RESUMO
Transcriptional enhancers have been extensively characterized, but cis-regulatory elements involved in acute gene repression have received less attention. Transcription factor GATA1 promotes erythroid differentiation by activating and repressing distinct gene sets. Here, we study the mechanism by which GATA1 silences the proliferative gene Kit during murine erythroid cell maturation and define stages from initial loss of activation to heterochromatinization. We find that GATA1 inactivates a potent upstream enhancer but concomitantly creates a discrete intronic regulatory region marked by H3K27ac, short noncoding RNAs, and de novo chromatin looping. This enhancer-like element forms transiently and serves to delay Kit silencing. The element is ultimately erased via the FOG1/NuRD deacetylase complex, as revealed by the study of a disease-associated GATA1 variant. Hence, regulatory sites can be self-limiting by dynamic co-factor usage. Genome-wide analyses across cell types and species uncover transiently active elements at numerous genes during repression, suggesting that modulation of silencing kinetics is widespread.
Assuntos
Estudo de Associação Genômica Ampla , Sequências Reguladoras de Ácido Nucleico , Animais , Camundongos , Íntrons , Diferenciação Celular , Inativação Gênica , Complexo Mi-2 de Remodelação de Nucleossomo e DesacetilaseRESUMO
Meningioma-1 (MN1) overexpression in AML is associated with poor prognosis, and forced expression of MN1 induces leukemia in mice. We sought to determine how MN1 causes AML. We found that overexpression of MN1 can be induced by translocations that result in hijacking of a downstream enhancer. Structure predictions revealed that the entire MN1 coding frame is disordered. We identified the myeloid progenitor-specific BAF complex as the key interaction partner of MN1. MN1 over-stabilizes BAF on enhancer chromatin, a function directly linked to the presence of a long polyQ-stretch within MN1. BAF over-stabilization at binding sites of transcription factors regulating a hematopoietic stem/progenitor program prevents the developmentally appropriate decommissioning of these enhancers and results in impaired myeloid differentiation and leukemia. Beyond AML, our data detail how the overexpression of a polyQ protein, in the absence of any coding sequence mutation, can be sufficient to cause malignant transformation.
Assuntos
Carcinogênese/genética , DNA Helicases/genética , Proteínas Intrinsicamente Desordenadas/genética , Leucemia Mieloide Aguda/genética , Proteínas Nucleares/genética , Transativadores/genética , Fatores de Transcrição/genética , Proteínas Supressoras de Tumor/genética , Animais , Sequência de Bases , Carcinogênese/metabolismo , Carcinogênese/patologia , Linhagem Celular Tumoral , Cromatina/genética , Cromatina/metabolismo , Cromatina/patologia , DNA Helicases/metabolismo , Elementos Facilitadores Genéticos , Feminino , Regulação Leucêmica da Expressão Gênica , Redes Reguladoras de Genes , Humanos , Proteínas Intrinsicamente Desordenadas/metabolismo , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/mortalidade , Leucemia Mieloide Aguda/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas Nucleares/metabolismo , Peptídeos/genética , Peptídeos/metabolismo , Mapeamento de Interação de Proteínas , Estabilidade Proteica , Transporte Proteico , Transdução de Sinais , Análise de Sobrevida , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/metabolismoRESUMO
Knowledge of locations and activities of cis-regulatory elements (CREs) is needed to decipher basic mechanisms of gene regulation and to understand the impact of genetic variants on complex traits. Previous studies identified candidate CREs (cCREs) using epigenetic features in one species, making comparisons difficult between species. In contrast, we conducted an interspecies study defining epigenetic states and identifying cCREs in blood cell types to generate regulatory maps that are comparable between species, using integrative modeling of eight epigenetic features jointly in human and mouse in our Validated Systematic Integration (VISION) Project. The resulting catalogs of cCREs are useful resources for further studies of gene regulation in blood cells, indicated by high overlap with known functional elements and strong enrichment for human genetic variants associated with blood cell phenotypes. The contribution of each epigenetic state in cCREs to gene regulation, inferred from a multivariate regression, was used to estimate epigenetic state regulatory potential (esRP) scores for each cCRE in each cell type, which were used to categorize dynamic changes in cCREs. Groups of cCREs displaying similar patterns of regulatory activity in human and mouse cell types, obtained by joint clustering on esRP scores, harbor distinctive transcription factor binding motifs that are similar between species. An interspecies comparison of cCREs revealed both conserved and species-specific patterns of epigenetic evolution. Finally, we show that comparisons of the epigenetic landscape between species can reveal elements with similar roles in regulation, even in the absence of genomic sequence alignment.
