RESUMEN
BACKGROUND: Although disease-causal genetic variants have been found within silencer sequences, we still lack a comprehensive analysis of the association of silencers with diseases. Here, we profiled GWAS variants in 2.8 million candidate silencers across 97 human samples derived from a diverse panel of tissues and developmental time points, using deep learning models. RESULTS: We show that candidate silencers exhibit strong enrichment in disease-associated variants, and several diseases display a much stronger association with silencer variants than enhancer variants. Close to 52% of candidate silencers cluster, forming silencer-rich loci, and, in the loci of Parkinson's-disease-hallmark genes TRIM31 and MAL, the associated SNPs densely populate clustered candidate silencers rather than enhancers displaying an overall twofold enrichment in silencers versus enhancers. The disruption of apoptosis in neuronal cells is associated with both schizophrenia and bipolar disorder and can largely be attributed to variants within candidate silencers. Our model permits a mechanistic explanation of causative SNP effects by identifying altered binding of tissue-specific repressors and activators, validated with a 70% of directional concordance using SNP-SELEX. Narrowing the focus of the analysis to individual silencer variants, experimental data confirms the role of the rs62055708 SNP in Parkinson's disease, rs2535629 in schizophrenia, and rs6207121 in type 1 diabetes. CONCLUSIONS: In summary, our results indicate that advances in deep learning models for the discovery of disease-causal variants within candidate silencers effectively "double" the number of functionally characterized GWAS variants. This provides a basis for explaining mechanisms of action and designing novel diagnostics and therapeutics.
Asunto(s)
Estudio de Asociación del Genoma Completo , Polimorfismo de Nucleótido Simple , Humanos , Enfermedad de Parkinson/genética , Predisposición Genética a la Enfermedad , Aprendizaje Profundo , Esquizofrenia/genética , Elementos Silenciadores Transcripcionales/genéticaRESUMEN
The action of cis-regulatory elements with either activation or repression functions underpins the precise regulation of gene expression during normal development and cell differentiation. Gene activation by the combined activities of promoters and distal enhancers has been extensively studied in normal and pathological contexts. In sharp contrast, gene repression by cis-acting silencers, defined as genetic elements that negatively regulate gene transcription in a position-independent fashion, is less well understood. Here, we repurpose the STARR-seq approach as a novel high-throughput reporter strategy to quantitatively assess silencer activity in mammals. We assessed silencer activity from DNase hypersensitive I sites in a mouse T cell line. Identified silencers were associated with either repressive or active chromatin marks and enriched for binding motifs of known transcriptional repressors. CRISPR-mediated genomic deletions validated the repressive function of distinct silencers involved in the repression of non-T cell genes and genes regulated during T cell differentiation. Finally, we unravel an association of silencer activity with short tandem repeats, highlighting the role of repetitive elements in silencer activity. Our results provide a general strategy for genome-wide identification and characterization of silencer elements.
Asunto(s)
Elementos Silenciadores Transcripcionales , Linfocitos T , Animales , Ratones , Elementos Silenciadores Transcripcionales/genética , Linfocitos T/metabolismo , Factores de Transcripción/metabolismo , Secuencias Reguladoras de Ácidos Nucleicos , Repeticiones de Microsatélite , Mamíferos/genéticaRESUMEN
Cell type- and differentiation-specific gene expression is precisely controlled by genomic non-coding regulatory elements (NCREs), which include promoters, enhancers, silencers and insulators. It is estimated that more than 90% of disease-associated sequence variants lie within the non-coding part of the genome, potentially affecting the activity of NCREs. Consequently, the functional annotation of NCREs is a major driver of genome research. Compared with our knowledge of other regulatory elements, our knowledge of silencers, which are NCREs that repress the transcription of genes, is largely lacking. Multiple recent studies have reported large-scale identification of transcription silencer elements, indicating their importance in homeostasis and disease. In this Review, we discuss the biology of silencers, including methods for their discovery, epigenomic and other characteristics, and modes of function of silencers. We also discuss important silencer-relevant considerations in assessing data from genome-wide association studies and shed light on potential future silencer-based therapeutic applications.
