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1.
Genes Dev ; 31(7): 674-687, 2017 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-28446596

RESUMO

MicroRNAs (miRNAs) are post-transcriptional regulators of gene expression critical for organismal viability. Changes in miRNA activity are common in cancer, but how these changes relate to subsequent alterations in transcription and the process of tumorigenesis is not well understood. Here, we report a deep transcriptional, oncogenic network regulated by miRNAs. We present analysis of the gene expression and phenotypic changes associated with global miRNA restoration in miRNA-deficient fibroblasts. This analysis uncovers a miRNA-repressed network containing oncofetal genes Imp1, Imp2, and Imp3 (Imp1-3) that is up-regulated primarily transcriptionally >100-fold upon Dicer loss and is resistant to resilencing by complete restoration of miRNA activity. This Dicer-resistant epigenetic switch confers tumorigenicity to these cells. Let-7 targets Imp1-3 are required for this tumorigenicity and feed back to reinforce and sustain expression of the oncogenic network. Together, these Dicer-resistant genes constitute an mRNA expression signature that is present in numerous human cancers and is associated with poor survival.


Assuntos
Antígenos de Neoplasias/genética , Transformação Celular Neoplásica/genética , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/fisiologia , MicroRNAs/genética , Ribonuclease III/genética , Ribonuclease III/fisiologia , Animais , Antígenos de Neoplasias/metabolismo , Células Cultivadas , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Camundongos , Camundongos Knockout , Oncogenes , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Ativação Transcricional
2.
Genes Dev ; 27(8): 941-54, 2013 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-23630078

RESUMO

MicroRNAs (miRNAs) are critical to proliferation, differentiation, and development. Here, we characterize gene expression in murine Dicer-null adult mesenchymal stem cell lines, a fibroblast cell type. Loss of Dicer leads to derepression of let-7 targets at levels that exceed 10-fold to 100-fold with increases in transcription. Direct and indirect targets of this miRNA belong to a mid-gestation embryonic program that encompasses known oncofetal genes as well as oncogenes not previously associated with an embryonic state. Surprisingly, this mid-gestation program represents a distinct period that occurs between the pluripotent state of the inner cell mass at embryonic day 3.5 (E3.5) and the induction of let-7 upon differentiation at E10.5. Within this mid-gestation program, we characterize the let-7 target Nr6a1, an embryonic transcriptional repressor that regulates gene expression in adult fibroblasts following miRNA loss. In total, let-7 is required for the continual suppression of embryonic gene expression in adult cells, a mechanism that may underlie its tumor-suppressive function.


Assuntos
Fibroblastos/citologia , Regulação da Expressão Gênica , MicroRNAs/genética , MicroRNAs/metabolismo , Membro 1 do Grupo A da Subfamília 6 de Receptores Nucleares/genética , Membro 1 do Grupo A da Subfamília 6 de Receptores Nucleares/metabolismo , Animais , Antígenos de Neoplasias/metabolismo , Linhagem Celular , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Fibroblastos/metabolismo , Deleção de Genes , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Inativação de Genes , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Camundongos , Ligação Proteica , Ribonuclease III/genética , Ribonuclease III/metabolismo
3.
Genes Dev ; 26(21): 2392-407, 2012 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-23073843

RESUMO

The MALAT1 (metastasis-associated lung adenocarcinoma transcript 1) locus is misregulated in many human cancers and produces an abundant long nuclear-retained noncoding RNA. Despite being transcribed by RNA polymerase II, the 3' end of MALAT1 is produced not by canonical cleavage/polyadenylation but instead by recognition and cleavage of a tRNA-like structure by RNase P. Mature MALAT1 thus lacks a poly(A) tail yet is expressed at a level higher than many protein-coding genes in vivo. Here we show that the 3' ends of MALAT1 and the MEN ß long noncoding RNAs are protected from 3'-5' exonucleases by highly conserved triple helical structures. Surprisingly, when these structures are placed downstream from an ORF, the transcript is efficiently translated in vivo despite the lack of a poly(A) tail. The triple helix therefore also functions as a translational enhancer, and mutations in this region separate this translation activity from simple effects on RNA stability or transport. We further found that a transcript ending in a triple helix is efficiently repressed by microRNAs in vivo, arguing against a major role for the poly(A) tail in microRNA-mediated silencing. These results provide new insights into how transcripts that lack poly(A) tails are stabilized and regulated and suggest that RNA triple-helical structures likely have key regulatory functions in vivo.


