RESUMO
R-loops are three-stranded nucleic acid structures found abundantly and yet often viewed as by-products of transcription. Studying cells from patients with a motor neuron disease (amyotrophic lateral sclerosis 4 [ALS4]) caused by a mutation in senataxin, we uncovered how R-loops promote transcription. In ALS4 patients, the senataxin mutation depletes R-loops with a consequent effect on gene expression. With fewer R-loops in ALS4 cells, the expression of BAMBI, a negative regulator of transforming growth factor ß (TGF-ß), is reduced; that then leads to the activation of the TGF-ß pathway. We uncovered that genome-wide R-loops influence promoter methylation of over 1,200 human genes. DNA methyl-transferase 1 favors binding to double-stranded DNA over R-loops. Thus, in forming R-loops, nascent RNA blocks DNA methylation and promotes further transcription. Hence, our results show that nucleic acid structures, in addition to sequences, influence the binding and activity of regulatory proteins.
Assuntos
Regulação da Expressão Gênica/genética , Regiões Promotoras Genéticas , RNA Helicases/genética , RNA Helicases/metabolismo , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/metabolismo , DNA/genética , DNA/ultraestrutura , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , DNA Helicases , Metilação de DNA/genética , Humanos , Proteínas de Membrana/metabolismo , Enzimas Multifuncionais , Mutação , Regiões Promotoras Genéticas/genética , Processamento de Proteína Pós-Traducional , RNA/genética , RNA/ultraestrutura , Motivos de Ligação ao RNA , Ativação Transcricional/genética , Fator de Crescimento Transformador beta/metabolismoRESUMO
Enhancers, critical regulatory elements within the human genome, are often transcribed into enhancer RNAs. The dysregulation of enhancers leads to diseases collectively termed enhanceropathies. While it is known that enhancers play a role in diseases by regulating gene expression, the specific mechanisms by which individual enhancers cause diseases are not well understood. Studies of individual enhancers are needed to fill this gap. This study delves into the role of APOE-activating noncoding RNA, AANCR, in the central nervous system, elucidating its function as a genetic modifier in Alzheimer's Disease. We employed RNA interference, RNaseH-mediated degradation, and single-molecule RNA fluorescence in situ hybridization to demonstrate that mere transcription of AANCR is insufficient; rather, its transcripts are crucial for promoting APOE expression. Our findings revealed that AANCR is induced by ATM-mediated ERK phosphorylation and subsequent AP-1 transcription factor activation. Once activated, AANCR enhances APOE expression, which in turn imparts an inflammatory phenotype to astrocytes. These findings demonstrate that AANCR is a key enhancer RNA in some cell types within the nervous system, pivotal for regulating APOE expression and influencing inflammatory responses, underscoring its potential as a therapeutic target in neurodegenerative diseases.
RESUMO
RNA is modified by hundreds of chemical reactions and folds into innumerable shapes. However, the regulatory role of RNA sequence and structure and how dysregulation leads to diseases remain largely unknown. Here, we uncovered a mechanism where RNA abasic sites in R-loops regulate transcription by pausing RNA polymerase II. We found an enhancer RNA, AANCR, that regulates the transcription and expression of apolipoprotein E (APOE). In some human cells such as fibroblasts, AANCR is folded into an R-loop and modified by N-glycosidic cleavage; in this form, AANCR is a partially transcribed nonfunctional enhancer and APOE is not expressed. In contrast, in other cell types including hepatocytes and under stress, AANCR does not form a stable R-loop as its sequence is not modified, so it is transcribed into a full-length enhancer that promotes APOE expression. DNA sequence variants in AANCR are associated significantly with APOE expression and Alzheimer's Disease, thus AANCR is a modifier of Alzheimer's Disease. Besides AANCR, thousands of noncoding RNAs are regulated by abasic sites in R-loops. Together our data reveal the essentiality of the folding and modification of RNA in cellular regulation and demonstrate that dysregulation underlies common complex diseases such as Alzheimer's disease.
