Structural and functional basis of PU.1-BAF interaction enables targeting of lineage-specific transcription [RNA-seq]

Transcription factors (TFs) regulate gene expression by engaging chromatin remodeling complexes, yet the structural principles governing these critical interactions remain poorly defined. Here, we uncover the molecular mechanism by which lineage-specific pioneer transcription factor PU.1 (encoded by SPI1) directly engages the BAF (mSWI/SNF) chromatin remodeling complex. First, using a variety of genomic approaches, we establish that BAF collaborates with PU.1 to regulate transcription in AML cells. Then, using a combination of biochemistry and biophysics, mass spectrometry-based protein footprinting, and crystallography, we map the PU.1-BAF60A interface to a disordered region of PU.1 that adopts a helical conformation upon binding to the YEATS-like domain of BAF60A. Disruption of this functionally critical interface via knockdown abrogates the ability of PU.1 to rescue cell viability. Finally, we conducted a high-throughput screen that yielded small molecules which selectively bind BAF60A and disrupt PU.1 binding. Co-crystal structures reveal distinct compound binding modes that converge on a critical PU.1-BAF60A interaction hotspot. These findings define, for the first time, the structural interface between a pioneer transcription factor and the BAF complex and establish a platform that enables targeting transcription factor-chromatin remodeling complex interactions in cancer. Comparative gene expression profiling analysis of RNA-seq data for THP-1 cells cells treated with dual SMARCA4/SMARCA2 inhibitor Compound 49 for 24h; MV-4-11 cells treated with dual SMARCA4/SMARCA2 inhibitor FHT-1015 for 24h or shPU.1 for 48h.

转录因子（Transcription factors, TFs）通过招募染色质重塑复合物调控基因表达，但介导这类关键相互作用的结构原则仍未得到明确阐释。本研究揭示了谱系特异性先锋转录因子（pioneer transcription factor）PU.1（由SPI1基因编码）直接结合BAF（mSWI/SNF）染色质重塑复合物的分子机制。首先，我们通过多种基因组学手段证实，BAF与PU.1在急性髓系白血病（Acute Myeloid Leukemia, AML）细胞中协同调控基因转录。随后，结合生物化学、生物物理学、基于质谱的蛋白质足迹法（protein footprinting）与晶体学技术，我们将PU.1与BAF60A的相互作用界面定位至PU.1的一个无序区域——该区域在结合BAF60A的YEATS样结构域时会形成螺旋构象。通过基因敲低手段破坏这一功能关键界面，会消除PU.1拯救细胞活力的能力。最后，我们开展了高通量筛选，获得了可选择性结合BAF60A并阻断PU.1结合的小分子化合物。共晶结构分析显示，不同化合物的结合模式均汇聚于PU.1与BAF60A相互作用的关键热点区域。本研究首次明确了先锋转录因子与BAF复合物之间的结构界面，并建立了可靶向癌症中转录因子-染色质重塑复合物相互作用的研究平台。本数据集为经双重SMARCA4/SMARCA2抑制剂Compound 49处理24小时的THP-1细胞、经双重SMARCA4/SMARCA2抑制剂FHT-1015处理24小时或经shPU.1处理48小时的MV-4-11细胞的RNA测序数据的比较基因表达谱分析结果。