Oncogene-induced transcriptional changes promote R-loop accumulation underlying genomic instability at fragile sites

Michal Irony Tur Sinai Dan Sarni ¹ Takayo Sasaki ² Sonia Barroso ³ Karin Miron ¹ Brian Magnuson ^4,5 Mats Ljungman ^5,6,7 Andres Aguilera ³ David M. Gilbert ² Batsheva Kerem ¹

¹Department of Genetics, The Hebrew University of Jerusalem, Israel
²Laboratory of Chromosome Replication and Epigenome Regulation, San Diego Biomedical Research Institute, USA
³Department of Genome Biology, Andalusian Center of Molecular Biology and Regenerative Medicine CABIMER, Seville University-CSIC, Spain
⁴Department of Biostatistics, School of Public Health, University of Michigan, USA
⁵Rogel Cancer Center and Center for RNA Biomedicine, University of Michigan, USA
⁶Department of Radiation Oncology, University of Michigan Medical School, USA
⁷Department of Environmental Health Sciences, School of Public Health, University of Michigan, USA

Fragile sites (FSs) are regions susceptible to replication stress and are hotspots for chromosomal instability in cancer. Chromosomal fragility is both cell-type and stress-inducer specific with a unique landscape of FSs in different conditions. While the molecular basis underlying the chemically-induced FSs was widely studied and key factors were identified, the nature of oncogene-induced chromosomal fragility has not been investigated yet. Here, we investigate the underpinning molecular mechanism of oncogene-induced fragility, by exploring the effect of overexpression of the mutated HRAS oncogene (RAS) on the replication timing program (RT), transcriptional program and chromosomal instability. The results show that mutated RAS affects both the RT and transcription of specific regions. Furthermore, we show that RAS cells are deficient in RNA-DNA hybrid (R-loop) processing or prevention, leading to accumulation of R-loops. We uncover that the fragility core is characterized by delayed RT of transcribed large genes, which accumulate R-loops. Thus, we propose a model underlying oncogene-induced chromosomal instability comprised of perturbed DNA replication as a result of transcription dependent R-loop accumulation. This suggests that the molecular basis underlying oncogene-induced fragility differs from the fragility induced by replication inhibitors. Furthermore, this model will allow a comprehensive mapping of oncogene-induced fragile sites and understanding of genomic instability in cancers.