Use this URL to cite or link to this record in EThOS: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.820773
Title: Time-lapse super-resolution imaging reveals key principles for cis-limited Xist RNA localisation
Author: Rodermund, Lisa
ISNI:       0000 0004 9356 6897
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 2020
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Abstract:
Dosage compensation in XX female mammals is achieved by X chromosome inactivation (XCI). The key player in this process is the long noncoding RNA Xist (X inactive specific transcript), which associates in cis with the inactive X chromosome elect (Xi) to mediate gene silencing and heterochromatization. Xist spreads across the entire Xi, but little is known about the dynamics of this process, particularly in the context of single cells. This thesis documents the development of a new, time-resolved super-resolution imaging methodology: RNA-SPLIT (Sequential Pulse Localisation Imaging over Time) combining Bgl-stem-loop labelling, HaloTag technology and 3D structured illumination microscopy (3D-SIM) to detect individual Xist RNA molecules in single mouse embryonic stem cells (mESC) with high spatial and temporal resolution. XY and XX mESCs expressing inducible autosomal transgenic or endogenous Bgl-stem-loop tagged Xist in tandem with BglG-Halo fusion protein were used to visualise distinct, functional Xist foci and analyse their spreading behaviour across the Xi during early XCI. RNA-SPLIT yielded important insights into Xist RNA kinetics, localisation and spreading behaviour during XCI and a feedback mechanism linking Xist RNA synthesis and degradation. RNA-SPLIT also uncovered novel coupling behaviour of Xist RNA molecules which, using perturbation experiments with CIZ-1 and SPEN, was shown to be independent of Xist localisation to the Xi, but dependent upon SPEN. In depth analysis demonstrated SPEN to have separable, SPOC domain independent functions in Xist RNA localisation, stability and coupling behaviour.
Supervisor: Coker, Heather ; Schermelleh, Lothar ; Brockdorff, Neil Sponsor: University of Oxford ; Medical Research Council
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.820773  DOI: Not available
Keywords: Developmental Biology ; Genetics ; Biochemistry
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