X Chromosome Dosage Modulates Multiple Molecular and Cellular Properties of Mouse Pluripotent Stem Cells Independently of Global DNA Methylation Levels

During early mammalian development, the two X-chromosomes in female cells are active. Dosage compensation between XX female and XY male cells is then achieved by X-chromosome inactivation in female cells. Reprogramming female mouse somatic cells into induced pluripotent stem cells (iPSCs) leads to X-chromosome reactivation. The extent to which increased X-chromosome dosage (X-dosage) in female iPSCs leads to differences in the molecular and cellular properties of XX and XY 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. We show that the transcriptome, growth and pluripotency exit are also modulated by X-dosage in iPSCs. To understand the mechanisms by which increased X-dosage modulates the molecular and cellular properties of mouse pluripotent stem cells, we used heterozygous deletions of the X-linked gene Dusp9 in XX embryonic stem cells. We show that X-dosage regulates the transcriptome, open chromatin landscape, growth and pluripotency exit largely independently of global DNA methylation. Our results uncover new insights into X-dosage in pluripotent stem cells, providing principles of how gene dosage modulates the epigenetic and genetic mechanisms regulating cell identity. Transcriptome profiles of early and late passage female induced Pluripotent Stem Cells (XX/XO iPSCs), early and late passage male induced Pluripotent Stem Cells (XY iPCSs), wild-type female Embryonic Stem Cells (WT XX ESCs), wild-type male Embryonic Stem Cells (WT XY ESCs), Dusp9 heterozygous mutants (Dusp9+/- XX ESCs) were generated by next generation sequencing using Illumina HiSeq 4000. In total, 32 samples were analyzed including: three early XX and three late passage XO iPSC lines, three early and three late passage XY iPSCs lines, three WT XX ESC lines, three WT XY ESC lines and two independent Dusp9 clones. Where indicated, samples originate from two different timepoints of LIF withdrawal differentiation experiment (0h, 24h), and samples are in technical replicate ("b"). Open chromatin profiles of early and late passage female induced Pluripotent Stem Cells (XX/XO iPSCs), early and late passage male induced Pluripotent Stem Cells (XY iPCSs), wild-type female Embryonic Stem Cells (WT XX ESCs), wild-type male Embryonic Stem Cells (WT XY ESCs), Dusp9 heterozygous mutants (Dusp9+/- XX ESCs) were generated by next generation sequencing using Illumina HiSeq 4000. In total, 20 samples were analyzed including: three early XX and three late passage XO iPSC lines, three early and three late passage XY iPSCs lines, three WT XX ESC lines, three WT XY ESC lines and two independent Dusp9 clones.