metabolismo , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Humanos , Masculino , Mutação de Sentido Incorreto , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Proteínas Ribossômicas/antagonistas & inibidores , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Ribossomos/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
3.
Cell ; 170(3): 522-533.e15, 2017 Jul 27.
Artigo em Inglês | MEDLINE | ID: mdl-28753427

RESUMO

Genome-wide association studies (GWASs) implicate the PHACTR1 locus (6p24) in risk for five vascular diseases, including coronary artery disease, migraine headache, cervical artery dissection, fibromuscular dysplasia, and hypertension. Through genetic fine mapping, we prioritized rs9349379, a common SNP in the third intron of the PHACTR1 gene, as the putative causal variant. Epigenomic data from human tissue revealed an enhancer signature at rs9349379 exclusively in aorta, suggesting a regulatory function for this SNP in the vasculature. CRISPR-edited stem cell-derived endothelial cells demonstrate rs9349379 regulates expression of endothelin 1 (EDN1), a gene located 600 kb upstream of PHACTR1. The known physiologic effects of EDN1 on the vasculature may explain the pattern of risk for the five associated diseases. Overall, these data illustrate the integration of genetic, phenotypic, and epigenetic analysis to identify the biologic mechanism by which a common, non-coding variant can distally regulate a gene and contribute to the pathogenesis of multiple vascular diseases.


Assuntos
Doença da Artéria Coronariana/genética , Endotelina-1/genética , Predisposição Genética para Doença , Polimorfismo de Nucleotídeo Único , Doenças Vasculares/genética , Acetilação , Células Cultivadas , Cromatina/metabolismo , Mapeamento Cromossômico , Cromossomos Humanos Par 6 , Células Endoteliais/citologia , Endotelina-1/sangue , Epigenômica , Edição de Genes , Expressão Gênica , Estudo de Associação Genômica Ampla , Histonas/metabolismo , Humanos , Músculo Liso Vascular/citologia
4.
Cell ; 171(2): 305-320.e24, 2017 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-28985562

RESUMO

The human genome folds to create thousands of intervals, called "contact domains," that exhibit enhanced contact frequency within themselves. "Loop domains" form because of tethering between two loci-almost always bound by CTCF and cohesin-lying on the same chromosome. "Compartment domains" form when genomic intervals with similar histone marks co-segregate. Here, we explore the effects of degrading cohesin. All loop domains are eliminated, but neither compartment domains nor histone marks are affected. Loss of loop domains does not lead to widespread ectopic gene activation but does affect a significant minority of active genes. In particular, cohesin loss causes superenhancers to co-localize, forming hundreds of links within and across chromosomes and affecting the regulation of nearby genes. We then restore cohesin and monitor the re-formation of each loop. Although re-formation rates vary greatly, many megabase-sized loops recovered in under an hour, consistent with a model where loop extrusion is rapid.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Núcleo Celular/genética , Proteínas Cromossômicas não Histona/metabolismo , Cromossomos/metabolismo , Genoma Humano , Proteínas Repressoras/metabolismo , Fator de Ligação a CCCTC , Linhagem Celular Tumoral , Proteínas de Ligação a DNA , Elementos Facilitadores Genéticos , Código das Histonas , Humanos , Proteínas Nucleares/metabolismo , Nucleossomos/metabolismo , Fosfoproteínas/metabolismo , Coesinas
5.
Nature ; 626(8000): 799-807, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38326615

