Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 65
Filtrar
1.
Cell ; 169(2): 216-228.e19, 2017 04 06.
Artigo em Inglês | MEDLINE | ID: mdl-28388407

RESUMO

Chromatin architecture is fundamental in regulating gene expression. To investigate when spatial genome organization is first established during development, we examined chromatin conformation during Drosophila embryogenesis and observed the emergence of chromatin architecture within a tight time window that coincides with the onset of transcription activation in the zygote. Prior to zygotic genome activation, the genome is mostly unstructured. Early expressed genes serve as nucleation sites for topologically associating domain (TAD) boundaries. Activation of gene expression coincides with the establishment of TADs throughout the genome and co-localization of housekeeping gene clusters, which remain stable in subsequent stages of development. However, the appearance of TAD boundaries is independent of transcription and requires the transcription factor Zelda for locus-specific TAD boundary insulation. These results offer insight into when spatial organization of the genome emerges and identify a key factor that helps trigger this architecture.


Assuntos
Cromatina/metabolismo , Drosophila melanogaster/embriologia , Drosophila melanogaster/genética , Genoma de Inseto , Ativação Transcricional , Zigoto/metabolismo , Animais , Proteínas de Drosophila/metabolismo , Embrião não Mamífero/metabolismo , Genes Essenciais , Proteínas Nucleares , RNA Polimerase II/metabolismo , Fatores de Tempo , Fatores de Transcrição/metabolismo , Transcrição Gênica
2.
Cell ; 152(1-2): 327-39, 2013 Jan 17.
Artigo em Inglês | MEDLINE | ID: mdl-23332764

RESUMO

Although the proteins that read the gene regulatory code, transcription factors (TFs), have been largely identified, it is not well known which sequences TFs can recognize. We have analyzed the sequence-specific binding of human TFs using high-throughput SELEX and ChIP sequencing. A total of 830 binding profiles were obtained, describing 239 distinctly different binding specificities. The models represent the majority of human TFs, approximately doubling the coverage compared to existing systematic studies. Our results reveal additional specificity determinants for a large number of factors for which a partial specificity was known, including a commonly observed A- or T-rich stretch that flanks the core motifs. Global analysis of the data revealed that homodimer orientation and spacing preferences, and base-stacking interactions, have a larger role in TF-DNA binding than previously appreciated. We further describe a binding model incorporating these features that is required to understand binding of TFs to DNA.


Assuntos
Imunoprecipitação da Cromatina , Modelos Biológicos , Técnica de Seleção de Aptâmeros , Fatores de Transcrição/metabolismo , Animais , DNA/química , Humanos , Cadeias de Markov , Camundongos , Filogenia , Fatores de Transcrição/genética
3.
Am J Hum Genet ; 111(6): 1125-1139, 2024 06 06.
Artigo em Inglês | MEDLINE | ID: mdl-38759652

RESUMO

Sperm production and function require the correct establishment of DNA methylation patterns in the germline. Here, we examined the genome-wide DNA methylation changes during human spermatogenesis and its alterations in disturbed spermatogenesis. We found that spermatogenesis is associated with remodeling of the methylome, comprising a global decline in DNA methylation in primary spermatocytes followed by selective remethylation, resulting in a spermatids/sperm-specific methylome. Hypomethylated regions in spermatids/sperm were enriched in specific transcription factor binding sites for DMRT and SOX family members and spermatid-specific genes. Intriguingly, while SINEs displayed differential methylation throughout spermatogenesis, LINEs appeared to be protected from changes in DNA methylation. In disturbed spermatogenesis, germ cells exhibited considerable DNA methylation changes, which were significantly enriched at transposable elements and genes involved in spermatogenesis. We detected hypomethylation in SVA and L1HS in disturbed spermatogenesis, suggesting an association between the abnormal programming of these regions and failure of germ cells progressing beyond meiosis.