Asunto(s)
Estudio de Asociación del Genoma Completo , Elementos Silenciadores Transcripcionales , Elementos Silenciadores Transcripcionales/genética , Secuencias Reguladoras de Ácidos Nucleicos/genética , Regiones Promotoras Genéticas , Regulación de la Expresión Génica/genéticaRESUMEN
Drosophila chromosomes are elongated by retrotransposon attachment, a process poorly understood. Here we characterized a mutation affecting the HipHop telomere-capping protein. In mutant ovaries and the embryos that they produce, telomere retrotransposons are activated and transposon RNP accumulates. Genetic results are consistent with that this hiphop mutation weakens the efficacy of HP1-mediated silencing while leaving piRNA-based mechanisms largely intact. Remarkably, mutant females display normal fecundity suggesting that telomere de-silencing is compatible with germline development. Moreover, unlike prior mutants with overactive telomeres, the hiphop stock does not over-accumulate transposons for hundreds of generations. This is likely due to the loss of HipHop's abilities both to silence transcription and to recruit transposons to telomeres in the mutant. Furthermore, embryos produced by mutant mothers experience a checkpoint activation, and a further loss of maternal HipHop leads to end-to-end fusion and embryonic arrest. Telomeric retroelements fulfill an essential function yet maintain a potentially conflicting relationship with their Drosophila host. Our study thus showcases a possible intermediate in this arm race in which the host is adapting to over-activated transposons while maintaining genome stability. Our results suggest that the collapse of such a relationship might only occur when the selfish element acquires the ability to target non-telomeric regions of the genome. HipHop is likely part of this machinery restricting the elements to the gene-poor region of telomeres. Lastly, our hiphop mutation behaves as a recessive suppressor of PEV that is mediated by centric heterochromatin, suggesting its broader effect on chromatin not limited to telomeres.
Asunto(s)
Elementos Transponibles de ADN/genética , Proteínas de Drosophila/genética , Retroelementos/genética , Elementos Silenciadores Transcripcionales/genética , Telómero/genética , Animales , Cromatina/genética , Proteínas Cromosómicas no Histona/genética , Drosophila melanogaster/genética , Femenino , Inestabilidad Genómica/genética , Células Germinativas/metabolismo , Heterocromatina/genética , Mutación , ARN Interferente Pequeño/genéticaRESUMEN
The mechanisms underlying gene repression and silencers are poorly understood. Here we investigate the hypothesis that H3K27me3-rich regions of the genome, defined from clusters of H3K27me3 peaks, may be used to identify silencers that can regulate gene expression via proximity or looping. We find that H3K27me3-rich regions are associated with chromatin interactions and interact preferentially with each other. H3K27me3-rich regions component removal at interaction anchors by CRISPR leads to upregulation of interacting target genes, altered H3K27me3 and H3K27ac levels at interacting regions, and altered chromatin interactions. Chromatin interactions did not change at regions with high H3K27me3, but regions with low H3K27me3 and high H3K27ac levels showed changes in chromatin interactions. Cells with H3K27me3-rich regions knockout also show changes in phenotype associated with cell identity, and altered xenograft tumor growth. Finally, we observe that H3K27me3-rich regions-associated genes and long-range chromatin interactions are susceptible to H3K27me3 depletion. Our results characterize H3K27me3-rich regions and their mechanisms of functioning via looping.
