Supplementary MaterialsDocument S1. to X-chromosome reactivation. The extent to which increased X-chromosome dosage (X-dosage) in female iPSCs compared with male iPSCs leads to differences in the properties of iPSCs is still unclear. We show that chromatin accessibility in mouse iPSCs is modulated by X-dosage. Specific sets of transcriptional regulator motifs are enriched in chromatin with increased accessibility in XX or XY iPSCs. The transcriptome, growth and pluripotency exit are also modulated by X-dosage in iPSCs. To understand how increased X-dosage modulates the properties of mouse pluripotent stem cells, we used heterozygous deletions of the X-linked gene (dual-specificity phosphatase 9) is in part responsible for inhibiting DNMT3A/B/L and global DNA methylation in XX ESCs (Choi et?al., 2017a). The expression level of is higher in XX ESCs than in XY ESCs, and overexpression of in XY ESCs induced female-like global DNA hypomethylation and a female-like proteome. Conversely, heterozygous deletion of in XX ESCs restored male-like global DNA methylation, suggesting that is responsible for MAPK-mediated DNMT3A/B repression. However, whether heterozygous deletion in XX ESCs has effects on the transcriptional regulatory network, open chromatin landscape, and pluripotency exit has not yet been explored. In addition, how and which X-linked genes modulate the pluripotency gene network of naive PSCs remains unclear. Furthermore, novel insights may be gained by identification of heterozygous XX ESCs maintain female-like chromatin accessibility, growth, and delayed exit from pluripotency in the presence of male-like global DNA methylation. Altogether, our study uncovers X-dosage as a previously unrecognized modulator of chromatin accessibility and of growth in PSCs. Our results clarify the effects of X-dosage on the pluripotency transcriptome, revealing the uncoupling of DNA methylation from chromatin accessibility. This provides principles for using gene dosage in designing experiments to understand the epigenetic and genetic mechanisms regulating cell identity. Results Differences in Transcriptional Landscapes and Pluripotency Exit Correlate with the Presence of XaXa in iPSCs To explore the importance of X-dosage on the transcriptome and pluripotency exit of mouse iPSCs, we derived XX and XY iPSC lines. We used isogenic mouse embryonic fibroblasts (MEFs) carrying a tetO inducible transgene encoding the reprogramming factors in the locus and the reverse tetracycline transactivator (M2rtTA) in the locus (Figure?1A and Table S1) (Carey et?al., 2010, Pasque et?al., 2018). After 16?days of doxycycline (dox) treatment to induce reprogramming, 10 female and 11 male iPSC lines were expanded on feeders in the presence of serum and leukemia inhibitory factor (LIF) SU 5416 enzyme inhibitor (S/L) in the absence of dox (Figure?1A), or adapted to dual ERK/GSK3 inhibition and LIF conditions (2i/L). This scheme allowed us to compare female and male iPSCs without the influence of differences in genetic background, reprogramming system, or derivation method. Both female and male iPSCs could be propagated over multiple passages while maintaining their morphology, indicative of self-renewal, and expressed pluripotency-associated factors NANOG and DPPA4 (Figures 1B, 1C, S1A, and S1B). As expected, the transcriptome of our iPSCs was similar to that of naive ESCs (Figure?S1C). Thus, derivation of isogenic female and male iPSCs allowed us to systematically compare SU 5416 enzyme inhibitor the transcriptome MSK1 and epigenome of these cells. Open in a separate window Figure?1 Two X chromosomes Modulate the Transcriptome, Cellular Growth, and Pluripotency Exit in Mouse iPSCs (A) Scheme of female and male SU 5416 enzyme inhibitor iPSCs derivation, characterization, and differentiation. (B) Representative images of female and male iPSCs/ESCs grown on feeders in S/L. Scale bar, 50?m. (C) Immunofluorescence analysis for NANOG/DPPA4 in iPSCs grown in S/L. Representative images of all lines examined for NANOG (red), DPPA4 (green), and DAPI (blue, nuclei counterstaining) are shown. Scale bar, 50?m. (D) (i) Mean expression ratio to autosomes for sex chromosomes and chromosomes 8 and 9. The dosage of X- and Y-linked genes was used to infer XX, XY, XO, and partial XO (pXO) genotypes. (ii) Representative karyotype images of XX and XO iPSC lines grown in S/L. (E) Unsupervised hierarchical clustering of top 200 most variable autosomal genes in XY, XX, pXO, and XO iPSCs. Early-passage iPSCs cluster by X-dosage, late-passage iPSCs do not. (F) DEG analysis, identifying clear differences between XX and XY iPSCs, but not XO and XY iPSCs (log2fold log21.5, FDR 0.05). (G) Western blot analysis for NANOG, DNMT3B, and DUSP9 protein in SU 5416 enzyme inhibitor iPSCs grown in.