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Quick Fluorescent In Situ Hybridization Protocol for Xist RNA Combined with Immunofluorescence of Histone Modification in X-chromosome Inactivation
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Xist RNA can silence genes that escape X-chromosome inactivation beyond early development. This process, dependent on SPEN, impacts gene activity and chromosome structure, with sustained upregulation leading to irreversible silencing.

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Area of Science:

  • Genetics
  • Epigenetics
  • Developmental Biology

Background:

  • X-chromosome inactivation (XCI) silences one X chromosome in XX females via Xist noncoding RNA.
  • Some X-linked genes escape XCI, maintaining expression on the inactive X chromosome.

Purpose of the Study:

  • To investigate if Xist RNA can silence XCI escapee genes beyond early embryogenesis.
  • To explore the role of Xist RNA in regulating the three-dimensional topology of the inactive X chromosome.

Main Methods:

  • Increasing endogenous Xist RNA levels in vitro and in vivo.
  • Analyzing differentiated cells and mouse pre- and post-implantation embryos.
  • Investigating the role of SPEN in Xist-mediated silencing and topological domain elimination.

Main Results:

  • Xist RNA can silence escapee genes in differentiated cells and in mouse embryos, extending beyond early development.
  • Xist RNA, dependent on SPEN, eliminates topologically associating domain-like structures in clusters of escapees.
  • Xist-mediated silencing and topological domain elimination are initially reversible but become irreversible with sustained Xist upregulation, leading to DNA methylation.

Conclusions:

  • Xist RNA controls gene activity and the 3D topology of the inactive X chromosome beyond early developmental stages.
  • The findings reveal a broader regulatory role for Xist RNA in maintaining X chromosome silencing and structure.