RESUMO

Linking variants from genome-wide association studies (GWAS) to underlying mechanisms of disease remains a challenge1-3. For some diseases, a successful strategy has been to look for cases in which multiple GWAS loci contain genes that act in the same biological pathway1-6. However, our knowledge of which genes act in which pathways is incomplete, particularly for cell-type-specific pathways or understudied genes. Here we introduce a method to connect GWAS variants to functions. This method links variants to genes using epigenomics data, links genes to pathways de novo using Perturb-seq and integrates these data to identify convergence of GWAS loci onto pathways. We apply this approach to study the role of endothelial cells in genetic risk for coronary artery disease (CAD), and discover 43 CAD GWAS signals that converge on the cerebral cavernous malformation (CCM) signalling pathway. Two regulators of this pathway, CCM2 and TLNRD1, are each linked to a CAD risk variant, regulate other CAD risk genes and affect atheroprotective processes in endothelial cells. These results suggest a model whereby CAD risk is driven in part by the convergence of causal genes onto a particular transcriptional pathway in endothelial cells. They highlight shared genes between common and rare vascular diseases (CAD and CCM), and identify TLNRD1 as a new, previously uncharacterized member of the CCM signalling pathway. This approach will be widely useful for linking variants to functions for other common polygenic diseases.


Assuntos
Doença da Artéria Coronariana , Células Endoteliais , Estudo de Associação Genômica Ampla , Hemangioma Cavernoso do Sistema Nervoso Central , Humanos , Doença da Artéria Coronariana/genética , Doença da Artéria Coronariana/patologia , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Predisposição Genética para Doença/genética , Hemangioma Cavernoso do Sistema Nervoso Central/genética , Hemangioma Cavernoso do Sistema Nervoso Central/patologia , Polimorfismo de Nucleotídeo Único , Epigenômica , Transdução de Sinais/genética , Herança Multifatorial
6.
Cell ; 159(1): 188-199, 2014 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-25259926

RESUMO

Intermolecular RNA-RNA interactions are used by many noncoding RNAs (ncRNAs) to achieve their diverse functions. To identify these contacts, we developed a method based on RNA antisense purification to systematically map RNA-RNA interactions (RAP-RNA) and applied it to investigate two ncRNAs implicated in RNA processing: U1 small nuclear RNA, a component of the spliceosome, and Malat1, a large ncRNA that localizes to nuclear speckles. U1 and Malat1 interact with nascent transcripts through distinct targeting mechanisms. Using differential crosslinking, we confirmed that U1 directly hybridizes to 5' splice sites and 5' splice site motifs throughout introns and found that Malat1 interacts with pre-mRNAs indirectly through protein intermediates. Interactions with nascent pre-mRNAs cause U1 and Malat1 to localize proximally to chromatin at active genes, demonstrating that ncRNAs can use RNA-RNA interactions to target specific pre-mRNAs and genomic sites. RAP-RNA is sensitive to lower abundance RNAs as well, making it generally applicable for investigating ncRNAs.


Assuntos
Técnicas Genéticas , RNA Mensageiro/metabolismo , Animais , Sequência de Bases , Reagentes de Ligações Cruzadas/metabolismo , Camundongos , Dados de Sequência Molecular , Motivos de Nucleotídeos , Sítios de Splice de RNA , RNA Longo não Codificante/química , RNA Longo não Codificante/metabolismo , RNA Mensageiro/química , RNA Nuclear Pequeno/metabolismo , RNA não Traduzido/química , RNA não Traduzido/metabolismo
7.
Cell ; 159(1): 148-162, 2014 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-25219674

RESUMO

Pseudouridine is the most abundant RNA modification, yet except for a few well-studied cases, little is known about the modified positions and their function(s). Here, we develop Ψ-seq for transcriptome-wide quantitative mapping of pseudouridine. We validate Ψ-seq with spike-ins and de novo identification of previously reported positions and discover hundreds of unique sites in human and yeast mRNAs and snoRNAs. Perturbing pseudouridine synthases (PUS) uncovers which pseudouridine synthase modifies each site and their target sequence features. mRNA pseudouridinylation depends on both site-specific and snoRNA-guided pseudouridine synthases. Upon heat shock in yeast, Pus7p-mediated pseudouridylation is induced at >200 sites, and PUS7 deletion decreases the levels of otherwise pseudouridylated mRNA, suggesting a role in enhancing transcript stability. rRNA pseudouridine stoichiometries are conserved but reduced in cells from dyskeratosis congenita patients, where the PUS DKC1 is mutated. Our work identifies an enhanced, transcriptome-wide scope for pseudouridine and methods to dissect its underlying mechanisms and function.


