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. 2016 Nov;48(11):1430-1435.
doi: 10.1038/ng.3678. Epub 2016 Sep 26.

Analysis of Allelic Expression Patterns in Clonal Somatic Cells by Single-Cell RNA-seq

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Free PMC article

Analysis of Allelic Expression Patterns in Clonal Somatic Cells by Single-Cell RNA-seq

Björn Reinius et al. Nat Genet. .
Free PMC article

Abstract

Cellular heterogeneity can emerge from the expression of only one parental allele. However, it has remained controversial whether, or to what degree, random monoallelic expression of autosomal genes (aRME) is mitotically inherited (clonal) or stochastic (dynamic) in somatic cells, particularly in vivo. Here we used allele-sensitive single-cell RNA-seq on clonal primary mouse fibroblasts and freshly isolated human CD8+ T cells to dissect clonal and dynamic monoallelic expression patterns. Dynamic aRME affected a considerable portion of the cells' transcriptomes, with levels dependent on the cells' transcriptional activity. Notably, clonal aRME was detected, but it was surprisingly scarce (<1% of genes) and mainly affected the most weakly expressed genes. Consequently, the overwhelming majority of aRME occurs transiently within individual cells, and patterns of aRME are thus primarily scattered throughout somatic cell populations rather than, as previously hypothesized, confined to patches of clonally related cells.

Conflict of interest statement

The authors declare no competing financial interests.

Figures

Figure 1
Figure 1. The vast majority of aRME in primary mouse fibroblasts is dynamic.
(a) Schematic overview of the experimental design. (b) Histogram showing percent aRME in randomly picked primary mouse fibroblasts (n=163). (c) Percent autosomal genes with bi- or monoallelic expression in single cells, after aggregating all cells of a clone, and expected levels from randomly pooling allelic calls of the same number of non-clonal cells. Dots represent percent monoallelic genes observed in single cells and bars depict the average. Observed allelic expression on the X-chromosome is shown to the right of each clonal set. Biallelic genes on female X-chromosomes are X-inactivation escapees (Supplementary Fig. 7–9). Sex and number of sequenced cells (out of all cells of the clone) are indicated above. (d) Percent of genes with aRME when sequencing transcriptomes of 5 or 15 pooled clonal cells (dots), and values obtained after in silico pooling of clonal or non-clonal cells shown as boxplots; indicating median (belt), interquartile range (box) and farthest points at maximum 1.5 times the interquartile rage (whiskers). Expression threshold in (bd): RPKM>20. (e) Percent clonal aRME in the seven clones (circles) (observed minus expected), for genes detected either above 20 or 1 RPKM. The P-value denotes a median significantly greater than zero (one-sided Wilcoxon test). “ns”: not significant.
Figure 2
Figure 2. Scarce clonal aRME in low-expressed genes
(a) Genes with parental-specific (imprinted) monoallelic expression, ordered and colored according to chromosomal localization, with log10 P-values (Fisher’s exact test) on y-axis. (b) Gene scatter with fraction cells having consistent maternal or paternal monoallelic expression (x-axis) plotted against the log10 P-values (Fisher’s exact test). Genes with P<0.05 and consistent monoallelic in ≥90% of expressing cells were classified parent-of-origin-specific monoallelic, all being known imprinted genes except the novel candidates in brackets (DLX5 is known imprinted in human). (c) Test on clonal aRME (as in (a)) for male primary fibroblast clone 6 (n=38 cells), and scatterplot (as in (b)). E-values denote expected number of false positives above thresholds. (d) Test on clonal aRME for female primary fibroblast clone 7 (n=60 cells) and scatterplot (as in (c)). (e) Expression-level boxplots of clonal aRME (colored) and other genes (gray) in clones 6 and 7. P-values from two-sided Wilcoxon test. (f) Boxplots of relative expression of clonal aRME genes, over whole cell populations (left), or single cells expressing monoallelic (clonal or dynamic aRME) in both clone and non-clonal cells. P-values signify deviation from equal expression (two-sided Wilcoxon test). Additional boxplots (right) show relative expression levels in cells with dynamic aRME and biallelic expression, demonstrating the expected ~1:2 expression-level when transcribing 1 versus 2 alleles. (af) Threshold: RPKM>1.
Figure 3
Figure 3. Dynamic and clonal aRME in human T-cells.
(a) Schematic representation of the experimental design. (b) Boxplots of percent aRME genes in HLA-B7 restricted T-cells, isolated at day 15 (red) or 136 (blue) after vaccination; and in all in vivo- (green) and ex vivo-expanded (orange) T-cells. Threshold: RPKM>20. (c) Upper panel: Percent aRME in 32 in vivo T-cell clones (circle: per cell, dot: median) and in all in vivo T-cells as a boxplot to the right. Lower panel: Percent clone-consistent aRME (observed minus expected, from sampled in silico pooling) as blue boxplots. The pink boxplot (lower right) and red solid line show median percent clonal aRME estimated over all clones. P-value for clonal aRME above zero according to a one-sided Wilcoxon test. Threshold: RPKM>1. (d) Example of gene-level identification on an ex vivo-expanded T-cell clone (clone C), showing highly significant clonal aRME of KLRB1 (Scatter plots as described in Fig. 2c–d).
Figure 4
Figure 4. Cellular size and cell-cycle phase affect the degree dynamic aRME.
(a) Observed allelic expression in primary fibroblasts of large and small cellular size. P-value: two-sided Wilcoxon test. (b) Percent monoallelic expression in large and small fibroblasts inferred by split-cell analysis. P-value: two-sided Wilcoxon test. (c) Scatter plot and Pearson correlation of percent aRME and cell size (estimated by FACS) in ex vivo-expanded T-cells. (d) Principal component analysis (scatterplot of component 1 and 2) of fibroblasts using the top 100 most-variable cell-cycle genes, with cells colored according to cell-cycle classification. (e) Boxplots of percent aRME in fibroblasts of different cell-cycle phases. P-values: two-sided Wilcoxon test. (ad) Expression threshold: RPKM>20.

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References

    1. Elowitz MB, Levine AJ, Siggia ED, Swain PS. Stochastic gene expression in a single cell. Science. 2002;297:1183–1186. - PubMed
    1. Suter DM, et al. Mammalian genes are transcribed with widely different bursting kinetics. Science. 2011;332:472–474. - PubMed
    1. Cook DL, Gerber AN, Tapscott SJ. Modeling stochastic gene expression: implications for haploinsufficiency. Proc Natl Acad Sci USA. 1998;95:15641–15646. - PMC - PubMed
    1. McAdams HH, Arkin A. Stochastic mechanisms in gene expression. Proc Natl Acad Sci USA. 1997;94:814–819. - PMC - PubMed
    1. Raj A, Rifkin SA, Andersen E, van Oudenaarden A. Variability in gene expression underlies incomplete penetrance. Nature. 2010;463:913–918. - PMC - PubMed

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