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RNA-seq in differentiating Xmas cells ES cells [Xmas Differentiation RNA-seq]

ABSTRACT: Major complications with in vitro culture of female embryonic stem cells (ESC) have impeded study of sex-specific pluripotency; however, from the published work female pluripotency significantly differs to male. We report a replenishable female ESC system that has enabled us to optimise a protocol for preserving the XX karyotype. Our protocol also improves male ESC fitness. To demonstrate the utility of the system, we screened for regulators of the female-specific process of X chromosome inactivation, revealing a new role for chromatin remodellers Smarcc1 and Smarca4 in establishment of X inactivation. The remodellers create a nucleosome depleted region at gene promotors on the inactive X during exit from pluripotency, without which gene silencing fails. Our female ESC system provides a tractable model for XX ESC culture that will expedite study of female pluripotency and has enabled us to discover new features of the female-specific process of X inactivation. This experiment is designed to test if Xmas ES cells are transcriptionally similar to published ES cell lines during differentiation.

ORGANISM(S): Mus musculus

PROVIDER: GSE137156 | GEO | 2022/02/02

REPOSITORIES: GEO

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DNA-seq in male ES cells cultured by either standard ES cell culture or our improved culture conditions [Male DNA-seq]

Project description:Major complications with in vitro culture of female embryonic stem cells (ESC) have impeded study of sex-specific pluripotency; however, from the published work female pluripotency significantly differs to male. We report a replenishable female ESC system that has enabled us to optimise a protocol for preserving the XX karyotype. Our protocol also improves male ESC fitness. To demonstrate the utility of the system, we screened for regulators of the female-specific process of X chromosome inactivation, revealing a new role for chromatin remodellers Smarcc1 and Smarca4 in establishment of X inactivation. The remodellers create a nucleosome depleted region at gene promotors on the inactive X during exit from pluripotency, without which gene silencing fails. Our female ESC system provides a tractable model for XX ESC culture that will expedite study of female pluripotency and has enabled us to discover new features of the female-specific process of X inactivation. This experiment is designed to test the effect of our improved ES culture conditions on the male ES cell karyotype.

2022-10-31 | GSE137158 | GEO

RNA-seq in male ES cells cultured by either standard ES cell culture or our improved culture conditions [Male RNA-seq]

2022-10-31 | GSE137157 | GEO

RNA-seq in differentiating female FVBxCAST F1 ES cells with Smarca4 gene knockdown [Female Smarca4 RNA-seq]

Project description:Major complications with in vitro culture of female embryonic stem cells (ESC) have impeded study of sex-specific pluripotency; however, from the published work female pluripotency significantly differs to male. We report a replenishable female ESC system that has enabled us to optimise a protocol for preserving the XX karyotype. Our protocol also improves male ESC fitness. To demonstrate the utility of the system, we screened for regulators of the female-specific process of X chromosome inactivation, revealing a new role for chromatin remodellers Smarcc1 and Smarca4 in establishment of X inactivation. The remodellers create a nucleosome depleted region at gene promotors on the inactive X during exit from pluripotency, without which gene silencing fails. Our female ESC system provides a tractable model for XX ESC culture that will expedite study of female pluripotency and has enabled us to discover new features of the female-specific process of X inactivation. This experiment is designed to test if X chromosome inactivation is altered upon Smarca4 gene knockdown.

2022-02-02 | GSE137162 | GEO

RNA-seq in differentiating female FVBxCAST F1 ES cells with Smarcc1 gene knockdown [Female Smarcc1 RNA-seq]

Project description:Major complications with in vitro culture of female embryonic stem cells (ESC) have impeded study of sex-specific pluripotency; however, from the published work female pluripotency significantly differs to male. We report a replenishable female ESC system that has enabled us to optimise a protocol for preserving the XX karyotype. Our protocol also improves male ESC fitness. To demonstrate the utility of the system, we screened for regulators of the female-specific process of X chromosome inactivation, revealing a new role for chromatin remodellers Smarcc1 and Smarca4 in establishment of X inactivation. The remodellers create a nucleosome depleted region at gene promotors on the inactive X during exit from pluripotency, without which gene silencing fails. Our female ESC system provides a tractable model for XX ESC culture that will expedite study of female pluripotency and has enabled us to discover new features of the female-specific process of X inactivation. This experiment is designed to test if X chromosome inactivation is altered upon Smarcc1 gene knockdown.

2022-02-02 | GSE137160 | GEO

NOMe-seq in differentiating FVBxCAST F1 ES cells with Smarcc1 gene knockdown [NOMe-Seq]

Project description:Major complications with in vitro culture of female embryonic stem cells (ESC) have impeded study of sex-specific pluripotency; however, from the published work female pluripotency significantly differs to male. We report a replenishable female ESC system that has enabled us to optimise a protocol for preserving the XX karyotype. Our protocol also improves male ESC fitness. To demonstrate the utility of the system, we screened for regulators of the female-specific process of X chromosome inactivation, revealing a new role for chromatin remodellers Smarcc1 and Smarca4 in establishment of X inactivation. The remodellers create a nucleosome depleted region at gene promotors on the inactive X during exit from pluripotency, without which gene silencing fails. Our female ESC system provides a tractable model for XX ESC culture that will expedite study of female pluripotency and has enabled us to discover new features of the female-specific process of X inactivation. This experiment is designed to test if nucleosome remodelling during X chromosome inactivation is altered upon Smarcc1 gene knockdown.