Asunto(s)
Cromatina/metabolismo , Represión Epigenética , Histonas/genética , Neoplasias/genética , Elementos Silenciadores Transcripcionales/genética , Animales , Línea Celular Tumoral , Cromatina/genética , Secuenciación de Inmunoprecipitación de Cromatina , Femenino , Factores de Crecimiento de Fibroblastos/genética , Regulación Neoplásica de la Expresión Génica , Técnicas de Silenciamiento del Gen , Técnicas de Inactivación de Genes , Histonas/metabolismo , Humanos , Factor II del Crecimiento Similar a la Insulina/genética , Ratones , RNA-Seq , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The study of gene regulation is dominated by a focus on the control of gene activation or increase in the level of expression. Just as critical is the process of gene repression or silencing. Chromatin signatures have identified enhancers, however, genome-wide identification of silencers by computational or experimental approaches are lacking. Here, we first define uncharacterized cis-regulatory elements likely containing silencers and find that 41.5% of ~7500 tested elements show silencer activity using massively parallel reporter assay (MPRA). We trained a support vector machine classifier based on MPRA data to predict candidate silencers in over 100 human and mouse cell or tissue types. The predicted candidate silencers exhibit characteristics expected of silencers. Leveraging promoter-capture HiC data, we find that over 50% of silencers are interacting with gene promoters having very low to no expression. Our results suggest a general strategy for genome-wide identification and characterization of silencer elements.
Asunto(s)
Regulación de la Expresión Génica/genética , Proteínas Represoras/genética , Elementos Silenciadores Transcripcionales/genética , Transcripción Genética/genética , Animales , Línea Celular , Elementos de Facilitación Genéticos/genética , Humanos , Ratones , Regiones Promotoras Genéticas/genética , Activación Transcripcional/genéticaRESUMEN
The majority of the human genome does not encode proteins. Many of these noncoding regions contain important regulatory sequences that control gene expression. To date, most studies have focused on activators such as enhancers, but regions that repress gene expression-silencers-have not been systematically studied. We have developed a system that identifies silencer regions in a genome-wide fashion on the basis of silencer-mediated transcriptional repression of caspase 9. We found that silencers are widely distributed and may function in a tissue-specific fashion. These silencers harbor unique epigenetic signatures and are associated with specific transcription factors. Silencers also act at multiple genes, and at the level of chromosomal domains and long-range interactions. Deletion of silencer regions linked to the drug transporter genes ABCC2 and ABCG2 caused chemo-resistance. Overall, our study demonstrates that tissue-specific silencing is widespread throughout the human genome and probably contributes substantially to the regulation of gene expression and human biology.
Asunto(s)
Silenciador del Gen , Variación Genética , Genoma Humano/genética , Proteínas Represoras/genética , Elementos Silenciadores Transcripcionales/genética , Eliminación de Gen , Humanos , Proteína 2 Asociada a Resistencia a Múltiples Medicamentos , Especificidad de Órganos , Transcripción GenéticaRESUMEN
Lineage-specific gene expression is modulated by a balance between transcriptional activation and repression during animal development. Knowledge about enhancer-centered transcriptional activation has advanced considerably, but silencers and their roles in normal development remain poorly understood. Here, we performed chromatin interaction analyses of Polycomb repressive complex 2 (PRC2), a key inducer of transcriptional gene silencing, to uncover silencers, their molecular identity and associated chromatin connectivity. Systematic analysis of cis-regulatory silencer elements reveals their chromatin features and gene-targeting specificity. Deletion of certain PRC2-bound silencers in mice results in transcriptional derepression of their interacting genes and pleiotropic developmental phenotypes, including embryonic lethality. While some PRC2-bound elements function as silencers in pluripotent cells, they can transition into active tissue-specific enhancers during development, highlighting their regulatory versatility. Our study characterizes the molecular profile of silencers and their associated chromatin architectures, and suggests the possibility of targeted reactivation of epigenetically silenced genes.