Assuntos
Metilação de DNA , Genoma Humano , Espermatogênese , Humanos , Espermatogênese/genética , Masculino , Espermátides/metabolismo , Espermatócitos/metabolismo , Elementos de DNA Transponíveis/genética , Espermatozoides/metabolismo , Meiose/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
4.
Genome Res ; 32(7): 1408-1423, 2022 07.
Artigo em Inglês | MEDLINE | ID: mdl-34987056

RESUMO

There is considerable interest in understanding the effect of transposable elements (TEs) on embryonic development. Studies in humans and mice are limited by the difficulty of working with mammalian embryos and by the relative scarcity of active TEs in these organisms. The zebrafish is an outstanding model for the study of vertebrate development, and over half of its genome consists of diverse TEs. However, zebrafish TEs remain poorly characterized. Here we describe the demography and genomic distribution of zebrafish TEs and their expression throughout embryogenesis using bulk and single-cell RNA sequencing data. These results reveal a highly dynamic genomic ecosystem comprising nearly 2000 distinct TE families, which vary in copy number by four orders of magnitude and span a wide range of ages. Longer retroelements tend to be retained in intergenic regions, whereas short interspersed nuclear elements (SINEs) and DNA transposons are more frequently found nearby or within genes. Locus-specific mapping of TE expression reveals extensive TE transcription during development. Although two-thirds of TE transcripts are likely driven by nearby gene promoters, we still observe stage- and tissue-specific expression patterns in self-regulated TEs. Long terminal repeat (LTR) retroelements are most transcriptionally active immediately following zygotic genome activation, whereas DNA transposons are enriched among transcripts expressed in later stages of development. Single-cell analysis reveals several endogenous retroviruses expressed in specific somatic cell lineages. Overall, our study provides a valuable resource for using zebrafish as a model to study the impact of TEs on vertebrate development.


Assuntos
Elementos de DNA Transponíveis , Peixe-Zebra , Animais , Elementos de DNA Transponíveis/genética , Ecossistema , Genômica/métodos , Humanos , Mamíferos/genética , Camundongos , Retroelementos/genética , Peixe-Zebra/genética
5.
Neurobiol Dis ; 194: 106463, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38485095

RESUMO

Mutations in NR2E3, a gene encoding an orphan nuclear transcription factor, cause two retinal dystrophies with a distinct phenotype, but the precise role of NR2E3 in rod and cone transcriptional networks remains unclear. To dissect NR2E3 function, we performed scRNA-seq in the retinas of wildtype and two different Nr2e3 mouse models that show phenotypes similar to patients carrying NR2E3 mutations. Our results reveal that rod and cone populations are not homogeneous and can be separated into different sub-classes. We identify a previously unreported cone pathway that generates hybrid cones co-expressing both cone- and rod-related genes. In mutant retinas, this hybrid cone subpopulation is more abundant and includes a subpopulation of rods transitioning towards a cone cell fate. Hybrid photoreceptors with high misexpression of cone- and rod-related genes are prone to regulated necrosis. Overall, our results shed light on the role of NR2E3 in modulating photoreceptor differentiation towards cone and rod fates and explain how different mutations in NR2E3 lead to distinct visual disorders in humans.


Assuntos
Receptores Nucleares Órfãos , Retina , Camundongos , Animais , Humanos , Receptores Nucleares Órfãos/metabolismo , Retina/metabolismo , Células Fotorreceptoras Retinianas Cones/metabolismo , Diferenciação Celular , Regulação da Expressão Gênica
6.
Genome Res ; 31(6): 981-994, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-34006569

