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Free keywords:
X chromosome
Gene dosage
Embryonic stem cells
Sex differences
Pluripotency
MAPK signaling
CRISPR screen
Dusp9
Klhl13
Abstract:
Background: X-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.
Results: To 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.
Conclusions: Here, 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.
