RESUMO
Establishing causal links between non-coding variants and human phenotypes is an increasing challenge. Here, we introduce a high-throughput mouse reporter assay for assessing the pathogenic potential of human enhancer variants in vivo and examine nearly a thousand variants in an enhancer repeatedly linked to polydactyly. We show that 71% of all rare non-coding variants previously proposed as causal lead to reporter gene expression in a pattern consistent with their pathogenic role. Variants observed to alter enhancer activity were further confirmed to cause polydactyly in knockin mice. We also used combinatorial and single-nucleotide mutagenesis to evaluate the in vivo impact of mutations affecting all positions of the enhancer and identified additional functional substitutions, including potentially pathogenic variants hitherto not observed in humans. Our results uncover the functional consequences of hundreds of mutations in a phenotype-associated enhancer and establish a widely applicable strategy for systematic in vivo evaluation of human enhancer variants.
Assuntos
Elementos Facilitadores Genéticos/genética , Ensaios de Triagem em Larga Escala/métodos , Polidactilia/genética , Animais , Elementos Facilitadores Genéticos/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/genética , Técnicas de Introdução de Genes/métodos , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Humanos , Camundongos , Mutação , Fenótipo , Polidactilia/metabolismo , RNA não Traduzido/genéticaRESUMO
Non-coding "ultraconserved" regions containing hundreds of consecutive bases of perfect sequence conservation across mammalian genomes can function as distant-acting enhancers. However, initial deletion studies in mice revealed that loss of such extraordinarily constrained sequences had no immediate impact on viability. Here, we show that ultraconserved enhancers are required for normal development. Focusing on some of the longest ultraconserved sites genome wide, located near the essential neuronal transcription factor Arx, we used genome editing to create an expanded series of knockout mice lacking individual or combinations of ultraconserved enhancers. Mice with single or pairwise deletions of ultraconserved enhancers were viable and fertile but in nearly all cases showed neurological or growth abnormalities, including substantial alterations of neuron populations and structural brain defects. Our results demonstrate the functional importance of ultraconserved enhancers and indicate that remarkably strong sequence conservation likely results from fitness deficits that appear subtle in a laboratory setting.
Assuntos
Sequência Conservada , Desenvolvimento Embrionário/genética , Elementos Facilitadores Genéticos , Animais , Encéfalo/anormalidades , Encéfalo/embriologia , Encéfalo/metabolismo , Feminino , Deleção de Genes , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Masculino , Camundongos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Common sequence variants in cis-regulatory elements (CREs) are suspected etiological causes of complex disorders. We previously identified an intronic enhancer variant in the RET gene disrupting SOX10 binding and increasing Hirschsprung disease (HSCR) risk 4-fold. We now show that two other functionally independent CRE variants, one binding Gata2 and the other binding Rarb, also reduce Ret expression and increase risk 2- and 1.7-fold. By studying human and mouse fetal gut tissues and cell lines, we demonstrate that reduced RET expression propagates throughout its gene regulatory network, exerting effects on both its positive and negative feedback components. We also provide evidence that the presence of a combination of CRE variants synergistically reduces RET expression and its effects throughout the GRN. These studies show how the effects of functionally independent non-coding variants in a coordinated gene regulatory network amplify their individually small effects, providing a model for complex disorders.
