RESUMO
The emergence of new structures can often be linked to the evolution of novel cell types that follows the rewiring of developmental gene regulatory subnetworks. Vertebrates are characterized by a complex body plan compared to the other chordate clades and the question remains of whether and how the emergence of vertebrate morphological innovations can be related to the appearance of new embryonic cell populations. We previously proposed, by studying mesoderm development in the cephalochordate amphioxus, a scenario for the evolution of the vertebrate head mesoderm. To further test this scenario at the cell population level, we used scRNA-seq to construct a cell atlas of the amphioxus neurula, stage at which the main mesodermal compartments are specified. Our data allowed us to validate the presence of a prechordal-plate like territory in amphioxus. Additionally, the transcriptomic profile of somite cell populations supports the homology between specific territories of amphioxus somites and vertebrate cranial/pharyngeal and lateral plate mesoderm. Finally, our work provides evidence that the appearance of the specific mesodermal structures of the vertebrate head was associated to both segregation of pre-existing cell populations, and co-option of new genes for the control of myogenesis.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Cabeça , Anfioxos , Mesoderma , Vertebrados , Animais , Mesoderma/citologia , Mesoderma/embriologia , Anfioxos/embriologia , Anfioxos/genética , Cabeça/embriologia , Vertebrados/embriologia , Vertebrados/genética , Somitos/embriologia , Somitos/citologia , Somitos/metabolismo , Evolução Biológica , TranscriptomaRESUMO
BACKGROUND: Vision depends on the interplay between photoreceptor cells of the neural retina and the underlying retinal pigment epithelium (RPE). Most genes involved in inherited retinal diseases display specific spatiotemporal expression within these interconnected retinal components through the local recruitment of cis-regulatory elements (CREs) in 3D nuclear space. RESULTS: To understand the role of differential chromatin architecture in establishing tissue-specific expression at inherited retinal disease loci, we mapped genome-wide chromatin interactions using in situ Hi-C and H3K4me3 HiChIP on neural retina and RPE/choroid from human adult donor eyes. We observed chromatin looping between active promoters and 32,425 and 8060 candidate CREs in the neural retina and RPE/choroid, respectively. A comparative 3D genome analysis between these two retinal tissues revealed that 56% of 290 known inherited retinal disease genes were marked by differential chromatin interactions. One of these was ABCA4, which is implicated in the most common autosomal recessive inherited retinal disease. We zoomed in on retina- and RPE-specific cis-regulatory interactions at the ABCA4 locus using high-resolution UMI-4C. Integration with bulk and single-cell epigenomic datasets and in vivo enhancer assays in zebrafish revealed tissue-specific CREs interacting with ABCA4. CONCLUSIONS: Through comparative 3D genome mapping, based on genome-wide, promoter-centric, and locus-specific assays of human neural retina and RPE, we have shown that gene regulation at key inherited retinal disease loci is likely mediated by tissue-specific chromatin interactions. These findings do not only provide insight into tissue-specific regulatory landscapes at retinal disease loci, but also delineate the search space for non-coding genomic variation underlying unsolved inherited retinal diseases.
Assuntos
Cromatina , Retina , Doenças Retinianas , Epitélio Pigmentado da Retina , Humanos , Epitélio Pigmentado da Retina/metabolismo , Cromatina/metabolismo , Doenças Retinianas/genética , Doenças Retinianas/metabolismo , Retina/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Regiões Promotoras Genéticas , Loci Gênicos , Peixe-Zebra/genética , Sequências Reguladoras de Ácido Nucleico , Genoma HumanoRESUMO
Retinoic acid (RA) is the ligand of RA receptors (RARs), transcription factors that bind to RA response elements. RA signaling is required for multiple processes during embryonic development, including body axis extension, hindbrain antero-posterior patterning and forelimb bud initiation. Although some RA target genes have been identified, little is known about the genome-wide effects of RA signaling during in vivo embryonic development. Here, we stimulate the RA pathway by treating zebrafish embryos with all-trans-RA (atRA) and use a combination of RNA-seq, ATAC-seq, ChIP-seq and HiChIP to gain insight into the molecular mechanisms by which exogenously induced RA signaling controls gene expression. We find that RA signaling is involved in anterior/posterior patterning, central nervous system development, and the transition from pluripotency to differentiation. AtRA treatment also alters chromatin accessibility during early development and promotes chromatin binding of RARαa and the RA targets Hoxb1b, Meis2b and Sox3, which cooperate in central nervous system development. Finally, we show that exogenous RA induces a rewiring of chromatin architecture, with alterations in chromatin 3D interactions involving target genes. Altogether, our findings identify genome-wide targets of RA signaling and provide a molecular mechanism by which developmental signaling pathways regulate target gene expression by altering chromatin topology.