Assuntos
Doença de Alzheimer , Estruturas R-Loop , Humanos , RNA/genética , Doença de Alzheimer/genética , Transcrição Gênica , Apolipoproteínas E/genéticaRESUMO
RNA abasic sites and the mechanisms involved in their regulation are mostly unknown; in contrast, DNA abasic sites are well-studied. We found surprisingly that, in yeast and human cells, RNA abasic sites are prevalent. When a base is lost from RNA, the remaining ribose is found as a closed-ring or an open-ring sugar with a reactive C1' aldehyde group. Using primary amine-based reagents that react with the aldehyde group, we uncovered evidence for abasic sites in nascent RNA, messenger RNA, and ribosomal RNA from yeast and human cells. Mass spectroscopic analysis confirmed the presence of RNA abasic sites. The RNA abasic sites were found to be coupled to R-loops. We show that human methylpurine DNA glycosylase cleaves N-glycosidic bonds on RNA and that human apurinic/apyrimidinic endonuclease 1 incises RNA abasic sites in RNA-DNA hybrids. Our results reveal that, in yeast and human cells, there are RNA abasic sites, and we identify a glycosylase that generates these sites and an AP endonuclease that processes them.
Assuntos
Sequência de Bases/genética , RNA/química , RNA/genética , Sítios de Ligação , DNA/química , Dano ao DNA/genética , DNA Glicosilases/metabolismo , Reparo do DNA/genética , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/genética , Desoxirribonuclease I/metabolismo , Humanos , Nucleotídeos/genética , Estruturas R-Loop/genética , Saccharomyces cerevisiae/genética , Especificidade por Substrato , Leveduras/genéticaRESUMO
Aberrant gene expression underlies many human diseases. RNA polymerase II (Pol II) pausing is a key regulatory step in transcription. Here, we mapped the locations of RNA Pol II in normal human cells and found that RNA Pol II pauses in a consistent manner across individuals and cell types. At more than 1,000 genes including MYO1E and SESN2, RNA Pol II pauses at precise nucleotide locations. Characterization of these sites shows that RNA Pol II pauses at GC-rich regions that are marked by a sequence motif. Sixty-five percent of the pause sites are cytosines. By differential allelic gene expression analysis, we showed in our samples and a population dataset from the Genotype-Tissue Expression (GTEx) consortium that genes with more paused polymerase have lower expression levels. Furthermore, mutagenesis of the pause sites led to a significant increase in promoter activities. Thus, our data uncover that RNA Pol II pauses precisely at sites with distinct sequence features that in turn regulate gene expression.
Assuntos
Regulação da Expressão Gênica , RNA Polimerase II/metabolismo , Adulto , Alelos , Células Cultivadas , Humanos , Recém-NascidoRESUMO
RNA/DNA hybrids form when RNA hybridizes with its template DNA generating a three-stranded structure known as the R-loop. Knowledge of how they form and resolve, as well as their functional roles, is limited. Here, by pull-down assays followed by mass spectrometry, we identified 803 proteins that bind to RNA/DNA hybrids. Because these proteins were identified using in vitro assays, we confirmed that they bind to R-loops in vivo. They include proteins that are involved in a variety of functions, including most steps of RNA processing. The proteins are enriched for K homology (KH) and helicase domains. Among them, more than 300 proteins preferred binding to hybrids than double-stranded DNA. These proteins serve as starting points for mechanistic studies to elucidate what RNA/DNA hybrids regulate and how they are regulated.
Assuntos
Proteínas de Ligação a DNA/química , DNA/química , Proteínas de Ligação a RNA/química , RNA/química , Linhagem Celular , DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Humanos , Conformação de Ácido Nucleico , Ligação Proteica , Domínios Proteicos , RNA/metabolismo , Proteínas de Ligação a RNA/metabolismoRESUMO
OBJECTIVE: To determine the clinical and molecular features in patients with amyotrophic lateral sclerosis 4 (ALS4) due to mutations in the senataxin (SETX) gene and to develop tools for evaluating SETX variants. METHODS: Our study involved 32 patients, including 31 with mutation in SETX at c.1166 T>C (p.Leu389Ser) and 1 with mutation at c.1153 G>A (p.Glu385Lys). Clinical characterization of the patients included neurological examination, blood tests, magnetic resonance imaging (MRI), and dual-energy x-ray absorptiometry (DEXA). Fibroblasts and motor neurons were obtained to model the disease and characterize the molecular alteration in senataxin function. RESULTS: We report key clinical features of ALS4. Laboratory analysis showed alteration of serum creatine kinase and creatinine in the Leu389Ser ALS4 cohort. MRI showed increased muscle fat fraction in the lower extremities, which correlates with disease duration (thigh fat fraction R2 = 0.35, p = 0.01; lower leg fat fraction R2 = 0.49, p < 0.01). DEXA measurements showed lower extremities are more affected than upper extremities (average fat z scores of 2.1 and 0.6, respectively). A cellular assay for SETX function confirmed that like the Leu389Ser mutation, the Glu385Lys variant leads to a decrease in R loops, likely from a gain of function. INTERPRETATION: We identified clinical laboratory and radiological features of ALS4, and hence they should be monitored for disease progression. The molecular characterization of R-loop levels in patient-derived cells provides insight into the disease pathology and assays to evaluate the pathogenicity of candidate mutations in the SETX gene. ANN NEUROL 2020;87:547-555.
