Time-lapse super-resolution imaging reveals key principles for cis-limited Xist RNA localization [ChrRNA-seq]

Xist RNA, which directs the process of X chromosome inactivation in mammals, localizes in cis over the chromosome from which it is transcribed, recruiting chromatin modifiers to silence underlying genes. To analyze cis-limited Xist RNA localization we have developed time-resolved super-resolution imaging of individual Xist RNA molecules in single cells. Using this approach, we quantify fundamental parameters underpinning Xist RNA dynamics, demonstrating a feedback mechanism linking Xist RNA synthesis and degradation, and an unusual coupling behavior that physically links temporally separated Xist RNA molecules. Additionally, we show that the protein SPEN, a key factor for Xist-mediated gene-silencing, has a separable function in Xist RNA localization, stability and in coupling behavior. Our results provide important insights towards understanding the unique and unusual behavior of Xist RNA. Overall design: We isolated chromatin-associated RNA (ChrRNA) to analyze the allelic ratio, aiming to understand Xist-mediated Silencing. Xist RNA is labeled.The ChrRNA was isolated from cells with trangenic Xist model (iXist-Chr15) or endogenous Xist model (iXist-ChrX) derived from mouse embryonic stem cells. Cell lines with different factor knockout (CIZ1, SpenRRM, SpenSPOCmut) were generated. The allelic ratios were systematically and comprehensively compared.