Assuntos
Elementos Facilitadores Genéticos , Regulação da Expressão Gênica , Redes Reguladoras de Genes , Doença de Hirschsprung/genética , Proteínas Proto-Oncogênicas c-ret/genética , Alelos , Animais , Sítios de Ligação , Modelos Animais de Doenças , Fator de Transcrição GATA2/genética , Fator de Transcrição GATA2/metabolismo , Trato Gastrointestinal/embriologia , Humanos , Camundongos , Camundongos Transgênicos , RNA não Traduzido/genética , Receptores do Ácido Retinoico/genética , Receptores do Ácido Retinoico/metabolismo , Fatores de Transcrição SOXE/genética , Fatores de Transcrição SOXE/metabolismoRESUMO
The evolution of body shape is thought to be tightly coupled to changes in regulatory sequences, but specific molecular events associated with major morphological transitions in vertebrates have remained elusive. We identified snake-specific sequence changes within an otherwise highly conserved long-range limb enhancer of Sonic hedgehog (Shh). Transgenic mouse reporter assays revealed that the in vivo activity pattern of the enhancer is conserved across a wide range of vertebrates, including fish, but not in snakes. Genomic substitution of the mouse enhancer with its human or fish ortholog results in normal limb development. In contrast, replacement with snake orthologs caused severe limb reduction. Synthetic restoration of a single transcription factor binding site lost in the snake lineage reinstated full in vivo function to the snake enhancer. Our results demonstrate changes in a regulatory sequence associated with a major body plan transition and highlight the role of enhancers in morphological evolution. PAPERCLIP.
Assuntos
Evolução Biológica , Elementos Facilitadores Genéticos , Extremidades/crescimento & desenvolvimento , Proteínas Hedgehog/genética , Serpentes/genética , Animais , Sequência de Bases , Evolução Molecular , Técnicas de Introdução de Genes , Camundongos , Camundongos Transgênicos , Mutação , Filogenia , Serpentes/classificaçãoRESUMO
Enhancers are distal regulatory elements that can activate tissue-specific gene expression and are abundant throughout mammalian genomes. Although substantial progress has been made toward genome-wide annotation of mammalian enhancers, their temporal activity patterns and global contributions in the context of developmental in vivo processes remain poorly explored. Here we used epigenomic profiling for H3K27ac, a mark of active enhancers, coupled to transgenic mouse assays to examine the genome-wide utilization of enhancers in three different mouse tissues across seven developmental stages. The majority of the â¼90,000 enhancers identified exhibited tightly temporally restricted predicted activity windows and were associated with stage-specific biological functions and regulatory pathways in individual tissues. Comparative genomic analysis revealed that evolutionary conservation of enhancers decreases following midgestation across all tissues examined. The dynamic enhancer activities uncovered in this study illuminate rapid and pervasive temporal in vivo changes in enhancer usage that underlie processes central to development and disease.
Assuntos
Elementos Facilitadores Genéticos , Regulação da Expressão Gênica no Desenvolvimento , Estudo de Associação Genômica Ampla , Acetilação , Animais , Epigênese Genética , Evolução Molecular , Histonas/metabolismo , Camundongos , Camundongos Transgênicos , Especificidade de ÓrgãosRESUMO
The mammalian telencephalon plays critical roles in cognition, motor function, and emotion. Though many of the genes required for its development have been identified, the distant-acting regulatory sequences orchestrating their in vivo expression are mostly unknown. Here, we describe a digital atlas of in vivo enhancers active in subregions of the developing telencephalon. We identified more than 4,600 candidate embryonic forebrain enhancers and studied the in vivo activity of 329 of these sequences in transgenic mouse embryos. We generated serial sets of histological brain sections for 145 reproducible forebrain enhancers, resulting in a publicly accessible web-based data collection comprising more than 32,000 sections. We also used epigenomic analysis of human and mouse cortex tissue to directly compare the genome-wide enhancer architecture in these species. These data provide a primary resource for investigating gene regulatory mechanisms of telencephalon development and enable studies of the role of distant-acting enhancers in neurodevelopmental disorders.