Assuntos
Esclerose Lateral Amiotrófica/metabolismo , DNA Helicases/metabolismo , Enzimas Multifuncionais/metabolismo , RNA Helicases/metabolismo , Absorciometria de Fóton , Tecido Adiposo/diagnóstico por imagem , Adulto , Idoso , Idoso de 80 Anos ou mais , Esclerose Lateral Amiotrófica/diagnóstico por imagem , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/fisiopatologia , Western Blotting , Creatina Quinase/metabolismo , Creatinina/metabolismo , DNA Helicases/genética , Eletromiografia , Feminino , Fibroblastos , Humanos , Células-Tronco Pluripotentes Induzidas , Lactente , Extremidade Inferior/diagnóstico por imagem , Imageamento por Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Enzimas Multifuncionais/genética , Músculo Esquelético/diagnóstico por imagem , Mutação , Condução Nervosa , Estruturas R-Loop/genética , RNA Helicases/genética , RNA Mensageiro , Extremidade Superior/diagnóstico por imagem , Adulto JovemRESUMO
Complex regulation of gene expression in mammals has evolved from simpler eukaryotic systems, yet the mechanistic features of this evolution remain elusive. Here, we compared the transcriptional landscapes of the distantly related budding and fission yeast. We adapted the Precision Run-On sequencing (PRO-seq) approach to map the positions of RNA polymerase active sites genome-wide in Schizosaccharomyces pombe and Saccharomyces cerevisiae. Additionally, we mapped preferred sites of transcription initiation in each organism using PRO-cap. Unexpectedly, we identify a pause in early elongation, specific to S. pombe, that requires the conserved elongation factor subunit Spt4 and resembles promoter-proximal pausing in metazoans. PRO-seq profiles in strains lacking Spt4 reveal globally elevated levels of transcribing RNA Polymerase II (Pol II) within genes in both species. Messenger RNA abundance, however, does not reflect the increases in Pol II density, indicating a global reduction in elongation rate. Together, our results provide the first base-pair resolution map of transcription elongation in S. pombe and identify divergent roles for Spt4 in controlling elongation in budding and fission yeast.
Assuntos
Fatores de Alongamento de Peptídeos/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Proteínas de Schizosaccharomyces pombe/genética , Schizosaccharomyces/genética , Evolução Molecular , Regulação Fúngica da Expressão Gênica , Nucleossomos/enzimologia , Nucleossomos/genética , Regiões Promotoras Genéticas , RNA Polimerase II/fisiologia , Transcrição GênicaRESUMO
Alterations of RNA sequences and structures, such as those from editing and alternative splicing, result in two or more RNA transcripts from a DNA template. It was thought that in yeast, RNA editing only occurs in tRNAs. Here, we found that Saccharomyces cerevisiae have all 12 types of RNA-DNA sequence differences (RDDs) in the mRNA. We showed these sequence differences are propagated to proteins, as we identified peptides encoded by the RNA sequences in addition to those by the DNA sequences at RDD sites. RDDs are significantly enriched at regions with R-loops. A screen of yeast mutants showed that RDD formation is affected by mutations in genes regulating R-loops. Loss-of-function mutations in ribonuclease H, senataxin, and topoisomerase I that resolve RNA-DNA hybrids lead to increases in RDD frequency. Our results demonstrate that RDD is a conserved process that diversifies transcriptomes and proteomes and provide a mechanistic link between R-loops and RDDs.
Assuntos
Pareamento Incorreto de Bases , DNA Fúngico/genética , RNA Fúngico/genética , RNA Mensageiro/genética , Saccharomyces cerevisiae/genética , DNA Topoisomerases Tipo I/genética , DNA Fúngico/química , Mutação com Perda de Função , RNA Fúngico/química , RNA Mensageiro/química , Ribonuclease H/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
Modifications to the global run-on and sequencing (GRO-seq) protocol that enrich for 5'-capped RNAs can be used to reveal active transcriptional regulatory elements (TREs) with high accuracy. Here, we introduce discriminative regulatory-element detection from GRO-seq (dREG), a sensitive machine learning method that uses support vector regression to identify active TREs from GRO-seq data without requiring cap-based enrichment (https://github.com/Danko-Lab/dREG/). This approach allows TREs to be assayed together with gene expression levels and other transcriptional features in a single experiment. Predicted TREs are more enriched for several marks of transcriptional activationincluding expression quantitative trait loci, disease-associated polymorphisms, acetylated histone 3 lysine 27 (H3K27ac) and transcription factor bindingthan those identified by alternative functional assays. Using dREG, we surveyed TREs in eight human cell types and provide new insights into global patterns of TRE function.
