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)

叉头盒蛋白 A1 （FOXA1） 是一种先驱转录因子，可在经典 DNA 基序处与染色质结合。这种结合促进染色质的开放，提供对其他转录因子的访问。用小分子靶向 FOXA1 等转录因子可以作为阐明转录调控快速动力学的有用工具，但由于缺乏可定义的结合口袋，因此仍然很困难。通过使用亲电化合物库的基于活性的蛋白质分析进行化学筛选，Won 等人将 WX-02-23 鉴定为 FOXA1 在 Wing2 区域的特定半胱氨酸残基 （C258） 处的共价结合物，该残基已知与次要 DNA 沟接触。WX-02-23 与 FOXA1 的结合需要 DNA 的存在和增强的 FOXA1-DNA 相互作用。ChIP-seq（染色质免疫沉淀测序）和 ATAC-seq（使用测序检测转座酶可及染色质）分析表明，WX-02-23 与 FOXA1 的 C258 依赖性结合可以增加或减少整个基因组中 FOXA1 的结合，这与染色质可及性的改变相关。研究小组发现，WX-02-23 改变了 10% 的 FOXA1 结合位点。基序分析显示，这些由 WX-02-23 介导的 FOXA1 结合位点增加，缺乏经典基序的辅助 3 bp 组分。他们提出 WX-02-23 可能会松弛 FOXA1 识别的 DNA 基序，以扩大其在细胞中的结合位点。定量 NanoBRET 测定证实，WX-02-23 增加了 FOXA1 与非经典基序的结合，这也见于 FOXA1 （R261G） 的 Wing2 区域的热点癌症突变。 尽管 WX-02-23 对 FOXA1 先锋活性影响的结构基础尚不清楚，但 WX-02-23 的鉴定为揭示 FOXA1 生物学和染色质调控的新见解提供了一种多功能工具。

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