Assuntos
Pseudouridina/análise , RNA Mensageiro/química , RNA não Traduzido/química , Animais , Candida albicans/genética , Candida albicans/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Disceratose Congênita/genética , Disceratose Congênita/metabolismo , Perfilação da Expressão Gênica , Humanos , Transferases Intramoleculares/química , Transferases Intramoleculares/metabolismo , Camundongos , Dados de Sequência Molecular , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Pseudouridina/metabolismo , RNA/química , RNA/genética , RNA Ribossômico/química , RNA Ribossômico/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Especificidade por Substrato , Telomerase/química , Telomerase/genética
8.
Nat Rev Mol Cell Biol ; 17(12): 756-770, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-27780979

RESUMO

Over the past decade, it has become clear that mammalian genomes encode thousands of long non-coding RNAs (lncRNAs), many of which are now implicated in diverse biological processes. Recent work studying the molecular mechanisms of several key examples - including Xist, which orchestrates X chromosome inactivation - has provided new insights into how lncRNAs can control cellular functions by acting in the nucleus. Here we discuss emerging mechanistic insights into how lncRNAs can regulate gene expression by coordinating regulatory proteins, localizing to target loci and shaping three-dimensional (3D) nuclear organization. We explore these principles to highlight biological challenges in gene regulation, in which lncRNAs are well-suited to perform roles that cannot be carried out by DNA elements or protein regulators alone, such as acting as spatial amplifiers of regulatory signals in the nucleus.


Assuntos
Núcleo Celular/ultraestrutura , Regulação da Expressão Gênica , RNA Longo não Codificante/fisiologia , Animais , Núcleo Celular/genética , Expressão Gênica , Humanos , Transporte de RNA
9.
Nature ; 607(7917): 176-184, 2022 07.
Artigo em Inglês | MEDLINE | ID: mdl-35594906

RESUMO

Gene regulation in the human genome is controlled by distal enhancers that activate specific nearby promoters1. A proposed model for this specificity is that promoters have sequence-encoded preferences for certain enhancers, for example, mediated by interacting sets of transcription factors or cofactors2. This 'biochemical compatibility' model has been supported by observations at individual human promoters and by genome-wide measurements in Drosophila3-9. However, the degree to which human enhancers and promoters are intrinsically compatible has not yet been systematically measured, and how their activities combine to control RNA expression remains unclear. Here we design a high-throughput reporter assay called enhancer × promoter self-transcribing active regulatory region sequencing (ExP STARR-seq) and applied it to examine the combinatorial compatibilities of 1,000 enhancer and 1,000 promoter sequences in human K562 cells. We identify simple rules for enhancer-promoter compatibility, whereby most enhancers activate all promoters by similar amounts, and intrinsic enhancer and promoter activities multiplicatively combine to determine RNA output (R2 = 0.82). In addition, two classes of enhancers and promoters show subtle preferential effects. Promoters of housekeeping genes contain built-in activating motifs for factors such as GABPA and YY1, which decrease the responsiveness of promoters to distal enhancers. Promoters of variably expressed genes lack these motifs and show stronger responsiveness to enhancers. Together, this systematic assessment of enhancer-promoter compatibility suggests a multiplicative model tuned by enhancer and promoter class to control gene transcription in the human genome.


Assuntos
Elementos Facilitadores Genéticos , Regiões Promotoras Genéticas , Elementos Facilitadores Genéticos/genética , Humanos , Regiões Promotoras Genéticas/genética , RNA/biossíntese , RNA/genética , Fatores de Transcrição/metabolismo
10.
Nat Methods ; 21(4): 723-734, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38504114

RESUMO

The ENCODE Consortium's efforts to annotate noncoding cis-regulatory elements (CREs) have advanced our understanding of gene regulatory landscapes. Pooled, noncoding CRISPR screens offer a systematic approach to investigate cis-regulatory mechanisms. The ENCODE4 Functional Characterization Centers conducted 108 screens in human cell lines, comprising >540,000 perturbations across 24.85 megabases of the genome. Using 332 functionally confirmed CRE-gene links in K562 cells, we established guidelines for screening endogenous noncoding elements with CRISPR interference (CRISPRi), including accurate detection of CREs that exhibit variable, often low, transcriptional effects. Benchmarking five screen analysis tools, we find that CASA produces the most conservative CRE calls and is robust to artifacts of low-specificity single guide RNAs. We uncover a subtle DNA strand bias for CRISPRi in transcribed regions with implications for screen design and analysis. Together, we provide an accessible data resource, predesigned single guide RNAs for targeting 3,275,697 ENCODE SCREEN candidate CREs with CRISPRi and screening guidelines to accelerate functional characterization of the noncoding genome.