Assuntos
Desenvolvimento Embrionário , Regulação da Expressão Gênica no Desenvolvimento , Tretinoína , Animais , Cromatina/metabolismo , Embrião não Mamífero/metabolismo , Embrião não Mamífero/efeitos dos fármacos , Desenvolvimento Embrionário/genética , Desenvolvimento Embrionário/efeitos dos fármacos , Epigenoma , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Tretinoína/farmacologia , Tretinoína/metabolismo , Peixe-Zebra/genética , Peixe-Zebra/embriologia , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
Skates are cartilaginous fish whose body plan features enlarged wing-like pectoral fins, enabling them to thrive in benthic environments1,2. However, the molecular underpinnings of this unique trait remain unclear. Here we investigate the origin of this phenotypic innovation by developing the little skate Leucoraja erinacea as a genomically enabled model. Analysis of a high-quality chromosome-scale genome sequence for the little skate shows that it preserves many ancestral jawed vertebrate features compared with other sequenced genomes, including numerous ancient microchromosomes. Combining genome comparisons with extensive regulatory datasets in developing fins-including gene expression, chromatin occupancy and three-dimensional conformation-we find skate-specific genomic rearrangements that alter the three-dimensional regulatory landscape of genes that are involved in the planar cell polarity pathway. Functional inhibition of planar cell polarity signalling resulted in a reduction in anterior fin size, confirming that this pathway is a major contributor to batoid fin morphology. We also identified a fin-specific enhancer that interacts with several hoxa genes, consistent with the redeployment of hox gene expression in anterior pectoral fins, and confirmed its potential to activate transcription in the anterior fin using zebrafish reporter assays. Our findings underscore the central role of genome reorganization and regulatory variation in the evolution of phenotypes, shedding light on the molecular origin of an enigmatic trait.
Assuntos
Nadadeiras de Animais , Evolução Biológica , Genoma , Genômica , Rajidae , Animais , Nadadeiras de Animais/anatomia & histologia , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Rajidae/anatomia & histologia , Rajidae/genética , Peixe-Zebra/genética , Genes Reporter/genéticaRESUMO
Metamorphosis is a widely studied post-embryonic process in which many tissues undergo dramatic modifications to adapt to the new adult lifestyle. Flatfishes represent a good example of metamorphosis in teleost fishes. During metamorphosis of flatfish, organ regression and neoformation occur, with one of the most notable changes being the migration of one of the eyes to the other side of the body. In order to create a useful and reliable tool to advance the molecular study of metamorphosis in flatfish, we generated a chromatin accessible atlas as well as gene expression profile during four developmental stages ranging from a phylotypic to a post-metamorphic stage. We identified 29,019 differentially accessible chromatin regions and 3,253 differentially expressed genes. We found stage-specific regulatory regions and gene expression profiles, supporting the quality of the results. Our work provides strongly reproducible data for further studies to elucidate the regulatory elements that ensure successful metamorphosis in flatfish species.
Assuntos
Cromatina , Linguados , Animais , Cromatina/genética , Cromatina/metabolismo , Linguados/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Metamorfose Biológica/genética , TranscriptomaRESUMO
Zebrafish, a popular organism for studying embryonic development and for modeling human diseases, has so far lacked a systematic functional annotation program akin to those in other animal models. To address this, we formed the international DANIO-CODE consortium and created a central repository to store and process zebrafish developmental functional genomic data. Our data coordination center ( https://danio-code.zfin.org ) combines a total of 1,802 sets of unpublished and re-analyzed published genomic data, which we used to improve existing annotations and show its utility in experimental design. We identified over 140,000 cis-regulatory elements throughout development, including classes with distinct features dependent on their activity in time and space. We delineated the distinct distance topology and chromatin features between regulatory elements active during zygotic genome activation and those active during organogenesis. Finally, we matched regulatory elements and epigenomic landscapes between zebrafish and mouse and predicted functional relationships between them beyond sequence similarity, thus extending the utility of zebrafish developmental genomics to mammals.