2022-02-02 | GSE137159 | GEO

X Chromosome Dosage Modulates Multiple Molecular and Cellular Properties of Mouse Pluripotent Stem Cells Independently of Global DNA Methylation Levels

Project description:During early mammalian development, the two X-chromosomes in female cells are active. Dosage compensation between XX female and XY male cells is then achieved by X-chromosome inactivation in female cells. Reprogramming female mouse somatic cells into induced pluripotent stem cells (iPSCs) leads to X-chromosome reactivation. The extent to which increased X-chromosome dosage (X-dosage) in female iPSCs leads to differences in the molecular and cellular properties of XX and XY iPSCs is still unclear. We show that chromatin accessibility in mouse iPSCs is modulated by X-dosage. Specific sets of transcriptional regulator motifs are enriched in chromatin with increased accessibility in XX or XY iPSCs. We show that the transcriptome, growth and pluripotency exit are also modulated by X-dosage in iPSCs. To understand the mechanisms by which increased X-dosage modulates the molecular and cellular properties of mouse pluripotent stem cells, we used heterozygous deletions of the X-linked gene Dusp9 in XX embryonic stem cells. We show that X-dosage regulates the transcriptome, open chromatin landscape, growth and pluripotency exit largely independently of global DNA methylation. Our results uncover new insights into X-dosage in pluripotent stem cells, providing principles of how gene dosage modulates the epigenetic and genetic mechanisms regulating cell identity.

2018-03-31 | GSE110215 | GEO

Reinforced STAT3 activity sustains self-renewal of human embryonic stem cells in a naive-like state

Project description:Only rodent embryonic stem (ES) cells can self-renew in the pristine state of pluripotency called the naive or ground state. Human ES (hES) cells self-renew in the so-called primed state of pluripotency, which is an obstacle to research, hindering cost-effective cultivation in media devoid of animal-derived products, genetic stability, and genome engineering. Here we show that forced expression of a hormone-dependent STAT3-ERT2, in combination with LIF and inhibitors of MEK and GSK3beta, allows hES cells to escape from the primed state, and enter a new state designated as TL2i, characterized by the activation of STAT3 target genes, regular passaging by single cell dissociation, and the expression of naive state-specific transcription factors. We used microarrays to analyse the gene expression changes occuring during the adaptation to the naive culture conditions. We then compared our dataset to previously published dataset of mouse ESc and EpiSc, human primed and naive ESc, and human embryos.

2014-12-01 | GSE55708 | GEO

An Alternative Splicing Switch Regulates Embryonic Stem Cell Pluripotency and Reprogramming [RNA-Seq]

Project description:Alternative splicing (AS) is a key process underlying the expansion of proteomic diversity and the regulation of gene expression. However, the contribution of AS to the control of embryonic stem cell (ESC) pluripotency is not well understood. Here, we identify an evolutionarily conserved ESC-specific AS event that changes the DNA binding preference of the forkhead family transcription factor FOXP1. We show that the ESC-specific isoform of FOXP1 stimulates the expression of transcription factor genes required for pluripotency including OCT4, NANOG, NR5A2 and GDF3, while concomitantly repressing genes required for ESC differentiation. Remarkably, this isoform also promotes the maintenance of ESC pluripotency and the efficient reprogramming of somatic cells to induced pluripotent stem cells. These results thus reveal that an AS switch plays a pivotal role in the regulation of pluripotency and functions by controlling critical ESC-specific transcriptional programs. Exons 18 and 18b form a mutually exclusive splicing event. The FOXP1 (non-ES) isoform contains only exon 18 and not 18b, while the FOXP1-ES isoform contains only exon 18b and not 18. To investigate whether FOXP1 and FOXP1-ES control different sets of genes, we performed knockdowns using custom siRNA pools targeting FOXP1 exons 18 or 18b in undifferentiated H9 cells, followed by RNA-Seq profiling.

2011-09-14 | E-GEOD-30995 | biostudies-arrayexpress

Genome wide nucleosome specifity and function of chromatin remodellers in embryonic stem cells [Chd8]

Project description:How various ATP-dependent chromatin remodellers bind to nucleosomes to regulate transcription is not well defined in mammalian cells. Here, we present genome-wide remodeller-interacting nucleosome profiles for Chd1, Chd2, Chd4, Chd6, Chd8, Chd9, Brg1 and Ep400 in mouse embryonic stem (ES) cells. These remodellers bind to nucleosomes at specific positions, either at one or both nucleosomes that flank each side of nucleosome-free promoter regions (NFRs), at enhancer elements, or within gene bodies. At promoters, bidirectional transcription commonly initiates on either side of remodeller-bound nucleosomes. Transcriptome analysis upon remodeller depletion reveals reciprocal mechanisms of transcriptional regulation by remodellers. At active genes, certain remodellers are positive regulators of transcription, whereas others act as repressors. At bivalent genes, which are bound by repressive Polycomb complexes, the same remodellers act in the opposite way. Together, these findings reveal how remodellers integrate promoter nucleosomal architecture to regulate ES cell transcription programs.

2016-01-26 | GSE64784 | GEO

Genome wide nucleosome specifity and function of chromatin remodellers in embryonic stem cells [Ep400]

2016-01-26 | GSE64786 | GEO

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