Assuntos
Elementos Facilitadores Genéticos , Telencéfalo/metabolismo , Animais , Embrião de Mamíferos/metabolismo , Feto/metabolismo , Estudo de Associação Genômica Ampla , Humanos , Camundongos , Telencéfalo/embriologia , Transcriptoma , Fatores de Transcrição de p300-CBP/metabolismoRESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMO
The Encyclopedia of DNA Elements (ENCODE) project has established a genomic resource for mammalian development, profiling a diverse panel of mouse tissues at 8 developmental stages from 10.5 days after conception until birth, including transcriptomes, methylomes and chromatin states. Here we systematically examined the state and accessibility of chromatin in the developing mouse fetus. In total we performed 1,128 chromatin immunoprecipitation with sequencing (ChIP-seq) assays for histone modifications and 132 assay for transposase-accessible chromatin using sequencing (ATAC-seq) assays for chromatin accessibility across 72 distinct tissue-stages. We used integrative analysis to develop a unified set of chromatin state annotations, infer the identities of dynamic enhancers and key transcriptional regulators, and characterize the relationship between chromatin state and accessibility during developmental gene regulation. We also leveraged these data to link enhancers to putative target genes and demonstrate tissue-specific enrichments of sequence variants associated with disease in humans. The mouse ENCODE data sets provide a compendium of resources for biomedical researchers and achieve, to our knowledge, the most comprehensive view of chromatin dynamics during mammalian fetal development to date.
Assuntos
Cromatina/genética , Cromatina/metabolismo , Conjuntos de Dados como Assunto , Desenvolvimento Fetal/genética , Histonas/metabolismo , Anotação de Sequência Molecular , Sequências Reguladoras de Ácido Nucleico/genética , Animais , Cromatina/química , Sequenciamento de Cromatina por Imunoprecipitação , Doença/genética , Elementos Facilitadores Genéticos/genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento/genética , Variação Genética , Histonas/química , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Especificidade de Órgãos/genética , Reprodutibilidade dos Testes , Transposases/metabolismoRESUMO
Distant-acting tissue-specific enhancers, which regulate gene expression, vastly outnumber protein-coding genes in mammalian genomes, but the functional importance of this regulatory complexity remains unclear. Here we show that the pervasive presence of multiple enhancers with similar activities near the same gene confers phenotypic robustness to loss-of-function mutations in individual enhancers. We used genome editing to create 23 mouse deletion lines and inter-crosses, including both single and combinatorial enhancer deletions at seven distinct loci required for limb development. Unexpectedly, none of the ten deletions of individual enhancers caused noticeable changes in limb morphology. By contrast, the removal of pairs of limb enhancers near the same gene resulted in discernible phenotypes, indicating that enhancers function redundantly in establishing normal morphology. In a genetic background sensitized by reduced baseline expression of the target gene, even single enhancer deletions caused limb abnormalities, suggesting that functional redundancy is conferred by additive effects of enhancers on gene expression levels. A genome-wide analysis integrating epigenomic and transcriptomic data from 29 developmental mouse tissues revealed that mammalian genes are very commonly associated with multiple enhancers that have similar spatiotemporal activity. Systematic exploration of three representative developmental structures (limb, brain and heart) uncovered more than one thousand cases in which five or more enhancers with redundant activity patterns were found near the same gene. Together, our data indicate that enhancer redundancy is a remarkably widespread feature of mammalian genomes that provides an effective regulatory buffer to prevent deleterious phenotypic consequences upon the loss of individual enhancers.
Assuntos
Elementos Facilitadores Genéticos/genética , Extremidades/embriologia , Regulação da Expressão Gênica no Desenvolvimento/genética , Fenótipo , Animais , Encéfalo/embriologia , Feminino , Genoma , Coração/embriologia , Deformidades Congênitas dos Membros/embriologia , Deformidades Congênitas dos Membros/genética , Masculino , Camundongos , Deleção de Sequência , Análise Espaço-TemporalRESUMO
Enhancers are important non-coding elements, but they have traditionally been hard to characterize experimentally. The development of massively parallel assays allows the characterization of large numbers of enhancers for the first time. Here, we developed a framework using Drosophila STARR-seq to create shape-matching filters based on meta-profiles of epigenetic features. We integrated these features with supervised machine-learning algorithms to predict enhancers. We further demonstrated that our model could be transferred to predict enhancers in mammals. We comprehensively validated the predictions using a combination of in vivo and in vitro approaches, involving transgenic assays in mice and transduction-based reporter assays in human cell lines (153 enhancers in total). The results confirmed that our model can accurately predict enhancers in different species without re-parameterization. Finally, we examined the transcription factor binding patterns at predicted enhancers versus promoters. We demonstrated that these patterns enable the construction of a secondary model that effectively distinguishes enhancers and promoters.
