Regulation of gene expression during development and stress response requires the concerted action of transcription factors and chromatin-binding proteins. Because this process is cell-type specific and varies with cellular conditions, mapping of chromatin factors at individual regulatory loci is crucial for understanding cis-regulatory control. Previous methods only characterize static protein binding. We present “TurboCas,” a method combining a proximity-labeling (PL) enzyme, miniTurbo, with CRISPR-dCas9 that allows for efficient and site-specific labeling of chromatin factors in mammalian cells. Validating TurboCas at the FOS promoter, we identify proteins recruited upon heat shock, cross-validated via RNA polymerase II and P-TEFb immunoprecipitation. These methodologies reveal canonical and uncharacterized factors that function to activate expression of heat-shock-responsive genes. Applying TurboCas to the MYC promoter, we identify two P-TEFb coactivators, the super elongation complex (SEC) and BRD4, as MYC co-regulators. TurboCas provides a genome-specific targeting PL, with the potential to deepen our molecular understanding of transcriptional regulatory pathways in development and stress response.

中文翻译：

---

TurboCas：一种基因组区域基因座特异性标记和分离其相关蛋白质相互作用组的方法


发育和应激反应过程中基因表达的调节需要转录因子和染色质结合蛋白的协同作用。由于此过程是细胞类型特异性的，并且随细胞条件而变化，因此在单个调节位点绘制染色质因子对于了解顺式调节控制至关重要。以前的方法仅表征静态蛋白质结合。我们提出了“TurboCas”，这是一种将邻近标记 （PL） 酶 miniTurbo 与 CRISPR-dCas9 相结合的方法，该方法允许对哺乳动物细胞中的染色质因子进行高效和位点特异性标记。在 FOS 启动子处验证 TurboCas，我们鉴定了热休克时募集的蛋白质，通过 RNA 聚合酶 II 和 P-TEFb 免疫沉淀进行交叉验证。这些方法揭示了激活热休克反应基因表达的经典和未表征因素。将 TurboCas 应用于 MYC 启动子，我们确定了两种 P-TEFb 共激活因子，即超级延伸复合物 （SEC） 和 BRD4，作为 MYC 共调节因子。TurboCas 提供了一种基因组特异性靶向 PL，有可能加深我们对发育和应激反应中转录调控途径的分子理解。