AND-1 is a critical regulator of R-loop dynamics and a target to overcome endocrine resistance.

R-loops are three-stranded DNA/RNA hybrids that are essential for various cellular pathways. However, when dysregulated, they lead to genomic instability and numerous human diseases. R-loops are tightly regulated, with RNase H1 acting as a key enzyme responsible for resolving DNA/RNA hybrids. Here, we identify the DNA-binding protein AND-1 as an essential factor in R-loop regulation through directly binding to R-loop structures, where it enhances the recruitment of RNase H1 and stimulates its endonuclease activity. We also provide in vivo evidence that R-loop accumulation occurs in the mammary gland tissue of AND-1-deficient mice. Furthermore, we demonstrate that inhibition of AND-1 decreases ESR1 expression by disrupting R-loop regulation at the enhancer region of the ESR1 gene in estrogen receptor-positive (ER(+)) breast cancer cells, thereby overcoming resistance to aromatase inhibitors. Collectively, our findings reveal a mechanism by which AND-1 modulates R-loop dynamics and present a promising therapeutic strategy to combat endocrine resistance in breast cancer.