Assuntos
Epigênese Genética/fisiologia , Reconhecimento Automatizado de Padrão/métodos , Animais , Linhagem Celular , Drosophila , Histonas/genética , Histonas/metabolismo , Humanos , Camundongos , Camundongos Transgênicos , Reprodutibilidade dos TestesRESUMO
BACKGROUND: Previous research in autism and other neurodevelopmental disorders (NDDs) has indicated an important contribution of protein-coding (coding) de novo variants (DNVs) within specific genes. The role of de novo noncoding variation has been observable as a general increase in genetic burden but has yet to be resolved to individual functional elements. In this study, we assessed whole-genome sequencing data in 2671 families with autism (discovery cohort of 516 families, replication cohort of 2155 families). We focused on DNVs in enhancers with characterized in vivo activity in the brain and identified an excess of DNVs in an enhancer named hs737. RESULTS: We adapted the fitDNM statistical model to work in noncoding regions and tested enhancers for excess of DNVs in families with autism. We found only one enhancer (hs737) with nominal significance in the discovery (p = 0.0172), replication (p = 2.5 × 10-3), and combined dataset (p = 1.1 × 10-4). Each individual with a DNV in hs737 had shared phenotypes including being male, intact cognitive function, and hypotonia or motor delay. Our in vitro assessment of the DNVs showed they all reduce enhancer activity in a neuronal cell line. By epigenomic analyses, we found that hs737 is brain-specific and targets the transcription factor gene EBF3 in human fetal brain. EBF3 is genome-wide significant for coding DNVs in NDDs (missense p = 8.12 × 10-35, loss-of-function p = 2.26 × 10-13) and is widely expressed in the body. Through characterization of promoters bound by EBF3 in neuronal cells, we saw enrichment for binding to NDD genes (p = 7.43 × 10-6, OR = 1.87) involved in gene regulation. Individuals with coding DNVs have greater phenotypic severity (hypotonia, ataxia, and delayed development syndrome [HADDS]) in comparison to individuals with noncoding DNVs that have autism and hypotonia. CONCLUSIONS: In this study, we identify DNVs in the hs737 enhancer in individuals with autism. Through multiple approaches, we find hs737 targets the gene EBF3 that is genome-wide significant in NDDs. By assessment of noncoding variation and the genes they affect, we are beginning to understand their impact on gene regulatory networks in NDDs.
Assuntos
Transtorno Autístico/genética , Predisposição Genética para Doença , Hipotonia Muscular/genética , Transtornos do Neurodesenvolvimento/genética , Fatores de Transcrição/genética , Transtorno Autístico/epidemiologia , Transtorno Autístico/patologia , Elementos Facilitadores Genéticos/genética , Exoma/genética , Feminino , Redes Reguladoras de Genes/genética , Humanos , Masculino , Hipotonia Muscular/epidemiologia , Hipotonia Muscular/patologia , Mutação/genética , Transtornos do Neurodesenvolvimento/epidemiologia , Transtornos do Neurodesenvolvimento/patologia , Neurônios/metabolismo , Neurônios/patologiaRESUMO
The progressive loss of midbrain (MB) dopaminergic (DA) neurons defines the motor features of Parkinson disease (PD), and modulation of risk by common variants in PD has been well established through genome-wide association studies (GWASs). We acquired open chromatin signatures of purified embryonic mouse MB DA neurons because we anticipated that a fraction of PD-associated genetic variation might mediate the variants' effects within this neuronal population. Correlation with >2,300 putative enhancers assayed in mice revealed enrichment for MB cis-regulatory elements (CREs), and these data were reinforced by transgenic analyses of six additional sequences in zebrafish and mice. One CRE, within intron 4 of the familial PD gene SNCA, directed reporter expression in catecholaminergic neurons from transgenic mice and zebrafish. Sequencing of this CRE in 986 individuals with PD and 992 controls revealed two common variants associated with elevated PD risk. To assess potential mechanisms of action, we screened >16,000 proteins for DNA binding capacity and identified a subset whose binding is impacted by these enhancer variants. Additional genotyping across the SNCA locus identified a single PD-associated haplotype, containing the minor alleles of both of the aforementioned PD-risk variants. Our work posits a model for how common variation at SNCA might modulate PD risk and highlights the value of cell-context-dependent guided searches for functional non-coding variation.