RESUMO
Recurrent chromosomal rearrangements found in rhabdomyosarcoma (RMS) produce the PAX3-FOXO1 fusion protein, which is an oncogenic driver and a dependency in this disease. One important function of PAX3-FOXO1 is to arrest myogenic differentiation, which is linked to the ability of RMS cells to gain an unlimited proliferation potential. Here, we developed a phenotypic screening strategy for identifying factors that collaborate with PAX3-FOXO1 to block myo-differentiation in RMS. Unlike most genes evaluated in our screen, we found that loss of any of the three subunits of the Nuclear Factor Y (NF-Y) complex leads to a myo-differentiation phenotype that resembles the effect of inactivating PAX3-FOXO1. While the transcriptomes of NF-Y- and PAX3-FOXO1-deficient RMS cells bear remarkable similarity to one another, we found that these two transcription factors occupy nonoverlapping sites along the genome: NF-Y preferentially occupies promoters, whereas PAX3-FOXO1 primarily binds to distal enhancers. By integrating multiple functional approaches, we map the PAX3 promoter as the point of intersection between these two regulators. We show that NF-Y occupies CCAAT motifs present upstream of PAX3 to function as a transcriptional activator of PAX3-FOXO1 expression in RMS. These findings reveal a critical upstream role of NF-Y in the oncogenic PAX3-FOXO1 pathway, highlighting how a broadly essential transcription factor can perform tumor-specific roles in governing cellular state.
Assuntos
Rabdomiossarcoma , Fator de Ligação a CCAAT/genética , Diferenciação Celular/genética , Aberrações Cromossômicas , Rabdomiossarcoma/genética , Fatores de TranscriçãoRESUMO
The human cerebral cortex contains many cell types that likely underwent independent functional changes during evolution. However, cell-type-specific regulatory landscapes in the cortex remain largely unexplored. Here we report epigenomic and transcriptomic analyses of the two main cortical neuronal subtypes, glutamatergic projection neurons and GABAergic interneurons, in human, chimpanzee, and rhesus macaque. Using genome-wide profiling of the H3K27ac histone modification, we identify neuron-subtype-specific regulatory elements that previously went undetected in bulk brain tissue samples. Human-specific regulatory changes are uncovered in multiple genes, including those associated with language, autism spectrum disorder, and drug addiction. We observe preferential evolutionary divergence in neuron subtype-specific regulatory elements and show that a substantial fraction of pan-neuronal regulatory elements undergoes subtype-specific evolutionary changes. This study sheds light on the interplay between regulatory evolution and cell-type-dependent gene-expression programs, and provides a resource for further exploration of human brain evolution and function.
Assuntos
Córtex Cerebral/metabolismo , Evolução Molecular , Neurônios/metabolismo , Animais , Transtorno do Espectro Autista/genética , Encéfalo/metabolismo , Epigênese Genética , Epigenômica , Expressão Gênica , Código das Histonas , Humanos , Interneurônios/metabolismo , Macaca mulatta/genética , Pan troglodytes/genética , Primatas/genética , Elementos Reguladores de Transcrição , Sequências Reguladoras de Ácido Nucleico , TranscriptomaRESUMO
Neuronal microexons represent the most highly conserved class of alternative splicing events and their timed expression shapes neuronal biology, including neuronal commitment and differentiation. The six-nt microexon 34' is included in the neuronal form of TAF1 mRNA, which encodes the largest subunit of the basal transcription factor TFIID. In this study, we investigate the tissue distribution of TAF1-34' mRNA and protein and the mechanism responsible for its neuronal-specific splicing. Using isoform-specific RNA probes and antibodies, we observe that canonical TAF1 and TAF1-34' have different distributions in the brain, which distinguish proliferating from post-mitotic neurons. Knockdown and ectopic expression experiments demonstrate that the neuronal-specific splicing factor SRRM4/nSR100 promotes the inclusion of microexon 34' into TAF1 mRNA, through the recognition of UGC sequences in the poly-pyrimidine tract upstream of the regulated microexon. These results show that SRRM4 regulates temporal and spatial expression of alternative TAF1 mRNAs to generate a neuronal-specific TFIID complex.
