FOXP1 phosphorylation antagonizes its O-GlcNAcylation in regulating ATR activation in response to replication stress.

ATR signaling is essential in sensing and responding to the replication stress; as such, any defects can impair cellular function and survival. ATR itself is activated via tightly regulated mechanisms. Here, we identify FOXP1, a forkhead-box-containing transcription factor, as a regulator coordinating ATR activation. We show that, unlike its role as a transcription factor, FOXP1 functions as a scaffold and directly binds to RPA–ssDNA and ATR–ATRIP complexes, facilitating the recruitment and activation of ATR. This process is regulated by FOXP1 O-GlcNAcylation, which represses its interaction with ATR, while CHK1-mediated phosphorylation of FOXP1 inhibits its O-GlcNAcylation upon replication stress. Supporting the physiological relevance of this loop, we find pathogenic FOXP1 mutants identified in various tumor tissues with compromised ATR activation and stalled replication fork stability. We thus conclude that FOXP1 may serve as a potential chemotherapeutic target in related tumors. Synopsis: Replication stress responses regulated by ATR are crucial in resolving genomic threats. This study identifies forkhead-box-containing transcription factor FOXP1 as a new regulator, which promotes ATR activation independent of its transcriptional activity. FOXP1 loads onto stalled replication forks by directly binding to RPA-ssDNA complexes. FOXP1 promotes chromatin loading of ATR and activation of ATR-CHK1. O-GlcNAcylation of FOXP1 represses its interaction with ATR. CHK1-mediated phosphorylation of FOXP1 antagonizes its O-GlcNAcylation. Direct binding to RPA-ssDNA complexes mediates FOXP1 loading onto stalled forks, where it aids ATR-CHK1 activation independent of its transcriptional activity. [ABSTRACT FROM AUTHOR]