Assuntos
Bases de Dados Genéticas , Regulação da Expressão Gênica no Desenvolvimento , Genoma , Genômica , Sequências Reguladoras de Ácido Nucleico , Proteínas de Peixe-Zebra , Peixe-Zebra , Animais , Cromatina/genética , Genoma/genética , Humanos , Camundongos , Anotação de Sequência Molecular , Organogênese/genética , Sequências Reguladoras de Ácido Nucleico/genética , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genéticaRESUMO
One of the central problems of vertebrate evolution is understanding the relationship among the distal portions of fins and limbs. Lacking comparable morphological markers of these regions in fish and tetrapods, these relationships have remained uncertain for the past century and a half. Here we show that Gli3 functions in controlling the proliferative expansion of distal progenitors are shared among dorsal and paired fins as well as tetrapod limbs. Mutant knockout gli3 fins in medaka (Oryzias latipes) form multiple radials and rays, in a pattern reminiscent of the polydactyly observed in Gli3-null mutant mice. In limbs, Gli3 controls both anterior-posterior patterning and cell proliferation, two processes that can be genetically uncoupled. In situ hybridization, quantification of proliferation markers, and analysis of regulatory regions reveal that in paired and dorsal fins, gli3 plays a main role in controlling proliferation but not in patterning. Moreover, gli3 down-regulation in shh mutant fins rescues fin loss in a manner similar to how Gli3 deficiency restores digits in the limbs of Shh mutant mouse embryos. We hypothesize that the Gli3/Shh gene pathway preceded the origin of paired appendages and was originally involved in modulating cell proliferation. Accordingly, the distal regions of dorsal fins, paired fins, and limbs retain a deep regulatory and functional homology that predates the origin of paired appendages.
Assuntos
Nadadeiras de Animais/crescimento & desenvolvimento , Redes Reguladoras de Genes/genética , Proteínas do Tecido Nervoso/genética , Oryzias/genética , Proteína Gli3 com Dedos de Zinco/genética , Animais , Evolução Biológica , Padronização Corporal/genética , Proliferação de Células/genética , Extremidades/crescimento & desenvolvimento , Proteínas de Peixes/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , CamundongosRESUMO
Sight depends on the tight cooperation between photoreceptors and pigmented cells, which derive from common progenitors through the bifurcation of a single gene regulatory network into the neural retina (NR) and retinal-pigmented epithelium (RPE) programs. Although genetic studies have identified upstream nodes controlling these networks, their regulatory logic remains poorly investigated. Here, we characterize transcriptome dynamics and chromatin accessibility in segregating NR/RPE populations in zebrafish. We analyze cis-regulatory modules and enriched transcription factor motives to show extensive network redundancy and context-dependent activity. We identify downstream targets, highlighting an early recruitment of desmosomal genes in the flattening RPE and revealing Tead factors as upstream regulators. We investigate the RPE specification network dynamics to uncover an unexpected sequence of transcription factors recruitment, which is conserved in humans. This systematic interrogation of the NR/RPE bifurcation should improve both genetic counseling for eye disorders and hiPSCs-to-RPE differentiation protocols for cell-replacement therapies in degenerative diseases.
Assuntos
Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica no Desenvolvimento , Redes Reguladoras de Genes , Morfogênese/genética , Epitélio Pigmentado da Retina/metabolismo , Peixe-Zebra/genética , Animais , Animais Geneticamente Modificados , Sequenciamento de Cromatina por Imunoprecipitação/métodos , Análise por Conglomerados , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , RNA-Seq/métodos , Epitélio Pigmentado da Retina/citologia , Epitélio Pigmentado da Retina/embriologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição/classificação , Fatores de Transcrição/genética , Peixe-Zebra/embriologiaRESUMO
The notochord is an evolutionary novelty in vertebrates that functions as an important signaling center during development. Notochord ablation in chicken has demonstrated that it is crucial for pancreas development; however, the molecular mechanism has not been fully described. Here, we show that in zebrafish, the loss of function of nog2, a Bmp antagonist expressed in the notochord, impairs ß cell differentiation, compatible with the antagonistic role of Bmp in ß cell differentiation. In addition, we show that nog2 expression in the notochord is induced by at least one notochord enhancer and its loss of function reduces the number of pancreatic progenitors and impairs ß cell differentiation. Tracing Nog2 diffusion, we show that Nog2 emanates from the notochord to the pancreas progenitor domain. Finally, we find a notochord enhancer in human and mice Nog genomic landscapes, suggesting that the acquisition of a Nog notochord enhancer occurred early in the vertebrate phylogeny and contributes to the development of complex organs like the pancreas.
