RESUMO
In skeletal myogenesis, the transcription factor MyoD activates distinct transcriptional programs in progenitors compared to terminally differentiated cells. Using ChIP-Seq and gene expression analyses, we show that in primary myoblasts, Snail-HDAC1/2 repressive complex binds and excludes MyoD from its targets. Notably, Snail binds E box motifs that are G/C rich in their central dinucleotides, and such sites are almost exclusively associated with genes expressed during differentiation. By contrast, Snail does not bind the A/T-rich E boxes associated with MyoD targets in myoblasts. Thus, Snai1-HDAC1/2 prevent MyoD occupancy on differentiation-specific regulatory elements, and the change from Snail to MyoD binding often results in enhancer switching during differentiation. Furthermore, we show that a regulatory network involving myogenic regulatory factors (MRFs), Snai1/2, miR-30a, and miR-206 acts as a molecular switch that controls entry into myogenic differentiation. Together, these results reveal a regulatory paradigm that directs distinct gene expression programs in progenitors versus terminally differentiated cells.
Assuntos
Elementos Facilitadores Genéticos/fisiologia , Desenvolvimento Muscular/genética , Proteína MyoD/metabolismo , Mioblastos Esqueléticos/fisiologia , Fatores de Transcrição/metabolismo , Animais , Sequência de Bases , Diferenciação Celular/genética , Camundongos , Dados de Sequência Molecular , Proteína MyoD/química , Proteína MyoD/genética , Mioblastos Esqueléticos/citologia , Cultura Primária de Células , Ligação Proteica/genética , Fatores de Transcrição da Família Snail , Fatores de Transcrição/química , Fatores de Transcrição/genética , Transcrição Gênica/fisiologiaRESUMO
Primary open-angle glaucoma (POAG) is a hereditary neurodegenerative disease, characterized by optic nerve changes including increased excavation, notching and optic disc hemorrhages. The excavation can be described by the vertical cup-disc ratio (VCDR). Previously, genome-wide significant evidence for the association of rs10483727 in SIX1-SIX6 locus with VCDR and subsequent POAG was found. Using 1000 genomes-based imputation of four independent population-based cohorts in the Netherlands, we identified a missense variant rs33912345 (His141Asn) in SIX6 associated with VCDR (Pmeta = 7.74 × 10(-7), n = 11 473) and POAG (Pmeta = 6.09 × 10(-3), n = 292). Exome sequencing analysis revealed another missense variant rs146737847 (Glu129Lys) also in SIX6 associated with VCDR (P = 5.09 × 10(-3), n = 1208). These two findings point to SIX6 as the responsible gene for the previously reported association signal. Functional characterization of SIX6 in zebrafish revealed that knockdown of six6b led to a small eye phenotype. Histological analysis showed retinal lamination, implying an apparent normal development of the eye, but an underdeveloped lens, and reduced optic nerve diameter. Expression analysis of morphants at 3 dpf showed a 5.5-fold up-regulation of cdkn2b, a cyclin-dependent kinase inhibitor, involved in cell cycle regulation and previously associated with VCDR and POAG in genome-wide association studies (GWASs). Since both six6b and cdkn2b play a key role in cell proliferation, we assessed the proliferative activity in the eye of morphants and found an alteration in the proliferative pattern of retinal cells. Our findings in humans and zebrafish suggest a functional involvement of six6b in early eye development, and open new insights into the genetic architecture of POAG.