Assuntos
Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Humanos , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Sistemas CRISPR-Cas/genética , Genoma , Células K562 , RNA Guia de Sistemas CRISPR-Cas
11.
Nature ; 593(7858): 238-243, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33828297

RESUMO

Genome-wide association studies (GWAS) have identified thousands of noncoding loci that are associated with human diseases and complex traits, each of which could reveal insights into the mechanisms of disease1. Many of the underlying causal variants may affect enhancers2,3, but we lack accurate maps of enhancers and their target genes to interpret such variants. We recently developed the activity-by-contact (ABC) model to predict which enhancers regulate which genes and validated the model using CRISPR perturbations in several cell types4. Here we apply this ABC model to create enhancer-gene maps in 131 human cell types and tissues, and use these maps to interpret the functions of GWAS variants. Across 72 diseases and complex traits, ABC links 5,036 GWAS signals to 2,249 unique genes, including a class of 577 genes that appear to influence multiple phenotypes through variants in enhancers that act in different cell types. In inflammatory bowel disease (IBD), causal variants are enriched in predicted enhancers by more than 20-fold in particular cell types such as dendritic cells, and ABC achieves higher precision than other regulatory methods at connecting noncoding variants to target genes. These variant-to-function maps reveal an enhancer that contains an IBD risk variant and that regulates the expression of PPIF to alter the membrane potential of mitochondria in macrophages. Our study reveals principles of genome regulation, identifies genes that affect IBD and provides a resource and generalizable strategy to connect risk variants of common diseases to their molecular and cellular functions.


Assuntos
Elementos Facilitadores Genéticos/genética , Predisposição Genética para Doença , Variação Genética/genética , Genoma Humano/genética , Estudo de Associação Genômica Ampla , Doenças Inflamatórias Intestinais/genética , Linhagem Celular , Cromossomos Humanos Par 10/genética , Ciclofilinas/genética , Células Dendríticas , Feminino , Humanos , Macrófagos/metabolismo , Masculino , Mitocôndrias/metabolismo , Especificidade de Órgãos/genética , Fenótipo
12.
Nature ; 595(7865): 107-113, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33915569

RESUMO

COVID-19, which is caused by SARS-CoV-2, can result in acute respiratory distress syndrome and multiple organ failure1-4, but little is known about its pathophysiology. Here we generated single-cell atlases of 24 lung, 16 kidney, 16 liver and 19 heart autopsy tissue samples and spatial atlases of 14 lung samples from donors who died of COVID-19. Integrated computational analysis uncovered substantial remodelling in the lung epithelial, immune and stromal compartments, with evidence of multiple paths of failed tissue regeneration, including defective alveolar type 2 differentiation and expansion of fibroblasts and putative TP63+ intrapulmonary basal-like progenitor cells. Viral RNAs were enriched in mononuclear phagocytic and endothelial lung cells, which induced specific host programs. Spatial analysis in lung distinguished inflammatory host responses in lung regions with and without viral RNA. Analysis of the other tissue atlases showed transcriptional alterations in multiple cell types in heart tissue from donors with COVID-19, and mapped cell types and genes implicated with disease severity based on COVID-19 genome-wide association studies. Our foundational dataset elucidates the biological effect of severe SARS-CoV-2 infection across the body, a key step towards new treatments.


