scholarly journals Identification of X-chromosomal genes that drive sex differences in embryonic stem cells through a hierarchical CRISPR screening approach

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Oriana Genolet ◽  
Anna A. Monaco ◽  
Ilona Dunkel ◽  
Michael Boettcher ◽  
Edda G. Schulz
Keyword(s):  
Stem Cells ◽  
Sex Differences ◽  
Embryonic Stem Cells ◽  
Genetic Basis ◽  
Embryonic Stem ◽  
Pluripotent Cells ◽  
Screening Approach ◽  
Factor Expression ◽  
Chromosomal Genes ◽  
Pluripotency Factor

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.

scholarly journals Identification of X-chromosomal genes that drive global X-dosage effects in mammals

2020 ◽  
Author(s):  
Oriana Genolet ◽  
Anna A. Monaco ◽  
Ilona Dunkel ◽  
Michael Boettcher ◽  
Edda G. Schulz
Keyword(s):  
Sex Differences ◽  
Stem Cell ◽  
Target Genes ◽  
Embryonic Stem ◽  
Adaptor Protein ◽  
Induced Pluripotent Stem Cell ◽  
Pluripotent Cells ◽  
Factor Expression ◽  
Chromosomal Genes ◽  
Pluripotency Factor

AbstractX-chromosomal genes contribute to sex differences, in particular during early development, when both X chromosomes are active in females. Here, 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) signalling pathway and delaying differentiation. To identify the genetic basis of these sex differences, we have performed a series of CRISPR knockout screens in murine embryonic stem cells to comprehensively identify X-linked genes that cause the female pluripotency phenotype. We found multiple genes that act in concert, among which Klhl13 plays a central role. We show that this E3 ubiquitin ligase substrate adaptor protein promotes pluripotency factor expression, delays differentiation and represses MAPK target genes, and we identify putative substrates. We thus elucidate the mechanisms that drive sex-induced differences in pluripotent cells with implications for gender medicine in the context of induced pluripotent stem cell based therapies.

scholarly journals Identification of Novel miRNAs in Mouse Embryonic Stem Cells, Embryonic Fibroblasts, and Reprogrammed Pluripotent Cells

2019 ◽  
Author(s):  
Botao Zhao ◽  
Chunsun Fan
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Embryonic Stem Cells ◽  
De Novo ◽  
Embryonic Stem ◽  
Induced Pluripotent Stem Cell ◽  
Mouse Embryonic Stem Cells ◽  
Pluripotent Cells ◽  
Embryonic Fibroblasts ◽  
Species Specific

AbstractMicroRNAs (miRNAs) are a class of non-coding small RNAs that function in almost every known cellular activity. MiRNAs play an important role in gene regulation that controls embryonic stem cell (ESC) pluripotency and differentiation, as well as induced pluripotent stem cell (iPSC) reprogramming. In this study, we identified nine novel miRNAs by mining the deep sequencing dataset from mouse embryonic stem cells, mouse embryonic fibroblasts (MEF) and three kinds of reprogrammed pluripotent cells. Most of them are non-conserved but species-specific and cell-specific miRNAs. Eight miRNAs are derived from gene introns, including a “mirtron” miRNA, miR-novel-41. We also showed that miR-novel-27 is a mouse-specific miRNA and the 5′ arm of its precursor hairpin, embedding the mature miR-novel-27, uniquely exists in mouse species but not in any other Placentalia animals. Notably, the 5′ arm of the pre-miR-novel-27 hairpin shows nearly perfect palindrome to the 3′ arm suggesting that it was generated by inverted duplication of the 3′ arm. By this mechanism, the pre-miR-novel-27 hairpin was de novo gained in the mouse genome. This is a new type of de novo miRNA emergence mechanism in animals, which we called “inverted local half hairpin duplication” here. In addition, very limited nucleotide mutants accumulated on the newly emerged 5′ arm since its birth suggesting an especially young evolutionary history of the miR-novel-27 gene.