Asunto(s)
Cromatina/genética , Elementos de Facilitación Genéticos/genética , Silenciador del Gen , Complejo Represivo Polycomb 2/metabolismo , Proteínas Represoras/metabolismo , Elementos Silenciadores Transcripcionales/genética , Animales , Línea Celular , Femenino , Masculino , Ratones , Ratones Noqueados , Células Madre Embrionarias de Ratones , Especificidad de Órganos , Fenotipo , Complejo Represivo Polycomb 2/genética , Proteínas Represoras/genética , Activación TranscripcionalRESUMEN
cis-Regulatory communication is crucial in mammalian development and is thought to be restricted by the spatial partitioning of the genome in topologically associating domains (TADs). Here, we discovered that the Xist locus is regulated by sequences in the neighboring TAD. In particular, the promoter of the noncoding RNA Linx (LinxP) acts as a long-range silencer and influences the choice of X chromosome to be inactivated. This is independent of Linx transcription and independent of any effect on Tsix, the antisense regulator of Xist that shares the same TAD as Linx. Unlike Tsix, LinxP is well conserved across mammals, suggesting an ancestral mechanism for random monoallelic Xist regulation. When introduced in the same TAD as Xist, LinxP switches from a silencer to an enhancer. Our study uncovers an unsuspected regulatory axis for X chromosome inactivation and a class of cis-regulatory effects that may exploit TAD partitioning to modulate developmental decisions.
Asunto(s)
Secuencia Conservada/genética , ARN Largo no Codificante/genética , Cromosoma X/genética , Animales , Línea Celular , Elementos de Facilitación Genéticos/genética , Ratones , Regiones Promotoras Genéticas/genética , ARN sin Sentido/genética , Elementos Silenciadores Transcripcionales/genética , Transcripción Genética/genéticaRESUMEN
A major challenge in biology is to understand how complex gene expression patterns are encoded in the genome. While transcriptional enhancers have been studied extensively, few transcriptional silencers have been identified, and they remain poorly understood. Here, we used a novel strategy to screen hundreds of sequences for tissue-specific silencer activity in whole Drosophila embryos. Almost all of the transcriptional silencers that we identified were also active enhancers in other cellular contexts. These elements are bound by more transcription factors than non-silencers. A subset of these silencers forms long-range contacts with promoters. Deletion of a silencer caused derepression of its target gene. Our results challenge the common practice of treating enhancers and silencers as separate classes of regulatory elements and suggest the possibility that thousands or more bifunctional CRMs remain to be discovered in Drosophila and 104-105 in humans.
Asunto(s)
Drosophila/genética , Elementos de Facilitación Genéticos/genética , Elementos Silenciadores Transcripcionales/genética , Transcripción Genética/genética , Animales , Animales Modificados Genéticamente/genética , MasculinoRESUMEN
DNA damage responses (DDR) to double-strand breaks (DSBs) alter cellular transcription programs at the genome-wide level. Through processes that are less well understood, DSBs also alter transcriptional responses locally, which may be important for efficient DSB repair. Here, we developed an approach to elucidate the cis-acting responses to DSBs in G1 phase cells. We found that DSBs within a gene body silence its expression, as well as the transcription of local undamaged genes at a distance defined by the spread of γ-H2AX from the DSB. Importantly, DSBs not only repress ongoing transcription but also block the inducible expression of regional genes. DSB-mediated transcriptional repression depends on DDR signaling but does not require the generation of inaccessible chromatin. Our findings demonstrate that in G1 phase cells, DDR signaling establishes a robust and extensive region of transcriptional repression spreading from DSB sites and introduce an approach to study the mechanistic impact of targeted DNA breaks in nearly any chromatin environment.
Asunto(s)
Reparación del ADN/genética , Fase G1/genética , Elementos Silenciadores Transcripcionales/genética , Animales , Ciclo Celular/genética , Línea Celular , ADN/genética , Roturas del ADN de Doble Cadena , Daño del ADN/fisiología , Reparación del ADN por Unión de Extremidades/genética , Reparación del ADN/fisiología , Proteínas de Unión al ADN/metabolismo , Fase G1/fisiología , Humanos , Ratones , Elementos Reguladores de la Transcripción/genética , Elementos Reguladores de la Transcripción/fisiología , Elementos Silenciadores Transcripcionales/fisiologíaRESUMEN
Programs of gene transcription are controlled by cis-acting DNA elements, including enhancers, silencers, and promoters. Local accessibility of chromatin has proven to be a highly informative structural feature for identifying such regulatory elements, which tend to be relatively open due to their interactions with proteins. Recently, ATAC-seq (assay for transposase-accessible chromatin using sequencing) has emerged as one of the most powerful approaches for genome-wide chromatin accessibility profiling. This method assesses DNA accessibility using hyperactive Tn5 transposase, which simultaneously cuts DNA and inserts sequencing adaptors, preferentially in regions of open chromatin. ATAC-seq is a relatively simple procedure which can be applied to only a few thousand cells. It is well-suited to developing embryos of sea urchins and other echinoderms, which are a prominent experimental model for understanding the genomic control of animal development. In this chapter, we present a protocol for applying ATAC-seq to embryonic cells of sea urchins.