Assuntos
Inteligência Artificial , Regulação da Expressão Gênica/fisiologia , Elementos Reguladores de Transcrição/fisiologia , Linhagem Celular , Estudo de Associação Genômica Ampla , Histonas , Humanos , Células K562 , Polimorfismo de Nucleotídeo Único , Locos de Características Quantitativas , Elementos Reguladores de Transcrição/genética , SoftwareRESUMO
DNA sequence variants influence gene expression and cellular phenotypes. In this study, we focused on natural variation in the gene encoding the histone demethylase, KDM4C, which promotes transcriptional activation by removing the repressive histone mark, H3K9me3, from its target genes. We uncovered cis-acting variants that contribute to extensive individual differences in KDM4C expression. We also identified the target genes of KDM4C and demonstrated that variation in KDM4C expression leads to differences in the growth of normal and some cancer cells. Together, our results from genetic mapping and molecular analysis provide an example of how genetic variation affects epigenetic regulation of gene expression and cellular phenotype.
Assuntos
Proliferação de Células , Regulação da Expressão Gênica , Histonas/metabolismo , Histona Desmetilases com o Domínio Jumonji/genética , Histona Desmetilases com o Domínio Jumonji/metabolismo , Neoplasias/genética , Células Cultivadas , Epigênese Genética , Técnicas de Silenciamento de Genes , Ligação Genética , Variação Genética , Humanos , Metilação , Neoplasias/metabolismo , Neoplasias/patologia , FenótipoRESUMO
Individual differences in sensitivity to insulin contribute to disease susceptibility including diabetes and metabolic syndrome. Cellular responses to insulin are well studied. However, which steps in these response pathways differ across individuals remains largely unknown. Such knowledge is needed to guide more precise therapeutic interventions. Here, we studied insulin response and found extensive individual variation in the activation of key signaling factors, including ERK whose induction differs by more than 20-fold among our subjects. This variation in kinase activity is propagated to differences in downstream gene expression response to insulin. By genetic analysis, we identified cis-acting DNA variants that influence signaling response, which in turn affects downstream changes in gene expression and cellular phenotypes, such as protein translation and cell proliferation. These findings show that polymorphic differences in signal transduction contribute to individual variation in insulin response, and suggest kinase modulators as promising therapeutics for diseases characterized by insulin resistance.
Assuntos
Variação Genética , Insulina/farmacologia , Sistema de Sinalização das MAP Quinases , Receptor de Insulina/metabolismo , Proteínas de Transporte/genética , Prepúcio do Pênis/metabolismo , Proteínas Ativadoras de GTPase/genética , Regulação da Expressão Gênica , Humanos , Recém-Nascido , Resistência à Insulina , Masculino , Proteínas de Neoplasias/genética , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
Cells respond to variable environments by changing gene expression and gene interactions. To study how human cells response to stress, we analyzed the expression of >5000 genes in cultured B cells from nearly 100 normal individuals following endoplasmic reticulum stress and exposure to ionizing radiation. We identified thousands of genes that are induced or repressed. Then, we constructed coexpression networks and inferred interactions among genes. We used coexpression and machine learning analyses to study how genes interact with each other in response to stress. The results showed that for most genes, their interactions with each other are the same at baseline and in response to different stresses; however, a small set of genes acquired new interacting partners to engage in stress-specific responses. These genes with altered interacting partners are associated with diseases in which endoplasmic reticulum stress response or sensitivity to radiation has been implicated. Thus, our findings showed that to understand disease-specific pathways, it is important to identify not only genes that change expression levels but also those that alter interactions with other genes.
Assuntos
Regulação da Expressão Gênica , Estresse Fisiológico/genética , Inteligência Artificial , Células Cultivadas , Estresse do Retículo Endoplasmático/genética , Redes Reguladoras de Genes , Radiação IonizanteRESUMO
Variation in DNA sequence contributes to individual differences in quantitative traits, but in humans the specific sequence variants are known for very few traits. We characterized variation in gene expression in cells from individuals belonging to three major population groups. This quantitative phenotype differs significantly between European-derived and Asian-derived populations for 1,097 of 4,197 genes tested. For the phenotypes with the strongest evidence of cis determinants, most of the variation is due to allele frequency differences at cis-linked regulators. The results show that specific genetic variation among populations contributes appreciably to differences in gene expression phenotypes. Populations differ in prevalence of many complex genetic diseases, such as diabetes and cardiovascular disease. As some of these are probably influenced by the level of gene expression, our results suggest that allele frequency differences at regulatory polymorphisms also account for some population differences in prevalence of complex diseases.
