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1.
Proc Natl Acad Sci U S A ; 114(4): E496-E505, 2017 01 24.
Artigo em Inglês | MEDLINE | ID: mdl-27994142

RESUMO

The microRNA miR-504 targets TP53 mRNA encoding the p53 tumor suppressor. miR-504 resides within the fibroblast growth factor 13 (FGF13) gene, which is overexpressed in various cancers. We report that the FGF13 locus, comprising FGF13 and miR-504, is transcriptionally repressed by p53, defining an additional negative feedback loop in the p53 network. Furthermore, we show that FGF13 1A is a nucleolar protein that represses ribosomal RNA transcription and attenuates protein synthesis. Importantly, in cancer cells expressing high levels of FGF13, the depletion of FGF13 elicits increased proteostasis stress, associated with the accumulation of reactive oxygen species and apoptosis. Notably, stepwise neoplastic transformation is accompanied by a gradual increase in FGF13 expression and increased dependence on FGF13 for survival ("nononcogene addiction"). Moreover, FGF13 overexpression enables cells to cope more effectively with the stress elicited by oncogenic Ras protein. We propose that, in cells in which activated oncogenes drive excessive protein synthesis, FGF13 may favor survival by maintaining translation rates at a level compatible with the protein quality-control capacity of the cell. Thus, FGF13 may serve as an enabler, allowing cancer cells to evade proteostasis stress triggered by oncogene activation.


Assuntos
Fatores de Crescimento de Fibroblastos/metabolismo , Neoplasias/metabolismo , Ribossomos/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular , Fatores de Crescimento de Fibroblastos/genética , Humanos , MicroRNAs/genética , Neoplasias/genética , Proteína Supressora de Tumor p53/genética
2.
Nat Protoc ; 10(4): 605-18, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25811895

RESUMO

4sUDRB-seq separately measures, on a genomic scale, the distinct contributions of transcription elongation speed and rate of RNA polymerase II (Pol II) transition into active elongation (TAE) to the overall mRNA production rate. It uses reversible inhibition of transcription elongation with 5,6-dichloro-1-ß-D-ribofuranosylbenzimidazole (DRB), combined with a pulse of 4-thiouridine (4sU), to tag newly transcribed RNA. After DRB removal, cells are collected at several time points, and tagged RNA is biotinylated, captured on streptavidin beads and sequenced. 4sUDRB-seq enables the comparison of elongation speeds between different developmental stages or different cell types, and it allows the impact of specific transcription factors on transcription elongation speed versus TAE to be studied. RNA preparation takes ∼4 d to complete, with deep sequencing requiring an additional ∼4-11 d plus 1-3 d for bioinformatics analysis. The experimental protocol requires basic molecular biology skills, whereas data analysis requires knowledge in bioinformatics, particularly MATLAB and the Linux environment.


Assuntos
Diclororribofuranosilbenzimidazol/química , RNA Polimerase II/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Análise de Sequência de RNA/métodos , Tiouridina/metabolismo , Elongação da Transcrição Genética , Biotina/química , Genoma , Células HeLa , Humanos , RNA/isolamento & purificação , RNA Polimerase II/química , RNA Polimerase II/genética , Reprodutibilidade dos Testes , Análise de Sequência de RNA/instrumentação , Estreptavidina/química , Tiouridina/química
3.
Genome Res ; 24(10): 1572-83, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25049226

RESUMO

Various histone modifications decorate nucleosomes within transcribed genes. Among these, monoubiquitylation of histone H2B (H2Bub1) and methylation of histone H3 on lysines 36 (H3K36me2/3) and 79 (H3K79me2/3) correlate positively with gene expression. By measuring the progression of the transcriptional machinery along genes within live cells, we now report that H2B monoubiquitylation occurs cotranscriptionally and accurately reflects the advance of RNA polymerase II (Pol II). In contrast, H3K36me3 and H3K79me2 are less dynamic and represent Pol II movement less faithfully. High-resolution ChIP-seq reveals that H2Bub1 levels are selectively reduced at exons and decrease in an exon-dependent stepwise manner toward the 3' end of genes. Exonic depletion of H2Bub1 in gene bodies is highly correlated with Pol II pausing at exons, suggesting elongation rate changes associated with intron-exon structure. In support of this notion, H2Bub1 levels were found to be significantly correlated with transcription elongation rates measured in various cell lines. Overall, our data shed light on the organization of H2Bub1 within transcribed genes and single out H2Bub1 as a reliable marker for ongoing transcription elongation.


