R-loop editing by DNA cytosine deaminase APOBEC3B modulates the activity of oestrogen receptor enhancers

Abstract Oestrogen receptor (ER) activation leads to the formation of DNA double strand breaks (DSB), promoting genomic instability and tumour heterogeneity. The single-stranded DNA cytosine deaminase APOBEC3B (A3B) serves as a co-activator of ER and is implicated in inducing DSBs at transcriptional enhancers regulated by ER. Using whole-genome sequencing in an engineered cell model lacking base excision repair (BER) function, we demonstrate that A3B preferentially targets transcriptionally active regulatory regions in an R-loop-dependent manner. Strand-specific DNA:RNA immunoprecipitation sequencing (ssDRIP-seq) and ssDNA-associated protein immunoprecipitation sequencing (SPI-seq) confirm that A3B binds to and deaminates ssDNA within R-loops, a process facilitated by ER transactivation. Furthermore, BER-mediated processing of A3B-induced uracil bases contributes to the formation of R-loop-associated DSBs, which are essential for ER-regulated gene activation. These findings establish a role for A3B in R-loop homeostasis and transcriptional regulation, with implications for understanding ER-driven genomic instability and potential therapeutic targeting of A3B.