Asunto(s)
Cromatina/genética , Equinodermos/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Análisis de Secuencia de ADN/métodos , Animales , Equinodermos/crecimiento & desarrollo , Elementos de Facilitación Genéticos/genética , Regiones Promotoras Genéticas/genética , Secuencias Reguladoras de Ácidos Nucleicos/genética , Elementos Silenciadores Transcripcionales/genética , Transposasas/química , Transposasas/genéticaRESUMEN
Transposable elements, also known as transposons, are now recognized not only as parasitic DNA, the spread of which in the genome must be controlled by the host, but also as major players in genome evolution and regulation. Long interspersed element-1 (LINE-1, also known as L1), the only currently autonomous mobile transposon in humans, occupies 17% of the genome and generates inter- and intra-individual genetic variation, in some cases resulting in disease. However, how L1 activity is controlled and the function of L1s in host gene regulation are not completely understood. Here we use CRISPR-Cas9 screening strategies in two distinct human cell lines to provide a genome-wide survey of genes involved in the control of L1 retrotransposition. We identify functionally diverse genes that either promote or restrict L1 retrotransposition. These genes, which are often associated with human diseases, control the L1 life cycle at the transcriptional or the post-transcriptional level in a manner that can depend on the endogenous L1 nucleotide sequence, underscoring the complexity of L1 regulation. We further investigate the restriction of L1 by the protein MORC2 and by the human silencing hub (HUSH) complex subunits MPP8 and TASOR. HUSH and MORC2 can selectively bind evolutionarily young, full-length L1s located within transcriptionally permissive euchromatic environments, and promote deposition of histone H3 Lys9 trimethylation (H3K9me3) for transcriptional silencing. Notably, these silencing events often occur within introns of transcriptionally active genes, and lead to the downregulation of host gene expression in a HUSH-, MORC2-, and L1-dependent manner. Together, these results provide a rich resource for studies of L1 retrotransposition, elucidate a novel L1 restriction pathway and illustrate how epigenetic silencing of transposable elements rewires host gene expression programs.
Asunto(s)
Eucromatina/genética , Silenciador del Gen , Genoma Humano/genética , Elementos de Nucleótido Esparcido Largo/genética , Elementos Silenciadores Transcripcionales/genética , Animales , Sistemas CRISPR-Cas/genética , Células Madre Embrionarias , Genómica , Humanos , Células K562 , Masculino , Ratones , Complejos Multiproteicos/metabolismo , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Subunidades de Proteína/metabolismo , Factores de Transcripción/metabolismo , Transcripción GenéticaRESUMEN
RNA-protein interactions with physiological outcomes usually rely on conserved sequences within the RNA element. By contrast, activity of the diverse gamma-interferon-activated inhibitor of translation (GAIT)-elements relies on the conserved RNA folding motifs rather than the conserved sequence motifs. These elements drive the translational silencing of a group of chemokine (CC/CXC) and chemokine receptor (CCR) mRNAs, thereby helping to resolve physiological inflammation. Despite sequence dissimilarity, these RNA elements adopt common secondary structures (as revealed by 2D-1H NMR spectroscopy), providing a basis for their interaction with the RNA-binding GAIT complex. However, many of these elements (e.g. those derived from CCL22, CXCL13, CCR4 and ceruloplasmin (Cp) mRNAs) have substantially different affinities for GAIT complex binding. Toeprinting analysis shows that different positions within the overall conserved GAIT element structure contribute to differential affinities of the GAIT protein complex towards the elements. Thus, heterogeneity of GAIT elements may provide hierarchical fine-tuning of the resolution of inflammation.
