A Primary Role for the Tsix lncRNA in Maintaining Random X-Chromosome Inactivation

Abstract

Differentiating pluripotent epiblast cells in eutherians undergo random X-inactivation, which equalizes X-linked gene expression between the sexes by silencing one of the two X-chromosomes in females. Tsix RNA is believed to orchestrate the initiation of X-inactivation, influencing the choice of which X remains active by preventing expression of the antisense Xist RNA, which is required to silence the inactive-X. Here we profile X-chromosome activity in Tsix-mutant (X(ΔTsix)) mouse embryonic epiblasts, epiblast stem cells, and embryonic stem cells. Unexpectedly, we find that Xist is stably repressed on the X(ΔTsix) in both sexes in undifferentiated epiblast cells in vivo and in vitro, resulting in stochastic X-inactivation in females despite Tsix-heterozygosity. Tsix is instead required to silence Xist on the active-X as epiblast cells differentiate in both males and females. Thus, Tsix is not required at the onset of random X-inactivation; instead, it protects the active-X from ectopic silencing once X-inactivation has commenced.

Figure 1. Xist is induced from the X^ΔTsix in E5.25 male epiblast cells

(A) Diagram illustrating WT Xist and Tsix loci and the ΔTsix mutation. Dotted lines indicate the locations of strand-specific (ss) RNA FISH probes. Filled arrowheads mark the locations of RT-PCR primer pairs. 1 (orange arrowheads), Xist RT-PCR amplicon; 2 (blue arrowheads), Tsix exon 4 RT-PCR amplicon; 3 (purple arrowheads), Tsix RT-PCR amplicon spanning exons 2–4. (B) RT-PCR amplification of Tsix (exon 4) and Xist RNAs in E5.25 epiblasts. M, marker; NTC, no template control; +, reaction with reverse transcriptase (RT); −, no RT control lane. (C) Strand-specific RNA FISH detection of Xist RNA (green) and Tsix RNA (red) coupled with IF staining for NANOG (purple) in isolated epiblasts and extra-embryonic ectoderm, which serves as a negative control for NANOG expression. Nuclei are stained blue with DAPI. Scale bar, 10 μm. Right, quantification of Xist and Tsix expression in NANOG-positive epiblast nuclei. The X-axis of each graph represents the average percentage of nuclei per embryo in each class (n = 3 embryos/genotype; 49–68 nuclei/embryo). Diagrams along the Y-axis depict all observed expression patterns. Error bars represent the standard deviation of data from 3 different embryos. *, p ≤ 0.001 (Chi-square test). (D) Xist RNA coating (green) and H3-K27me3 enrichment (purple) in E5.25 X^ΔTsixY epiblast nuclei. (E) Silencing of the X-linked gene Pgk1 (red) in E5.25 X^ΔTsixY epiblast nuclei upon ectopic Xist RNA coating (green).

Figure 2. Ectopic Xist RNA induction in differentiated but not undifferentiated X^ΔTsixY EpiSCs and ESCs

(A) RT-PCR amplification of Xist and Tsix RNAs in undifferentiated and differentiated XY and X^ΔTsixY EpiSC lines (2 and 4 cell lines, respectively). β-actin amplification serves as control. M, marker; +, reaction with reverse transcriptase (RT); −, no RT control lane. (B) RNA FISH detection of Xist RNA (green) and Tsix RNA (red) in representative undifferentiated and day (d) 10 differentiated EpiSC lines (XY line number [no.] 1; X^ΔTsixY line no. 2). Nuclei are stained blue with DAPI. Scale bar, 10 μm. (C) Quantification of Xist RNA coated nuclei in undifferentiated (d0) and d5 and d10 differentiated EpiSC lines. Scale bar, 10 μm. Only cells with a single Xist locus detected by DNA FISH (left) following RNA FISH were counted; n=100 nuclei/cell line. (D) RNA FISH detection of Xist (green) combined with IF detection of H3-K27me3 (red) in d10 differentiated X^ΔTsixY EpiSCs. Data from two different lines are shown. (E) Silencing of Pgk1 (red) upon Xist RNA (green) coating in representative d10 differentiated X^ΔTsixY EpiSCs. (F–G) RT-PCR (F) and RNA FISH (G) detection of Xist and Tsix RNAs in undifferentiated or embryoid body-differentiated XY and X^ΔTsixY ESC lines (2 and 3 lines, respectively). Scale bar, 10 μm. (H) Quantification of Xist RNA coated nuclei in the differentiated ESC lines. Only cells with one Xist locus detected by DNA FISH (left) following RNA FISH were counted; n=100 nuclei/cell line. Scale bar, 10 μm. See also Figs. S1–S2 and Table S1.

Figure 3. Xist expression in E6.5 and E5.25 WT and Tsix-heterozygous female epiblast cells