Assuntos
COVID-19/patologia , COVID-19/virologia , Rim/patologia , Fígado/patologia , Pulmão/patologia , Miocárdio/patologia , SARS-CoV-2/patogenicidade , Adulto , Idoso , Idoso de 80 Anos ou mais , Atlas como Assunto , Autopsia , Bancos de Espécimes Biológicos , COVID-19/genética , COVID-19/imunologia , Células Endoteliais , Células Epiteliais/patologia , Células Epiteliais/virologia , Feminino , Fibroblastos , Estudo de Associação Genômica Ampla , Coração/virologia , Humanos , Inflamação/patologia , Inflamação/virologia , Rim/virologia , Fígado/virologia , Pulmão/virologia , Masculino , Pessoa de Meia-Idade , Especificidade de Órgãos , Fagócitos , Alvéolos Pulmonares/patologia , Alvéolos Pulmonares/virologia , RNA Viral/análise , Regeneração , SARS-CoV-2/imunologia , Análise de Célula Única , Carga Viral
13.
Cell ; 147(1): 81-94, 2011 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-21962509

RESUMO

The let-7 tumor suppressor microRNAs are known for their regulation of oncogenes, while the RNA-binding proteins Lin28a/b promote malignancy by inhibiting let-7 biogenesis. We have uncovered unexpected roles for the Lin28/let-7 pathway in regulating metabolism. When overexpressed in mice, both Lin28a and LIN28B promote an insulin-sensitized state that resists high-fat-diet induced diabetes. Conversely, muscle-specific loss of Lin28a or overexpression of let-7 results in insulin resistance and impaired glucose tolerance. These phenomena occur, in part, through the let-7-mediated repression of multiple components of the insulin-PI3K-mTOR pathway, including IGF1R, INSR, and IRS2. In addition, the mTOR inhibitor, rapamycin, abrogates Lin28a-mediated insulin sensitivity and enhanced glucose uptake. Moreover, let-7 targets are enriched for genes containing SNPs associated with type 2 diabetes and control of fasting glucose in human genome-wide association studies. These data establish the Lin28/let-7 pathway as a central regulator of mammalian glucose metabolism.


Assuntos
Glucose/metabolismo , MicroRNAs/metabolismo , Animais , Diabetes Mellitus Tipo 2/metabolismo , Regulação da Expressão Gênica , Estudo de Associação Genômica Ampla , Humanos , Resistência à Insulina , Camundongos , Camundongos Knockout , Camundongos Transgênicos , MicroRNAs/genética , Obesidade/genética , Obesidade/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo
14.
Hum Mol Genet ; 31(12): 1946-1961, 2022 06 22.
Artigo em Inglês | MEDLINE | ID: mdl-34970970

RESUMO

BACKGROUND: FCGR2A binds antibody-antigen complexes to regulate the abundance of circulating and deposited complexes along with downstream immune and autoimmune responses. Although the abundance of FCRG2A may be critical in immune-mediated diseases, little is known about whether its surface expression is regulated through cis genomic elements and non-coding variants. In the current study, we aimed to characterize the regulation of FCGR2A expression, the impact of genetic variation and its association with autoimmune disease. METHODS: We applied CRISPR-based interference and editing to scrutinize 1.7 Mb of open chromatin surrounding the FCGR2A gene to identify regulatory elements. Relevant transcription factors (TFs) binding to these regions were defined through public databases. Genetic variants affecting regulation were identified using luciferase reporter assays and were verified in a cohort of 1996 genotyped healthy individuals using flow cytometry. RESULTS: We identified a complex proximal region and five distal enhancers regulating FCGR2A. The proximal region split into subregions upstream and downstream of the transcription start site, was enriched in binding of inflammation-regulated TFs, and harbored a variant associated with FCGR2A expression in primary myeloid cells. One distal enhancer region was occupied by CCCTC-binding factor (CTCF) whose binding site was disrupted by a rare genetic variant, altering gene expression. CONCLUSIONS: The FCGR2A gene is regulated by multiple proximal and distal genomic regions, with links to autoimmune disease. These findings may open up novel therapeutic avenues where fine-tuning of FCGR2A levels may constitute a part of treatment strategies for immune-mediated diseases.


