Screens In Embryonic Stem Cells Research Articles

A CRISPR/Cas9 screen in embryonic stem cells reveals that Mdm2 regulates totipotency exit

During early embryonic development, the transition from totipotency to pluripotency is a fundamental and critical process for proper development. However, the regulatory mechanisms governing this transition remain elusive. Here, we conducted a comprehensive genome-wide CRISPR/Cas9 screen to investigate the 2-cell-like cells (2CLCs) phenotype in mouse embryonic stem cells (mESCs). This effort led to the identification of ten regulators that play a pivotal role in determining cell fate during this transition. Notably, our study revealed Mdm2 as a significant negative regulator of 2CLCs, as perturbation of Mdm2 resulted in a higher proportion of 2CLCs. Mdm2 appears to influence cell fate through its impact on cell cycle progression and H3K27me3 epigenetic modifications. In summary, the results of our CRISPR/Cas9 screen have uncovered several genes with distinct functions in regulating totipotency and pluripotency at various levels, offering a valuable resource for potential targets in future molecular studies.

Functional redundancy among Polycomb complexes in maintaining the pluripotent state of embryonic stem cells.

SummaryPolycomb group proteins assemble into multi-protein complexes, known as Polycomb repressive complexes 1 and 2 (PRC1 and PRC2), that guide cell fate decisions during embryonic development. PRC1 forms an array of biochemically distinct canonical PRC1 (cPRC1) or non-canonical PRC1 (ncPRC1) complexes characterized by the mutually exclusive presence of PCGF (PCGF1-PCGF6) paralog subunit; however, whether each one of these subcomplexes fulfills a distinct role remains largely controversial. Here, by performing a CRISPR-based loss-of-function screen in embryonic stem cells (ESCs), we uncovered a previously unappreciated functional redundancy among PRC1 subcomplexes. Disruption of ncPRC1, but not cPRC1, displayed severe defects in ESC pluripotency. Remarkably, coablation of non-canonical and canonical PRC1 in ESCs resulted in exacerbation of the phenotype observed in the non-canonical PRC1-null ESCs, highlighting the importance of functional redundancy among PRC1 subcomplexes. Together, our studies demonstrate that PRC1 subcomplexes act redundantly to silence lineage-specific genes and ensure robust maintenance of ESC identity.

Potential Role of Ubiquitin E3 Ligase, FBXO9, As a Tumor Suppressor in AML

Hematopoietic stem cells (HSCs) give rise to all cells of the blood. The balance between HSC self-renewal and differentiation is critical to maintaining hematopoiesis throughout the life of the organism, and disruptions to this balance often result in malignancies, including acute myeloid leukemia (AML). Multiple transcription factor networks are known to be important regulators of HSCs and, when mutated, can lead to leukemia development. More recently, protein degradation by the ubiquitin proteasome system (UPS) has been shown to regulate HSC self-renewal, differentiation, and survival. The role of this pathway in leukemogenesis is less clear.We recently performed a screen in embryonic stem cells to identify members of the UPS involved in cell fate decisions. From this, we identified the Ubiquitin E3 ligase FBXO9, which is a substrate recognition component of the Skp1-Cullin-F-box (SCF)-type E3 ligase complex. Interestingly, FBXO9 is highly expressed in hematopoietic stem and progenitor cells, but is decreased in patients with AML including patients with Inv(16) chromosomal abnormality. In order to study FBXO9 in normal and malignant hematopoiesis, we utilized a novel method using CRISPR/Cas9 to generate a conditional knockout model in collaboration with the Mouse Transgenic Core at UNMC. We bred our mice to transgenic mice expressing Cre Recombinase from the Mx1 promoter, which allows for PolyIC inducible expression of Cre in all hematopoietic cells including HSC. Our preliminary data shows alterations in hematopoiesis, including less competitive repopulation capability of the HSC in vivo and decreased differentiation potential of the hematopoietic progenitor population (HPC). To test if Fbxo9 plays a role in leukemia development, we crossed mice with the conditional allele of Fbxo9 with a knockin mouse model of AML that expresses the leukemia fusion gene Cbfb-MYH11 . In a small cohort of mice, we found that deletion of Fbxo9 in the context of Cbfb-MYH11 caused markedly accelerated leukemia development with >50% myeloid blasts in the peripheral blood by week 6. These findings suggest that the ubiquitin E3 ligase, Fbxo9, is a novel tumor suppressor and can regulate self-renewal, differentiation, and malignant transformation. In addition The dynamic reversibility of the ubiquitin modification (by kinases, phosphatases, E3 ligases, and de-ubiquitinases) and the recent development of both general proteasome inhibitors as well as specific antagonists of ligase function suggest that manipulation of proteins by ubiquitin E3 ligases could provide potential targets for drug discovery and stem cell therapy. DisclosuresNo relevant conflicts of interest to declare.