Assuntos
Cromatina/genética , Neurônios Dopaminérgicos/patologia , Elementos Facilitadores Genéticos/genética , Predisposição Genética para Doença/genética , Doença de Parkinson/genética , alfa-Sinucleína/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Alelos , Animais , Modelos Animais de Doenças , Feminino , Genótipo , Humanos , Íntrons/genética , Masculino , Camundongos , Camundongos Transgênicos , Pessoa de Meia-Idade , Gravidez , Peixe-ZebraRESUMO
Hmx1 encodes a homeodomain transcription factor expressed in the developing lateral craniofacial mesenchyme, retina and sensory ganglia. Mutation or mis-regulation of Hmx1 underlies malformations of the eye and external ear in multiple species. Deletion or insertional duplication of an evolutionarily conserved region (ECR) downstream of Hmx1 has recently been described in rat and cow, respectively. Here, we demonstrate that the impact of Hmx1 loss is greater than previously appreciated, with a variety of lateral cranioskeletal defects, auriculofacial nerve deficits, and duplication of the caudal region of the external ear. Using a transgenic approach, we demonstrate that a 594â bp sequence encompassing the ECR recapitulates specific aspects of the endogenous Hmx1 lateral facial expression pattern. Moreover, we show that Hoxa2, Meis and Pbx proteins act cooperatively on the ECR, via a core 32â bp sequence, to regulate Hmx1 expression. These studies highlight the conserved role for Hmx1 in BA2-derived tissues and provide an entry point for improved understanding of the causes of the frequent lateral facial birth defects in humans.
Assuntos
Pareamento de Bases/genética , Pavilhão Auricular/metabolismo , Evolução Molecular , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Morfogênese/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Animais , Sequência de Bases , Sequência Conservada/genética , Anormalidades Craniofaciais/genética , Pavilhão Auricular/anormalidades , Pavilhão Auricular/patologia , Elementos Facilitadores Genéticos/genética , Face/embriologia , Regulação da Expressão Gênica no Desenvolvimento , Genes Reporter , Camundongos Mutantes , Camundongos Transgênicos , Especificidade de Órgãos/genética , Estimulação Física , Fator de Transcrição 1 de Leucemia de Células Pré-B , Ligação Proteica/genética , Células Receptoras Sensoriais/patologiaRESUMO
Retinal development requires precise temporal and spatial coordination of cell cycle exit, cell fate specification, cell migration and differentiation. When this process is disrupted, retinoblastoma, a developmental tumor of the retina, can form. Epigenetic modulators are central to precisely coordinating developmental events, and many epigenetic processes have been implicated in cancer. Studying epigenetic mechanisms in development is challenging because they often regulate multiple cellular processes; therefore, elucidating the primary molecular mechanisms involved can be difficult. Here we explore the role of Brg1 (Smarca4) in retinal development and retinoblastoma in mice using molecular and cellular approaches. Brg1 was found to regulate retinal size by controlling cell cycle length, cell cycle exit and cell survival during development. Brg1 was not required for cell fate specification but was required for photoreceptor differentiation and cell adhesion/polarity programs that contribute to proper retinal lamination during development. The combination of defective cell differentiation and lamination led to retinal degeneration in Brg1-deficient retinae. Despite the hypocellularity, premature cell cycle exit, increased cell death and extended cell cycle length, retinal progenitor cells persisted in Brg1-deficient retinae, making them more susceptible to retinoblastoma. ChIP-Seq analysis suggests that Brg1 might regulate gene expression through multiple mechanisms.
