The Program in Cellular and Molecular Oncology, University of New Mexico Comprehensive Cancer Center, Albuquerque, NM, USA.
Department of Internal Medicine, Division of Molecular Medicine, University of New Mexico Health Science Center, Albuquerque, NM, USA.
Methods Mol Biol. 2022;2472:95-108. doi: 10.1007/978-1-0716-2201-8_9.
The sequence-specific transcription factor RBPJ, also known as CSL (CBF1, Su(H), Lag1), is an evolutionarily conserved protein that mediates Notch signaling to guide cell fates. When cells enter mitosis, DNA is condensed and most transcription factors dissociate from chromatin; however, a few, select transcription factors, termed bookmarking factors, remain associated. These mitotic chromatin-bound factors are believed to play important roles in maintaining cell fates through cell division. RBPJ is one such factor that remains mitotic chromatin associated and therefore could function as a bookmarking factor. Here, we describe how to obtain highly purified mitotic cells from the mouse embryonal carcinoma cell line F9, perform chromatin immunoprecipitation with mitotic cells, and measure the first run of RNA synthesis upon mitotic exit. These methods serve as basis to understand the roles of mitotic bookmarking by RBPJ in propagating Notch signals through cell division.
序列特异性转录因子 RBPJ，也称为 CSL（CBF1、Su(H)、Lag1），是一种进化上保守的蛋白质，它介导 Notch 信号以指导细胞命运。当细胞进入有丝分裂时，DNA 被浓缩，大多数转录因子从染色质上解离；然而，有少数特定的转录因子，称为书签因子，仍然与染色质结合。这些有丝分裂染色质结合的因子被认为在通过细胞分裂维持细胞命运方面发挥重要作用。RBPJ 就是这样一种仍然与有丝分裂染色质相关联的因子，因此它可以作为一个书签因子。在这里，我们描述了如何从小鼠胚胎癌细胞系 F9 中获得高度纯化的有丝分裂细胞，用有丝分裂细胞进行染色质免疫沉淀，并测量有丝分裂退出后 RNA 合成的第一次运行。这些方法为理解 RBPJ 通过细胞分裂传播 Notch 信号的有丝分裂书签的作用提供了基础。
