RESUMEN
Cell fitness and survival upon exposure to DNA damage depends on the repair of DNA lesions. Interestingly, cellular identity does affect and finetunes such response, although the molecular basis of such differences between tissues and cell types is not well understood. Thus, a possibility is that DNA repair itself is controlled by the mechanisms that govern cell identity. Here we show that the KLF4, involved in cellular homeostasis, proliferation, cell reprogramming and cancer development, directly regulates resection and homologous recombination proficiency. Indeed, resection efficiency follows KLF4 protein levels, i.e. decreases upon KLF4 downregulation and increases when is overexpressed. Moreover, KLF4 role in resection requires its methylation by the methyl-transferase PRMT5. Thus, PRMT5 depletion not only mimics KLF4 downregulation, but also showed an epistatic genetic relationship. Our data support a model in which the methylation of KLF4 by PRMT5 is a priming event required to license DNA resection and homologous recombination.
Asunto(s)
Reparación del ADN por Unión de Extremidades , Epistasis Genética , Factores de Transcripción de Tipo Kruppel/metabolismo , Procesamiento Proteico-Postraduccional , Proteína-Arginina N-Metiltransferasas/metabolismo , Reparación del ADN por Recombinación , Línea Celular Tumoral , ADN/metabolismo , Roturas del ADN de Doble Cadena , Regulación de la Expresión Génica , Humanos , Factor 4 Similar a Kruppel , Factores de Transcripción de Tipo Kruppel/genética , Metilación , Proteína-Arginina N-Metiltransferasas/genéticaRESUMEN
THO/TREX connects transcription with genome integrity in yeast, but a role of mammalian THO in these processes is uncertain, which suggests a differential implication of mRNP biogenesis factors in genome integrity in yeast and humans. We show that human THO depletion impairs transcription elongation and mRNA export and increases instability associated with DNA breaks, leading to hyper-recombination and γH2AX and 53BP1 foci accumulation. This is accompanied by replication alteration as determined by DNA combing. Genome instability is R-loop-dependent, as deduced from the ability of the AID enzyme to increase DNA damage and of RNaseH to reduce it, or from the enhancement of R-loop-dependent class-switching caused by THOC1-depletion in CH12 murine cells. Therefore, mammalian THO prevents R-loop formation and has a role in genome dynamics and function consistent with an evolutionary conservation of the functional connection between these mRNP biogenesis factors and genome integrity that had not been anticipated.
Asunto(s)
Proteínas de Ciclo Celular/genética , Exodesoxirribonucleasas/genética , Inestabilidad Genómica/genética , Proteínas Nucleares/genética , Fosfoproteínas/genética , Recombinación Genética/genética , Transcripción Genética/genética , Animales , Apoptosis , Citidina Desaminasa/genética , Daño del ADN/genética , Proteínas de Unión al ADN , Silenciador del Gen , Células HeLa , Histonas/genética , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Ratones , ARN Mensajero/genética , Proteínas de Unión al ARN , Ribonucleasa H/genética , Ribonucleoproteínas/genética , Proteína 1 de Unión al Supresor Tumoral P53RESUMEN
BACKGROUND: One key step in gene expression is the biogenesis of mRNA ribonucleoparticle complexes (mRNPs). Formation of the mRNP requires the participation of a number of conserved factors such as the THO complex. THO interacts physically and functionally with the Sub2/UAP56 RNA-dependent ATPase, and the Yra1/REF1/ALY RNA-binding protein linking transcription, mRNA export and genome integrity. Given the link between genome instability and cancer, we have performed a comparative analysis of the expression patterns of THOC1, a THO complex subunit, and ALY in tumor samples. METHODS: The mRNA levels were measured by quantitative real-time PCR and hybridization of a tumor tissue cDNA array; and the protein levels and distribution by immunostaining of a custom tissue array containing a set of paraffin-embedded samples of different tumor and normal tissues followed by statistical analysis. RESULTS: We show that the expression of two mRNP factors, THOC1 and ALY are altered in several tumor tissues. THOC1 mRNA and protein levels are up-regulated in ovarian and lung tumors and down-regulated in those of testis and skin, whereas ALY is altered in a wide variety of tumors. In contrast to THOC1, ALY protein is highly detected in normal proliferative cells, but poorly in high-grade cancers. CONCLUSIONS: These results suggest a differential connection between tumorogenesis and the expression levels of human THO and ALY. This study opens the possibility of defining mRNP biogenesis factors as putative players in cell proliferation that could contribute to tumor development.