Assuntos
Sequência Conservada/genética , Elementos Facilitadores Genéticos , Notocorda/embriologia , Pâncreas/embriologia , Vertebrados/embriologia , Vertebrados/genética , Animais , Regulação da Expressão Gênica no Desenvolvimento , Genoma , Modelos Biológicos , Tamanho do Órgão/genética , Pâncreas/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
We investigated how the two rounds of whole-genome duplication that occurred at the base of the vertebrate lineage have impacted ancient microsyntenic associations involving developmental regulators (known as genomic regulatory blocks, GRBs). We showed that the majority of GRBs identified in the last common ancestor of chordates have been maintained as a single copy in humans. We found evidence that dismantling of the duplicated GRB copies occurred early in vertebrate evolution often through the differential retention of the regulatory gene but loss of the bystander gene's exonic sequences. Despite the large evolutionary scale, the presence of duplicated highly conserved noncoding regions provided unambiguous proof for this scenario for multiple ancient GRBs. Remarkably, the dismantling of ancient GRB duplicates has contributed to the creation of large gene deserts associated with regulatory genes in vertebrates, providing a potentially widespread mechanism for the origin of these enigmatic genomic traits.
Assuntos
Evolução Molecular , Regulação da Expressão Gênica no Desenvolvimento , Genes Reguladores , Poliploidia , Vertebrados/genética , Animais , Duplicação Cromossômica , Genoma Humano , Humanos , Elementos Reguladores de TranscriçãoRESUMO
Vertebrates have greatly elaborated the basic chordate body plan and evolved highly distinctive genomes that have been sculpted by two whole-genome duplications. Here we sequence the genome of the Mediterranean amphioxus (Branchiostoma lanceolatum) and characterize DNA methylation, chromatin accessibility, histone modifications and transcriptomes across multiple developmental stages and adult tissues to investigate the evolution of the regulation of the chordate genome. Comparisons with vertebrates identify an intermediate stage in the evolution of differentially methylated enhancers, and a high conservation of gene expression and its cis-regulatory logic between amphioxus and vertebrates that occurs maximally at an earlier mid-embryonic phylotypic period. We analyse regulatory evolution after whole-genome duplications, and find that-in vertebrates-over 80% of broadly expressed gene families with multiple paralogues derived from whole-genome duplications have members that restricted their ancestral expression, and underwent specialization rather than subfunctionalization. Counter-intuitively, paralogues that restricted their expression increased the complexity of their regulatory landscapes. These data pave the way for a better understanding of the regulatory principles that underlie key vertebrate innovations.
Assuntos
Regulação da Expressão Gênica , Genômica , Anfioxos/genética , Vertebrados/genética , Animais , Padronização Corporal/genética , Metilação de DNA , Humanos , Anfioxos/embriologia , Anotação de Sequência Molecular , Regiões Promotoras Genéticas , Transcriptoma/genéticaRESUMO
Despite their evolutionary, developmental and functional importance, the origin of vertebrate paired appendages remains uncertain. In mice, a single enhancer termed ZRS is solely responsible for Shh expression in limbs. Here, zebrafish and mouse transgenic assays trace the functional equivalence of ZRS across the gnathostome phylogeny. CRISPR/Cas9-mediated deletion of the medaka (Oryzias latipes) ZRS and enhancer assays identify the existence of ZRS shadow enhancers in both teleost and human genomes. Deletion of both ZRS and shadow ZRS abolishes shh expression and completely truncates pectoral fin formation. Strikingly, deletion of ZRS results in an almost complete ablation of the dorsal fin. This finding indicates that a ZRS-Shh regulatory module is shared by paired and median fins and that paired fins likely emerged by the co-option of developmental programs established in the median fins of stem gnathostomes. Shh function was later reinforced in pectoral fin development with the recruitment of shadow enhancers, conferring additional robustness.
