Figure 2. Rlim is required for the maintenance of Xist expression and Xist clouds at blastocyst stages.
(A) Expression profiles from the Xist locus in preimplantation embryos WT and KO for Rlim. Error bars indicate standard error of the mean (SEM). Significant differences of Xist levels in WT and KO females P<0.01 are indicated (Student’s t-test). (B) WT and RlimKO embryos at E2.5 were co-stained with probes recognizing Xist (red) and Rlim (green) via RNA FISH. Two representative RlimΔ/Δ embryos are shown. The boxed area is magnified in the panel below. Arrows point at Xist cloud and Rlim transcription focus. (C) RNA FISH on WT and RlimKO embryos at E3.5. The boxed area is magnified in the panels below. Arrows point at Xist and Rlim transcription foci. (D) Tabular summary of Xist clouds detected in B and C.
Figure 2—figure supplement 1. Rlim regulates Xist levels and Xist clouds at blastocyst stages.
(A) Modified from the UCSC Genome Browser: Cumulative mapped raw reads of pooled E4.5 female embryos WT/WT (blue) or Δ/Δ (red) for Rlim on the Tsix/Xist gene locus (variable scales). Structure of the Tsix and Xist genes is shown below in blue. Exons are boxed. Arrows indicate direction of transcription. Two alternatively spliced forms of Xist are shown. Note that >90% of reads in the locus map within the Xist transcription unit. (B) Random downsizing each library to 200.000 reads does not significantly change/affect Xist expression profiles. Representative results (n = 3) obtained from the WT/RlimKO dataset with each library randomly adjusted to 200k reads. (C) RT-qPCR analysis of Xist expression in RlimWT/WTand RlimΔ/Δ at E2.5, E3.5 and E4.5. Data represent values obtained from three embryos for each stage and genotype. Gender of each embryo was determined via Zfy. Xist values were normalized against actin and are shown relative to WT/WT control. Error bars indicate SEM.