Asunto(s)
Quimiocinas/genética , Regulación de la Expresión Génica , ARN Mensajero/genética , Elementos Silenciadores Transcripcionales/genética , Regiones no Traducidas 3'/genética , Animales , Secuencia de Bases , Quimiocina CCL22/genética , Quimiocina CCL22/metabolismo , Quimiocinas/metabolismo , Secuencia Conservada/genética , Humanos , Inflamación/genética , Inflamación/metabolismo , Ratones Noqueados , Ratones Transgénicos , Conformación de Ácido Nucleico , Operón , ARN Mensajero/química , ARN Mensajero/metabolismo , Células U937RESUMEN
Cell-type-specific and inducible alternative splicing has a fundamental impact on regulating gene expression and cellular function in a variety of settings, including activation and differentiation. We have recently shown that activation-induced skipping of TRAF3 exon 8 activates noncanonical NF-κB signaling upon T cell stimulation, but the regulatory basis for this splicing event remains unknown. Here we identify cis- and trans-regulatory elements rendering this splicing switch activation dependent and cell type specific. The cis-acting element is located 340 to 440 nucleotides upstream of the regulated exon and acts in a distance-dependent manner, since altering the location reduces its activity. A small interfering RNA screen, followed by cross-link immunoprecipitation and mutational analyses, identified CELF2 and hnRNP C as trans-acting factors that directly bind the regulatory sequence and together mediate increased exon skipping in activated T cells. CELF2 expression levels correlate with TRAF3 exon skipping in several model systems, suggesting that CELF2 is the decisive factor, with hnRNP C being necessary but not sufficient. These data suggest an interplay between CELF2 and hnRNP C as the mechanistic basis for activation-dependent alternative splicing of TRAF3 exon 8 and additional exons and uncover an intronic splicing silencer whose full activity depends on the precise location more than 300 nucleotides upstream of the regulated exon.
Asunto(s)
Empalme Alternativo/genética , Proteínas CELF/metabolismo , Exones/genética , Ribonucleoproteína Heterogénea-Nuclear Grupo C/metabolismo , Intrones/genética , Activación de Linfocitos/genética , Proteínas del Tejido Nervioso/metabolismo , Factor 3 Asociado a Receptor de TNF/genética , Sitios de Unión , Células HEK293 , Humanos , Poli U/metabolismo , Unión Proteica/genética , ARN Interferente Pequeño/metabolismo , Elementos Silenciadores Transcripcionales/genética , Linfocitos T/inmunología , Factor 3 Asociado a Receptor de TNF/metabolismoRESUMEN
Fabry disease is an X-linked recessive inborn disorder of the glycosphingolipid metabolism, caused by total or partial deficiency of the lysosomal α-galactosidase A enzyme due to mutations in the GLA gene. The prevalent c.639+919 G>A mutation in GLA leads to pathogenic insertion of a 57bp pseudoexon sequence from intron 4, which is responsible for the cardiac variant phenotype. In this study we investigate the splicing regulatory mechanism leading to GLA pseudoexon activation. Splicing analysis of GLA minigenes revealed that pseudoexon activation is influenced by cell-type. We demonstrate that the wild-type sequence harbors an hnRNP A1 and hnRNP A2/B1-binding exonic splicing silencer (ESS) overlapping the 5'splice site (5'ss) that prevents pseudoexon inclusion. The c.639+919 G>A mutation disrupts this ESS allowing U1 snRNP recognition of the 5'ss. We show that the wild-type GLA 5'ss motif with the ESS is also able to inhibit inclusion of an unrelated pseudoexon in the FGB gene, and that also in the FGB context inactivation of the ESS by the c.639+919 G>A mutation causes pseudoexon activation, underscoring the universal nature of the ESS. Finally, we demonstrate that splice switching oligonucleotide (SSO) mediated blocking of the pseudoexon 3'ss and 5'ss effectively restores normal GLA splicing. This indicates that SSO based splicing correction may be a therapeutic alternative in the treatment of Fabry disease.