Assuntos
Éxons , Regulação da Expressão Gênica , Histona Acetiltransferases/genética , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Splicing de RNA , RNA Mensageiro/genética , Fatores Associados à Proteína de Ligação a TATA/genética , Fator de Transcrição TFIID/genética , Animais , Encéfalo/metabolismo , Diferenciação Celular , Imuno-Histoquímica , Camundongos , Neurogênese/genética , Neurônios/citologiaRESUMO
Cell-autonomous circadian oscillations strongly influence tissue physiology and pathophysiology of peripheral organs including the heart, in which the circadian clock is known to determine cardiac metabolism and the outcome of for instance ischemic stress. Human pluripotent stem cells represent a powerful tool to study developmental processes in vitro, but the extent to which human embryonic stem (ES) cell-derived cardiomyocytes establish circadian rhythmicity in the absence of a systemic context is unknown. Here we demonstrate that while undifferentiated human ES cells do not possess an intrinsic functional clock, oscillatory expression of known core clock genes emerges spontaneously during directed cardiac differentiation. We identify a set of clock-controlled output genes that contain an oscillatory network of stress-related transcripts. Furthermore, we demonstrate that this network results in a time-dependent functional response to doxorubicin, a frequently used anti-cancer drug with known cardiotoxic side effects. Taken together, our data provide a framework from which the effect of oscillatory gene expression on cardiomyocyte physiology can be modeled in vitro, and demonstrate the influence of a functional clock on experimental outcome.
Assuntos
Proteínas CLOCK/genética , Relógios Circadianos , Células-Tronco Embrionárias Humanas/fisiologia , Miócitos Cardíacos/fisiologia , Proteínas Circadianas Period/genética , Diferenciação Celular , Ritmo Circadiano , Doxorrubicina/farmacologia , Expressão Gênica , Humanos , Miócitos Cardíacos/efeitos dos fármacos , Proteínas Circadianas Period/metabolismo , Inibidores da Topoisomerase II/farmacologiaRESUMO
BACKGROUND: Cardiac ischemic injury induces a pathological remodeling response, which can ultimately lead to heart failure. Detailed mechanistic insights into molecular signaling pathways relevant for different aspects of cardiac remodeling will support the identification of novel therapeutic targets. METHODS: Although genome-wide transcriptome analysis on diseased tissues has greatly advanced our understanding of the regulatory networks that drive pathological changes in the heart, this approach has been disadvantaged by the fact that the signals are derived from tissue homogenates. Here we used tomo-seq to obtain a genome-wide gene expression signature with high spatial resolution spanning from the infarcted area to the remote to identify new regulators of cardiac remodeling. Cardiac tissue samples from patients suffering from ischemic heart disease were used to validate our findings. RESULTS: Tracing transcriptional differences with a high spatial resolution across the infarcted heart enabled us to identify gene clusters that share a comparable expression profile. The spatial distribution patterns indicated a separation of expressional changes for genes involved in specific aspects of cardiac remodeling, such as fibrosis, cardiomyocyte hypertrophy, and calcium handling (Col1a2, Nppa, and Serca2). Subsequent correlation analysis allowed for the identification of novel factors that share a comparable transcriptional regulation pattern across the infarcted tissue. The strong correlation between the expression levels of these known marker genes and the expression of the coregulated genes could be confirmed in human ischemic cardiac tissue samples. Follow-up analysis identified SOX9 as common transcriptional regulator of a large portion of the fibrosis-related genes that become activated under conditions of ischemic injury. Lineage-tracing experiments indicated that the majority of COL1-positive fibroblasts stem from a pool of SOX9-expressing cells, and in vivo loss of Sox9 blunted the cardiac fibrotic response on ischemic injury. The colocalization between SOX9 and COL1 could also be confirmed in patients suffering from ischemic heart disease. CONCLUSIONS: Based on the exact local expression cues, tomo-seq can serve to reveal novel genes and key transcription factors involved in specific aspects of cardiac remodeling. Using tomo-seq, we were able to unveil the unknown relevance of SOX9 as a key regulator of cardiac fibrosis, pointing to SOX9 as a potential therapeutic target for cardiac fibrosis.