Assuntos
Doenças Autoimunes , Elementos Facilitadores Genéticos , Receptores de IgG , Doenças Autoimunes/genética , Sítios de Ligação , Genômica , Genótipo , Humanos , Receptores de IgG/genética
15.
Nature ; 561(7721): 132-136, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-30150775

RESUMO

The human genome contains thousands of long non-coding RNAs1, but specific biological functions and biochemical mechanisms have been discovered for only about a dozen2-7. A specific long non-coding RNA-non-coding RNA activated by DNA damage (NORAD)-has recently been shown to be required for maintaining genomic stability8, but its molecular mechanism is unknown. Here we combine RNA antisense purification and quantitative mass spectrometry to identify proteins that directly interact with NORAD in living cells. We show that NORAD interacts with proteins involved in DNA replication and repair in steady-state cells and localizes to the nucleus upon stimulation with replication stress or DNA damage. In particular, NORAD interacts with RBMX, a component of the DNA-damage response, and contains the strongest RBMX-binding site in the transcriptome. We demonstrate that NORAD controls the ability of RBMX to assemble a ribonucleoprotein complex-which we term NORAD-activated ribonucleoprotein complex 1 (NARC1)-that contains the known suppressors of genomic instability topoisomerase I (TOP1), ALYREF and the PRPF19-CDC5L complex. Cells depleted for NORAD or RBMX display an increased frequency of chromosome segregation defects, reduced replication-fork velocity and altered cell-cycle progression-which represent phenotypes that are mechanistically linked to TOP1 and PRPF19-CDC5L function. Expression of NORAD in trans can rescue defects caused by NORAD depletion, but rescue is significantly impaired when the RBMX-binding site in NORAD is deleted. Our results demonstrate that the interaction between NORAD and RBMX is important for NORAD function, and that NORAD is required for the assembly of the previously unknown topoisomerase complex NARC1, which contributes to maintaining genomic stability. In addition, we uncover a previously unknown function for long non-coding RNAs in modulating the ability of an RNA-binding protein to assemble a higher-order ribonucleoprotein complex.


Assuntos
DNA Topoisomerases Tipo I/metabolismo , Instabilidade Genômica , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , RNA Longo não Codificante/metabolismo , Proteínas de Ligação a RNA/metabolismo , Sítios de Ligação , Ciclo Celular , Proteínas de Ciclo Celular/metabolismo , Núcleo Celular/metabolismo , Sobrevivência Celular , Segregação de Cromossomos , Dano ao DNA , Reparo do DNA , Enzimas Reparadoras do DNA/metabolismo , Replicação do DNA , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Humanos , Espectrometria de Massas , Proteínas Nucleares/metabolismo , Ligação Proteica , Fatores de Processamento de RNA/metabolismo , RNA Longo não Codificante/genética , Ribonucleoproteínas/metabolismo , Fatores de Transcrição/metabolismo
17.
Nature ; 548(7667): 343-346, 2017 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-28792927

RESUMO

Mammalian genomes contain thousands of loci that transcribe long noncoding RNAs (lncRNAs), some of which are known to carry out critical roles in diverse cellular processes through a variety of mechanisms. Although some lncRNA loci encode RNAs that act non-locally (in trans), there is emerging evidence that many lncRNA loci act locally (in cis) to regulate the expression of nearby genes-for example, through functions of the lncRNA promoter, transcription, or transcript itself. Despite their potentially important roles, it remains challenging to identify functional lncRNA loci and distinguish among these and other mechanisms. Here, to address these challenges, we developed a genome-scale CRISPR-Cas9 activation screen that targets more than 10,000 lncRNA transcriptional start sites to identify noncoding loci that influence a phenotype of interest. We found 11 lncRNA loci that, upon recruitment of an activator, mediate resistance to BRAF inhibitors in human melanoma cells. Most candidate loci appear to regulate nearby genes. Detailed analysis of one candidate, termed EMICERI, revealed that its transcriptional activation resulted in dosage-dependent activation of four neighbouring protein-coding genes, one of which confers the resistance phenotype. Our screening and characterization approach provides a CRISPR toolkit with which to systematically discover the functions of noncoding loci and elucidate their diverse roles in gene regulation and cellular function.


