Cenik Bercin K, Aoi Yuki, Iwanaszko Marta, Howard Benjamin C, Morgan Marc A, Andersen Grant D, Bartom Elizabeth T, Shilatifard Ali
Simpson Querrey Institute for Epigenetics, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA; Department of Biochemistry and Molecular Genetics, Northwestern University Feinberg School of Medicine, 303 E. Superior St., Chicago, IL 60611, USA.
Simpson Querrey Institute for Epigenetics, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA; Department of Biochemistry and Molecular Genetics, Northwestern University Feinberg School of Medicine, 303 E. Superior St., Chicago, IL 60611, USA; Robert H. Lurie NCI Comprehensive Cancer Center, Northwestern University Feinberg School of Medicine, 303 E. Superior St., Chicago, IL 60611, USA.
Mol Cell. 2024 Dec 19;84(24):4929-4944.e8. doi: 10.1016/j.molcel.2024.11.007.
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.
在发育和应激反应过程中，基因表达的调控需要转录因子和染色质结合蛋白的协同作用。由于这个过程具有细胞类型特异性，并且会随细胞条件而变化，因此在单个调控位点绘制染色质因子图谱对于理解顺式调控控制至关重要。以前的方法只能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，可在哺乳动物细胞中对染色质因子进行高效且位点特异性的标记。通过在FOS启动子处验证TurboCas，我们鉴定了热休克时招募的蛋白质，并通过RNA聚合酶II和P-TEFb免疫沉淀进行了交叉验证。这些方法揭示了激活热休克反应基因表达的典型和未表征的因子。将TurboCas应用于MYC启动子，我们鉴定了两个P-TEFb共激活因子，即超级延伸复合物（SEC）和BRD4，作为MYC的共调节因子。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。
