Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 12 de 12
Filtrar
1.
Mol Cell ; 82(19): 3598-3612.e7, 2022 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-36113480

RESUMO

Gene transcription is a highly regulated process in all animals. In Drosophila, two major transcriptional programs, housekeeping and developmental, have promoters with distinct regulatory compatibilities and nucleosome organization. However, it remains unclear how the differences in chromatin structure relate to the distinct regulatory properties and which chromatin remodelers are required for these programs. Using rapid degradation of core remodeler subunits in Drosophila melanogaster S2 cells, we demonstrate that developmental gene transcription requires SWI/SNF-type complexes, primarily to maintain distal enhancer accessibility. In contrast, wild-type-level housekeeping gene transcription requires the Iswi and Ino80 remodelers to maintain nucleosome positioning and phasing at promoters. These differential remodeler dependencies relate to different DNA-sequence-intrinsic nucleosome affinities, which favor a default ON state for housekeeping but a default OFF state for developmental gene transcription. Overall, our results demonstrate how different transcription-regulatory strategies are implemented by DNA sequence, chromatin structure, and remodeler activity.


Assuntos
Cromatina , Nucleossomos , Animais , Cromatina/genética , Cromatina/metabolismo , Montagem e Desmontagem da Cromatina , DNA/metabolismo , Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Zeladoria , Nucleossomos/genética , Nucleossomos/metabolismo
2.
Nat Methods ; 15(2): 141-149, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29256496

RESUMO

The identification of transcriptional enhancers in the human genome is a prime goal in biology. Enhancers are typically predicted via chromatin marks, yet their function is primarily assessed with plasmid-based reporter assays. Here, we show that such assays are rendered unreliable by two previously reported phenomena relating to plasmid transfection into human cells: (i) the bacterial plasmid origin of replication (ORI) functions as a conflicting core promoter and (ii) a type I interferon (IFN-I) response is activated. These cause confounding false positives and negatives in luciferase assays and STARR-seq screens. We overcome both problems by employing the ORI as core promoter and by inhibiting two IFN-I-inducing kinases, enabling genome-wide STARR-seq screens in human cells. In HeLa-S3 cells, we uncover strong enhancers, IFN-I-induced enhancers, and enhancers endogenously silenced at the chromatin level. Our findings apply to all episomal enhancer activity assays in mammalian cells and are key to the characterization of human enhancers.


Assuntos
Cromatina/genética , Elementos Facilitadores Genéticos , Regulação da Expressão Gênica , Genes Reporter , Regiões Promotoras Genéticas , Mapeamento Cromossômico , Reações Falso-Negativas , Genoma Humano , Células HeLa , Humanos
3.
Nature ; 525(7570): 543-547, 2015 Sep 24.
Artigo em Inglês | MEDLINE | ID: mdl-26367798

RESUMO

Following the discovery of BRD4 as a non-oncogene addiction target in acute myeloid leukaemia (AML), bromodomain and extra terminal protein (BET) inhibitors are being explored as a promising therapeutic avenue in numerous cancers. While clinical trials have reported single-agent activity in advanced haematological malignancies, mechanisms determining the response to BET inhibition remain poorly understood. To identify factors involved in primary and acquired BET resistance in leukaemia, here we perform a chromatin-focused RNAi screen in a sensitive MLL-AF9;Nras(G12D)-driven AML mouse model, and investigate dynamic transcriptional profiles in sensitive and resistant mouse and human leukaemias. Our screen shows that suppression of the PRC2 complex, contrary to effects in other contexts, promotes BET inhibitor resistance in AML. PRC2 suppression does not directly affect the regulation of Brd4-dependent transcripts, but facilitates the remodelling of regulatory pathways that restore the transcription of key targets such as Myc. Similarly, while BET inhibition triggers acute MYC repression in human leukaemias regardless of their sensitivity, resistant leukaemias are uniformly characterized by their ability to rapidly restore MYC transcription. This process involves the activation and recruitment of WNT signalling components, which compensate for the loss of BRD4 and drive resistance in various cancer models. Dynamic chromatin immunoprecipitation sequencing and self-transcribing active regulatory region sequencing of enhancer profiles reveal that BET-resistant states are characterized by remodelled regulatory landscapes, involving the activation of a focal MYC enhancer that recruits WNT machinery in response to BET inhibition. Together, our results identify and validate WNT signalling as a driver and candidate biomarker of primary and acquired BET resistance in leukaemia, and implicate the rewiring of transcriptional programs as an important mechanism promoting resistance to BET inhibitors and, potentially, other chromatin-targeted therapies.