scholarly journals Reciprocal Transcriptional Regulation of Pou5f1 and Sox2 via the Oct4/Sox2 Complex in Embryonic Stem Cells

2005 ◽  
Vol 25 (14) ◽  
pp. 6031-6046 ◽  
Author(s):  
Joon-Lin Chew ◽  
Yuin-Han Loh ◽  
Wensheng Zhang ◽  
Xi Chen ◽  
Wai-Leong Tam ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Regulatory Element ◽  
Embryonic Stem ◽  
Preimplantation Embryos ◽  
Distal Enhancer ◽  
Human Escs ◽  
Pluripotent Cells ◽  
Specific Expression ◽  
Endogenous Expression

ABSTRACT Embryonic stem cells (ESCs) are pluripotent cells that can either self-renew or differentiate into many cell types. Oct4 and Sox2 are transcription factors essential to the pluripotent and self-renewing phenotypes of ESCs. Both factors are upstream in the hierarchy of the transcription regulatory network and are partners in regulating several ESC-specific genes. In ESCs, Sox2 is transcriptionally regulated by an enhancer containing a composite sox-oct element that Oct4 and Sox2 bind in a combinatorial interaction. It has previously been shown that Pou5f1, the Oct4 gene, contains a distal enhancer imparting specific expression in both ESCs and preimplantation embryos. Here, we identify a composite sox-oct element within this enhancer and show that it is involved in Pou5f1 transcriptional activity in ESCs. In vitro experiments with ESC nuclear extracts demonstrate that Oct4 and Sox2 interact specifically with this regulatory element. More importantly, by chromatin immunoprecipitation assay, we establish that both Oct4 and Sox2 bind directly to the composite sox-oct elements in both Pou5f1 and Sox2 in living mouse and human ESCs. Specific knockdown of either Oct4 or Sox2 by RNA interference leads to the reduction of both genes' enhancer activities and endogenous expression levels in addition to ESC differentiation. Our data uncover a positive and potentially self-reinforcing regulatory loop that maintains Pou5f1 and Sox2 expression via the Oct4/Sox2 complex in pluripotent cells.

scholarly journals Pluripotency factor binding and Tsix expression act synergistically to repress Xist in undifferentiated embryonic stem cells

2011 ◽  
Vol 4 (1) ◽  
pp. 17 ◽  
Author(s):  
Tatyana B Nesterova ◽  
Claire E Senner ◽  
Janina Schneider ◽  
Tilly Alcayna-Stevens ◽  
Anna Tattermusch ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Embryonic Stem ◽  
Factor Binding ◽  
Pluripotency Factor

scholarly journals X-chromosome dosage as a modulator of pluripotency, signalling and differentiation?

2017 ◽  
Vol 372 (1733) ◽  
pp. 20160366 ◽  
Author(s):  
Edda G. Schulz
Keyword(s):  
Stem Cells ◽  
Sex Differences ◽  
Embryonic Stem Cells ◽  
X Chromosome ◽  
Mammalian Species ◽  
Embryonic Stem ◽  
X Chromosome Inactivation ◽  
Chromosome Inactivation ◽  
Hormone Production ◽  
Similar Phenotype

Already during early embryogenesis, before sex-specific hormone production is initiated, sex differences in embryonic development have been observed in several mammalian species. Typically, female embryos develop more slowly than their male siblings. A similar phenotype has recently been described in differentiating murine embryonic stem cells, where a double dose of the X-chromosome halts differentiation until dosage-compensation has been achieved through X-chromosome inactivation. On the molecular level, several processes associated with early differentiation of embryonic stem cells have been found to be affected by X-chromosome dosage, such as the transcriptional state of the pluripotency network, the activity pattern of several signal transduction pathways and global levels of DNA-methylation. This review provides an overview of the sex differences described in embryonic stem cells from mice and discusses a series of X-linked genes that are associated with pluripotency, signalling and differentiation and their potential involvement in mediating the observed X-dosage–dependent effects. This article is part of the themed issue ‘X-chromosome inactivation: a tribute to Mary Lyon’.

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