Assuntos
Regulação da Expressão Gênica , Proteínas Musculares/biossíntese , Isquemia Miocárdica/metabolismo , Miocárdio/metabolismo , Fatores de Transcrição SOX9/biossíntese , Colágeno Tipo I/biossíntese , Colágeno Tipo I/genética , Feminino , Fibrose , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Proteínas Musculares/genética , Isquemia Miocárdica/genética , Fatores de Transcrição SOX9/genéticaRESUMO
BACKGROUND AND PURPOSE: Genome-wide association studies significantly link intracranial aneurysm (IA) to single-nucleotide polymorphisms (SNPs) in 6 genomic loci. To gain insight into the relevance of these IA-associated SNPs, we aimed to identify regulatory regions and analyze overall gene expression in the human circle of Willis (CoW), on which these aneurysms develop. METHODS: We performed chromatin immunoprecipitation and sequencing for histone modifications H3K4me1 and H3K27ac to identify regulatory regions, including distal enhancers and active promoters, in postmortem specimens of the human CoW. These experiments were complemented with RNA sequencing on the same specimens. We determined whether these regulatory regions overlap with IA-associated SNPs, using computational methods. By combining our results with publicly available data, we investigated the effect of IA-associated SNPs on the newly identified regulatory regions and linked them to potential target genes. RESULTS: We find that IA-associated SNPs are significantly enriched in CoW regulatory regions. Some of the IA-associated SNPs that overlap with a regulatory region are likely to alter transcription factor binding, and in proximity to these regulatory regions are 102 genes that are expressed in the CoW. In addition, gene expression in the CoW is enriched for genes related to cell adhesion and the extracellular matrix. CONCLUSIONS: CoW regulatory regions link IA-associated SNPs to genes with a potential role in the development of IAs. Our data refine previous predictions on SNPs associated with IA and provide a substantial resource from which candidates for follow-up studies can be prioritized.
Assuntos
Círculo Arterial do Cérebro/diagnóstico por imagem , DNA/metabolismo , Predisposição Genética para Doença , Aneurisma Intracraniano/genética , Polimorfismo de Nucleotídeo Único/genética , Adulto , Feminino , Loci Gênicos/genética , Genoma Humano , Estudo de Associação Genômica Ampla , Humanos , Masculino , Pessoa de Meia-Idade , Fatores de RiscoRESUMO
Glial fibrillary acidic protein (GFAP) is the main intermediate filament in astrocytes and is regulated by epigenetic mechanisms during development. We demonstrate that histone acetylation also controls GFAP expression in mature astrocytes. Inhibition of histone deacetylases (HDACs) with trichostatin A or sodium butyrate reduced GFAP expression in primary human astrocytes and astrocytoma cells. Because splicing occurs co-transcriptionally, we investigated whether histone acetylation changes the ratio between the canonical isoform GFAPα and the alternative GFAPδ splice variant. We observed that decreased transcription of GFAP enhanced alternative isoform expression, as HDAC inhibition increased the GFAPδâ¶GFAPα ratio. Expression of GFAPδ was dependent on the presence and binding of splicing factors of the SR protein family. Inhibition of HDAC activity also resulted in aggregation of the GFAP network, reminiscent of our previous findings of a GFAPδ-induced network collapse. Taken together, our data demonstrate that HDAC inhibition results in changes in transcription, splicing and organization of GFAP. These data imply that a tight regulation of histone acetylation in astrocytes is essential, because dysregulation of gene expression causes the aggregation of GFAP, a hallmark of human diseases like Alexander's disease.
