Double X-chromosomal dosage leads to substantial sex differences during early development, before the process of X chromosome inactivation has occurred. Since these observations occur before fetal hormones are produced, they have been attributed to variations in X-chromosomal dosage. Sex differences can be also observed by using mESCs as an in vitro model, where female cells display lower levels of the differentiationinducing MAPK signaling pathway leading to higher expression of pluripotency factors and slower differentiation kinetics and are therefore found in a more naïve state of pluripotency compared to their male counterparts. These effects are underlied by X-linked MAPK inhibitors present in a double dosage in female mESCs, whose identity, until now, has remained obscured. We have therefore carried out a series of CRISPR knockout screens to identify the genes behind the female pluripotency phenotype, finding in an unbiased and high-throughput manner several genes that act together to shift female mESCs to a more naive state of pluripotency. Among these genes, we find the E3 ubiquitin ligase adaptor protein Klhl13, which induces higher pluripotency factor expression, slower differentiation kinetics and lower MAPK target gene expression. Klhl13 acts together with Dusp9, a known Erk phosphatase and MAPK inhibitor, to mediate the sex differences observed in mESC, as double Klhl13 and Dusp9 heterozygous mutants qualitatively recapitulate the male pluripotency phenotype. Dusp9, however, seems to act primarily on the MAPK signaling pathway as an Erk phosphatase, as its effects on pluripotency factor expression and differentiation kinetics are considerably weaker compared to the effects mediated by Klhl13. We have additionally determined, through Klhl13 pull-down experiments and proteomics profiling of Klhl13 homozygous mutants, several putative Klhl13 target proteins: Alg13, Scml2, Peg10, Larp1 and Cct3. Alg13 might represent a pluripotency-destabilizing factor, as it was found depleted in screens implemented for the identification of X-linked genes that increase pluripotency factor expression and lead to slower differentiation kinetics. The role of the other putative candidates in mediating the effects observed in Klhl13 mutants remains, however, subject of further study. We have thus identified several genes behind the sex differences observed in mESCs in an unbiased manner, and we have validated the top two candidates: Klhl13 and Dusp9. This work is the first to link Klhl13 to the modulation of pluripotency and to the sex differences observed in mESCs. Additionally, it has begun to shed some light into possible mechanisms through which Klhl13 might mediate the reported female pluripotency phenotype.