Assuntos
Diferenciação Celular/genética , Proteínas de Homeodomínio/genética , Degeneração Neural/genética , Nervo Óptico/metabolismo , Nervo Óptico/patologia , Transativadores/genética , Adolescente , Adulto , Fatores Etários , Idoso , Idoso de 80 Anos ou mais , Animais , Proliferação de Células , Mapeamento Cromossômico , Exoma , Olho/embriologia , Olho/metabolismo , Perfilação da Expressão Gênica , Estudo de Associação Genômica Ampla , Glaucoma de Ângulo Aberto/genética , Glaucoma de Ângulo Aberto/metabolismo , Glaucoma de Ângulo Aberto/patologia , Sequenciamento de Nucleotídeos em Larga Escala , Proteínas de Homeodomínio/metabolismo , Humanos , Pessoa de Meia-Idade , Modelos Biológicos , Organogênese/genética , Fenótipo , Locos de Características Quantitativas , Transativadores/metabolismo , Adulto Jovem , Peixe-ZebraRESUMO
Spatiotemporal control of gene expression is central to animal development. Core promoters represent a previously unanticipated regulatory level by interacting with cis-regulatory elements and transcription initiation in different physiological and developmental contexts. Here, we provide a first and comprehensive description of the core promoter repertoire and its dynamic use during the development of a vertebrate embryo. By using cap analysis of gene expression (CAGE), we mapped transcription initiation events at single nucleotide resolution across 12 stages of zebrafish development. These CAGE-based transcriptome maps reveal genome-wide rules of core promoter usage, structure, and dynamics, key to understanding the control of gene regulation during vertebrate ontogeny. They revealed the existence of multiple classes of pervasive intra- and intergenic post-transcriptionally processed RNA products and their developmental dynamics. Among these RNAs, we report splice donor site-associated intronic RNA (sRNA) to be specific to genes of the splicing machinery. For the identification of conserved features, we compared the zebrafish data sets to the first CAGE promoter map of Tetraodon and the existing human CAGE data. We show that a number of features, such as promoter type, newly discovered promoter properties such as a specialized purine-rich initiator motif, as well as sRNAs and the genes in which they are detected, are conserved in mammalian and Tetraodon CAGE-defined promoter maps. The zebrafish developmental promoterome represents a powerful resource for studying developmental gene regulation and revealing promoter features shared across vertebrates.
Assuntos
Desenvolvimento Embrionário/genética , Regulação da Expressão Gênica no Desenvolvimento , Purinas/metabolismo , Sítio de Iniciação de Transcrição , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Animais , Evolução Molecular , Perfilação da Expressão Gênica , Genes , Genoma , Filogenia , Regiões Promotoras Genéticas , RNA/genética , RNA/metabolismo , Capuzes de RNA/genética , Splicing de RNA , Transcriptoma , Vertebrados/genéticaRESUMO
BACKGROUND: Tuberous sclerosis complex (TSC) is an autosomal dominant disorder caused by mutations in TSC1 and TSC2. Conventional DNA diagnostic screens identify a TSC1 or TSC2 mutation in 75 - 90% of individuals categorised with definite TSC. The remaining individuals either have a mutation that is undetectable using conventional methods, or possibly a mutation in another as yet unidentified gene. METHODS: Here we apply a targeted Next Generation Sequencing (NGS) approach to screen the complete TSC1 and TSC2 genomic loci in 7 individuals fulfilling the clinical diagnostic criteria for definite TSC in whom no TSC1 or TSC2 mutations were identified using conventional screening methods. RESULTS: We identified and confirmed pathogenic mutations in 3 individuals. In the remaining individuals we identified variants of uncertain clinical significance. The identified variants included mosaic changes, changes located deep in intronic sequences and changes affecting promoter regions that would not have been identified using exon-only based analyses. CONCLUSIONS: Targeted NGS of the TSC1 and TSC2 loci is a suitable method to increase the yield of mutations identified in the TSC patient population.