Assuntos
Resistencia a Medicamentos Antineoplásicos/genética , Loci Gênicos/genética , Genoma Humano/genética , Indóis/farmacologia , Melanoma/genética , RNA Longo não Codificante/genética , Sulfonamidas/farmacologia , Ativação Transcricional/genética , Sistemas CRISPR-Cas/genética , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Loci Gênicos/efeitos dos fármacos , Via de Sinalização Hippo , Humanos , Indóis/uso terapêutico , Melanoma/tratamento farmacológico , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Fenótipo , Regiões Promotoras Genéticas/genética , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Sulfonamidas/uso terapêutico , Sítio de Iniciação de Transcrição , Vemurafenib
19.
Nature ; 547(7661): 55-60, 2017 07 06.
Artigo em Inglês | MEDLINE | ID: mdl-28658208

RESUMO

Genomic analysis of tumours has led to the identification of hundreds of cancer genes on the basis of the presence of mutations in protein-coding regions. By contrast, much less is known about cancer-causing mutations in non-coding regions. Here we perform deep sequencing in 360 primary breast cancers and develop computational methods to identify significantly mutated promoters. Clear signals are found in the promoters of three genes. FOXA1, a known driver of hormone-receptor positive breast cancer, harbours a mutational hotspot in its promoter leading to overexpression through increased E2F binding. RMRP and NEAT1, two non-coding RNA genes, carry mutations that affect protein binding to their promoters and alter expression levels. Our study shows that promoter regions harbour recurrent mutations in cancer with functional consequences and that the mutations occur at similar frequencies as in coding regions. Power analyses indicate that more such regions remain to be discovered through deep sequencing of adequately sized cohorts of patients.


Assuntos
Neoplasias da Mama/genética , Regulação Neoplásica da Expressão Gênica/genética , Mutação , Regiões Promotoras Genéticas/genética , Estudos de Coortes , Fatores de Transcrição E2F/metabolismo , Exoma/genética , Fator 3-alfa Nuclear de Hepatócito/genética , Fator 3-alfa Nuclear de Hepatócito/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Ligação Proteica/genética , RNA Longo não Codificante/genética , Receptores de Estrogênio/antagonistas & inibidores
20.
Proc Natl Acad Sci U S A ; 117(52): 33404-33413, 2020 12 29.
Artigo em Inglês | MEDLINE | ID: mdl-33376219

RESUMO

Single-cell quantification of RNAs is important for understanding cellular heterogeneity and gene regulation, yet current approaches suffer from low sensitivity for individual transcripts, limiting their utility for many applications. Here we present Hybridization of Probes to RNA for sequencing (HyPR-seq), a method to sensitively quantify the expression of hundreds of chosen genes in single cells. HyPR-seq involves hybridizing DNA probes to RNA, distributing cells into nanoliter droplets, amplifying the probes with PCR, and sequencing the amplicons to quantify the expression of chosen genes. HyPR-seq achieves high sensitivity for individual transcripts, detects nonpolyadenylated and low-abundance transcripts, and can profile more than 100,000 single cells. We demonstrate how HyPR-seq can profile the effects of CRISPR perturbations in pooled screens, detect time-resolved changes in gene expression via measurements of gene introns, and detect rare transcripts and quantify cell-type frequencies in tissue using low-abundance marker genes. By directing sequencing power to genes of interest and sensitively quantifying individual transcripts, HyPR-seq reduces costs by up to 100-fold compared to whole-transcriptome single-cell RNA-sequencing, making HyPR-seq a powerful method for targeted RNA profiling in single cells.


Assuntos
Sondas de DNA/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Hibridização de Ácido Nucleico , RNA/metabolismo , Análise de Célula Única , Animais , Sistemas CRISPR-Cas/genética , Expressão Gênica , Humanos , Íntrons/genética , Células K562 , Rim/citologia , Camundongos , Poliadenilação , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Células THP-1 , Fatores de Tempo
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