RESUMO
The resurrection of pseudogenes during evolution produced lncRNAs with new biological function. Here we show that pseudogene-evolution created an Oct4 pseudogene lncRNA that is able to direct epigenetic silencing of the parental Oct4 gene via a 2-step, lncRNA dependent mechanism. The murine Oct4 pseudogene 4 (mOct4P4) lncRNA recruits the RNA binding protein FUS to allow the binding of the SUV39H1 HMTase to a defined mOct4P4 lncRNA sequence element. The mOct4P4-FUS-SUV39H1 silencing complex holds target site specificity for the parental Oct4 promoter and interference with individual components results in loss of Oct4 silencing. SUV39H1 and FUS do not bind parental Oct4 mRNA, confirming the acquisition of a new biological function by the mOct4P4 lncRNA. Importantly, all features of mOct4P4 function are recapitulated by the human hOCT4P3 pseudogene lncRNA, indicating evolutionary conservation. Our data highlight the biological relevance of rapidly evolving lncRNAs that infiltrate into central epigenetic regulatory circuits in vertebrate cells.
Assuntos
Metiltransferases/metabolismo , Fator 3 de Transcrição de Octâmero/genética , Pseudogenes , RNA Longo não Codificante/genética , Proteína FUS de Ligação a RNA/genética , Proteínas Repressoras/metabolismo , Animais , Linhagem Celular Tumoral , Epigênese Genética , Feminino , Inativação Gênica , Humanos , Metiltransferases/genética , Camundongos , Complexos Multiproteicos/genética , Neoplasias Ovarianas/genética , Regiões Promotoras Genéticas , Proteína FUS de Ligação a RNA/metabolismo , Proteínas Repressoras/genéticaRESUMO
Pseudogene-derived, long non-coding RNAs (lncRNAs) act as epigenetic regulators of gene expression. Here we present a panel of new mouse Oct4 pseudogenes and demonstrate that the X-linked Oct4 pseudogene Oct4P4 critically impacts mouse embryonic stem cells (mESCs) self-renewal. Sense Oct4P4 transcription produces a spliced, nuclear-restricted lncRNA that is efficiently upregulated during mESC differentiation. Oct4P4 lncRNA forms a complex with the SUV39H1 HMTase to direct the imposition of H3K9me3 and HP1α to the promoter of the ancestral Oct4 gene, located on chromosome 17, leading to gene silencing and reduced mESC self-renewal. Targeting Oct4P4 expression in primary mouse embryonic fibroblasts causes the re-acquisition of self-renewing features of mESC. We demonstrate that Oct4P4 lncRNA plays an important role in inducing and maintaining silencing of the ancestral Oct4 gene in differentiating mESCs. Our data introduces a sense pseudogene-lncRNA-based mechanism of epigenetic gene regulation that controls the cross-talk between pseudogenes and their ancestral genes.
Assuntos
Epigênese Genética/genética , Regulação da Expressão Gênica no Desenvolvimento , Metiltransferases/metabolismo , Células-Tronco Embrionárias Murinas/metabolismo , Fator 3 de Transcrição de Octâmero/genética , Pseudogenes/genética , RNA Longo não Codificante/genética , Proteínas Repressoras/metabolismo , Animais , Linhagem Celular , Linhagem Celular Tumoral , Autorrenovação Celular/genética , Imunoprecipitação da Cromatina , Homólogo 5 da Proteína Cromobox , Proteínas Cromossômicas não Histona/metabolismo , Histonas/metabolismo , Imunoprecipitação , Camundongos , Células-Tronco Embrionárias Murinas/citologia , Células NIH 3T3RESUMO
The pluripotency of mouse embryonic stem cells (mESCs) is controlled by a network of transcription factors, mi-RNAs, and signaling pathways. Here, we present a new regulatory circuit that connects miR-335, Oct4, and the Retinoblastoma pathway to control mESC self-renewal and differentiation. Oct4 drives the expression of Nipp1 and Ccnf that inhibit the activity of the protein phosphatase 1 (PP1) complex to establish hyperphosphorylation of the retinoblastoma protein 1 (pRb) as a hallmark feature of self-renewing mESCs. The Oct4-Nipp1/Ccnf-PP1-pRb axis promoting mESC self-renewal is under control of miR-335 that regulates Oct4 and Rb expression. During mESC differentiation, miR-335 upregulation co-operates with the transcriptional repression of Oct4 to facilitate the collapse of the Oct4-Nipp1/Ccnf-PP1-pRb axis, pRb dephosphorylation, the exit from self-renewal, and the establishment of a pRb-regulated cell cycle program. Our results introduce Oct4-dependent control of the Rb pathway as novel regulatory circuit controlling mESC self-renewal and differentiation.
Assuntos
Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , MicroRNAs/metabolismo , Fator 3 de Transcrição de Octâmero/metabolismo , Proteína do Retinoblastoma/metabolismo , Animais , Western Blotting , Pontos de Checagem do Ciclo Celular/genética , Pontos de Checagem do Ciclo Celular/fisiologia , Células Cultivadas , Imunoprecipitação da Cromatina , Citometria de Fluxo , Imunoprecipitação , Camundongos , MicroRNAs/genética , Fator 3 de Transcrição de Octâmero/genética , Ligação Proteica , Proteína do Retinoblastoma/genéticaRESUMO
Tumor associated fibroblasts (TAFs) are considered a microenvironmental element critical for tumor growth and progression. Experimental studies suggest that their origin could be from mesenchymal stem cells (MSCs) derived from the bone marrow. However, the role played by TAFs in cirrhosis, hepatocellular carcinoma development, and progression is largely unknown, and in vitro human models are missing. This paper for the first time demonstrates that (1) human neoplastic livers possess a population of multipotent adult stem cells (MASCs) with properties of TAFs; (2) a population of MASC-derived TAFs is already present in cirrhotic, not yet neoplastic, livers; (3) MASCs isolated from nonneoplastic and noncirrhotic liver scan acquire a TAF phenotype when grown in a medium conditioned by tumor cell lines, supporting the notion that TAF could originate from resident primitive cells (MASCs), possibly through a paracrine mechanism.