Assuntos
Análise Mutacional de DNA , Sequenciamento de Nucleotídeos em Larga Escala , Mutação , Proteínas Supressoras de Tumor/genética , Adolescente , Criança , Loci Gênicos/genética , Genômica , Humanos , Pessoa de Meia-Idade , Esclerose Tuberosa/genética , Proteína 1 do Complexo Esclerose Tuberosa , Proteína 2 do Complexo Esclerose TuberosaRESUMO
Current RNA expression profiling methods rely on enrichment steps for specific RNA classes, thereby not detecting all RNA species in an unperturbed manner. We report strand-specific RNAome sequencing that determines expression of small and large RNAs from rRNA-depleted total RNA in a single sequence run. Since current analysis pipelines cannot reliably analyze small and large RNAs simultaneously, we developed TRAP, Total Rna Analysis Pipeline, a robust interface that is also compatible with existing RNA sequencing protocols. RNAome sequencing quantitatively preserved all RNA classes, allowing cross-class comparisons that facilitates the identification of relationships between different RNA classes. We demonstrate the strength of RNAome sequencing in mouse embryonic stem cells treated with cisplatin. MicroRNA and mRNA expression in RNAome sequencing significantly correlated between replicates and was in concordance with both existing RNA sequencing methods and gene expression arrays generated from the same samples. Moreover, RNAome sequencing also detected additional RNA classes such as enhancer RNAs, anti-sense RNAs, novel RNA species and numerous differentially expressed RNAs undetectable by other methods. At the level of complete RNA classes, RNAome sequencing also identified a specific global repression of the microRNA and microRNA isoform classes after cisplatin treatment whereas all other classes such as mRNAs were unchanged. These characteristics of RNAome sequencing will significantly improve expression analysis as well as studies on RNA biology not covered by existing methods.
Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/métodos , RNA/metabolismo , Análise de Sequência de RNA/métodos , Transcriptoma , Animais , Humanos , CamundongosRESUMO
Uveal melanoma is a lethal cancer with a strong propensity to metastasize. Limited therapeutic options are available once the disease has disseminated. A strong predictor for metastasis is the loss of chromosome 3. Inactivating mutations in BAP1 encoding the BRCA1-associated protein 1 and located on chromosome 3p21.1, have been described in uveal melanoma and other types of cancer. In this study, we determined the prevalence of somatic BAP1 mutations and examined whether these mutations correlate with the functional expression of BAP1 in uveal melanoma tissue and with other clinical, histopathological and chromosomal parameters. We screened a cohort of 74 uveal melanomas for BAP1 mutations, using different deep sequencing methods. The frequency of BAP1 mutations in our study group was 47%. The expression of BAP1 protein was studied using immunohistochemistry. BAP1 staining was absent in 43% of the cases. BAP1 mutation status was strongly associated with BAP1 protein expression (P<0.001), loss of chromosome 3 (P<0.001), and other aggressive prognostic factors. Patients with a BAP1 mutation and absent BAP1 expression had an almost eightfold higher chance of developing metastases compared with those without these changes (P=0.002). We found a strong correlation between the immunohistochemical and sequencing data and therefore propose that, immunohistochemical screening for BAP1 should become routine in the histopathological work-up of uveal melanoma. Furthermore, our analysis indicates that loss of BAP1 may be particularly involved in the progression of uveal melanoma to an aggressive, metastatic phenotype.
Assuntos
Biomarcadores Tumorais/genética , Imuno-Histoquímica , Melanoma/genética , Mutação , Proteínas Supressoras de Tumor/genética , Ubiquitina Tiolesterase/genética , Neoplasias Uveais/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/análise , Análise Mutacional de DNA , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Hibridização in Situ Fluorescente , Estimativa de Kaplan-Meier , Masculino , Melanoma/mortalidade , Melanoma/patologia , Pessoa de Meia-Idade , Modelos de Riscos Proporcionais , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Neoplasias Uveais/mortalidade , Neoplasias Uveais/patologiaRESUMO
Neonatal hyperbilirubinemia (NH) is a common finding in newborn babies in Indonesia. Common and rare variants of UGT1A1 have been known to contribute to NH etiology. This study aims to identify UGT1A1 genetic variation and haplotype associated with NH in Indonesian population. DNA was isolated from 116 cases and 115 controls and a targeted-deep sequencing approach was performed on the promoter, UTRs, and exonic regions of UGT1A1. Determining association of common variants and haplotype analysis were performed using PLINK and Haploview. Ten and 4 rare variants were identified in cases and controls, respectively. The UGT1A1 rare variants frequency in cases (5.17%) was higher than that in controls (1.7%). Four of those rare variants in cases (p.Ala61Thr, p.His300Arg, p.Lys407Asn, and p.Tyr514Asn) and three in controls (p.Tyr79X, p.Ala346Val, and p.Thr412Ser) are novel variants. The frequencies of p.Gly71Arg, p.Pro229Gln, and TA7 common variants were not significantly different between cases and controls. A haplotype, consisting of 3 major alleles of 3' UTRs common variants (rs8330C>G, rs10929303C>T, and rs1042640C>G), was associated with NH incidence (p = 0.025) in this population. Using targeted-deep sequencing and haplotype analysis, we identified novel UGT1A1 rare variants and disease-associated haplotype in NH in Indonesian population.