Assuntos
Azepinas/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Leucemia Mieloide Aguda/genética , Proteínas Nucleares/antagonistas & inibidores , Fatores de Transcrição/antagonistas & inibidores , Transcrição Gênica/efeitos dos fármacos , Triazóis/farmacologia , Animais , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , Cromatina/genética , Cromatina/metabolismo , Elementos Facilitadores Genéticos/genética , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Genes myc/genética , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Masculino , Camundongos , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica/genética , Via de Sinalização Wnt/efeitos dos fármacos
4.
Mol Cell ; 50(5): 736-48, 2013 Jun 06.
Artigo em Inglês | MEDLINE | ID: mdl-23665228

RESUMO

A large fraction of our genome consists of mobile genetic elements. Governing transposons in germ cells is critically important, and failure to do so compromises genome integrity, leading to sterility. In animals, the piRNA pathway is the key to transposon constraint, yet the precise molecular details of how piRNAs are formed and how the pathway represses mobile elements remain poorly understood. In an effort to identify general requirements for transposon control and components of the piRNA pathway, we carried out a genome-wide RNAi screen in Drosophila ovarian somatic sheet cells. We identified and validated 87 genes necessary for transposon silencing. Among these were several piRNA biogenesis factors. We also found CG3893 (asterix) to be essential for transposon silencing, most likely by contributing to the effector step of transcriptional repression. Asterix loss leads to decreases in H3K9me3 marks on certain transposons but has no effect on piRNA levels.


Assuntos
Elementos de DNA Transponíveis , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , RNA Interferente Pequeno/metabolismo , Animais , Proteínas de Drosophila/genética , Drosophila melanogaster/metabolismo , Feminino , Técnicas de Silenciamento de Genes , Inativação Gênica , Genoma de Inseto , Ovário/fisiologia , Interferência de RNA , RNA Interferente Pequeno/genética , Reprodutibilidade dos Testes , Proteína SUMO-1/genética , Proteína SUMO-1/metabolismo
5.
Genes Dev ; 24(22): 2499-504, 2010 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-20966049

RESUMO

Combining RNAi in cultured cells and analysis of mutant animals, we probed the roles of known Piwi-interacting RNA (piRNA) pathway components in the initiation and effector phases of transposon silencing. Squash associated physically with Piwi, and reductions in its expression led to modest transposon derepression without effects on piRNAs, consistent with an effector role. Alterations in Zucchini or Armitage reduced both Piwi protein and piRNAs, indicating functions in the formation of a stable Piwi RISC (RNA-induced silencing complex). Notably, loss of Zucchini or mutations within its catalytic domain led to accumulation of unprocessed precursor transcripts from flamenco, consistent with a role for this putative nuclease in piRNA biogenesis.


Assuntos
Drosophila melanogaster/metabolismo , RNA Interferente Pequeno/biossíntese , Animais , Células Cultivadas , Elementos de DNA Transponíveis/fisiologia , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Endorribonucleases/genética , Endorribonucleases/metabolismo , Feminino , Mutação , RNA Helicases/genética , RNA Helicases/metabolismo , Interferência de RNA , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo
6.
Genomics ; 106(3): 145-150, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26072434

RESUMO

Differential gene expression is the basis for cell type diversity in multicellular organisms and the driving force of development and differentiation. It is achieved by cell type-specific transcriptional enhancers, which are genomic DNA sequences that activate the transcription of their target genes. Their identification and characterization is fundamental to our understanding of gene regulation. Features that are associated with enhancer activity, such as regulatory factor binding or histone modifications can predict the location of enhancers. Nonetheless, enhancer activity can only be assessed by transcriptional reporter assays. Over the past years massively parallel reporter assays have been developed for large scale testing of enhancers. In this review we focus on the principles and applications of STARR-seq, a functional assay that quantifies enhancer strengths in complex candidate libraries and thus allows activity-based enhancer identification in entire genomes. We explain how STARR-seq works, discuss current uses and give an outlook to future applications.


Assuntos
Elementos Facilitadores Genéticos , Genoma Humano , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Regiões Promotoras Genéticas , Mapeamento Cromossômico , Regulação da Expressão Gênica , Genes Reporter , Humanos , Análise de Sequência de DNA/métodos
7.
RNA ; 19(8): 1064-77, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23788724

RESUMO

Piwi proteins and their associated small RNAs are essential for fertility in animals. In part, this is due to their roles in guarding germ cell genomes against the activity of mobile genetic elements. piRNA populations direct Piwi proteins to silence transposon targets and, as such, form a molecular code that discriminates transposons from endogenous genes. Information ultimately carried by piRNAs is encoded within genomic loci, termed piRNA clusters. These give rise to long, single-stranded, primary transcripts that are processed into piRNAs. Despite the biological importance of this pathway, neither the characteristics that define a locus as a source of piRNAs nor the mechanisms that catalyze primary piRNA biogenesis are well understood. We searched an EMS-mutant collection annotated for fertility phenotypes for genes involved in the piRNA pathway. Twenty-seven homozygous sterile strains showed transposon-silencing defects. One of these, which strongly impacted primary piRNA biogenesis, harbored a causal mutation in CG5508, a member of the Drosophila glycerol-3-phosphate O-acetyltransferase (GPAT) family. These enzymes catalyze the first acylation step on the path to the production of phosphatidic acid (PA). Though this pointed strongly to a function for phospholipid signaling in the piRNA pathway, a mutant form of CG5508, which lacks the GPAT active site, still functions in piRNA biogenesis. We have named this new biogenesis factor Minotaur.


Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Glicerol-3-Fosfato O-Aciltransferase/metabolismo , RNA Interferente Pequeno/biossíntese , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Domínio Catalítico , Elementos de DNA Transponíveis/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Feminino , Genes de Insetos , Glicerol-3-Fosfato O-Aciltransferase/genética , Masculino , Dados de Sequência Molecular , Mutação , Fosfolipídeos/metabolismo , RNA Interferente Pequeno/genética , Transdução de Sinais
9.
RNA ; 18(8): 1446-57, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22753781

RESUMO

In animals, the piRNA pathway preserves the integrity of gametic genomes, guarding them against the activity of mobile genetic elements. This innate immune mechanism relies on distinct genomic loci, termed piRNA clusters, to provide a molecular definition of transposons, enabling their discrimination from genes. piRNA clusters give rise to long, single-stranded precursors, which are processed into primary piRNAs through an unknown mechanism. These can engage in an adaptive amplification loop, the ping-pong cycle, to optimize the content of small RNA populations via the generation of secondary piRNAs. Many proteins have been ascribed functions in either primary biogenesis or the ping-pong cycle, though for the most part the molecular functions of proteins implicated in these pathways remain obscure. Here, we link shutdown (shu), a gene previously shown to be required for fertility in Drosophila, to the piRNA pathway. Analysis of knockdown phenotypes in both the germline and somatic compartments of the ovary demonstrate important roles for shutdown in both primary biogenesis and the ping-pong cycle. shutdown is a member of the FKBP family of immunophilins. Shu contains domains implicated in peptidyl-prolyl cis-trans isomerase activity and in the binding of HSP90-family chaperones, though the relevance of these domains to piRNA biogenesis is unknown.


Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Fertilidade/genética , Inativação Gênica , Ovário/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Animais , Proteínas de Drosophila/genética , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/metabolismo , Feminino , Imunofluorescência , Masculino , Ovário/citologia , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Retroelementos/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas de Ligação a Tacrolimo/genética , Proteínas de Ligação a Tacrolimo/metabolismo
10.
RNA ; 18(1): 42-52, 2012 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-22096018

RESUMO

In animals a discrete class of small RNAs, the piwi-interacting RNAs (piRNAs), guard germ cell genomes against the activity of mobile genetic elements. piRNAs are generated, via an unknown mechanism, from apparently single-stranded precursors that arise from discrete genomic loci, termed piRNA clusters. Presently, little is known about the signals that distinguish a locus as a source of piRNAs. It is also unknown how individual piRNAs are selected from long precursor transcripts. To address these questions, we inserted new artificial sequence information into piRNA clusters and introduced these marked clusters as transgenes into heterologous genomic positions in mice and flies. Profiling of piRNA from transgenic animals demonstrated that artificial sequences were incorporated into the piRNA repertoire. Transgenic piRNA clusters are functional in non-native genomic contexts in both mice and flies, indicating that the signals that define piRNA generative loci must lie within the clusters themselves rather than being implicit in their genomic position. Comparison of transgenic animals that carry insertions of the same artificial sequence into different ectopic piRNA-generating loci showed that both local and long-range sequence environments inform the generation of individual piRNAs from precursor transcripts.


Assuntos
Drosophila melanogaster/metabolismo , RNA Interferente Pequeno/biossíntese , Animais , Drosophila melanogaster/genética , Camundongos , Camundongos Transgênicos , RNA Interferente Pequeno/química , RNA Interferente Pequeno/genética
11.
Curr Biol ; 26(19): R895-R898, 2016 10 10.
Artigo em Inglês | MEDLINE | ID: mdl-27728794

RESUMO

A recent study visualizes nascent RNAs in live Drosophila embryos to establish a connection between enhancer strength and the frequency of transcriptional bursts. Interestingly, one enhancer can simultaneously activate two core promoters, challenging models of enhancer-core-promoter communication via direct protein-protein contacts.


Assuntos
Drosophila/genética , Elementos Facilitadores Genéticos , Animais , Proteínas de Drosophila/genética , Regulação da Expressão Gênica , Regiões Promotoras Genéticas , Ativação Transcricional
12.
Curr Opin Genet Dev ; 23(1): 44-52, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23317515

RESUMO

Piwi proteins, together with their bound Piwi-interacting RNAs, constitute an evolutionarily conserved, germline-specific innate immune system. The piRNA pathway is one of the key mechanisms for silencing transposable elements in the germline, thereby preserving genome integrity between generations. Recent work from several groups has significantly advanced our understanding of how piRNAs arise from discrete genomic loci, termed piRNA clusters, and how these Piwi-piRNA complexes enforce transposon silencing. Here, we discuss these recent findings, as well as highlight some aspects of piRNA biology that continue to escape our understanding.


Assuntos
Proteínas de Drosophila/genética , RNA Interferente Pequeno/genética , Transdução de Sinais , Animais , Elementos de DNA Transponíveis , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/crescimento & desenvolvimento , Feminino , Inativação Gênica , Loci Gênicos , Células Germinativas/metabolismo , Ovário/metabolismo , RNA Interferente Pequeno/metabolismo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA