TurboCas: A method for locus-specific labeling of genomic regions and isolating their associated protein interactome

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 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 heat shock-responsive genes. Applying TurboCas to the MYC promoter, we identify two P-TEFb coactivators, Super Elongation Complex and BRD4, as MYC regulators. TurboCas significantly improves locus-targeted proximity labeling, with potential to deepen understanding of regulatory pathways in development and stress response.

These are uncropped and/or unprocessed imaging files from this study.

发育过程中基因表达的调控及应激反应的应答，需转录因子与染色质结合蛋白的协同作用。鉴于此过程具有细胞类型特异性，并随细胞状态而异，故对单个调控位点的染色质因子进行映射，对于理解顺式调控控制至关重要。以往的方法仅对静态蛋白质结合进行表征。我们提出了 TurboCas 方法，该方法将 miniTurbo 邻近标记酶与 CRISPR/dCas9 相结合，使得在哺乳细胞中高效且特异性地标记染色质因子成为可能。通过在 FOS 启动子中验证 TurboCas，我们识别出在热休克过程中被招募的蛋白质，并通过 RNA 聚合酶 II 和 P-TEFb 免疫沉淀进行交叉验证。这些方法揭示了经典及未表征的因子，它们的功能在于激活热休克响应基因。将 TurboCas 应用于 MYC 启动子，我们鉴定出两种 P-TEFb 协同激活因子，即超级延伸复合体和 BRD4，它们是 MYC 的调控因子。TurboCas 显著提升了定位靶向邻近标记，有望深化对发育和应激反应中调控途径的理解。这些图像文件为本研究中的未裁剪和/或未处理的成像文件。