RESUMO

Chromatin architecture mapping in 3D formats has increased our understanding of how regulatory sequences and gene expression are connected and regulated in a genome. The 3D chromatin genome shows extensive remodeling during embryonic development, and although the cleavage-stage embryos of most species lack structure before zygotic genome activation (pre-ZGA), zebrafish has been reported to have structure. Here, we aimed to determine the chromosomal architecture in paternal/sperm zebrafish gamete cells to discern whether it either resembles or informs early pre-ZGA zebrafish embryo chromatin architecture. First, we assessed the higher-order architecture through advanced low-cell in situ Hi-C. The structure of zebrafish sperm, packaged by histones, lacks topological associated domains and instead displays "hinge-like" domains of ∼150 kb that repeat every 1-2 Mbs, suggesting a condensed repeating structure resembling mitotic chromosomes. The pre-ZGA embryos lacked chromosomal structure, in contrast to prior work, and only developed structure post-ZGA. During post-ZGA, we find chromatin architecture beginning to form at small contact domains of a median length of ∼90 kb. These small contact domains are established at enhancers, including super-enhancers, and chemical inhibition of Ep300a (p300) and Crebbpa (CBP) activity, lowering histone H3K27ac, but not transcription inhibition, diminishes these contacts. Together, this study reveals hinge-like domains in histone-packaged zebrafish sperm chromatin and determines that the initial formation of high-order chromatin architecture in zebrafish embryos occurs after ZGA primarily at enhancers bearing high H3K27ac.


Assuntos
Cromatina , Peixe-Zebra , Animais , Cromatina/genética , Cromatina/metabolismo , Cromossomos/genética , Desenvolvimento Embrionário/genética , Regulação da Expressão Gênica no Desenvolvimento , Masculino , Espermatozoides/metabolismo , Peixe-Zebra/genética , Zigoto
7.
EMBO Rep ; 22(11): e53048, 2021 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-34515391

RESUMO

During implantation, the murine embryo transitions from a "quiet" into an active metabolic/proliferative state, which kick-starts the growth and morphogenesis of the post-implantation conceptus. Such transition is also required for embryonic stem cells to be established from mouse blastocysts, but the factors regulating this process are poorly understood. Here, we show that Ronin plays a critical role in the process by enabling active energy production, and the loss of Ronin results in the establishment of a reversible quiescent state in which naïve pluripotency is promoted. In addition, Ronin fine-tunes the expression of genes that encode ribosomal proteins and is required for proper tissue-scale organisation of the pluripotent lineage during the transition from blastocyst to egg cylinder stage. Thus, Ronin function is essential for governing the metabolic capacity so that it can support the pluripotent lineage's high-energy demands for cell proliferation and morphogenesis.


Assuntos
Desenvolvimento Embrionário , Células-Tronco Embrionárias , Animais , Blastocisto/metabolismo , Implantação do Embrião/fisiologia , Embrião de Mamíferos/metabolismo , Desenvolvimento Embrionário/genética , Células-Tronco Embrionárias/metabolismo , Camundongos
8.
Cell ; 133(5): 813-28, 2008 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-18510926

RESUMO

Dosage compensation, mediated by the MSL complex, regulates X-chromosomal gene expression in Drosophila. Here we report that the histone H4 lysine 16 (H4K16) specific histone acetyltransferase MOF displays differential binding behavior depending on whether the target gene is located on the X chromosome versus the autosomes. More specifically, on the male X chromosome, where MSL1 and MSL3 are preferentially associated with the 3' end of dosage compensated genes, MOF displays a bimodal distribution binding to promoters and the 3' ends of genes. In contrast, on MSL1/MSL3 independent X-linked genes and autosomal genes in males and females, MOF binds primarily to promoters. Binding of MOF to autosomes is functional, as H4K16 acetylation and the transcription levels of a number of genes are affected upon MOF depletion. Therefore, MOF is not only involved in the onset of dosage compensation, but also acts as a regulator of gene expression in the Drosophila genome.


Assuntos
Mecanismo Genético de Compensação de Dose , Proteínas de Drosophila/metabolismo , Regulação da Expressão Gênica , Histona Acetiltransferases/metabolismo , Proteínas Nucleares/metabolismo , Região 3'-Flanqueadora , Acetilação , Animais , Linhagem Celular , Feminino , Genoma de Inseto , Histonas/genética , Histonas/metabolismo , Masculino , Regiões Promotoras Genéticas , Cromossomo X
9.
Development ; 146(19)2019 09 26.
Artigo em Inglês | MEDLINE | ID: mdl-31558569

RESUMO

The three-dimensional organisation of the genome plays a crucial role in developmental gene regulation. In recent years, techniques to investigate this organisation have become more accessible to labs worldwide due to improvements in protocols and decreases in the cost of high-throughput sequencing. However, the resulting datasets are complex and can be challenging to analyse and interpret. Here, we provide a guide to visualisation approaches that can aid the interpretation of such datasets and the communication of biological results.


Assuntos
Genoma , Imageamento Tridimensional , Algoritmos , Animais , Cromatina/genética , Humanos
10.
Trends Genet ; 34(12): 903-914, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30292539

RESUMO

The 3D structure of chromatin in the nucleus is important for the regulation of gene expression and the correct deployment of developmental programs. The differentiation of germ cells and early embryonic development (when the zygotic genome is activated and transcription is taking place for the first time) are accompanied by dramatic changes in gene expression and the epigenetic landscape. Recent studies used Hi-C to investigate the 3D chromatin organization during these developmental transitions, uncovering remarkable remodeling of the 3D genome. Here, we highlight the changes described so far and discuss some of the implications that these findings have for our understanding of the mechanisms and functionality of 3D chromatin architecture.


Assuntos
Cromatina/ultraestrutura , Desenvolvimento Embrionário/genética , Epigênese Genética/genética , Zigoto/ultraestrutura , Animais , Cromatina/genética , Drosophila melanogaster/genética , Drosophila melanogaster/crescimento & desenvolvimento , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Masculino , Camundongos , Conformação Molecular , Oócitos/crescimento & desenvolvimento , Oócitos/ultraestrutura , Espermatozoides/crescimento & desenvolvimento , Espermatozoides/ultraestrutura , Zigoto/crescimento & desenvolvimento
11.
Mol Cell ; 38(6): 827-41, 2010 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-20620954

RESUMO

Here, we report the biochemical characterization of the nonspecific lethal (NSL) complex (NSL1, NSL2, NSL3, MCRS2, MBD-R2, and WDS) that associates with the histone acetyltransferase MOF in both Drosophila and mammals. Chromatin immunoprecipitation-Seq analysis revealed association of NSL1 and MCRS2 with the promoter regions of more than 4000 target genes, 70% of these being actively transcribed. This binding is functional, as depletion of MCRS2, MBD-R2, and NSL3 severely affects gene expression genome wide. The NSL complex members bind to their target promoters independently of MOF. However, depletion of MCRS2 affects MOF recruitment to promoters. NSL complex stability is interdependent and relies mainly on the presence of NSL1 and MCRS2. Tethering of NSL3 to a heterologous promoter leads to robust transcription activation and is sensitive to the levels of NSL1, MCRS2, and MOF. Taken together, we conclude that the NSL complex acts as a major transcriptional regulator in Drosophila.


Assuntos
Proteínas de Drosophila/metabolismo , Drosophila/metabolismo , Histona Acetiltransferases/metabolismo , Proteínas Nucleares/metabolismo , Animais , Drosophila/genética , Proteínas de Drosophila/genética , Genoma de Inseto , Histona Acetiltransferases/genética , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Proteínas Nucleares/genética , Regiões Promotoras Genéticas , Ligação Proteica , Transcrição Gênica
12.
Bioinformatics ; 32(20): 3190-3192, 2016 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-27318199

RESUMO

Eukaryotic genomes are hierarchically organized into topologically associating domains (TADs). The computational identification of these domains and their associated properties critically depends on the choice of suitable parameters of TAD-calling algorithms. To reduce the element of trial-and-error in parameter selection, we have developed TADtool: an interactive plot to find robust TAD-calling parameters with immediate visual feedback. TADtool allows the direct export of TADs called with a chosen set of parameters for two of the most common TAD calling algorithms: directionality and insulation index. It can be used as an intuitive, standalone application or as a Python package for maximum flexibility. AVAILABILITY AND IMPLEMENTATION: TADtool is available as a Python package from GitHub (https://github.com/vaquerizaslab/tadtool) or can be installed directly via PyPI, the Python package index (tadtool). CONTACT: kai.kruse@mpi-muenster.mpg.de, jmv@mpi-muenster.mpg.deSupplementary information: Supplementary data are available at Bioinformatics online.