Assuntos
Histonas/genética , Histonas/metabolismo , RNA Polimerase II/metabolismo , Elongação da Transcrição Genética , Linhagem Celular Tumoral , Éxons , Células HeLa , Humanos , Dados de Sequência Molecular , RNA Mensageiro/metabolismo , Análise de Sequência de DNA , Ubiquitinação
4.
Genome Biol ; 15(5): R69, 2014 May 09.
Artigo em Inglês | MEDLINE | ID: mdl-24887486

RESUMO

Although transcriptional elongation by RNA polymerase II is coupled with many RNA-related processes, genomewide elongation rates remain unknown. We describe a method, called 4sUDRB-seq, based on reversible inhibition of transcription elongation coupled with tagging newly transcribed RNA with 4-thiouridine and high throughput sequencing to measure simultaneously with high confidence genome-wide transcription elongation rates in cells. We find that most genes are transcribed at about 3.5 Kb/min, with elongation rates varying between 2 Kb/min and 6 Kb/min. 4sUDRB-seq can facilitate genomewide exploration of the involvement of specific elongation factors in transcription and the contribution of deregulated transcription elongation to various pathologies.


Assuntos
Diclororribofuranosilbenzimidazol/farmacologia , RNA Polimerase II/metabolismo , Tiouridina/metabolismo , Elongação da Transcrição Genética , Genoma Humano , Células HeLa , Humanos , Análise de Sequência de RNA/métodos
5.
Biochim Biophys Acta ; 1839(8): 694-701, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24412854

RESUMO

Monoubiquitylation of histone H2B (H2Bub1), catalyzed by the heterodimeric ubiquitin ligase complex RNF20/40, regulates multiple molecular and biological processes. The addition of a large ubiquitin moiety to the small H2B is believed to change the biochemical features of the chromatin. H2B monoubiquitylation alters nucleosome stability, nucleosome reassembly and higher order compaction of the chromatin. While these effects explain some of the direct roles of H2Bub1, there is growing evidence that H2Bub1 can also regulate multiple DNA-templated processes indirectly, by recruitment of specific factors ("readers") to the chromatin. H2Bub1 readers mediate much of the effect of H2Bub1 on histone crosstalk, transcriptional outcome and probably other chromatin-related activities. Here we summarize the current knowledge about H2Bub1-specific readers and their role in various biological processes. This article is part of a Special Issue entitled: Molecular mechanisms of histone modification function.


Assuntos
Proteínas Cromossômicas não Histona/química , Epigênese Genética , Histonas/química , Nucleossomos/química , Processamento de Proteína Pós-Traducional , Fatores de Transcrição/química , Proteínas Cromossômicas não Histona/antagonistas & inibidores , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/genética , Histonas/metabolismo , Humanos , Metilação , Nucleossomos/genética , Nucleossomos/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição Gênica , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação
6.
Mol Cell ; 46(5): 662-73, 2012 Jun 08.
Artigo em Inglês | MEDLINE | ID: mdl-22681888

RESUMO

Embryonic stem cells (ESCs) maintain high genomic plasticity, which is essential for their capacity to enter diverse differentiation pathways. Posttranscriptional modifications of chromatin histones play a pivotal role in maintaining this plasticity. We now report that one such modification, monoubiquitylation of histone H2B on lysine 120 (H2Bub1), catalyzed by the E3 ligase RNF20, increases during ESC differentiation and is required for efficient execution of this process. This increase is particularly important for the transcriptional induction of relatively long genes during ESC differentiation. Furthermore, we identify the deubiquitinase USP44 as a negative regulator of H2B ubiquitylation, whose downregulation during ESC differentiation contributes to the increase in H2Bub1. Our findings suggest that optimal ESC differentiation requires dynamic changes in H2B ubiquitylation patterns, which must occur in a timely and well-coordinated manner.


Assuntos
Diferenciação Celular/genética , Células-Tronco Embrionárias/citologia , Endopeptidases/fisiologia , Histonas/metabolismo , Ubiquitina-Proteína Ligases/fisiologia , Animais , Montagem e Desmontagem da Cromatina , Regulação para Baixo , Células-Tronco Embrionárias/metabolismo , Endopeptidases/metabolismo , Epigênese Genética , Humanos , Camundongos , Modelos Genéticos , Ubiquitina-Proteína Ligases/metabolismo , Proteases Específicas de Ubiquitina , Ubiquitinação
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