RESUMEN
DNA-protein crosslinks (DPCs) arise from enzymatic intermediates, metabolism or chemicals like chemotherapeutics. DPCs are highly cytotoxic as they impede DNA-based processes such as replication, which is counteracted through proteolysis-mediated DPC removal by spartan (SPRTN) or the proteasome. However, whether DPCs affect transcription and how transcription-blocking DPCs are repaired remains largely unknown. Here we show that DPCs severely impede RNA polymerase II-mediated transcription and are preferentially repaired in active genes by transcription-coupled DPC (TC-DPC) repair. TC-DPC repair is initiated by recruiting the transcription-coupled nucleotide excision repair (TC-NER) factors CSB and CSA to DPC-stalled RNA polymerase II. CSA and CSB are indispensable for TC-DPC repair; however, the downstream TC-NER factors UVSSA and XPA are not, a result indicative of a non-canonical TC-NER mechanism. TC-DPC repair functions independently of SPRTN but is mediated by the ubiquitin ligase CRL4CSA and the proteasome. Thus, DPCs in genes are preferentially repaired in a transcription-coupled manner to facilitate unperturbed transcription.
Asunto(s)
ADN Helicasas , Enzimas Reparadoras del ADN , Reparación del ADN , Proteínas de Unión a Poli-ADP-Ribosa , Proteolisis , ARN Polimerasa II , Transcripción Genética , Enzimas Reparadoras del ADN/metabolismo , Enzimas Reparadoras del ADN/genética , Humanos , Proteínas de Unión a Poli-ADP-Ribosa/metabolismo , Proteínas de Unión a Poli-ADP-Ribosa/genética , ADN Helicasas/metabolismo , ADN Helicasas/genética , ARN Polimerasa II/metabolismo , ARN Polimerasa II/genética , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/genética , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/genética , ADN/metabolismo , ADN/genética , Células HEK293 , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Daño del ADN , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteínas Portadoras , Receptores de Interleucina-17RESUMEN
Human brain organoid technology has the potential to generate unprecedented insight into normal and aberrant brain development. It opens up a developmental time window in which the effects of gene or environmental perturbations can be experimentally tested. However, detection sensitivity and correct interpretation of phenotypes are hampered by notable batch-to-batch variability and low reproducibility of cell and regional identities. Here, we describe a detailed, simplified protocol for the robust and reproducible generation of brain organoids with cortical identity from feeder-independent induced pluripotent stem cells (iPSCs). This self-patterning approach minimizes media supplements and handling steps, resulting in cortical brain organoids that can be maintained over prolonged periods and that contain radial glial and intermediate progenitors, deep and upper layer neurons, and astrocytes.
RESUMEN
Transcription pause-release is an important, highly regulated step in the control of gene expression. Modulated by various factors, it enables signal integration and fine-tuning of transcriptional responses. Mutations in regulators of pause-release have been identified in a range of neurodevelopmental disorders that have several common features affecting multiple organ systems. This review summarizes current knowledge on this novel subclass of disorders, including an overview of clinical features, mechanistic details, and insight into the relevant neurodevelopmental processes.