Forkhead box protein A1 (FOXA1) is a pioneer transcription factor that binds to chromatin at a canonical DNA motif. This binding promotes the opening of chromatin, providing access to additional transcription factors. Targeting transcription factors such as FOXA1 with small molecules can serve as a useful tool to elucidate the rapid dynamics of transcriptional regulation, but remains difficult owing to a lack of definable binding pockets. Through a chemical screen using activity-based protein profiling with a library of electrophilic compounds, Won et al. identified WX-02-23 as a covalent binder of FOXA1 at a specific cysteine residue (C258) in the Wing2 region that is known to make contacts with the minor DNA groove. Binding of WX-02-23 to FOXA1 required the presence of DNA and enhanced FOXA1–DNA interactions. ChIP–seq (chromatin immunoprecipitation with sequencing) and ATAC-seq (assay for transposase-accessible chromatin using sequencing) analysis demonstrated that C258-dependent binding of WX-02-23 to FOXA1 can either increase or decrease FOXA1 binding throughout the genome, correlating with alterations in chromatin accessibility. The team found that WX-02-23 altered 10% of FOXA1 binding sites. Motif analysis revealed that these increased FOXA1 binding sites, mediated by WX-02-23, lack an ancillary 3 bp component of the canonical motif. They proposed that WX-02-23 may relax the DNA motif recognized by FOXA1 to expand its binding sites in cells. Quantitative NanoBRET assays confirmed that WX-02-23 increased FOXA1 binding to non-canonical motifs, which was also seen with a hotspot cancer mutation in the Wing2 region of FOXA1 (R261G). Although the structural basis for the effects of WX-02-23 on FOXA1 pioneering activity remain unclear, the identification of WX-02-23 offers a versatile tool to reveal new insights into FOXA1 biology and chromatin regulation.

Original reference: Mol. Cell https://doi.org/10.1016/j.molcel.2024.09.024 (2024)