Assuntos
Algoritmos , Genoma , Animais , Humanos , Software
13.
Methods ; 107: 89-97, 2016 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-27450428

RESUMO

Protein translation is at the heart of cellular metabolism and its in-depth characterization is key for many lines of research. Recently, ribosome profiling became the state-of-the-art method to quantitatively characterize translation dynamics at a transcriptome-wide level. However, the strategy of library generation affects its outcomes. Here, we present a modified ribosome-profiling protocol starting from yeast, human cells and vertebrate brain tissue. We use a DNA linker carrying four randomized positions at its 5' end and a reverse-transcription (RT) primer with three randomized positions to reduce artifacts during library preparation. The use of seven randomized nucleotides allows to efficiently detect library-generation artifacts. We find that the effect of polymerase chain reaction (PCR) artifacts is relatively small for global analyses when sufficient input material is used. However, when input material is limiting, our strategy improves the sensitivity of gene-specific analyses. Furthermore, randomized nucleotides alleviate the skewed frequency of specific sequences at the 3' end of ribosome-protected fragments (RPFs) likely resulting from ligase specificity. Finally, strategies that rely on dual ligation show a high degree of gene-coverage variation. Taken together, our approach helps to remedy two of the main problems associated with ribosome-profiling data. This will facilitate the analysis of translational dynamics and increase our understanding of the influence of RNA modifications on translation.


Assuntos
Perfilação da Expressão Gênica/métodos , Engenharia Genética/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Ribossomos/genética , Humanos , Oligonucleotídeos/genética , Biossíntese de Proteínas/genética , Ribossomos/química , Transcriptoma/genética
14.
15.
Nat Rev Genet ; 10(4): 252-63, 2009 04.
Artigo em Inglês | MEDLINE | ID: mdl-19274049

RESUMO

Transcription factors are key cellular components that control gene expression: their activities determine how cells function and respond to the environment. Currently, there is great interest in research into human transcriptional regulation. However, surprisingly little is known about these regulators themselves. For example, how many transcription factors does the human genome contain? How are they expressed in different tissues? Are they evolutionarily conserved? Here, we present an analysis of 1,391 manually curated sequence-specific DNA-binding transcription factors, their functions, genomic organization and evolutionary conservation. Much remains to be explored, but this study provides a solid foundation for future investigations to elucidate regulatory mechanisms underlying diverse mammalian biological processes.


Assuntos
Evolução Molecular , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Expressão Gênica , Genoma Humano , Humanos
16.
PLoS Genet ; 8(6): e1002736, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22723752

RESUMO

MOF is the major histone H4 lysine 16-specific (H4K16) acetyltransferase in mammals and Drosophila. In flies, it is involved in the regulation of X-chromosomal and autosomal genes as part of the MSL and the NSL complexes, respectively. While the function of the MSL complex as a dosage compensation regulator is fairly well understood, the role of the NSL complex in gene regulation is still poorly characterized. Here we report a comprehensive ChIP-seq analysis of four NSL complex members (NSL1, NSL3, MBD-R2, and MCRS2) throughout the Drosophila melanogaster genome. Strikingly, the majority (85.5%) of NSL-bound genes are constitutively expressed across different cell types. We find that an increased abundance of the histone modifications H4K16ac, H3K4me2, H3K4me3, and H3K9ac in gene promoter regions is characteristic of NSL-targeted genes. Furthermore, we show that these genes have a well-defined nucleosome free region and broad transcription initiation patterns. Finally, by performing ChIP-seq analyses of RNA polymerase II (Pol II) in NSL1- and NSL3-depleted cells, we demonstrate that both NSL proteins are required for efficient recruitment of Pol II to NSL target gene promoters. The observed Pol II reduction coincides with compromised binding of TBP and TFIIB to target promoters, indicating that the NSL complex is required for optimal recruitment of the pre-initiation complex on target genes. Moreover, genes that undergo the most dramatic loss of Pol II upon NSL knockdowns tend to be enriched in DNA Replication-related Element (DRE). Taken together, our findings show that the MOF-containing NSL complex acts as a major regulator of housekeeping genes in flies by modulating initiation of Pol II transcription.


