The role of Xist‐mediated Polycomb recruitment in the initiation of X‐chromosome inactivation

Abstract Xist RNA has been established as the master regulator of X‐chromosome inactivation (XCI) in female eutherian mammals, but its mechanism of action remains unclear. By creating novel Xist‐inducible mutants at the endogenous locus in male mouse embryonic stem (ES) cells, we dissect the role of the conserved A‐B‐C‐F repeats in the initiation of XCI. We find that transcriptional silencing can be largely uncoupled from Polycomb repressive complex 1 and complex 2 (PRC1/2) recruitment, which requires B and C repeats. Xist ΔB+C RNA specifically loses interaction with PCGF3/5 subunits of PRC1, while binding of other Xist partners is largely unaffected. However, a slight relaxation of transcriptional silencing in Xist ΔB+C indicates a role for PRC1/2 proteins in early stabilization of gene repression. Distinct modules within the Xist RNA are therefore involved in the convergence of independent chromatin modification and gene repression pathways. In this context, Polycomb recruitment seems to be of moderate relevance in the initiation of silencing.

. Lack of PRC2 and PRC1 recruitment to the Xist DB+C-bound X chromosome is not caused by deficient chromosomal RNA coating.
A Representative images of combined IF for JARID2 (green) on the left and for RING1B (green) on the right with RNA FISH for Xist (red) in Xist-TetOP lines (for clone 1 of each mutant type) upon D2 in the presence of DOX; DAPI in blue; scale bar: 10 lm. B Graph representing the mean % + SEM of Xist-coated chromosomes enriched for JARID2, EZH2, and RING1B in the different Xist-TetOP mutants (for clone 1 of each mutant type) from 2 to 4 independent experiments. A minimum of 50 Xist-coated chromosomes were counted per experiment. Only P-values corresponding to significant differences from unpaired Student's t-test, comparing mutants to Xist FL, are indicated as *P < 0.05. C Normalized Xist RNA levels retrieved after actinomycin D treatment for 2, 4, and 6 h measured by RT-qPCR compared to 18S rRNA; shown are the mean of three independent biological replicates normalized to the non-treated conditions (0 h); errors bars represent SEM. D Graph represents the mean area (above) and total intensity (below) of Xist RNA FISH signal in Xist FL and Xist DB+C-induced cells at day 2 of differentiation and in female MEFs; error bars represent SEM; a minimum of 71 Xist signals were counted per cell line; significant differences from unpaired Student's t-test, comparing mutants to Xist FL, are indicated as *P < 0.05 or ***P < 0.01. E Representative deconvoluted images from Z-projection images of RNA FISH using Stellaris fluorescent-labeled oligonucleotides targeting Xist in MEFs, Xist FL, and Xist DB+C-induced cells used for the quantification used in Fig EV3B; Scale bar: 10 lm.
Source data are available online for this figure. Xist FL (noDOX)

Xist FL DOX
Xist ΔB+C DOX C Figure EV3. Quality check of ChIRP procedure in Xist FL and Xist DB+C cells.
A RT-qPCR with three primer pairs along Xist to evaluate RNA retrieval after ChIRP procedure for Xist FL (in noDOX and DOX conditions) and Xist DB+C (DOX) at day 3 of differentiation. B Table showing  ▸ Figure EV4. Normalization of nChIP-seq to the percentage of Xist-induced cells in Xist FL and Xist DB+C confirms residual enrichment of PcG marks at initially active X-linked genes.
A Normalized signal of H3K27me3 and H2AK119ub around HoxC cluster (chr15: 102,840,000-103,110,000); shown is the signal of each sample around these cluster, normalized by the size of the library. B Barplot representing percentages of H3K27me3 and H2AK119ub reads mapping on X chromosome (chrX) in each sample. C Violin plots quantifying H3K27me3 and H2AK119ub enrichment over intergenic regions, initially active promoters, and initially active gene bodies on chrX and on autosomes in Xist FL and Xist DB+C cell lines upon DOX induction at day 2 of differentiation; shown is the distribution of the calculated log2 fold change in DOX versus noDOX conditions, the horizontal band is the median, and the lower and upper hinges correspond to the 25 th and 75 th percentiles; P-values were calculated using unilateral Wilcoxon test, comparing chrX and autosomal enrichment of PcG marks for each genomic region. D Table showing     A Genome browser plots showing RNA-seq reads on Xist/Tsix genes for Xist FL, Xist DA, and Xist DB+C mutants in DOX and noDOX conditions at day 2 of differentiation; yellow boxes display the deleted regions in both Xist DA and Xist DB+C. B Barplot representing percentages of RNA-seq reads mapping on X chromosome (chrX) in each sample. C Table showing the percentage of cells exhibiting an Xist-coated chrX for the different duplicates of Xist FL, Xist DA, and Xist DB+C in DOX and noDOX conditions as determined by Xist RNA FISH; at least 500 cells were counted to estimate the percentage of cells with a Xist-coated chrX. D Violin plots displaying the distribution of the average log2(fold change) in gene expression between DOX and noDOX conditions on chrX and autosomes in Xist FL, Xist DA, and Xist DB+C after normalization for the percentage of cells with a Xist-coated chrX; the horizontal band is the median of the values, and the lower and upper hinges correspond to the 25 th and 75 th percentiles; n = indicates the number of genes analyzed; P-values for chrX were calculated using a paired Wilcoxon test. E Plots display the comparison of log2(fold change) in X-linked gene silencing upon DOX induction between Xist FL and Xist DB+C at day 2 of differentiation; Limma ttest did not find any gene differentially expressed between Xist FL and Xist DB+C. F Box plots displaying the normalized read enrichment at promoters for H3K27me3 and H2AK119ub upon DOX induction for distinct categories of X-linked genes with different degrees of gene silencing between DOX and noDOX conditions in both Xist FL and Xist DB+C; the horizontal band of the box plot is the median of the values, the lower and upper hinges correspond to the 25 th and 75 th percentiles, the upper whisker extends from the hinge to the largest value not further than 1.5 interquartile range from the hinge, and the lower whisker extends from the hinge to the smallest value at most 1.5 interquartile range of the hinge; P-values were calculated using a Wilcoxon test; numbers inside the box plots indicate the number of genes analyzed. G Box plots displaying H3K27me3 and H2AK119ub normalized enrichment levels at promoters upon induction in two categories of X-linked genes: with no or little accumulation versus with accumulation of these PcG marks in induced Xist DB+C cells; the horizontal band of the box plot is the median of the values, the lower and upper hinges correspond to the 25 th and 75 th percentiles, the upper whisker extends from the hinge to the largest value not further than 1.5 interquartile range from the hinge, and the lower whisker extends from the hinge to the smallest value at most 1.5 interquartile range of the hinge; P-values were calculated using a Wilcoxon test; n = indicates the number of genes analyzed. H Box plots displaying the CpG content of promoters that accumulate or not H3K27me3/H2AK119ub between noDOX and DOX conditions in Xist DB+C at day 2 of differentiation; the horizontal band of the box plot is the median of the values, and the lower and upper hinges correspond to the 25 th and 75 th percentiles; the upper whisker extends from the hinge to the largest value not further than 1.5 interquartile range from the hinge, and the lower whisker extends from the hinge to the smallest value at most 1.5 interquartile range of the hinge; P-values were calculated using a Wilcoxon test; numbers inside the box plots indicate the number of promoters analyzed.
Source data are available online for this figure.