Assuntos
Nadadeiras de Animais/crescimento & desenvolvimento , Nadadeiras de Animais/metabolismo , Padronização Corporal/genética , Proteínas Hedgehog/genética , Animais , Animais Geneticamente Modificados , Sistemas CRISPR-Cas , Sequência Conservada , Elementos Facilitadores Genéticos , Evolução Molecular , Extremidades/crescimento & desenvolvimento , Proteínas de Peixes/genética , Humanos , Camundongos , Camundongos Transgênicos , Oryzias/genética , Oryzias/crescimento & desenvolvimento , Peixe-Zebra/genética , Peixe-Zebra/crescimento & desenvolvimento , Proteínas de Peixe-Zebra/genéticaRESUMO
BACKGROUND: The organisation of vertebrate genomes into topologically associating domains (TADs) is believed to facilitate the regulation of the genes located within them. A remaining question is whether TAD organisation is achieved through the interactions of the regulatory elements within them or if these interactions are favoured by the pre-existence of TADs. If the latter is true, the fusion of two independent TADs should result in the rewiring of the transcriptional landscape and the generation of ectopic contacts. RESULTS: We show that interactions within the PAX3 and FOXO1 domains are restricted to their respective TADs in normal conditions, while in a patient-derived alveolar rhabdomyosarcoma cell line, harbouring the diagnostic t(2;13)(q35;q14) translocation that brings together the PAX3 and FOXO1 genes, the PAX3 promoter interacts ectopically with FOXO1 sequences. Using a combination of 4C-seq datasets, we have modelled the three-dimensional organisation of the fused landscape in alveolar rhabdomyosarcoma. CONCLUSIONS: The chromosomal translocation that leads to alveolar rhabdomyosarcoma development generates a novel TAD that is likely to favour ectopic PAX3:FOXO1 oncogene activation in non-PAX3 territories. Rhabdomyosarcomas may therefore arise from cells which do not normally express PAX3. The borders of this novel TAD correspond to the original 5'- and 3'- borders of the PAX3 and FOXO1 TADs, respectively, suggesting that TAD organisation precedes the formation of regulatory long-range interactions. Our results demonstrate that, upon translocation, novel regulatory landscapes are formed allowing new intra-TAD interactions between the original loci involved.
Assuntos
Proteína Forkhead Box O1/genética , Fator de Transcrição PAX3/genética , Mapas de Interação de Proteínas/genética , Rabdomiossarcoma Alveolar/genética , Regulação Neoplásica da Expressão Gênica , Genoma Humano , Humanos , Proteínas de Fusão Oncogênica/genética , Regiões Promotoras Genéticas , Domínios Proteicos/genética , Sequências Reguladoras de Ácido Nucleico/genética , Rabdomiossarcoma Alveolar/patologia , Translocação Genética/genéticaRESUMO
Increasing evidence in the last years indicates that the vast amount of regulatory information contained in mammalian genomes is organized in precise 3D chromatin structures. However, the impact of this spatial chromatin organization on gene expression and its degree of evolutionary conservation is still poorly understood. The Six homeobox genes are essential developmental regulators organized in gene clusters conserved during evolution. Here, we reveal that the Six clusters share a deeply evolutionarily conserved 3D chromatin organization that predates the Cambrian explosion. This chromatin architecture generates two largely independent regulatory landscapes (RLs) contained in two adjacent topological associating domains (TADs). By disrupting the conserved TAD border in one of the zebrafish Six clusters, we demonstrate that this border is critical for preventing competition between promoters and enhancers located in separated RLs, thereby generating different expression patterns in genes located in close genomic proximity. Moreover, evolutionary comparison of Six-associated TAD borders reveals the presence of CCCTC-binding factor (CTCF) sites with diverging orientations in all studied deuterostomes. Genome-wide examination of mammalian HiC data reveals that this conserved CTCF configuration is a general signature of TAD borders, underscoring that common organizational principles underlie TAD compartmentalization in deuterostome evolution.
