Extensive cellular heterogeneity of X inactivation revealed by single-cell allele-specific expression in human fibroblasts

Abstract

X-chromosome inactivation (XCI) provides a dosage compensation mechanism where, in each female cell, one of the two X chromosomes is randomly silenced. However, some genes on the inactive X chromosome and outside the pseudoautosomal regions escape from XCI and are expressed from both alleles (escapees). We investigated XCI at single-cell resolution combining deep single-cell RNA sequencing with whole-genome sequencing to examine allelic-specific expression in 935 primary fibroblast and 48 lymphoblastoid single cells from five female individuals. In this framework we integrated an original method to identify and exclude doublets of cells. In fibroblast cells, we have identified 55 genes as escapees including five undescribed escapee genes. Moreover, we observed that all genes exhibit a variable propensity to escape XCI in each cell and cell type and that each cell displays a distinct expression profile of the escapee genes. A metric, the Inactivation Score-defined as the mean of the allelic expression profiles of the escapees per cell-enables us to discover a heterogeneous and continuous degree of cellular XCI with extremes represented by "inactive" cells, i.e., cells exclusively expressing the escaping genes from the active X chromosome and "escaping" cells expressing the escapees from both alleles. We found that this effect is associated with cell-cycle phases and, independently, with the XIST expression level, which is higher in the quiescent phase (G0). Single-cell allele-specific expression is a powerful tool to identify novel escapees in different tissues and provide evidence of an unexpected cellular heterogeneity of XCI.

Keywords: X inactivation; XIST; cell cycle; cell heterogeneity; single-cell transcriptomics.

Fig. 1.

Identification and elimination of confounding doublets. (A) Flowchart of the study. Whole- genome sequencing for each individual identified the respective heterozygous sites. Single-cell RNA-seq provided RNA abundance for each single cell. (B) Heat map of unsupervised hierarchical clustering using cell–cell pairwise Pearson correlation coefficients of AR from single cells from individual 3 before (Left) and after (Right), excluding the doublets. The heat map separated the cells expressing one haplotype (blue and green cluster) from cells expressing two haplotypes (black cluster, doublets). Pearson correlations range from −1 (red) to 1 (white).

Fig. 2.

Single-cell ASE profile of 22 robust escapee genes in human female fibroblasts. Composite figure of individual ARs per gene per cell (Top). AR profile of robust escapee genes (listed the rows) with a detectable expression in single cells (ordered along the columns) is shown. Every dot represents the AR of the respective gene in a cell. AR ranges from 0 (light orange) to 1 (dark blue). The size of the dot is proportional to the respective number of reads detected per cell. %, the percentage of cells where the respective gene is escaping XCI. (Bottom) Bar plot representing the IS (see text for details) per cell. IS ranges from 0 (light orange) to 1 (dark blue).

Fig. 3.

Cellular propensity to escape XCI. (A) Cells ranked by the IS calculated on all escapees in the robust set (Escapee) and on all inactivated genes (Inactive) per individual (color coded; see legend). Each dot represents a fibroblast cell. (B) Y axis: cells ordered according to the percentage of escapees displaying an inactive status (AR ≥ 0.95, with respect to all detectable escapees) per individual (x axis). Each dot represents a fibroblast cell.

Fig. 4.

XIST expression association and correlation with IS and its repressive role for X-chromosome genes. Manhattan-like plot for Pearson correlation of X-linked gene expression with IS. The plot represents −log₁₀ P value against the X-chromosome position. Vertical lines represent Pseudoautosomal Region (PAR) limits. Red horizontal lines represent Bonferroni significance thresholds for positively and negatively correlated genes, respectively.

Fig. 5.

XIST expression and IS dependency on the cell cycle. Distribution of XIST expression (Left) and of ISs per single cells (Right) according to cell-cycle phases (n = number of cells per stage). P values calculated with Mann–Whitney U test (see Materials and Methods for details).