Assuntos
Doença de Alexander/metabolismo , Astrócitos/metabolismo , Proteína Glial Fibrilar Ácida/metabolismo , Histona Desacetilases/metabolismo , Acetilação/efeitos dos fármacos , Doença de Alexander/genética , Processamento Alternativo/efeitos dos fármacos , Astrócitos/efeitos dos fármacos , Ácido Butírico/farmacologia , Linhagem Celular Tumoral , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/metabolismo , Epigênese Genética , Regulação da Expressão Gênica/efeitos dos fármacos , Proteína Glial Fibrilar Ácida/genética , Inibidores de Histona Desacetilases/farmacologia , Histonas/metabolismo , Humanos , Ácidos Hidroxâmicos/farmacologia , Agregados Proteicos , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Multimerização Proteica/efeitos dos fármacosRESUMO
Over the last decade, the noncoding part of the genome has been shown to harbour thousands of cis-regulatory elements, such as enhancers, that activate well-defined gene expression programs. Driven by the development of numerous techniques, many of these elements are now identified in multiple tissues and cell types, and their characteristics as well as importance in development and disease are becoming increasingly clear. Here, we provide an overview of the insights that were gained from the analysis of noncoding gene regulatory elements in the brain and describe their potential contribution to cell type specialization, brain function and neurodegenerative disease.
Assuntos
Encéfalo/metabolismo , Elementos Facilitadores Genéticos/fisiologia , Epigênese Genética/fisiologia , Regulação da Expressão Gênica/fisiologia , Doenças Neurodegenerativas/genética , Animais , HumanosRESUMO
Knowledge of locations and activities of cis -regulatory elements (CREs) is needed to decipher basic mechanisms of gene regulation and to understand the impact of genetic variants on complex traits. Previous studies identified candidate CREs (cCREs) using epigenetic features in one species, making comparisons difficult between species. In contrast, we conducted an interspecies study defining epigenetic states and identifying cCREs in blood cell types to generate regulatory maps that are comparable between species, using integrative modeling of eight epigenetic features jointly in human and mouse in our V al i dated S ystematic I ntegrati on (VISION) Project. The resulting catalogs of cCREs are useful resources for further studies of gene regulation in blood cells, indicated by high overlap with known functional elements and strong enrichment for human genetic variants associated with blood cell phenotypes. The contribution of each epigenetic state in cCREs to gene regulation, inferred from a multivariate regression, was used to estimate epigenetic state Regulatory Potential (esRP) scores for each cCRE in each cell type, which were used to categorize dynamic changes in cCREs. Groups of cCREs displaying similar patterns of regulatory activity in human and mouse cell types, obtained by joint clustering on esRP scores, harbored distinctive transcription factor binding motifs that were similar between species. An interspecies comparison of cCREs revealed both conserved and species-specific patterns of epigenetic evolution. Finally, we showed that comparisons of the epigenetic landscape between species can reveal elements with similar roles in regulation, even in the absence of genomic sequence alignment.
RESUMO
Transcription at most promoters is divergent, initiating at closely spaced oppositely oriented core promoters to produce sense transcripts along with often unstable upstream antisense transcripts (uasTrx). How antisense transcription is regulated and to what extent it is coordinated with sense transcription is not well understood. Here, by combining acute degradation of the multi-functional transcription factor CTCF and nascent transcription measurements, we find that CTCF specifically suppresses antisense but not sense transcription at hundreds of divergent promoters. Primary transcript RNA-FISH shows that CTCF lowers burst fraction but not burst intensity of uasTrx and that co-bursting of sense and antisense transcripts is disfavored. Genome editing, chromatin conformation studies and high-resolution transcript mapping revealed that precisely positioned CTCF directly suppresses the initiation of uasTrx, in a manner independent of its architectural function. In sum, CTCF shapes the transcriptional landscape in part by suppressing upstream antisense transcription.
Assuntos
Cromatina , Transcrição Gênica , Regiões Promotoras Genéticas , Cromatina/genética , RNA Antissenso/genética , Regulação da Expressão GênicaRESUMO
The heart is a highly metabolic organ that uses multiple energy sources to meet its demand for ATP production. Diurnal feeding-fasting cycles result in substrate availability fluctuations which, together with increased energetic demand during the active period, impose a need for rhythmic cardiac metabolism. The nuclear receptors REV-ERBα and ß are essential repressive components of the molecular circadian clock and major regulators of metabolism. To investigate their role in the heart, here we generated mice with cardiomyocyte (CM)-specific deletion of both Rev-erbs, which died prematurely due to dilated cardiomyopathy. Loss of Rev-erbs markedly downregulated fatty acid oxidation genes prior to overt pathology, which was mediated by induction of the transcriptional repressor E4BP4, a direct target of cardiac REV-ERBs. E4BP4 directly controls circadian expression of Nampt and its biosynthetic product NAD+ via distal cis-regulatory elements. Thus, REV-ERB-mediated E4BP4 repression is required for Nampt expression and NAD+ production by the salvage pathway. Together, these results highlight the indispensable role of circadian REV-ERBs in cardiac gene expression, metabolic homeostasis and function.