Assuntos
Etnicidade/genética , Perfilação da Expressão Gênica , Expressão Gênica , Frequência do Gene , Variação Genética , Genética Populacional , Humanos , Japão , Fenótipo , Polimorfismo de Nucleotídeo Único , População BrancaRESUMO
Radiation exposure through environmental, medical, and occupational settings is increasingly common. While radiation has harmful effects, it has utility in many applications such as radiotherapy for cancer. To increase the efficacy of radiation treatment and minimize its risks, a better understanding of the individual differences in radiosensitivity and the molecular basis of radiation response is needed. Here, we integrated human genetic and functional genomic approaches to study the response of human cells to radiation. We measured radiation-induced changes in gene expression and cell death in B cells from normal individuals. We found extensive individual variation in gene expression and cellular responses. To understand the genetic basis of this variation, we mapped the DNA sequence variants that influence expression response to radiation. We also identified radiation-responsive genes that regulate cell death; silencing of these genes by small interfering RNA led to an increase in radiation-induced cell death in human B cells, colorectal and prostate cancer cells. Together these results uncovered DNA variants that contribute to radiosensitivity and identified genes that can be targeted to increase the sensitivity of tumors to radiation.
Assuntos
Morte Celular/genética , Variação Genética , Tolerância a Radiação/genética , Apoptose , Linhagem Celular Tumoral , Regulação da Expressão Gênica/efeitos da radiação , Inativação Gênica , Ligação Genética , Genética Populacional , Humanos , Polimorfismo de Nucleotídeo ÚnicoRESUMO
There is extensive natural variation in human gene expression. As quantitative phenotypes, expression levels of genes are heritable. Genetic linkage and association mapping have identified cis- and trans-acting DNA variants that influence expression levels of human genes. New insights into human gene regulation are emerging from genetic analyses of gene expression in cells at rest and following exposure to stimuli. The integration of these genetic mapping results with data from co-expression networks is leading to a better understanding of how expression levels of individual genes are regulated and how genes interact with each other. These findings are important for basic understanding of gene regulation and of diseases that result from disruption of normal gene regulation.
Assuntos
Mapeamento Cromossômico , Regulação da Expressão Gênica/genética , Variação Genética/fisiologia , Mapeamento Cromossômico/métodos , Epistasia Genética/genética , Ligação Genética , Humanos , Modelos BiológicosRESUMO
Humans are exposed to radiation through the environment and in medical settings. To deal with radiation-induced damage, cells mount complex responses that rely on changes in gene expression. These gene expression responses differ greatly between individuals and contribute to individual differences in response to radiation. Here we identify regulators that influence expression levels of radiation-responsive genes. We treated radiation-induced changes in gene expression as quantitative phenotypes, and conducted genetic linkage and association studies to map their regulators. For more than 1,200 of these phenotypes there was significant evidence of linkage to specific chromosomal regions. Nearly all of the regulators act in trans to influence the expression of their target genes; there are very few cis-acting regulators. Some of the trans-acting regulators are transcription factors, but others are genes that were not known to have a regulatory function in radiation response. These results have implications for our basic and clinical understanding of how human cells respond to radiation.
Assuntos
Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos da radiação , Variação Genética/genética , Toxicogenética , Alelos , Linfócitos B/metabolismo , Linfócitos B/efeitos da radiação , Caspases/metabolismo , Linhagem Celular , Cromossomos Humanos/genética , Técnicas de Silenciamento de Genes , Ligação Genética , Genoma Humano/genética , Genótipo , Humanos , Fenótipo , Reação em Cadeia da Polimerase , Polimorfismo de Nucleotídeo Único/genética , Fatores de Transcrição/metabolismoAssuntos
Pessoas com Deficiência , Médicas , Preconceito , Animais , Feminino , Humanos , Grupos Minoritários , Animais de TrabalhoRESUMO
Our genotype inference method combines sparse marker data from a linkage scan and high-resolution SNP genotypes for several individuals to infer genotypes for related individuals. We illustrate the method's utility by inferring over 53 million SNP genotypes for 78 children in the Centre d'Etude du Polymorphisme Humain families. The method can be used to obtain high-density genotypes in different family structures, including nuclear families commonly used in complex disease gene mapping studies.