Nuclear functions of CFAP20 in transcription and replication [ChIP-seq TY-CFAP20]
Fine-tuning DNA replication and transcription is crucial to prevent collisions between their machineries. This is particularly important near promoters, where RNA polymerase II (RNAPII) initiates transcription and frequently arrests, forming R-loops. Arrested RNAPII can obstruct DNA replication, which often initiates near promoters. The mechanisms that rescue arrested RNAPII during elongation to avoid conflicts with co-directional replisomes remain unclear. Here, using genome-wide approaches and genetic screens, we identify CFAP20 as part of a protective pathway that salvages arrested RNAPII in promoter-proximal regions, diverting it from the path of co-directional replisomes. CFAP20-deficient cells accumulate R-loops near promoters, which leads to defects in replication timing and dynamics. These defects stem from accelerated replication-fork speeds that cause a secondary reduction in origin activity. Co-depletion of the Mediator complex or removal of R-loop-engaged RNAPII restores normal replication. Our findings suggest that transcription-dependent fork stalling in cis induces accelerated fork progression in trans, generating single-stranded DNA gaps. We propose that CFAP20 facilitates RNAPII elongation under high levels of Mediator-driven transcription, thereby preventing replisome collisions. This study provides a transcription-centred view of transcription-replication encounters, revealing how locally arrested transcription complexes propagate genome-wide replication phenotypes and defining CFAP20 as a key factor that safeguards genome stability. Overall design: To explore spatial connections between transcription and replication at the genome-wide level, as well as the role of CFAP20 in these processes, we mapped RNA polymerase II (RNAPII) and CFAP20 occupancy using chromatin immunoprecipitation (ChIP)-seq, nascent transcription by bromouridine (BrU)-seq and TTchem-seq, and R-loops by DNA-RNA hybrid immunoprecipitation (DRIP)-seq in RPE1 cells being either wild type (WT), CFAP20-KO, CFAP20-KO rescued with CFAP20-GFP, or CFAP20/CCNC-dKO.