Assuntos
Alelos , Variação Genética , Glucuronosiltransferase/genética , Haplótipos , Hiperbilirrubinemia Neonatal/genética , Regiões 3' não Traduzidas , Éxons , Feminino , Humanos , Hiperbilirrubinemia Neonatal/epidemiologia , Indonésia/epidemiologia , Masculino , Regiões Promotoras GenéticasRESUMO
Chromosome conformation capture (3C) and its derivatives (e.g., 4C, 5C and Hi-C) are used to analyze the 3D organization of genomes. We recently developed targeted chromatin capture (T2C), an inexpensive method for studying the 3D organization of genomes, interactomes and structural changes associated with gene regulation, the cell cycle, and cell survival and development. Here, we present the protocol for T2C based on capture, describing all experimental steps and bio-informatic tools in full detail. T2C offers high resolution, a large dynamic interaction frequency range and a high signal-to-noise ratio. Its resolution is determined by the resulting fragment size of the chosen restriction enzyme, which can lead to sub-kilobase-pair resolution. T2C's high coverage allows the identification of the interactome of each individual DNA fragment, which makes binning of reads (often used in other methods) basically unnecessary. Notably, T2C requires low sequencing efforts. T2C also allows multiplexing of samples for the direct comparison of multiple samples. It can be used to study topologically associating domains (TADs), determining their position, shape, boundaries, and intra- and inter-domain interactions, as well as the composition of aggregated loops, interactions between nucleosomes, individual transcription factor binding sites, and promoters and enhancers. T2C can be performed by any investigator with basic skills in molecular biology techniques in â¼7-8 d. Data analysis requires basic expertise in bioinformatics and in Linux and Python environments.
Assuntos
Biologia Computacional/métodos , Mapeamento Físico do Cromossomo/métodos , Análise de Sequência de DNA/métodos , Animais , Cromatina/ultraestrutura , Montagem e Desmontagem da Cromatina/fisiologia , Mapeamento Cromossômico/métodos , DNA , Regulação da Expressão Gênica , Genoma/genética , Genoma Humano/genética , Genoma Humano/fisiologia , Genômica , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Camundongos , Nucleossomos , SoftwareRESUMO
Mycoplasma pneumoniae is a common cause of respiratory tract infections (RTIs) in children. We recently demonstrated that this bacterium can be carried asymptomatically in the respiratory tract of children. To identify potential genetic differences between M. pneumoniae strains that are carried asymptomatically and those that cause symptomatic infections, we performed whole-genome sequence analysis of 20 M. pneumoniae strains. The analyzed strains included 3 reference strains, 3 strains isolated from asymptomatic children, 13 strains isolated from clinically well-defined patients suffering from an upper (n = 4) or lower (n = 9) RTI, and one strain isolated from a follow-up patient who recently recovered from an RTI. The obtained sequences were each compared to the sequences of the reference strains. To find differences between strains isolated from asymptomatic and symptomatic individuals, a variant comparison was performed between the different groups of strains. Irrespective of the group (asymptomatic vs. symptomatic) from which the strains originated, subtype 1 and subtype 2 strains formed separate clusters. We could not identify a specific genotype associated with M. pneumoniae virulence. However, we found marked genetic differences between clinical isolates and the reference strains, which indicated that the latter strains may not be regarded as appropriate representatives of circulating M. pneumoniae strains.