Assuntos
Proteínas de Drosophila , Drosophila melanogaster/genética , Histona-Lisina N-Metiltransferase/genética , Proteínas Nucleares , RNA Polimerase II , Fatores de Transcrição , Acetilação , Animais , Sítios de Ligação , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Regulação da Expressão Gênica , Genoma de Inseto , Histona-Lisina N-Metiltransferase/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Fator de Transcrição TFIIB/genética , Fator de Transcrição TFIIB/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas de Transporte Vesicular
17.
Cell Rep ; 43(8): 114498, 2024 Aug 27.
Artigo em Inglês | MEDLINE | ID: mdl-39084219

RESUMO

Cohesin shapes the chromatin architecture, including enhancer-promoter interactions. Its components, especially STAG2, but not its paralog STAG1, are frequently mutated in myeloid malignancies. To elucidate the underlying mechanisms of leukemogenesis, we comprehensively characterized genetic, transcriptional, and chromatin conformational changes in acute myeloid leukemia (AML) patient samples. Specific loci displayed altered cohesin occupancy, gene expression, and local chromatin activation, which were not compensated by the remaining STAG1-cohesin. These changes could be linked to disrupted spatial chromatin looping in cohesin-mutated AMLs. Complementary depletion of STAG2 or STAG1 in primary human hematopoietic progenitors (HSPCs) revealed effects resembling STAG2-mutant AML-specific changes following STAG2 knockdown, not invoked by the depletion of STAG1. STAG2-deficient HSPCs displayed impaired differentiation capacity and maintained HSPC-like gene expression. This work establishes STAG2 as a key regulator of chromatin contacts, gene expression, and differentiation in the hematopoietic system and identifies candidate target genes that may be implicated in human leukemogenesis.


Assuntos
Proteínas de Ciclo Celular , Cromatina , Proteínas Cromossômicas não Histona , Coesinas , Leucemia Mieloide Aguda , Mutação , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Leucemia Mieloide Aguda/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Proteínas Cromossômicas não Histona/genética , Mutação/genética , Células-Tronco Hematopoéticas/metabolismo , Diferenciação Celular/genética , Regulação Leucêmica da Expressão Gênica , Antígenos Nucleares/metabolismo , Antígenos Nucleares/genética , Proteínas Nucleares
18.
Nat Commun ; 15(1): 5693, 2024 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-38972954

RESUMO

Leukemias with ambiguous lineage comprise several loosely defined entities, often without a clear mechanistic basis. Here, we extensively profile the epigenome and transcriptome of a subgroup of such leukemias with CpG Island Methylator Phenotype. These leukemias exhibit comparable hybrid myeloid/lymphoid epigenetic landscapes, yet heterogeneous genetic alterations, suggesting they are defined by their shared epigenetic profile rather than common genetic lesions. Gene expression enrichment reveals similarity with early T-cell precursor acute lymphoblastic leukemia and a lymphoid progenitor cell of origin. In line with this, integration of differential DNA methylation and gene expression shows widespread silencing of myeloid transcription factors. Moreover, binding sites for hematopoietic transcription factors, including CEBPA, SPI1 and LEF1, are uniquely inaccessible in these leukemias. Hypermethylation also results in loss of CTCF binding, accompanied by changes in chromatin interactions involving key transcription factors. In conclusion, epigenetic dysregulation, and not genetic lesions, explains the mixed phenotype of this group of leukemias with ambiguous lineage. The data collected here constitute a useful and comprehensive epigenomic reference for subsequent studies of acute myeloid leukemias, T-cell acute lymphoblastic leukemias and mixed-phenotype leukemias.


