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. 2021 Feb 19;22(3):e51989. doi: 10.15252/embr.202051989

Figure EV4. System to simultaneously visualise H3K27me3 or H4K20me1 and Xist RNA in living cells.

Figure EV4

  1. Measurement of H4K20me1 (by IF‐RNA FISH) and H4K20me2/3 (by IF in TX‐Xist‐mCherry line) enrichment on the Xi. Xist‐inducible female cells were treated with DOX for 24 h prior to fixation. Scale bar = 2 μm.
  2. Schematic representation of the TX‐BglSL parental line obtained previously (Dossin et al, 2020). This system is based on the TX1072 female hybrid ESC line where Xist is induced by DOX treatment from the endogenous B6 allele. In TX‐BglSL 18 Bgl stem loops have been knocked‐in into the 7th exon of Xist.
  3. Schematic representation of the TX‐Xist‐EGFP; H4K20me1‐mCherry ESC line. This line is derived from TX‐BglSL by knocking‐in BglG‐EGFP expression cassette into Rosa 26 locus. Homologous recombination was aided by using nickase Cas9 protein and two gRNAs targeting Rosa 26. Knock‐in can occur only at one allele as the other one contains the rTTA element allowing for DOX‐inducible expression of Xist RNA. Stable lines expressing H4K20me1 specific mintbody fused to mCherry were generated by random insertion of a PiggyBac vector (bottom).
  4. Schematic representation of the TX‐Xist‐mCherry; H3K27me3‐sfGFP ESC line. This line is derived from TX‐BglSL by knocking‐in BglG‐mCherry expression cassette into Tigre locus. Homologous recombination was aided by using wild‐type Cas9 protein and a single gRNA targeting Tigre. Stable lines expressing H3K27me3 specific mintbody fused to sfGFP were generated by random insertion of a PiggyBac vector (bottom).
  5. Validation of efficient X chromosome inactivation in TX‐Xist‐EGFP; H4K20me1‐mCherry and TX‐Xist‐mCherry; H3K27me3‐sfGFP ESC lines. Cells were treated with DOX for 0 or 24 h; pyrosequencing was performed from cDNAs. Allelic ratios of two X‐linked genes (Rnf12 and AtrX) were measured. Note comparable levels of gene silencing in both clones.
  6. Quantification of Xist induction efficiency during live imaging analysis upon DOX treatment. Percentage Xist‐Bgl‐EGFP clouds was manually assessed in at least 30 cells.