RESUMO
BACKGROUND: The dynamic three-dimensional chromatin architecture of genomes and its co-evolutionary connection to its function-the storage, expression, and replication of genetic information-is still one of the central issues in biology. Here, we describe the much debated 3D architecture of the human and mouse genomes from the nucleosomal to the megabase pair level by a novel approach combining selective high-throughput high-resolution chromosomal interaction capture (T2C), polymer simulations, and scaling analysis of the 3D architecture and the DNA sequence. RESULTS: The genome is compacted into a chromatin quasi-fibre with ~5 ± 1 nucleosomes/11 nm, folded into stable ~30-100 kbp loops forming stable loop aggregates/rosettes connected by similar sized linkers. Minor but significant variations in the architecture are seen between cell types and functional states. The architecture and the DNA sequence show very similar fine-structured multi-scaling behaviour confirming their co-evolution and the above. CONCLUSIONS: This architecture, its dynamics, and accessibility, balance stability and flexibility ensuring genome integrity and variation enabling gene expression/regulation by self-organization of (in)active units already in proximity. Our results agree with the heuristics of the field and allow "architectural sequencing" at a genome mechanics level to understand the inseparable systems genomic properties.
RESUMO
The locations of transcriptional enhancers and promoters were recently mapped in many mammalian cell types. Proteins that bind those regulatory regions can determine cell identity but have not been systematically identified. Here we purify native enhancers, promoters or heterochromatin from embryonic stem cells by chromatin immunoprecipitations (ChIP) for characteristic histone modifications and identify associated proteins using mass spectrometry (MS). 239 factors are identified and predicted to bind enhancers or promoters with different levels of activity, or heterochromatin. Published genome-wide data indicate a high accuracy of location prediction by ChIP-MS. A quarter of the identified factors are important for pluripotency and includes Oct4, Esrrb, Klf5, Mycn and Dppa2, factors that drive reprogramming to pluripotent stem cells. We determined the genome-wide binding sites of Dppa2 and find that Dppa2 operates outside the classical pluripotency network. Our ChIP-MS method provides a detailed read-out of the transcriptional landscape representative of the investigated cell type.
Assuntos
Imunoprecipitação da Cromatina/métodos , Histonas/química , Animais , Sítios de Ligação , Domínio Catalítico , Células-Tronco Embrionárias/citologia , Elementos Facilitadores Genéticos , Genoma , Código das Histonas , Fatores de Transcrição Kruppel-Like/química , Espectrometria de Massas/métodos , Camundongos , Proteína Proto-Oncogênica N-Myc , Proteínas Nucleares/química , Fator 3 de Transcrição de Octâmero/metabolismo , Células-Tronco Pluripotentes/citologia , Regiões Promotoras Genéticas , Ligação Proteica , Proteínas Proto-Oncogênicas/química , Receptores de Estrogênio/química , Sequências Reguladoras de Ácido Nucleico , Reprodutibilidade dos Testes , Fatores de TranscriçãoRESUMO
Implementation of next-generation DNA sequencing (NGS) technology into routine diagnostic genome care requires strategic choices. Instead of theoretical discussions on the consequences of such choices, we compared NGS-based diagnostic practices in eight clinical genetic centers in the Netherlands, based on genetic testing of nine pre-selected patients with cardiomyopathy. We highlight critical implementation choices, including the specific contributions of laboratory and medical specialists, bioinformaticians and researchers to diagnostic genome care, and how these affect interpretation and reporting of variants. Reported pathogenic mutations were consistent for all but one patient. Of the two centers that were inconsistent in their diagnosis, one reported to have found 'no causal variant', thereby underdiagnosing this patient. The other provided an alternative diagnosis, identifying another variant as causal than the other centers. Ethical and legal analysis showed that informed consent procedures in all centers were generally adequate for diagnostic NGS applications that target a limited set of genes, but not for exome- and genome-based diagnosis. We propose changes to further improve and align these procedures, taking into account the blurring boundary between diagnostics and research, and specific counseling options for exome- and genome-based diagnostics. We conclude that alternative diagnoses may infer a certain level of 'greediness' to come to a positive diagnosis in interpreting sequencing results. Moreover, there is an increasing interdependence of clinic, diagnostics and research departments for comprehensive diagnostic genome care. Therefore, we invite clinical geneticists, physicians, researchers, bioinformatics experts and patients to reconsider their role and position in future diagnostic genome care.