Assuntos
Ilhas de CpG , Metilação de DNA , Epigênese Genética , Redes Reguladoras de Genes , Humanos , Metilação de DNA/genética , Ilhas de CpG/genética , Proteínas Estimuladoras de Ligação a CCAAT/genética , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Fator 1 de Ligação ao Facilitador Linfoide/genética , Fator 1 de Ligação ao Facilitador Linfoide/metabolismo , Fator de Ligação a CCCTC/metabolismo , Fator de Ligação a CCCTC/genética , Regulação Leucêmica da Expressão Gênica , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Cromatina/metabolismo , Cromatina/genética , Masculino , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patologia , Feminino , Hematopoese/genética , Criança , Transcriptoma , Proteínas Proto-Oncogênicas , Transativadores
19.
Genome Res ; 20(6): 861-73, 2010 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-20378718

RESUMO

The genetic code-the binding specificity of all transfer-RNAs--defines how protein primary structure is determined by DNA sequence. DNA also dictates when and where proteins are expressed, and this information is encoded in a pattern of specific sequence motifs that are recognized by transcription factors. However, the DNA-binding specificity is only known for a small fraction of the approximately 1400 human transcription factors (TFs). We describe here a high-throughput method for analyzing transcription factor binding specificity that is based on systematic evolution of ligands by exponential enrichment (SELEX) and massively parallel sequencing. The method is optimized for analysis of large numbers of TFs in parallel through the use of affinity-tagged proteins, barcoded selection oligonucleotides, and multiplexed sequencing. Data are analyzed by a new bioinformatic platform that uses the hundreds of thousands of sequencing reads obtained to control the quality of the experiments and to generate binding motifs for the TFs. The described technology allows higher throughput and identification of much longer binding profiles than current microarray-based methods. In addition, as our method is based on proteins expressed in mammalian cells, it can also be used to characterize DNA-binding preferences of full-length proteins or proteins requiring post-translational modifications. We validate the method by determining binding specificities of 14 different classes of TFs and by confirming the specificities for NFATC1 and RFX3 using ChIP-seq. Our results reveal unexpected dimeric modes of binding for several factors that were thought to preferentially bind DNA as monomers.


Assuntos
Técnica de Seleção de Aptâmeros , Fatores de Transcrição/metabolismo , Marcadores de Afinidade , Sequência de Bases , Sítios de Ligação , DNA , Humanos , Dados de Sequência Molecular
20.
PLoS Genet ; 6(2): e1000846, 2010 Feb 12.
Artigo em Inglês | MEDLINE | ID: mdl-20174442

RESUMO

Transcriptional regulation is one of the most important processes for modulating gene expression. Though much of this control is attributed to transcription factors, histones, and associated enzymes, it is increasingly apparent that the spatial organization of chromosomes within the nucleus has a profound effect on transcriptional activity. Studies in yeast indicate that the nuclear pore complex might promote transcription by recruiting chromatin to the nuclear periphery. In higher eukaryotes, however, it is not known whether such regulation has global significance. Here we establish nucleoporins as a major class of global regulators for gene expression in Drosophila melanogaster. Using chromatin-immunoprecipitation combined with microarray hybridisation, we show that Nup153 and Megator (Mtor) bind to 25% of the genome in continuous domains extending 10 kb to 500 kb. These Nucleoporin-Associated Regions (NARs) are dominated by markers for active transcription, including high RNA polymerase II occupancy and histone H4K16 acetylation. RNAi-mediated knock-down of Nup153 alters the expression of approximately 5,700 genes, with a pronounced down-regulatory effect within NARs. We find that nucleoporins play a central role in coordinating dosage compensation-an organism-wide process involving the doubling of expression of the male X chromosome. NARs are enriched on the male X chromosome and occupy 75% of this chromosome. Furthermore, Nup153-depletion abolishes the normal function of the male-specific dosage compensation complex. Finally, by extensive 3D imaging, we demonstrate that NARs contribute to gene expression control irrespective of their sub-nuclear localization. Therefore, we suggest that NAR-binding is used for chromosomal organization that enables gene expression control.


Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Genoma de Inseto/genética , Proteínas Associadas à Matriz Nuclear/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Transcrição Gênica , Animais , Cromatina/metabolismo , Drosophila melanogaster/citologia , Drosophila melanogaster/metabolismo , Masculino , Ligação Proteica , Cromossomo X/metabolismo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA