The Genetic Interaction Between RB1 and THOC1 in Mouse Embryonic Fibroblasts: A Model for Transcription Stress
Cedeño, Carlos Daniel
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Genome instability is a common feature of human cancer, contributing both to tumorigenesis itself, and to disease outcome. Transcription of the genome directly contributes to genome instability, particularly under conditions producing transcription stress. Various transcriptional impediments promote genome instability, in part, by leading to aberrant accumulation of RNA:DNA hybrid structures (R-loops) and associated DNA damage. The RB1 tumor suppressor actively maintains genome stability by suppressing inappropriate DNA replication, by facilitating DNA repair and by promoting mitotic fidelity. Although Rb1 broadly regulates transcription, it is unknown whether RB1 also maintains genome stability in part by limiting the impact of transcription stress. Here, acute genetic deletion of the RNA processing factor Thoc1 is used as a model for inducing transcription stress in the presence or absence of Rb1 deletion. The effects of deletion are investigated in Rosa26 CreERT2/+ transgenic mouse embryonic fibroblasts (MEFs) using tamoxifen-mediated recombination of floxed alleles. In spontaneously immortalized MEFs, acute Rb1 deletion increases global transcription rate and R-loop accumulation, suggesting that RB1 normally suppresses accumulation of R-loops, in part, by repressing transcription. These data support the possibility that RB1 defends genome stability in part by limiting the potential impact of transcription-related stress. Combined deletion of Rb1 and Thoc1 in an immortal MEF cell line leads to an early loss of proliferative capacity and an exaggerated DNA damage response to THOC1 loss compared to Thoc1 deletion alone. Thus, in immortal MEFs, RB1 may limit both the potential for R-loop-mediated transcription stress, and the toxicity of genetically induced transcription stress. Unlike immortal MEFs, early passage primary MEFs possess normal checkpoint function and maintain a diploid genome. In primary MEFs, Rb1 deletion alone confers a proliferative advantage, facilitating G1/S progression and early tetraploidization. Deletion of Thoc1 alone impedes proliferation, inducing a DNA damage response and cell death, without inducing early tetraploidization. Deleting Rb1 and Thoc1 in combination leads to more widespread tetraploidization than Rb1 deletion alone and more rapid cell loss than Thoc1 deletion alone. Overexpression of the R-loop-resolving enzyme RNASEH1 appears to suppress early tetraploidization after Rb1 deletion alone, and after Rb1/Thoc1 co-deletion. Thus, in primary MEFs, RB1 is required to suppress early tetraploidization due to acute transcription stress. Since tetraploidy facilitates genomic instability and tumor evolution, these data encourage the hypothesis that RB1 suppresses tumorigenesis in part by limiting the genomic impact of transcription stress. Altogether, the present findings further support a growing body of evidence that transcription stress is a central player in the genomic instability enabling oncogenesis and cancer progression.