Figure 1.
X:autosome expression ratios in triploid cultures.
The mean (± standard deviation) X:A expression ratios are shown for each genotype indicated at the bottom (Xa: active X chromosome, Xi: inactive X chromosome, Y: Y chromosome, A: autosome). Three individual triploid fibroblast cultures of each genotype were assayed alongside two female (black square) and two male (gray diamond) diploid fibroblast cultures. The X:A expression ratios calculated from published microarray data for adult fibroblasts from 12 female and 11 male individuals are represented by white square and white diamond, respectively [10].
Figure 2.
Global absolute autosomal gene expression is similar between triploid and diploid cultures.
(A) Example of absolute gene expression levels determined by tiling array hybridization with gDNA and cDNA along a genomic region that includes three autosomal genes (PDE4A (phosphodiesterase 4A), KEAP1 (kelch-like ECH-associated protein 1), and EDG8 (endothelial differentiation sphingolipid G-protein-coupled receptor 8) in triploid (XXX;AAA) and diploid (XX;AA) cultures. Location of exons (dark blue) and introns (light blue) shown on top are from the UCSC browser [26]. Probes with a high cDNA/gDNA signal ratio are associated with exons, especially at the 3′ end of genes, while probes with a low signal ratio are associated with introns, intergenic regions and low-expressed genes (e.g. EDG8). (B) Scatter plot of absolute expression values for 5,473 autosomal genes in triploid XXX;AAA versus diploid XX;AA cultures. The trend had a high R2 value (0.90), indicating a highly correlated expression between triploid and diploid cultures. (C) Distributions of absolute expression of autosomal genes show a similar pattern in triploid and diploid cultures. Absolute expression levels were transformed into log2 and binned before graphing the data (see also Figure S4).
Figure 3.
Distributions of X-linked and autosomal gene expression in human triploid cultures.
(A) Normalized distribution curves representing expression of 362 X-linked genes differ between triploid and diploid cultures. The average distribution curve for three XaXaXi cultures is shifted to the right (higher values) compared to the average for two female (XaXi) diploid cultures, whereas the average curves for three XaXiXi cultures and three XYY cultures are both shifted left compared to the average for two female (XaXi) and two male (XY) diploid cultures. (B) Normalized distribution curves representing expression of 10,267 autosomal genes are similar between triploid and diploid cultures. Normalized expression levels were transformed into log2 and binned before graphing the data. The distribution curves were smoothed.
Figure 4.
Expression of individual X-linked genes in human triploid and diploid cultures.
Scatter plots of X-linked gene expression normalized to autosomal expression (in log2 scale). Red symbols indicate genes with more than two-fold higher expression, pink symbols, genes with one- to two-fold higher expression, light blue symbols, genes with one- to two-fold lower expression and blue symbols, genes with more than two-fold lower expression. The dotted lines represent two-fold (black), one-fold (gray) and 0.5-fold (black) cutoffs, respectively. (A) Higher expression of X-linked genes in XaXaXi versus XaXiXi cultures. (B) Similar expression of X-linked genes in XYY versus XaXiXi cultures. (C) Higher expression of X-linked genes in XaXaXi versus XaXi cultures. (D) Lower expression of X-linked genes in XaXiXi versus XaXi cultures. (E) Lower expression of X-linked genes in XYY versus XY cultures.
Figure 5.
Identification of dosage-sensitive X-linked genes and effects of the X inactive status on gene expression.