Assuntos
Evolução Molecular , Proteínas Repressoras/química , Proteínas Repressoras/genética , Animais , Animais Geneticamente Modificados , Sequência de Bases , Sítios de Ligação/genética , Fator de Ligação a CCCTC , Cromatina/química , Cromatina/genética , Cromatina/metabolismo , Sequência Conservada , DNA/genética , Elementos Facilitadores Genéticos , Genes Homeobox , Proteínas de Homeodomínio/química , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Camundongos , Modelos Genéticos , Família Multigênica , Regiões Promotoras Genéticas , Domínios e Motivos de Interação entre Proteínas , Proteínas Repressoras/metabolismo , Strongylocentrotus purpuratus/genética , Peixe-Zebra/genéticaRESUMO
The self-organized morphogenesis of the vertebrate optic cup entails coupling the activation of the retinal gene regulatory network to the constriction-driven infolding of the retinal epithelium. Yet the genetic mechanisms underlying this coordination remain largely unexplored. Through phylogenetic footprinting and transgenesis in zebrafish, here we examine the cis-regulatory landscape of opo, an endocytosis regulator essential for eye morphogenesis. Among the different conserved enhancers identified, we isolate a single retina-specific element (H6_10137) and show that its activity depends on binding sites for the retinal determinant Vsx2. Gain- and loss-of-function experiments and ChIP analyses reveal that Vsx2 regulates opo expression through direct binding to this retinal enhancer. Furthermore, we show that vsx2 knockdown impairs the primary optic cup folding. These data support a model by which vsx2, operating through the effector gene opo, acts as a central transcriptional node that coordinates neural retina patterning and optic cup invagination in zebrafish.
Assuntos
Proteínas do Olho/metabolismo , Olho/embriologia , Proteínas de Homeodomínio/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Animais , Animais Geneticamente Modificados , Elementos Facilitadores Genéticos , Epigênese Genética , Proteínas do Olho/genética , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Técnicas de Silenciamento de Genes , Genômica , Proteínas de Homeodomínio/genética , Humanos , Filogenia , Ligação Proteica , Pegadas de Proteínas , Splicing de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Neurônios Retinianos , Peixe-Zebra , Proteínas de Peixe-Zebra/genéticaRESUMO
Genome-wide association studies (GWAS) have reproducibly associated variants within introns of FTO with increased risk for obesity and type 2 diabetes (T2D). Although the molecular mechanisms linking these noncoding variants with obesity are not immediately obvious, subsequent studies in mice demonstrated that FTO expression levels influence body mass and composition phenotypes. However, no direct connection between the obesity-associated variants and FTO expression or function has been made. Here we show that the obesity-associated noncoding sequences within FTO are functionally connected, at megabase distances, with the homeobox gene IRX3. The obesity-associated FTO region directly interacts with the promoters of IRX3 as well as FTO in the human, mouse and zebrafish genomes. Furthermore, long-range enhancers within this region recapitulate aspects of IRX3 expression, suggesting that the obesity-associated interval belongs to the regulatory landscape of IRX3. Consistent with this, obesity-associated single nucleotide polymorphisms are associated with expression of IRX3, but not FTO, in human brains. A direct link between IRX3 expression and regulation of body mass and composition is demonstrated by a reduction in body weight of 25 to 30% in Irx3-deficient mice, primarily through the loss of fat mass and increase in basal metabolic rate with browning of white adipose tissue. Finally, hypothalamic expression of a dominant-negative form of Irx3 reproduces the metabolic phenotypes of Irx3-deficient mice. Our data suggest that IRX3 is a functional long-range target of obesity-associated variants within FTO and represents a novel determinant of body mass and composition.
