Abstract
ABSTRACTWe previously identified the cyclin dependent kinase Cdk8 as a putative silencing factor for Xist. To investigate its role in X inactivation, we engineered a Cdk8 mutation in mouse embryonic stem cells (ESCs) carrying an inducible system for studying Xist function. We found that Xist repressed X-linked genes at half of the expression level in Cdk8 mutant cells, whereas they were almost completely silenced in the controls. Lack of Cdk8 impaired Ezh2 recruitment and the establishment of histone H3 lysine 27 tri-methylation but not PRC1 recruitment by Xist. Transgenic expression of wild-type but not catalytically inactive Cdk8 restored efficient gene repression and PRC2 recruitment. Mutation of the paralogous kinase Cdk19 did not affect Xist function, and combined mutations of Cdk8 and Cdk19 resembled the Cdk8 mutation. In mice, a Cdk8 mutation caused post-implantation lethality. We observed that homozygous Cdk8 mutant female embryos showed a greater developmental delay than males on day 10.5. Together with the inefficient repression of X-linked genes in differentiating Cdk8 mutant female ESCs, these data show a requirement for Cdk8 in the initiation of X inactivation.
Highlights
Mammals achieve dosage compensation for the different number of X chromosomes in male and female cells by silencing of the transcription of one of the two X chromosomes (Lyon, 1962)
Cdk8 is required for efficient gene repression by Xist For investigating the effect of Cdk8 on Xist function, we engineered a mutation of Cdk8 in mouse HATX3 embryonic stem cells (ESCs) that carry a doxycyclineinducible Xist allele (Fig. 1A)
Biochemical fractionation showed that Cdk8 is localized to the nucleus in ESCs and can be detected in the chromatin-associated fraction (Fig. 1H), which is consistent with its function in gene regulation
Summary
Mammals achieve dosage compensation for the different number of X chromosomes in male and female cells by silencing of the transcription of one of the two X chromosomes (Lyon, 1962). X chromosome inactivation (XCI) is initiated by the expression of the long noncoding Xist RNA, which localizes to the future inactive X chromosome (Xi), and triggers chromatin modifications and gene repression in an almost chromosome-wide manner (Galupa and Heard, 2018). The process of chromosomal silencing has been studied in mice and mouse embryonic stem cells (ESCs). In female mouse embryos, Xist is expressed from the paternally inherited X chromosome at the four-cell stage and imprinted XCI is observed. In the cells of the inner cell mass of the blastocyst that form the epiblast lineage, imprinted XCI is reversed and two active X chromosomes are present in the female embryos.
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