(A) Allele-specific RT-PCR detection of Tsix (exon 4) and Xist RNAs in epiblasts of three individual WT (X^LabX^JF1) and Tsix-heterozygous (X^ΔTsixX^JF1) E6.5 embryos. M, marker; NTC, no template control; +, reaction with reverse transcriptase (RT); −, no RT control lane. Bottom, Sanger sequencing of the amplified cDNAs. Blue highlights mark a SNP that differs between the X^Lab / X^ΔTsix and X^JF1 mouse strains. (B) RT-PCR followed by Pyrosequencing-based quantification of allelic Xist expression in epiblasts of individual E6.5 embryos. Error bars represent the standard deviation of data from 3 different embryos. (C–D) RNA FISH detection of Xist and Tsix RNAs coupled with IF detection of NANOG in isolated E6.5 (C) and E5.25 (D) epiblasts. Nuclei are stained blue with DAPI. Scale bars, 10 μm. Bottom, quantification of Xist and Tsix expression. The X-axis of each graph represents the average percentage of nuclei in each class (n = 3 embryos/genotype; 100 nuclei/E6.5 embryo and 45–71 nuclei/E5.25 embryo). Diagrams along the Y-axis depict all observed expression patterns. Error bars represent the standard deviation of data from 3 different embryos. *, p ≤0.01 (Chi-square test). (E) RT-PCR amplification of Tsix (exon 4) and Xist RNAs in WT and Tsix-heterozygous epiblasts. Bottom, Sanger sequencing of the Tsix and Xist cDNAs. (F) RT-PCR followed by Pyrosequencing-based quantification of allelic Xist expression in epiblasts of individual E5.25 embryos. Error bars represent the standard deviation of data from 3 different embryos. No significant differences in allelic Xist expression were observed between WT and Tsix-mutant embryos (p = 0.44, E5.25 X^LabX^JF1 vs. X^ΔTsixX^JF1; p = 0.46, E5.25 X^JF1X^Lab vs. X^JF1X^ΔTsix; Welch’s two-sample T-test.). See also Figure S3.

Figure 4. Lack of biased X-inactivation in undifferentiated Tsix-heterozygous EpiSC lines

(A) RT-PCR amplification of Tsix RNA from WT and Tsix-heterozygous EpiSC lines. M, marker; NTC, no template control; +, reaction with reverse transcriptase (RT); −, no RT control lane. (B) Representative Sanger sequencing chromatograms of Tsix cDNAs. (C) RT-PCR followed by Pyrosequencing-based quantification of allelic expression of Xist and the X-linked genes Rnf12 and Atrx. Each bar represents an individual EpiSC line. X_m, maternal X-chromosome; X_p, paternal X-chromosome. Error bars represent the standard deviation of ≥3 independent results. The mean and median of allelic expression of Xist, Rnf12, and Atrx lack significant difference (p > 0.1, Welch’s two-sample T-test and Mood’s Median test) between parent-of-origin matched WT and mutant EpiSCs. See also Figure S1 and Table S1.

Figure 5. Change in allelic Xist expression in differentiating Tsix heterozygous EpiSC lines

(A) RT-PCR followed by Pyrosequencing-based quantification of Xist expression in EpiSC lines (cell line numbers in parentheses) differentiated for 0, 5, 10, 15, and 20 days (d). X_m, maternal X-chromosome; X_p, paternal X-chromosome. Each bar represents an individual EpiSC line. Error bars represent the standard deviation of ≥3 independent results. See also Figures S4 and S5.

Figure 6. Ectopic Xist RNA coating in differentiated Tsix-heterozygous EpiSC lines

(A) RNA FISH detection of Xist RNA (green) and Tsix RNA (red) in representative undifferentiated and d10 differentiated WT and Tsix-heterozygous EpiSC lines (X^JF1X^Lab cell line no. 1; X^ΔTsixX^JF1 cell line no. 14). Nuclei are stained blue with DAPI. Scale bars, 10 μm. (B) RNA FISH detection of Xist RNA coat using an exonic probe (white) and nascent Xist RNA with an intronic probe (red), demonstrating that in cells with two Xist RNA coats both Xist alleles are transcribed. (C) Quantification of EpiSC nuclei displaying single vs. double Xist RNA coats during differentiation. Scale bar, 10 μm. Only cells with two Xist loci detected by DNA FISH (left) following RNA FISH were counted; n=100 nuclei/cell line. (D) Enrichment of H3-K27me3 on Xist RNA coated X-chromosomes in d10 differentiated X^ΔTsixX^JF1 EpiSCs. Data from two different lines (nos. 5 and 14) are shown. (E) Silencing of Pgk1 (red) upon ectopic Xist RNA coating (green) in d10 differentiated X^ΔTsixX^JF1 EpiSCs. (F) Reduced phospho-H3 staining, a marker of cell proliferation, in d10 differentiated X^ΔTsixX^JF1 EpiSCs (cell line 14) with two Xist RNA coats compared to nuclei with a single Xist coat (p<0.001, Fisher’s exact test). (G) Increased death of cells with two inactive-Xs compared to cells with one inactive-X in d10 differentiated X^ΔTsixX^JF1 EpiSCs (p<0.001, Welch’s two-sample T-test). The inactive-X is marked by H3-K27me3 accumulation (purple). Ethd-1 (red) marks dead cells and Calcein AM (green) marks live cells. (H) Reduced cell counts during differentiation of Tsix-heterozygous compared to WT EpiSCs. (I–J) Reduced viability of adherent (I) and non-adherent cells in suspension (J) during differentiation of Tsix-heterozygous compared to WT EpiSCs. See Table S2 for statistical comparisons; see also Figs. S6–S7.

Figure 7. A model of Tsix function in X-inactivation

At the onset of X-inactivation, Tsix heterozygous epiblast cells undergo stochastic X-inactivation indistinguishable from WT epiblasts. Upon continued differentiation of the epiblast cells, the X^ΔTsix ectopically induces Xist RNA. In female cells that had originally inactivated the WT X-chromosome, ectopic Xist induction accompanies the initiation of X-inactivation a second time (of the X^ΔTsix), resulting in two inactive-Xs. As a result of a paucity of X-linked gene expression, these cells are selected away due both to reduced proliferation and induced cell death. Thus, the developing embryo is ultimately populated only with cells that had originally inactivated the X^ΔTsix.