RESUMO
During mitosis, transcription is globally attenuated and chromatin architecture is dramatically reconfigured. We exploited the M- to G1-phase progression to interrogate the contributions of the architectural factor CTCF and the process of transcription to genome re-sculpting in newborn nuclei. Depletion of CTCF during the M- to G1-phase transition alters short-range compartmentalization after mitosis. Chromatin domain boundary re-formation is impaired upon CTCF loss, but a subset of boundaries, characterized by transitions in chromatin states, is established normally. Without CTCF, structural loops fail to form, leading to illegitimate contacts between cis-regulatory elements (CREs). Transient CRE contacts that are normally resolved after telophase persist deeply into G1-phase in CTCF-depleted cells. CTCF loss-associated gains in transcription are often linked to increased, normally illegitimate enhancer-promoter contacts. In contrast, at genes whose expression declines upon CTCF loss, CTCF seems to function as a conventional transcription activator, independent of its architectural role. CTCF-anchored structural loops facilitate formation of CRE loops nested within them, especially those involving weak CREs. Transcription inhibition does not significantly affect global architecture or transcription start site-associated boundaries. However, ongoing transcription contributes considerably to the formation of gene domains, regions of enriched contacts along gene bodies. Notably, gene domains emerge in ana/telophase prior to completion of the first round of transcription, suggesting that epigenetic features in gene bodies contribute to genome reconfiguration prior to transcription. The focus on the de novo formation of nuclear architecture during G1 entry yields insights into the contributions of CTCF and transcription to chromatin architecture dynamics during the mitosis to G1-phase progression.
Assuntos
Fator de Ligação a CCCTC/metabolismo , Cromatina/metabolismo , Mitose , Transcrição Gênica , Animais , Fator de Ligação a CCCTC/genética , Divisão Celular , Linhagem Celular , Cromatina/genética , Montagem e Desmontagem da Cromatina , Elementos Facilitadores Genéticos , Camundongos , Regiões Promotoras GenéticasRESUMO
CCCTC-binding factor (CTCF) is a conserved zinc finger transcription factor implicated in a wide range of functions, including genome organization, transcription activation, and elongation. To explore the basis for CTCF functional diversity, we coupled an auxin-induced degron system with precision nuclear run-on. Unexpectedly, oriented CTCF motifs in gene bodies are associated with transcriptional stalling in a manner independent of bound CTCF. Moreover, CTCF at different binding sites (CBSs) displays highly variable resistance to degradation. Motif sequence does not significantly predict degradation behavior, but location at chromatin boundaries and chromatin loop anchors, as well as co-occupancy with cohesin, are associated with delayed degradation. Single-molecule tracking experiments link chromatin residence time to CTCF degradation kinetics, which has ramifications regarding architectural CTCF functions. Our study highlights the heterogeneity of CBSs, uncovers properties specific to architecturally important CBSs, and provides insights into the basic processes of genome organization and transcription regulation.
