Abstract
BackgroundX-chromosomal genes contribute to sex differences, in particular during early development, when both X chromosomes are active in females. Double X-dosage shifts female pluripotent cells towards the naive stem cell state by increasing pluripotency factor expression, inhibiting the differentiation-promoting MAP kinase (MAPK) signaling pathway, and delaying differentiation.ResultsTo identify the genetic basis of these sex differences, we use a two-step CRISPR screening approach to comprehensively identify X-linked genes that cause the female pluripotency phenotype in murine embryonic stem cells. A primary chromosome-wide CRISPR knockout screen and three secondary screens assaying for different aspects of the female pluripotency phenotype allow us to uncover multiple genes that act in concert and to disentangle their relative roles. Among them, we identify Dusp9 and Klhl13 as two central players. While Dusp9 mainly affects MAPK pathway intermediates, Klhl13 promotes pluripotency factor expression and delays differentiation, with both factors jointly repressing MAPK target gene expression.ConclusionsHere, we elucidate the mechanisms that drive sex-induced differences in pluripotent cells and our approach serves as a blueprint to discover the genetic basis of the phenotypic consequences of other chromosomal effects.
Highlights
X-chromosomal genes contribute to sex differences, in particular during early development, when both X chromosomes are active in females
Pooled CRISPR knockout screen identifies X-chromosomal MAP kinase (MAPK) regulators The X chromosome encodes ~ 1000 genes, any of which could potentially mediate the sex differences observed in murine pluripotent stem cells with respect to pluripotency factor expression, MAPK pathway activity, and differentiation efficiency [11, 12]
We reasoned that the Klhl13mediated sex differences we have identified might be due to reduced protein abundance of Klhl13 substrate proteins in female compared to male cells, which affect pluripotency factors, differentiation, and MAPK target gene expression
Summary
X-chromosomal genes contribute to sex differences, in particular during early development, when both X chromosomes are active in females. Since no fetal hormones are produced at this stage, these observations have been attributed to variations in sex-chromosomal dosage, which in mice has been confirmed by the analysis of X-monosomic XO embryos [9, 10] These sex differences have been investigated at the molecular level in female mouse embryonic stem cells (mESC), which are derived from early blastocyst embryos and carry two active X chromosomes. Similar patterns have been observed in induced pluripotent stem cells (iPSCs) [12] These Xdosage effects are likely mediated by X-encoded genes that modulate the stem cell state, the identity of which remains mostly unknown. They might pose a biological checkpoint to ensure that only cells that have successfully inactivated one of their X chromosomes contribute to the differentiated adult organism
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