Assuntos
Cardiomiopatias/diagnóstico , Testes Genéticos/normas , Genoma Humano , Sequenciamento de Nucleotídeos em Larga Escala/normas , Mutação , Proteínas de Ligação ao Cálcio/genética , Miosinas Cardíacas/genética , Cardiomiopatias/genética , Proteínas de Transporte/genética , Exoma , Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala/instrumentação , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Consentimento Livre e Esclarecido/legislação & jurisprudência , Ensaio de Proficiência Laboratorial/estatística & dados numéricos , MAP Quinase Quinase Quinases/genética , Cadeias Pesadas de Miosina/genética , Países Baixos , Proteínas Serina-Treonina QuinasesRESUMO
ß-Thalassaemia is one of the most common autosomal recessive single-gene disorder worldwide, with a carrier frequency of 12% in Cyprus. Prenatal tests for at risk pregnancies use invasive methods and development of a non-invasive prenatal diagnostic (NIPD) method is of paramount importance to prevent unnecessary risks inherent to invasive methods. Here, we describe such a method by assessing a modified version of next generation sequencing (NGS) using the Illumina platform, called 'targeted sequencing', based on the detection of paternally inherited fetal alleles in maternal plasma. We selected four single-nucleotide polymorphisms (SNPs) located in the ß-globin locus with a high degree of heterozygosity in the Cypriot population. Spiked genomic samples were used to determine the specificity of the platform. We could detect the minor alleles in the expected ratio, showing the specificity of the platform. We then developed a multiplexed format for the selected SNPs and analysed ten maternal plasma samples from pregnancies at risk. The presence or absence of the paternal mutant allele was correctly determined in 27 out of 34 samples analysed. With haplotype analysis, NIPD was possible on eight out of ten families. This is the first study carried out for the NIPD of ß-thalassaemia using targeted NGS and haplotype analysis. Preliminary results show that NGS is effective in detecting paternally inherited alleles in the maternal plasma.
Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/métodos , Polimorfismo de Nucleotídeo Único/genética , Diagnóstico Pré-Natal/métodos , Talassemia beta/genética , Alelos , Feminino , Haplótipos/genética , Humanos , Mutação/genética , GravidezRESUMO
Pax3 and Pax7 regulate stem cell function in skeletal myogenesis. However, molecular insight into their distinct roles has remained elusive. Using gene expression data combined with genome-wide binding-site analysis, we show that both Pax3 and Pax7 bind identical DNA motifs and jointly activate a large panel of genes involved in muscle stem cell function. Surprisingly, in adult myoblasts Pax3 binds a subset (6.4%) of Pax7 targets. Despite a significant overlap in their transcriptional network, Pax7 regulates distinct panels of genes involved in the promotion of proliferation and inhibition of myogenic differentiation. We show that Pax7 has a higher binding affinity to the homeodomain-binding motif relative to Pax3, suggesting that intrinsic differences in DNA binding contribute to the observed functional difference between Pax3 and Pax7 binding in myogenesis. Together, our data demonstrate distinct attributes of Pax7 function and provide mechanistic insight into the nonredundancy of Pax3 and Pax7 in muscle development.