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Identification of X-linked genes that drive sex differences in murine embryonic stem cells

ABSTRACT: Dosage imbalance of X-chromosomal genes contributes to sex differences, in particular during early development, when both X chromosomes are active in females. X-encoded inhibitors of the differentiation-promoting MAP kinase (MAPK) signalling pathway slow down development, increase levels of naive pluripotency factors and decrease MAPK target gene expression. Through a hierarchical CRISPR screening approach in murine embryonic stem cells (mESC) we have comprehensively identified X-linked genes that modulate MAPK signalling, pluripotency factor expression and differentiation. We show that multiple genes act in concert to drive sex differences in mESC. Dusp9, a known negative regulator of the MAPK pathway alters phosphorylation of MAPK pathway intermediates in female cells, while Klhl13 underlies differences in pluripotency factor expression and differentiation by targeting differentiation-promoting factors for degradation. Klhl13 is a member of the Cullin 3 E3 ubiquitin ligase complex, where it acts as a substrate adaptor to target proteins for proteasomal degradation (Dhanoa et al., 2013). We reasoned that the Klhl13-mediated 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. To identify Klhl13 substrates in mESCs, we comprehensively profiled Klhl13 interaction partners and then selected those with increased protein levels in K13-HOM mutant cells. To identify interaction partners, we ectopically expressed either full-length Klhl13 or the substrate-binding Kelch domain, tagged with a green fluorescent protein (GFP), and identified binding partners by Immunoprecipitation-Mass Spectrometry (IP-MS) using a GFP-specific antibody. These experiments were performed in female mESCs with a homozygous Klhl13 deletion. By using this approach, we identify Scml2 and Alg13 as Klhl13 substrates.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Embryonic Stem Cell

SUBMITTER: David Meierhofer

LAB HEAD: Edda G. Schulz

PROVIDER: PXD016729 | Pride | 2021-03-18

REPOSITORIES: Pride

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Project description:Dosage imbalance of X-chromosomal genes contributes to sex differences, in particular during early development, when both X chromosomes are active in females. X-encoded inhibitors of the differentiation-promoting MAP kinase (MAPK) signalling pathway slow down development, increase levels of naive pluripotency factors and decrease MAPK target gene expression. Through a hierarchical CRISPR screening approach in murine embryonic stem cells (mESC) we have comprehensively identified X-linked genes that modulate MAPK signalling, pluripotency factor expression and differentiation. We show that multiple genes act in concert to drive sex differences in mESC. Dusp9, a known negative regulator of the MAPK pathway alters phosphorylation of MAPK pathway intermediates in female cells, while Klhl13 underlies differences in pluripotency factor expression and differentiation by targeting differentiation-promoting factors for degradation. Klhl13 is a member of the Cullin 3 E3 ubiquitin ligase complex, where it acts as a substrate adaptor to target proteins for proteasomal degradation (Dhanoa et al., 2013). We reasoned that the Klhl13-mediated 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. To identify Klhl13 substrates in mESCs, we comprehensively profiled Klhl13 interaction partners and then selected those with increased protein levels in K13-HOM mutant cells. To identify interaction partners, we ectopically expressed either full-length Klhl13 or the substrate-binding Kelch domain, tagged with a green fluorescent protein (GFP), and identified binding partners by Immunoprecipitation-Mass Spectrometry (IP-MS) using a GFP-specific antibody. These experiments were performed in female mESCs with a homozygous Klhl13 deletion. By using this approach, we identify Scml2 and Alg13 as Klhl13 substrates.

Project description:Recently, (in vitro) pluripotent EpiSCs were derived from the post-implantation egg cylinder stage epiblasts of mouse and rat. These EpiSCs resemble and correspond very closely to the conventional human embryonic stem cells (hESCs) in the colony morphology and culture/signaling requirements for maintaining pluripotency, but exhibit a range of significant phenotypic and signaling response differences from the conventional mouse ES cells (mESCs). These observations strongly support the notion that EpiSCs and hESCs are intrinsically similar, and raise an attractive hypothesis: as mESCs and EpiSCs/hESCs represent two distinct pluripotency states: the mESC-like state representing the ICM of pre-implantation blastcyst and the EpiSC-like state representing the post-implantation epiblasts, whether the epiblast state (including conventional hESCs) can be converted back to the ICM state. Despite studies providing evidence that epiblast-like cells exist and transition back and forth within colony of conventional mESCs; mESCs and EpiSCs share substantial set of pluripotency transcriptional factors, including Oct4, Sox2 and Nanog; and mESCs are more stable in culture, in the present study we found that EpiSCs differentiated rapidly under mESC culture conditions and no âspontaneouslyâ converted mESC could be readily identified and isolated over serial passages at the population or clonal level. Remarkably, we found that blockage of the TGFÎ² pathway or inhibition of the H3K4 demethylase LSD1 with small molecule inhibitors induced dramatic morphological changes of EpiSCs towards mESC phenotypes with activation of some ICM-specific gene expression. However, full conversion of EpiSCs to a mESC-like state with competence to chimeric contribution can only be readily generated with a combination of inhibitors of LSD1 and ALK. These observations underscore a powerful and direct induction of reprogramming from the developmentally later-stage EpiSCs to a mESC-like stage by a synergy of signaling and direct epigenetic modulations. It also highlights a significant role for TGFÎ² pathway inhibition in promoting reprogramming to and sustaining true pluripotency, which further supports our recent studies in generating chimerism-competent rat pluripotent cells. Collectively, our studies provide a proof-of-concept demonstration that pluripotency-restricted EpiSCs can be readily converted to a mESC-like state in the absence of any genetic manipulation by precise pharmacological control of signaling pathways that distinguish the two pluripotency states and an epigenetic target simultaneously, and offer a convenient experimental system to further study the mechanism. Such method and concept may also provide an avenue for generating a new type of mESC-like human pluripotent cell. Global gene-expression analyses of the parnate/mAMFGi cells

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Identification of X-linked genes that drive sex differences in murine embryonic stem cells

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