Assuntos
DNA Helicases/genética , DNA Helicases/fisiologia , Proteínas Nucleares/genética , Proteínas Nucleares/fisiologia , Retina/metabolismo , Retinoblastoma/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/fisiologia , Animais , Apoptose , Padronização Corporal , Adesão Celular , Ciclo Celular , Diferenciação Celular , Linhagem da Célula , Movimento Celular , Proliferação de Células , Modelos Animais de Doenças , Epigênese Genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Regulação Neoplásica da Expressão Gênica , Genes Supressores de Tumor , Camundongos , Microftalmia/genética , Retina/patologia , Fatores de Tempo , TransgenesRESUMO
Epigenomic mapping of enhancer-associated chromatin modifications facilitates the genome-wide discovery of tissue-specific enhancers in vivo. However, reliance on single chromatin marks leads to high rates of false-positive predictions. More sophisticated, integrative methods have been described, but commonly suffer from limited accessibility to the resulting predictions and reduced biological interpretability. Here we present the Limb-Enhancer Genie (LEG), a collection of highly accurate, genome-wide predictions of enhancers in the developing limb, available through a user-friendly online interface. We predict limb enhancers using a combination of >50 published limb-specific datasets and clusters of evolutionarily conserved transcription factor binding sites, taking advantage of the patterns observed at previously in vivo validated elements. By combining different statistical models, our approach outperforms current state-of-the-art methods and provides interpretable measures of feature importance. Our results indicate that including a previously unappreciated score that quantifies tissue-specific nuclease accessibility significantly improves prediction performance. We demonstrate the utility of our approach through in vivo validation of newly predicted elements. Moreover, we describe general features that can guide the type of datasets to include when predicting tissue-specific enhancers genome-wide, while providing an accessible resource to the general biological community and facilitating the functional interpretation of genetic studies of limb malformations.
Assuntos
Elementos Facilitadores Genéticos/genética , Extremidades/crescimento & desenvolvimento , Genômica/métodos , Crescimento e Desenvolvimento/genética , Software , Animais , Genoma/genética , Aprendizado de Máquina , CamundongosRESUMO
The SMARCA4 (also known as BRG1 in humans) chromatin remodeling factor is critical for establishing lineage-specific chromatin states during early mammalian development. However, the role of SMARCA4 in tissue-specific gene regulation during embryogenesis remains poorly defined. To investigate the genome-wide binding landscape of SMARCA4 in differentiating tissues, we engineered a Smarca4(FLAG) knock-in mouse line. Using ChIP-seq, we identified â¼51,000 SMARCA4-associated regions across six embryonic mouse tissues (forebrain, hindbrain, neural tube, heart, limb, and face) at mid-gestation (E11.5). The majority of these regions was distal from promoters and showed dynamic occupancy, with most distal SMARCA4 sites (73%) confined to a single or limited subset of tissues. To further characterize these regions, we profiled active and repressive histone marks in the same tissues and examined the intersection of informative chromatin states and SMARCA4 binding. This revealed distinct classes of distal SMARCA4-associated elements characterized by activating and repressive chromatin signatures that were associated with tissue-specific up- or down-regulation of gene expression and relevant active/repressed biological pathways. We further demonstrate the predicted active regulatory properties of SMARCA4-associated elements by retrospective analysis of tissue-specific enhancers and direct testing of SMARCA4-bound regions in transgenic mouse assays. Our results indicate a dual active/repressive function of SMARCA4 at distal regulatory sequences in vivo and support its role in tissue-specific gene regulation during embryonic development.
