RNA sequencing of Dusp9 and Klhl13 mutants in 1.8 female mouse embryonic stem cells (mESCs)
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA596761 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR10741300.fastq.gz | Fastqsanger.gz | |||
| SRR10741301.fastq.gz | Fastqsanger.gz | |||
| SRR10741302.fastq.gz | Fastqsanger.gz | |||
| SRR10741303.fastq.gz | Fastqsanger.gz | |||
| SRR10741304.fastq.gz | Fastqsanger.gz |
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Project description:Dosage imbalance for 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. Here, we carried out transcriptional profiling of the two top hits, Dusp9 and Klhl13, and observe that Klhl13 contributes more to the X-dosage induced transcriptome changes than Dusp9, and that a combined effect of both can explain about 50% of the observed sex differences.
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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.
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