Asunto(s)
Enfermedad de Fabry/genética , Ribonucleoproteína Nuclear Heterogénea A1/genética , Ribonucleoproteína Heterogénea-Nuclear Grupo A-B/genética , alfa-Galactosidasa/genética , Exones/genética , Enfermedad de Fabry/patología , Células HeLa , Humanos , Intrones , Mutación , Sitios de Empalme de ARN , Empalme del ARN/genética , ARN Mensajero/genética , Elementos Silenciadores Transcripcionales/genéticaRESUMEN
BACKGROUND: Genesis of novel gene regulatory modules is largely responsible for morphological and functional evolution. De novo generation of novel cis-regulatory elements (CREs) is much rarer than genomic events that alter existing CREs such as transposition, promoter switching or co-option. Only one case of de novo generation has been reported to date, in fish and without involvement of phenotype alteration. Yet, this event likely occurs in other animals and helps drive genetic/phenotypic variation. RESULTS: Using a porcine model of spontaneous hearing loss not previously characterized we performed gene mapping and mutation screening to determine the genetic foundation of the phenotype. We identified a mutation in the non-regulatory region of the melanocyte-specific promoter of microphthalmia-associated transcription factor (MITF) gene that generated a novel silencer. The consequent elimination of expression of the MITF-M isoform led to early degeneration of the intermediate cells of the cochlear stria vascularis and profound hearing loss, as well as depigmentation, all of which resemble the typical phenotype of Waardenburg syndrome in humans. The mutation exclusively affected MITF-M and no other isoforms. The essential function of Mitf-m in hearing development was further validated using a knock-out mouse model. CONCLUSIONS: Elimination of the MITF-M isoform alone is sufficient to cause deafness and depigmentation. To our knowledge, this study provides the first evidence of a de novo CRE in mammals that produces a systemic functional effect.
Asunto(s)
Pérdida Auditiva/genética , Factor de Transcripción Asociado a Microftalmía/genética , Elementos Silenciadores Transcripcionales/genética , Sus scrofa/genética , Animales , Secuencia de Bases , Mapeo Cromosómico , Cóclea/patología , Cóclea/fisiopatología , Modelos Animales de Enfermedad , Fenómenos Electrofisiológicos , Regulación de la Expresión Génica , Pruebas Genéticas , Estudio de Asociación del Genoma Completo , Pérdida Auditiva/fisiopatología , Factor de Transcripción Asociado a Microftalmía/metabolismo , Mutación/genética , Fenotipo , Regiones Promotoras Genéticas , Isoformas de Proteínas/genética , Transcripción GenéticaRESUMEN
Transposable elements are driving forces for establishing genetic innovations such as transcriptional regulatory networks in eukaryotic genomes. Here, we describe a silencer situated in the last 300 bp of the Mos1 transposase open reading frame (ORF) which functions in vertebrate and arthropod cells. Functional silencers are also found at similar locations within three other animal mariner elements, i.e. IS630-Tc1-mariner (ITm) DD34D elements, Himar1, Hsmar1 and Mcmar1. These silencers are able to impact eukaryotic promoters monitoring strong, moderate or low expression as well as those of mariner elements located upstream of the transposase ORF. We report that the silencing involves at least two transcription factors (TFs) that are conserved within animal species, NFAT-5 and Alx1. These cooperatively act with YY1 to trigger the silencing activity. Four other housekeeping transcription factors (TFs), neuron restrictive silencer factor (NRSF), GAGA factor (GAF) and GTGT factor (GTF), were also found to have binding sites within mariner silencers but their impact in modulating the silencer activity remains to be further specified. Interestingly, an NRSF binding site was found to overlap a 30 bp motif coding a highly conserved PHxxYSPDLAPxD peptide in mariner transposases. We also present experimental evidence that silencing is mainly achieved by co-opting the host Polycomb Repressive Complex 2 pathway. However, we observe that when PRC2 is impaired another host silencing pathway potentially takes over to maintain weak silencer activity. Mariner silencers harbour features of Polycomb Response Elements, which are probably a way for mariner elements to self-repress their transcription and mobility in somatic and germinal cells when the required TFs are expressed. At the evolutionary scale, mariner elements, through their exaptation, might have been a source of silencers playing a role in the chromatin configuration in eukaryotic genomes.