Scatter plots of X-linked gene expression normalized to autosomal expression (in log2 scale). (A) XaXiXi versus XaXi. (B) XYY versus XY. (C) XaXaXi versus XaXiXi. (D) XYY versus XaXiXi. (A,B) X-linked genes with similar expression (<1.2 fold change, p>0.2) in triploid (XaXiXi or XYY) versus diploid cultures are highlighted in (A,B): pink symbols: 82 X-linked genes with similar expression in XaXiXi versus XaXi; green symbols: 62 X-linked genes with similar expression in XYY versus XY; black symbols: 26 X-linked genes with similar expression both in XaXiXi versus XaXi and in XYY versus XY; gray symbols: 192 X-linked genes with no evidence of similar expression either in XaXiXi versus XaXi or in XYY versus XY. (C,D) X-linked genes that escape X inactivation are highlighted in (C,D). The escape status of X-linked genes were obtained from a previous study [14]: gray symbols (0Xi): X-linked genes subject to X inactivation (not expressed in rodent x human hybrid cells that retain an inactive human X); light blue symbols (1-6Xi): X-linked genes with variable escape (expressed in 1-6/9 hybrid lines); purple symbols (7-9Xi): X-linked genes that consistently escape (expressed in 7-9/9 hybrid lines). The dotted lines represent two-fold (black), one-fold (gray) and 0.5-fold (black) cutoffs, respectively.
Table 1.
Average fold change in expression of X-linked genes grouped either by sex-matched triploid/diploid comparisons or by X inactivation status.
Figure 6.
Expression of PAR1 genes and non-pseudoautosomal X/Y gene pairs in triploid and diploid cultures.
(A,B) Expression of PAR1 genes is similar between triploid and diploid cultures. (A) Average expression levels (± standard deviation) normalized to autosomal gene expression are shown in log2 for nine PAR1genes [PLCXD1: phosphatidylinositol-specific phospholipase C, X domain containing 1; GTPBP6: GTP binding protein 6; SLC25A6:solute carrier family 25 (mitochondrial carrier; adenine nucleotide translocator), member 6; CXYorf2: chromosome X and Y open reading frame 2; ASMTL: acetylserotonin O-methyltransferase-like; DHRSX: dehydrogenase/reductase (SDR family) X-linked; CD99: CD99 molecule; ZBED1: zinc finger, BED-type containing 1; SFRS17A: splicing factor, arginine/serine-rich 17A] in XaXaXi, XaXiXi, XYY triploid cultures, and in XaXi and XY diploid controls. (B) Average expression fold changes between triploid cultures with one or two Xa's and between female and male diploid cultures for nine PAR1 genes were not significantly different from 1.0 (p = 0.43, 0.29, 0.24, and 0.07 respectively; independent one-sample t-test, n = 9). (C) Cumulative expression of X- and Y-linked paralogs of XY gene pairs in human triploid and diploid cultures. Expression of the X- and Y-linked genes was normalized to autosomal gene expression. Stacked bars show the cumulative expression of X (solid bar) and Y paralogs (striped bar) in XaXaXi, XaXiXi, XYY triploid cultures, and XaXi and XY diploid controls. The X-linked paralogs are shown with (+) and (−) error bars in XXX and XX cell cultures and only with (−) error bars in XYY and XY cell cultures. The Y-linked paralogs are shown with (+) error bars. UTX/UTY: ubiquitously transcribed tetratricopeptide repeat, X chromosome/Y-linked; CXorf15/CYorf15A & B: chromosome X open reading frame 15/chromosome Y open reading frame 15A & chromosome Y open reading frame 15B; EIF1AX/EIF1AY: eukaryotic translation initiation factor 1A, X-linked/Y-linked; ZFX/ZFY: zinc finger protein, X-linked/Y-linked; DDX3X/DDX3Y: DEAD (Asp-Glu-Ala-Asp) box polypeptide 3, X-linked/Y-linked; RPS4X/RPS4Y1:ribosomal protein S4, X-linked/Y-linked 1; USP9X/USP9Y: ubiquitin specific peptidase 9, X-linked/Y-linked; JARID1C/JARID1D: jumonji, AT rich interactive domain 1C/jumonji, AT rich interactive domain 1D; TMSB4X/TMSB4Y: thymosin beta 4, X-linked/Y-linked; TBL1X/TBL1Y: transducin (beta)-like 1X-linked/Y-linked.