RESUMEN
The DNA damage response (DDR) preserves genomic integrity. Small non-coding RNAs termed DDRNAs are generated at DNA double-strand breaks (DSBs) and are critical for DDR activation. Here we show that active DDRNAs specifically localize to their damaged homologous genomic sites in a transcription-dependent manner. Following DNA damage, RNA polymerase II (RNAPII) binds to the MRE11-RAD50-NBS1 complex, is recruited to DSBs and synthesizes damage-induced long non-coding RNAs (dilncRNAs) from and towards DNA ends. DilncRNAs act both as DDRNA precursors and by recruiting DDRNAs through RNA-RNA pairing. Together, dilncRNAs and DDRNAs fuel DDR focus formation and associate with 53BP1. Accordingly, inhibition of RNAPII prevents DDRNA recruitment, DDR activation and DNA repair. Antisense oligonucleotides matching dilncRNAs and DDRNAs impair site-specific DDR focus formation and DNA repair. We propose that DDR signalling sites, in addition to sharing a common pool of proteins, individually host a unique set of site-specific RNAs necessary for DDR activation.
Asunto(s)
Roturas del ADN de Doble Cadena , Daño del ADN , Reparación del ADN , ARN Largo no Codificante/metabolismo , Transportadoras de Casetes de Unión a ATP/metabolismo , Ácido Anhídrido Hidrolasas , Animales , Proteínas de Ciclo Celular/metabolismo , Línea Celular , Sistema Libre de Células , Daño del ADN/genética , Daño del ADN/fisiología , Reparación del ADN/genética , Reparación del ADN/fisiología , Proteínas de Unión al ADN , Proteína Homóloga de MRE11/metabolismo , Ratones , Modelos Biológicos , Proteínas Nucleares/metabolismo , Oligonucleótidos Antisentido/genética , ARN Polimerasa II/metabolismo , ARN Largo no Codificante/genética , Transcripción Genética , Proteína 1 de Unión al Supresor Tumoral P53/metabolismoRESUMEN
Of the many types of DNA damage, DNA double-strand breaks (DSBs) are probably the most deleterious. Mounting evidence points to an intricate relationship between DSBs and transcription. A cell system in which the impact on transcription can be investigated at precisely mapped genomic DSBs is essential to study this relationship. Here in a human cell line, we map genome-wide and at high resolution the DSBs induced by a restriction enzyme, and we characterize their impact on gene expression by four independent approaches by monitoring steady-state RNA levels, rates of RNA synthesis, transcription initiation and RNA polymerase II elongation. We consistently observe transcriptional repression in proximity to DSBs. Downregulation of transcription depends on ATM kinase activity and on the distance from the DSB. Our study couples for the first time, to the best of our knowledge, high-resolution mapping of DSBs with multilayered transcriptomics to dissect the events shaping gene expression after DSB induction at multiple endogenous sites.
Asunto(s)
Roturas del ADN de Doble Cadena , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Animales , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Línea Celular Tumoral , Análisis por Conglomerados , ADN/metabolismo , Daño del ADN , Reparación del ADN , Proteínas de Unión al ADN/metabolismo , Genoma Humano , Humanos , Ratones , Células 3T3 NIH , Fosforilación , Análisis de Secuencia de ADN , Análisis de Secuencia de ARN , Transcripción Genética , TranscriptomaRESUMEN
The fine modulation of transcriptional activity around DNA lesions is essential to carefully regulate the crosstalk between the activation of the DNA damage response, DNA repair and transcription, particularly when the lesion occurs next to actively transcribed genes. Recently, several studies have been carried out to investigate how DNA lesions impact on local transcription, but the emerging model remains incomplete. Transcription of genes around damaged DNA is actively downregulated by the DNA damage response through different mechanisms, which appear specific to the chromatin context, the type of DNA damage or its complexity. Intriguingly, emerging evidence also indicates that transcription of noncoding RNAs (ncRNAs) is induced at sites of DNA damage, producing small ncRNAs that are, in turn, required for a full DNA damage response activation. We discuss here these recent findings, highlighting the major unresolved questions in the field, and propose ways to reconcile these apparently contradictory observations.