Assuntos
Fator de Ligação a CCCTC/metabolismo , Sequenciamento de Cromatina por Imunoprecipitação , Cromatina/metabolismo , Eritroblastos/metabolismo , Imagem Individual de Molécula , Animais , Sítios de Ligação , Fator de Ligação a CCCTC/genética , Sistemas CRISPR-Cas , Linhagem Celular , Cromatina/genética , Montagem e Desmontagem da Cromatina , Edição de Genes , Cinética , Camundongos , Simulação de Dinâmica Molecular , Ligação Proteica , Proteólise , RNA Polimerase II/metabolismo , Transcrição GênicaRESUMO
X-linked dystonia-parkinsonism (XDP) is a monogenic neurodegenerative disorder of the basal ganglia, which presents as a combination of hyperkinetic movements and parkinsonian features. The underlying genetic mechanism involves the insertion of a SINE-VNTR-Alu retrotransposon within the TAF1 gene. Interestingly, alterations of TAF1 have been involved in multiple neurological diseases. In XDP, the SINE-VNTR-Alu insertion in TAF1 has been proposed to result in alternative splicing defects, including the decreased incorporation of a neuron-specific microexon annotated as 34'. This mechanism has become controversial as recent studies failed to provide support. In order to resolve this conundrum, we examined the alternative splicing patterns of TAF1 mRNAs in XDP and control brains. The impact of the disease-associated SINE-VNTR-Alu on alternative splicing of microexon 34' was further investigated in cellular assays. Subsequently, microexon 34' incorporation was explored by RT-PCR and Nanopore long-read sequencing of TAF1 mRNAs from XDP and control brains tissues. Using cell-based splicing assays, we demonstrate that presence of the disease-associated SINE-VNTR-Alu does not affect the inclusion of microexon 34'. In addition, we show that (1) microexon 34'-containing TAF1 mRNAs are detected at similar levels in XDP as in controls and that (2) the architecture of TAF1 transcripts is remarkably similar between XDP and controls brains. These results indicate that microexon 34' incorporation into TAF1 mRNA is not affected in XDP brains. Our findings shift the current paradigm of XDP by discounting alternative splicing of TAF1 microexon 34' as the molecular basis for this disease.
RESUMO
Mutations in non-coding regulatory DNA such as enhancers underlie a wide variety of diseases including developmental disorders and cancer. As enhancers rapidly evolve, understanding their function and configuration in non-human disease models can have important clinical applications. Here, we analyze enhancer configurations in tissues isolated from the common marmoset, a widely used primate model for human disease. Integrating these data with human and mouse data, we find that enhancers containing trait-associated variants are preferentially conserved. In contrast, most human-specific enhancers are highly variable between individuals, with a subset failing to contact promoters. These are located further away from genes and more often reside in inactive B-compartments. Our data show that enhancers typically emerge as instable elements with minimal biological impact prior to their integration in a transcriptional program. Furthermore, our data provide insight into which trait variations in enhancers can be faithfully modeled using the common marmoset.
Assuntos
Doença/genética , Elementos Facilitadores Genéticos , Evolução Molecular , Predisposição Genética para Doença , Animais , Callithrix/genética , Sequência Conservada/genética , Humanos , Camundongos , Anotação de Sequência Molecular , Filogenia , Regiões Promotoras Genéticas , Característica Quantitativa HerdávelRESUMO
Speciation is associated with substantial rewiring of the regulatory circuitry underlying the expression of genes. Determining which changes are relevant and underlie the emergence of the human brain or its unique susceptibility to neural disease has been challenging. Here we annotate changes to gene regulatory elements (GREs) at cell type resolution in the brains of multiple primate species spanning most of primate evolution. We identify a unique set of regulatory elements that emerged in hominins prior to the separation of humans and chimpanzees. We demonstrate that these hominin gains perferentially affect oligodendrocyte function postnatally and are preferentially affected in the brains of autism patients. This preference is also observed for human-specific GREs suggesting this system is under continued selective pressure. Our data provide a roadmap of regulatory rewiring across primate evolution providing insight into the genomic changes that underlie the emergence of the brain and its susceptibility to neural disease.
Assuntos
Transtorno Autístico/metabolismo , Encéfalo/metabolismo , Hominidae/metabolismo , Oligodendroglia/metabolismo , Sequências Reguladoras de Ácido Nucleico/fisiologia , Animais , Transtorno Autístico/genética , Callithrix , Cromatina , Imunoprecipitação da Cromatina , Cromossomos/química , Suscetibilidade a Doenças , Evolução Molecular , Feminino , Regulação da Expressão Gênica , Genômica , Hominidae/genética , Humanos , Macaca mulatta , Pan troglodytesRESUMO
Imaging studies, high-resolution chromatin conformation maps, and genome-wide occupancy data of architectural proteins have revealed that genome topology is tightly intertwined with gene expression. Cross-talk between gene-regulatory elements is often organized within insulated neighborhoods, and regulatory cues that induce transcriptional changes can reshape chromatin folding patterns and gene positioning within the nucleus. The cause-consequence relationship of genome architecture and gene expression is intricate, and its molecular mechanisms are under intense investigation. Here, we review the interdependency of transcription and genome organization with emphasis on enhancer-promoter contacts in gene regulation.