Asunto(s)
Elementos Transponibles de ADN/genética , Proteínas de Unión al ADN/genética , Complejo Represivo Polycomb 2/genética , Elementos Silenciadores Transcripcionales/genética , Transposasas/genética , Secuencias de Aminoácidos/genética , Animales , Cromatina/genética , Proteínas de Unión al ADN/metabolismo , Genoma , Células HeLa , Proteínas de Homeodominio/genética , Humanos , Factores de Transcripción NFATC/genética , Complejo Represivo Polycomb 2/metabolismo , Transposasas/metabolismoRESUMEN
Splicing of the Drosophila P-element third intron (IVS3) is repressed in somatic tissues due to the function of an exonic splicing silencer (ESS) complex present on the 5' exon RNA. To comprehensively characterize the mechanisms of this alternative splicing regulation, we used biochemical fractionation and affinity purification to isolate the silencer complex assembled in vitro and identify the constituent proteins by mass spectrometry. Functional assays using splicing reporter minigenes identified the proteins hrp36 and hrp38 and the cytoplasmic poly(A)-binding protein PABPC1 as novel functional components of the splicing silencer. hrp48, PSI, and PABPC1 have high-affinity RNA-binding sites on the P-element IVS3 5' exon, whereas hrp36 and hrp38 proteins bind with low affinity to the P-element silencer RNA. RNA pull-down and immobilized protein assays showed that hrp48 protein binding to the silencer RNA can recruit hrp36 and hrp38. These studies identified additional components that function at the P-element ESS and indicated that proteins with low-affinity RNA-binding sites can be recruited in a functional manner through interactions with a protein bound to RNA at a high-affinity binding site. These studies have implications for the role of heterogeneous nuclear ribonucleoproteins (hnRNPs) in the control of alternative splicing at cis-acting regulatory sites.
Asunto(s)
Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Empalme del ARN/fisiología , Animales , Proteínas de Drosophila/química , Proteínas de Drosophila/aislamiento & purificación , Exones/genética , Regulación de la Expresión Génica , Genes Reporteros/genética , Ribonucleoproteínas Nucleares Heterogéneas/metabolismo , Espectrometría de Masas , Proteínas Nucleares , Unión Proteica , Interferencia de ARN , Precursores del ARN/metabolismo , Empalme del ARN/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ribonucleoproteínas/metabolismo , Elementos Silenciadores Transcripcionales/genéticaRESUMEN
Genetic information is organized in a complex structure composed of DNA and proteins together designated chromatin. Chromatin plays a dynamic role in transcriptional processes in that alteration of the interaction between its components results in the deregulation of cellular transcriptional program. Modification of epigenetic marks, variation in the precise positioning of nucleosomes, and consequent mobilization of nucleosomes regulate the access of various transcriptional factors to its underlying DNA template. Nucleosome-depleted regions, also designated open chromatin domains, are associated with active DNA regulatory elements, including promoters, enhancers, silencers, and insulators. Here, we describe the protocol of a rapid and simple technique entitled FAIRE (formaldehyde-assisted isolation of regulatory elements). Combined with high-throughput sequencing (FAIRE-seq), this procedure allows isolation of nucleosome-free regions and their mapping along the genome, thereby providing a global view of cell-specific regulatory elements.