Assuntos
DNA Helicases/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas Nucleares/metabolismo , Elementos Reguladores de Transcrição , Fatores de Transcrição/metabolismo , Animais , Encéfalo/embriologia , Encéfalo/metabolismo , Cromatina/genética , Cromatina/metabolismo , DNA Helicases/genética , Extremidades/embriologia , Genoma , Coração/embriologia , Histonas/genética , Histonas/metabolismo , Camundongos , Miocárdio/metabolismo , Proteínas Nucleares/genética , Especificidade de Órgãos , Ligação Proteica , Fatores de Transcrição/genéticaRESUMO
The accurate and comprehensive identification of functional regulatory sequences in mammalian genomes remains a major challenge. Here we describe site-specific integration fluorescence-activated cell sorting followed by sequencing (SIF-seq), an unbiased, medium-throughput functional assay for the discovery of distant-acting enhancers. Targeted single-copy genomic integration into pluripotent cells, reporter assays and flow cytometry are coupled with high-throughput DNA sequencing to enable parallel screening of large numbers of DNA sequences. By functionally interrogating >500 kilobases (kb) of mouse and human sequence in mouse embryonic stem cells for enhancer activity we identified enhancers at pluripotency loci including NANOG. In in vitro-differentiated cardiomyocytes and neural progenitor cells, we identified cardiac enhancers and neuronal enhancers, respectively. SIF-seq is a powerful and flexible method for de novo functional identification of mammalian enhancers in a potentially wide variety of cell types.
Assuntos
Células-Tronco Embrionárias/citologia , Elementos Facilitadores Genéticos , Miócitos Cardíacos/citologia , Células-Tronco Neurais/citologia , Animais , Diferenciação Celular , Separação Celular , Cromossomos Artificiais Bacterianos/genética , Citometria de Fluxo , Regulação da Expressão Gênica , Biblioteca Gênica , Genes Reporter , Vetores Genéticos , Genômica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Camundongos , Camundongos Transgênicos , Plasmídeos/metabolismo , Análise de Sequência de DNARESUMO
Short non-coding transcripts can be transcribed from distant-acting transcriptional enhancer loci, but the prevalence of such enhancer RNAs (eRNAs) within the transcriptome, and the association of eRNA expression with tissue-specific enhancer activity in vivo remain poorly understood. Here, we investigated the expression dynamics of tissue-specific non-coding RNAs in embryonic mouse tissues via deep RNA sequencing. Overall, approximately 80% of validated in vivo enhancers show tissue-specific RNA expression that correlates with tissue-specific enhancer activity. Globally, we identified thousands of tissue-specifically transcribed non-coding regions (TSTRs) displaying various genomic hallmarks of bona fide enhancers. In transgenic mouse reporter assays, over half of tested TSTRs functioned as enhancers with reproducible activity in the predicted tissue. Together, our results demonstrate that tissue-specific eRNA expression is a common feature of in vivo enhancers, as well as a major source of extragenic transcription, and that eRNA expression signatures can be used to predict tissue-specific enhancers independent of known epigenomic enhancer marks.
Assuntos
Elementos Facilitadores Genéticos , Regulação da Expressão Gênica no Desenvolvimento , RNA não Traduzido/genética , Animais , Perfilação da Expressão Gênica , Genômica/métodos , Camundongos , Camundongos Transgênicos , Especificidade de Órgãos/genética , Reprodutibilidade dos Testes , Transcrição GênicaRESUMO
Chromatin-based functional genomic analyses and genomewide association studies (GWASs) together implicate enhancers as critical elements influencing gene expression and risk for common diseases. Here, we performed systematic chromatin and transcriptome profiling in human pancreatic islets. Integrated analysis of islet data with those from nine cell types identified specific and significant enrichment of type 2 diabetes and related quantitative trait GWAS variants in islet enhancers. Our integrated chromatin maps reveal that most enhancers are short (median = 0.8 kb). Each cell type also contains a substantial number of more extended (≥ 3 kb) enhancers. Interestingly, these stretch enhancers are often tissue-specific and overlap locus control regions, suggesting that they are important chromatin regulatory beacons. Indeed, we show that (i) tissue specificity of enhancers and nearby gene expression increase with enhancer length; (ii) neighborhoods containing stretch enhancers are enriched for important cell type-specific genes; and (iii) GWAS variants associated with traits relevant to a particular cell type are more enriched in stretch enhancers compared with short enhancers. Reporter constructs containing stretch enhancer sequences exhibited tissue-specific activity in cell culture experiments and in transgenic mice. These results suggest that stretch enhancers are critical chromatin elements for coordinating cell type-specific regulatory programs and that sequence variation in stretch enhancers affects risk of major common human diseases.