Innovative high throughput screen in embryonic stem cells reports stress-forced imbalanced differentiation, important to analyze stress in IVF and drug development; analyses using bulk and single cell RNAseq

Innovative high throughput screen in embryonic stem cells reports stress-forced imbalanced differentiation, important to analyze stress in IVF and drug development; analyses using bulk and single cell RNAseq

CRISPR Activation Screens Systematically Identify Factors that Drive Neuronal Fate and Reprogramming

Comprehensive identification of factors that can specify neuronal fate could provide valuable insights into lineage specification and reprogramming, but systematic interrogation of transcription factors, and their interactions with each other, has proven technically challenging. We developed a CRISPR activation (CRISPRa) approach to systematically identify regulators of neuronal-fate specification. We activated expression of all endogenous transcription factors and other regulators via a pooled CRISPRa screen in embryonic stem cells, revealing genes including epigenetic regulators such as Ezh2 that can induce neuronal fate. Systematic CRISPR-based activation of factor pairs allowed us to generate a genetic interaction map for neuronal differentiation, with confirmation of top individual and combinatorial hits as bona fide inducers of neuronal fate. Several factor pairs could directly reprogram fibroblasts into neurons, which shared similar transcriptional programs with endogenous neurons. This study provides an unbiased discovery approach for systematic identification of genes that drive cell-fate acquisition.

Construction and Application of Random dsRNA Interference Library for Functional Genetic Screens in Embryonic Stem Cells.

RNA interference (RNAi) libraries have been proven to be a powerful tool for large-scale functional genetic screens. To facilitate high-throughput functional genetic screens in embryonic stem cells, a system for construction of random dsRNA-expressing RNAi libraries was developed. Previous studies have demonstrated that sequence-specific gene silencing could be induced by long dsRNA in mouse embryos, mouse oocytes, embryonic stem cells, and some other mammalian cells. Our study demonstrated that the dsRNA interference library can be used for functional genetic screens of genes involved in self-renewal of embryonic stem cells (ES cells). The random RNAi library is easy to construct and provides a useful tool for investigation of molecular mechanisms of cellular development and differentiation.

Forward Genetic Approach to Uncover Stress Resistance Genes in Mice — A High-throughput Screen in ES Cells

Phenotype-driven genetic screens in mice is a powerful technique to uncover gene functions, but are often hampered by extremely high costs, which severely limits its potential. We describe here the use of mouse embryonic stem (ES) cells as surrogate cells to screen for a phenotype of interest and subsequently introduce these cells into a host embryo to develop into a living mouse carrying the phenotype. This method provides (1) a cost effective, high-throughput platform for genetic screen in mammalian cells; (2) a rapid way to identify the mutated genes and verify causality; and (3) a short-cut to develop mouse mutants directly from these selected ES cells for whole animal studies. We demonstrated the use of paraquat (PQ) to select resistant mutants and identify mutations that confer oxidative stress resistance. Other stressors or cytotoxic compounds may also be used to screen for resistant mutants to uncover novel genetic determinants of a variety of cellular stress resistance.

Forward Genetic Approach to Uncover Stress Resistance Genes in Mice - A High-throughput Screen in ES Cells.

Phenotype-driven genetic screens in mice is a powerful technique to uncover gene functions, but are often hampered by extremely high costs, which severely limits its potential. We describe here the use of mouse embryonic stem (ES) cells as surrogate cells to screen for a phenotype of interest and subsequently introduce these cells into a host embryo to develop into a living mouse carrying the phenotype. This method provides (1) a cost effective, high-throughput platform for genetic screen in mammalian cells; (2) a rapid way to identify the mutated genes and verify causality; and (3) a short-cut to develop mouse mutants directly from these selected ES cells for whole animal studies. We demonstrated the use of paraquat (PQ) to select resistant mutants and identify mutations that confer oxidative stress resistance. Other stressors or cytotoxic compounds may also be used to screen for resistant mutants to uncover novel genetic determinants of a variety of cellular stress resistance.

Genome-wide shRNA screening to identify factors mediating Gata6 repression in mouse embryonic stem cells

The use of whole-genome pooled shRNA libraries in loss-of-function screening in tissue culture models provides an effective means to identify novel factors acting in pathways of interest. Embryonic stem cells (ESCs) offer a unique opportunity to study processes involved in stem cell pluripotency and differentiation. Here, we report a genome-wide shRNA screen in ESCs to identify novel components involved in repression of the Gata6 locus, using a cell viability-based screen, which offers the benefits of stable shRNA integration and a robust and simple protocol for hit identification. Candidate factors identified were enriched for transcription factors and included known Polycomb proteins and other chromatin-modifying factors. We identified the protein Bcor, which is known to associate in complexes with the Polycomb protein Ring1B, and verified its importance in Gata6 repression in ESCs. Potential further applications of such a screening strategy could allow the identification of factors important for regulation of gene expression and pluripotency.

Transmission of mutant phenotypes from ES cells to adult mice.