Assuntos
Proteínas de Homeodomínio/genética , Íntrons/genética , Oxigenases de Função Mista/genética , Obesidade/genética , Oxo-Ácido-Liases/genética , Proteínas/genética , Fatores de Transcrição/genética , Tecido Adiposo/metabolismo , Dioxigenase FTO Dependente de alfa-Cetoglutarato , Animais , Metabolismo Basal/genética , Índice de Massa Corporal , Peso Corporal/genética , Encéfalo/metabolismo , Diabetes Mellitus Tipo 2/genética , Dieta , Genes Dominantes/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Hipotálamo/metabolismo , Masculino , Camundongos , Fenótipo , Polimorfismo de Nucleotídeo Único/genética , Regiões Promotoras Genéticas/genética , Magreza/genética , Fatores de Transcrição/deficiência , Fatores de Transcrição/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/genéticaRESUMO
In multicellular organisms, cis-regulation controls gene expression in space and time. Despite the essential implication of cis-regulation in the development and evolution of organisms and in human diseases, our knowledge about regulatory sequences largely derives from analyzing their activity individually and outside their genomic context. Indeed, the contribution of these sequences to the expression of their target genes in their genomic context is still largely unknown. Here we present a novel genetic screen designed to visualize and interrupt gene regulatory landscapes in vertebrates. In this screen, based on the random insertion of an engineered Tol2 transposon carrying a strong insulator separating two fluorescent reporter genes, we isolated hundreds of zebrafish lines containing insertions that disrupt the cis-regulation of tissue-specific expressed genes. We therefore provide a new easy-to-handle tool that will help to disrupt and chart the regulatory activity spread through the vast noncoding regions of the vertebrate genome.
Assuntos
Elementos de DNA Transponíveis/genética , Elementos Facilitadores Genéticos , Elementos Isolantes , Mutagênese Insercional/métodos , Vertebrados/genética , Animais , Animais Geneticamente Modificados , Fluorescência , Genes Reporter/fisiologia , Genoma , Humanos , Camundongos , Peixe-Zebra/genéticaRESUMO
Common genetic variation at human 8q23.3 is significantly associated with colorectal cancer (CRC) risk. To elucidate the basis of this association we compared the frequency of common variants at 8q23.3 in 1,964 CRC cases and 2,081 healthy controls. Reporter gene studies showed that the single nucleotide polymorphism rs16888589 acts as an allele-specific transcriptional repressor. Chromosome conformation capture (3C) analysis demonstrated that the genomic region harboring rs16888589 interacts with the promoter of gene for eukaryotic translation initiation factor 3, subunit H (EIF3H). We show that increased expression of EIF3H gene increases CRC growth and invasiveness thereby providing a biological mechanism for the 8q23.3 association. These data provide evidence for a functional basis for the non-coding risk variant rs16888589 at 8q23.3 and provides novel insight into the etiological basis of CRC.
Assuntos
Alelos , Cromossomos Humanos Par 8/genética , Neoplasias Colorretais/genética , Fator de Iniciação 3 em Eucariotos/genética , Predisposição Genética para Doença , Variação Genética , Sequências Reguladoras de Ácido Nucleico/genética , Linhagem Celular Tumoral , Proliferação de Células , Neoplasias Colorretais/patologia , Ensaio de Desvio de Mobilidade Eletroforética , Feminino , Regulação Neoplásica da Expressão Gênica , Genes Reporter/genética , Loci Gênicos/genética , Humanos , Desequilíbrio de Ligação/genética , Masculino , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único/genética , Ligação Proteica , Fatores de RiscoRESUMO
POU3F4 encodes a POU-domain transcription factor required for inner ear development. Defects in POU3F4 function are associated with X-linked deafness type 3 (DFN3). Multiple deletions affecting up to ~900-kb upstream of POU3F4 are found in DFN3 patients, suggesting the presence of essential POU3F4 enhancers in this region. Recently, an inner ear enhancer was reported that is absent in most DFN3 patients with upstream deletions. However, two indications suggest that additional enhancers in the POU3F4 upstream region are required for POU3F4 function during inner ear development. First, there is at least one DFN3 deletion that does not eliminate the reported enhancer. Second, the expression pattern driven by this enhancer does not fully recapitulate Pou3f4 expression in the inner ear. Here, we screened a 1-Mb region upstream of the POU3F4 gene for additional cis-regulatory elements and searched for novel DFN3 mutations in the identified POU3F4 enhancers. We found several novel enhancers for otic vesicle expression. Some of these also drive expression in kidney, pancreas and brain, tissues that are known to express Pou3f4. In addition, we report a new and smallest deletion identified so far in a DFN3 family which eliminates 3.9 kb, comprising almost exclusively the previous reported inner ear enhancer. We suggest that multiple enhancers control the expression of Pou3f4 in the inner ear and these may contribute to the phenotype observed in DFN3 patients. In addition, the novel deletion demonstrates that the previous reported enhancer, although not sufficient, is essential for POU3F4 function during inner ear development.