Time-resolved structured illumination microscopy reveals key principles of Xist RNA spreading – Science Magazine

Visualizing Xist RNA dynamics

The noncoding RNA Xist, which controls the process of X chromosome inactivation in mammals, accumulates and spreads over the chromosome from which it is transcribed. The underlying basis for this unusual behavior is poorly understood. Using a new imaging approach called RNA-SPLIT for time-resolved analysis of Xist RNA molecules at super-resolution, Rodermund et al. analyzed fundamental parameters of Xist RNA behavior in normal cells and after the perturbation of factors implicated in Xist RNA function. The authors provide new insights into the basis of Xist RNA localization and confinement within the territory of a single X chromosome.

Science, abe7500, this issue p. eabe7500

X chromosome inactivation (XCI) in mammals equalizes levels of X-linked gene expression in XX females relative to XY males. The XCI process is governed by the noncoding RNA Xist (X-inactive specific transcript), which is expressed from the inactive X chromosome elect, where it accumulates in cis and recruits factors required for chromatin-based gene silencing. The basis for cis-confinement to a single chromosome territory is poorly understood but is thought to involve interactions between specific Xist RNA binding proteins (RBPs)for example, CIZ1 and SPENand a nuclear protein scaffold.

To better understand the distinct and unusual behavior of Xist RNA, we developed RNA-SPLIT (sequential pulse localization imaging over time)a method for time-resolved imaging of individual Xist molecules in the context of cells undergoing XCI. Here, we made use of the BglG/Bgl stem-loop system for tagging specific RNAs and a BglG-HaloTag fusion protein. Sequential pulsing with different fluorescent HaloTag ligands in living cells (mouse XX embryonic stem cells engineered with doxycycline-inducible Xist RNA) enabled us to image temporally resolved single Xist RNA molecules using super-resolution three-dimensional structured illumination microscopy (3D-SIM).

Consistent with previous work, we observed territories comprising around 50 to 100 single Xist molecules per cell. We identified a time window from 1.5 to 5 hours postinduction, during which we could quantify a gradual increase of Xist molecules within the volume of the Xist territory, referred to as expansion phase. A later time point (24 hours postinduction) was taken as representative of Xist RNA behavior at steady state. The application of RNA-SPLIT allowed us to measure key parameters of Xist RNA dynamics in both phases. Xist molecules turned over approximately every 2 or 4 hours in expansion and steady state phases, respectively. Inhibition of Xist transcription by doxycycline washout resulted in a marked increase in Xist RNA stability, which suggests a feedback mechanism that balances rates of transcription and turnover. We observed a progressive radial enlargement of Xist RNA territories during expansion phase, with an estimated rate of ~8 m3/hour. Radial enlargement was not evident at steady state, which suggests that Xist RNA molecules localize in a more stochastic manner once X inactivation has been established. RNA-SPLIT further revealed that newly synthesized Xist molecules predominantly localize to sites where presynthesized molecules are presenta phenomenon we refer to as coupling. We extended our analysis to examine the effect of ablating CIZ1 and SPEN, factors implicated in the localization and cis-confinement of Xist RNA. Loss of function of CIZ1, which results in widespread nuclear dispersal of Xist RNA in differentiated cells, led to increased levels of transcription and increased stability of Xist RNA. In spite of widespread Xist RNA dispersal, coupling of pre- and newly synthesized Xist molecules was retained. SPEN loss of function led to major defects in the long-range localization of Xist RNA, reduced Xist RNA stability, and a disruption of Xist RNA coupling. The function of SPEN in Xist RNA behavior is genetically separable from its function in chromatin-based gene silencing in XCI.

The development of RNA-SPLIT has enabled quantitative spatial analysis of single Xist molecules over time during the establishment of XCI. Application of this methodology has revealed fundamental principles of Xist RNA behavior, including feedback control of Xist turnover and transcription, the kinetics of Xist RNA spreading, and the coupling of pre- and newly synthesized molecules. Beyond these insights, RNA-SPLIT has progressed our understanding of how the factors CIZ1 and SPEN affect different aspects of Xist RNA behavior and localization.

Temporally distinct populations of Xist RNA molecules labeled in embryonic stem cell lines engineered with BglG stem-looptagged doxycycline-inducible Xist RNA and BglG-HaloTag fusion protein (top left). Unlike conventional widefield microscopy, visualization with super-resolution 3D-SIM imaging allows discrimination of individual Xist molecules transcribed at different time points (top right). This approach enabled quantification of the dynamic behavior of Xist RNA molecules characterized by jump and coupling (bottom left) and provided insights into functions of Xist-interacting proteins SPEN and CIZ1, which bind to A- and E-repeat domains of Xist RNA, respectively (bottom right). Tet-on, tetracycline-induced activation; Xi, inactive X chromosome; Xa, active X chromosome; mESC, mouse embryonic stem cell; DAPI, 4,6-diamidino-2-phenylindole.

X-inactive specific transcript (Xist) RNA directs the process of X chromosome inactivation in mammals by spreading in cis along the chromosome from which it is transcribed and recruiting chromatin modifiers to silence gene transcription. To elucidate mechanisms of Xist RNA cis-confinement, we established a sequential dual-color labeling, super-resolution imaging approach to trace individual Xist RNA molecules over time, which enabled us to define fundamental parameters of spreading. We demonstrate a feedback mechanism linking Xist RNA synthesis and degradation and an unexpected physical coupling between preceding and newly synthesized Xist RNA molecules. Additionally, we find that the protein SPEN, a key factor for Xist-mediated gene silencing, has a distinct function in Xist RNA localization, stability, and coupling behaviors. Our results provide insights toward understanding the distinct dynamic properties of Xist RNA.

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Time-resolved structured illumination microscopy reveals key principles of Xist RNA spreading - Science Magazine