Genetic manipulation of embryonic stem (ES) cells has been used to produce genetically engineered mice modeling human disorders. Here we describe a novel, additional application: selection for a phenotype of interest and subsequent transmission of that phenotype to a living mouse. We show, for the first time, that a cellular phenotype induced by ENU mutagenesis in ES cells can be transmitted and recapitulated in adult mice derived from these cells. We selected for paraquat-resistant (PQ(R)) ES clones. Subsequent injection of these cells into blastocysts resulted in the production of germline chimeras, from which tail skin fibroblasts exhibited enhanced PQ(R). This trait was also recovered in progeny of the chimera. We avoided PQ toxicity, which blocks the ability to involve the germline, by developing a sib-selection method, one that could be widely applied wherever the selection itself might diminish the pluripotency of the ES cells. Thus, phenotype-driven screens in ES cells are both feasible and efficient in producing intact mouse models for in vivo studies.

A cDNA-Based Random RNA Interference Library for Functional Genetic Screens in Embryonic Stem Cells

To facilitate high-throughput functional genetic screens in embryonic stem cells, a simple and efficient system to construct cDNA-based random RNA interference (RNAi) library was developed in the study. Previous studies have demonstrated that sequence-specific gene silencing could be induced by long double-stranded RNA (dsRNA) in mouse embryos, mouse oocytes, embryonic stem cells, and other mammalian cells. Based on these findings, a dsRNA-expressing RNAi vector system was designed. This study provided evidence that the vector design could induce efficient knockdown of expression of both exogenous egfp gene and endogenous MTM1 gene in mouse embryonic stem cells. A random RNAi library was established by cloning enzyme-digested cDNA of mouse embryonic stem (ES) cells into the BamHI site of the convergent dual promoter RNAi vector. Sequencing of 20 randomly selected clones from the library showed that 17 contained inserts and that all of them were unique sequences. A functional genetic screen of genes involving in self-renewal and differentiation with the random RNAi library identified ubiquitin. The ubiquitin knockdown ES cell line generated 20%-30% of undifferentiated colonies in the absence of leukemia inhibitor factor, whereas parental ES cells and control vector pDCont transfectants produced less than 5% of colonies of undifferentiated cells, suggesting that ubiquitin plays a role in ES cell differentiation. The random RNAi library provides a useful tool for investigation of molecular mechanisms of cellular development and differentiation.

Mismatch repair genes identified using genetic screens in Blm-deficient embryonic stem cells.

Phenotype-driven recessive genetic screens in diploid organisms require a strategy to render the mutation homozygous. Although homozygous mutant mice can be generated by breeding, a reliable method to make homozygous mutations in cultured cells has not been available, limiting recessive screens in culture. Cultured embryonic stem (ES) cells provide access to all of the genes required to elaborate the fundamental components and physiological systems of a mammalian cell. Here we have exploited the high rate of mitotic recombination in Bloom's syndrome protein (Blm)-deficient ES cells to generate a genome-wide library of homozygous mutant cells from heterozygous mutations induced with a revertible gene trap retrovirus. We have screened this library for cells with defects in DNA mismatch repair (MMR), a system that detects and repairs base-base mismatches. We demonstrate the recovery of cells with homozygous mutations in known and novel MMR genes. We identified Dnmt1(ref. 5) as a novel MMR gene and confirmed that Dnmt1-deficient ES cells exhibit micro-satellite instability, providing a mechanistic explanation for the role of Dnmt1 in cancer. The combination of insertional mutagenesis in Blm-deficient ES cells establishes a new approach for phenotype-based recessive genetic screens in ES cells.

The Secretory Protein Sec8 Is Required for Paraxial Mesoderm Formation in the Mouse

Thesec8gene, isolated in a gene trap screen in embryonic stem cells, is required for paraxial mesoderm formation in the mouse. Homozygoussec8mutant embryos initiate gastrulation but are unable to progress beyond the primitive streak stage and die shortly afterward. The genomic locus and cDNA of thesec8gene have been cloned. An open reading frame in the cDNA encodes a 971-amino-acid leucine-rich protein, similar to rat rSec8. A description of the mutant phenotype and the cloning of the gene is presented here and the results are considered in light of the possibility that the Sec8 protein is involved in secretion.

An induction gene trap screen in embryonic stem cells: Identification of genes that respond to retinoic acid in vitro.

We have developed a novel induction gene trap approach that preselects in vitro for integrations into genes that lie downstream of receptor/ligand-mediated signaling pathways. Using this approach, we have identified 20 gene trap integrations in embryonic stem cells, 9 of which were induced and 11 of which were repressed after exposure to exogenous retinoic acid (RA). All but one of these integrations showed unique spatially restricted or tissue-specific patterns of expression between 8.5 and 11.5 days of embryogenesis. Interestingly, expression was observed in tissues that are affected by alterations in RA levels during embryogenesis. Sequence analysis of fusion transcripts from six integrations revealed five novel gene sequences and the previously identified protooncogene c-fyn. To date, germ-line transmission and breeding has uncovered one homozygous embryonic lethal and three homozygous viable insertions. These studies demonstrate the potential of this induction gene trap approach for identifying and mutating genes downstream of signal transduction pathways.