Loss of ATM causes R-loop-associated transcriptional dysregulation and attenuates the related response to DNA damage [RNA-Seq]

An early childhood onset neurodegenerative disorder, Ataxia Telangiectasia (AT) affects 1 in 40,000 to 100,000 individuals worldwide and is caused by mutations in the ataxia telangiectasia mutated (ATM) threonine-serine kinase, which regulates the DNA damage response (DDR). While the cause of AT has been known for years, the exact molecular mechanisms underlying disease progression, particularly at the transcriptomic level, remain poorly understood. Three stranded structures, known as R-loops, have recently emerged as important players in the DDR via regulating key gene expression. Here, we utilized neuronal progenitor cells (NPCs) derived from induced pluripotent stem cells reprogrammed from patient-derived somatic cells to identify how loss of ATM impacts R-loop and transcriptional dynamics, both at baseline and in response to acute DNA damage. AT-derived NPCs (AT-NPCs) exhibited elevated spontaneous R-loop levels compared to controls (C-NPCs), as well as a strong positive correlation between R-loop accumulation and gene expression on a subset of dysregulated genes. Upon acute damage, loss of ATM resulted in an attenuated response, characterized by the impaired R-loop and transcriptional response to irradiation. Both C- and AT-NPCs underwent a similar cell cycle arrest, but AT-NPCs displayed an attenuated R-loop and transcriptional response, failing to activate proper DDR response. Importantly, R-loop formation is required for many key genes to properly respond to DNA damage, supporting a direct and causal role in this process. Overall, our data reveal an underappreciated mechanistic link between ATM, R-loop regulation, and transcription, the disruption of which may contribute to the impaired DDR observed in AT-NPCs. Overall design: RNA-seq data of control and Ataxia Telangiectasia (AT) patient-derived neuronal progenitor cells either untreated or irradiated with 10Gy and given 6h recovery. RNA-seq data of control and AT patient-derived neuronal progenitor cells infected with a lentivirus overexpressing FLAG-tagged RNaseH1 which were then irradiated with 10Gy after 42 hours and placed in an incubator for 6 hours.