Assuntos
RNA Longo não Codificante/genética , RNA Mensageiro/genética , Motivos de Aminoácidos , Sequência de Bases , Análise Mutacional de DNA , Regulação da Expressão Gênica , Células HeLa , Humanos , MicroRNAs/metabolismo , Dados de Sequência Molecular , Plasmídeos/genética , Desnaturação Proteica , Estrutura Secundária de Proteína , Processamento de Terminações 3' de RNA/genética , Estabilidade de RNA , RNA Longo não Codificante/química , RNA Longo não Codificante/metabolismo , Alinhamento de Sequência
5.
Genome Res ; 23(6): 907-16, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23539139

RESUMO

In human transcriptional regulation, DNA-sequence-specific factors can associate with intermediaries that orchestrate interactions with a diverse set of chromatin-modifying enzymes. One such intermediary is HCFC1 (also known as HCF-1). HCFC1, first identified in herpes simplex virus transcription, has a poorly defined role in cellular transcriptional regulation. We show here that, in HeLa cells, HCFC1 is observed bound to 5400 generally active CpG-island promoters. Examination of the DNA sequences underlying the HCFC1-binding sites revealed three sequence motifs associated with the binding of (1) ZNF143 and THAP11 (also known as Ronin), (2) GABP, and (3) YY1 sequence-specific transcription factors. Subsequent analysis revealed colocalization of HCFC1 with these four transcription factors at ∼90% of the 5400 HCFC1-bound promoters. These studies suggest that a relatively small number of transcription factors play a major role in HeLa-cell transcriptional regulation in association with HCFC1.


Assuntos
Ilhas de CpG , Fator de Transcrição de Proteínas de Ligação GA/metabolismo , Fator C1 de Célula Hospedeira/metabolismo , Regiões Promotoras Genéticas , Proteínas Repressoras/metabolismo , Transativadores/metabolismo , Fator de Transcrição YY1/metabolismo , Sequência de Bases , Sítios de Ligação , Regulação da Expressão Gênica , Células HeLa , Humanos , Motivos de Nucleotídeos , Matrizes de Pontuação de Posição Específica , Ligação Proteica , RNA Mensageiro/genética , Transdução de Sinais , Sítio de Iniciação de Transcrição , Ativação Transcricional
6.
Cell Rep ; 14(2): 310-9, 2016 Jan 12.
Artigo em Inglês | MEDLINE | ID: mdl-26748710

RESUMO

MicroRNAs (miRNAs) regulate diverse biological processes by repressing mRNAs, but their modest effects on direct targets, together with their participation in larger regulatory networks, make it challenging to delineate miRNA-mediated effects. Here, we describe an approach to characterizing miRNA-regulatory networks by systematically profiling transcriptional, post-transcriptional and epigenetic activity in a pair of isogenic murine fibroblast cell lines with and without Dicer expression. By RNA sequencing (RNA-seq) and CLIP (crosslinking followed by immunoprecipitation) sequencing (CLIP-seq), we found that most of the changes induced by global miRNA loss occur at the level of transcription. We then introduced a network modeling approach that integrated these data with epigenetic data to identify specific miRNA-regulated transcription factors that explain the impact of miRNA perturbation on gene expression. In total, we demonstrate that combining multiple genome-wide datasets spanning diverse regulatory modes enables accurate delineation of the downstream miRNA-regulated transcriptional network and establishes a model for studying similar networks in other systems.


Assuntos
Código das Histonas/genética , MicroRNAs/genética , Fatores de Transcrição/genética , Redes Reguladoras de Genes , Humanos , MicroRNAs/metabolismo
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