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

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.

The Pluripotency Factor Nanog Protects against Neuronal Amyloid β-Induced Toxicity and Oxidative Stress through Insulin Sensitivity Restoration

Amyloid β (Aβ) is a peptide fragment of the amyloid precursor protein that triggers the progression of Alzheimer’s Disease (AD). It is believed that Aβ contributes to neurodegeneration in several ways, including mitochondria dysfunction, oxidative stress and brain insulin resistance. Therefore, protecting neurons from Aβ-induced neurotoxicity is an effective strategy for attenuating AD pathogenesis. Recently, applications of stem cell-based therapies have demonstrated the ability to reduce the progression and outcome of neurodegenerative diseases. Particularly, Nanog is recognized as a stem cell-related pluripotency factor that enhances self-renewing capacities and helps reduce the senescent phenotypes of aged neuronal cells. However, whether the upregulation of Nanog can be an effective approach to alleviate Aβ-induced neurotoxicity and senescence is not yet understood. In the present study, we transiently overexpressed Nanog—both in vitro and in vivo—and investigated the protective effects and underlying mechanisms against Aβ. We found that overexpression of Nanog is responsible for attenuating Aβ-triggered neuronal insulin resistance, which restores cell survival through reducing intracellular mitochondrial superoxide accumulation and cellular senescence. In addition, upregulation of Nanog expression appears to increase secretion of neurotrophic factors through activation of the Nrf2 antioxidant defense pathway. Furthermore, improvement of memory and learning were also observed in rat model of Aβ neurotoxicity mediated by upregulation of Nanog in the brain. Taken together, our study suggests a potential role for Nanog in attenuating the neurotoxic effects of Aβ, which in turn, suggests that strategies to enhance Nanog expression may be used as a novel intervention for reducing Aβ neurotoxicity in the AD brain.

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

X-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.