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A genome wide RNAi screen in mouse embryonic stem cells identifies Mp1 as a key mediator of differentiation

ABSTRACT: Despite intense investigation of intrinsic and extrinsic factors that regulate pluripotency, the process of initial fate commitment of embryonic stem (ES) cells is still poorly understood. Here, we used a genome wide shRNA screen in mouse ES cells to identify genes that are essential for initiation of differentiation. Knockdown of the scaffolding protein Mek binding protein 1 (Mp1, also known as Lamtor3, Map2k1ip1) stimulated self-renewal of ES cells, blocked differentiation and promoted proliferation. Fibroblast growth factor 4 (FGF4) signaling is required for initial fate commitment of ES cells. Knockdown of Mp1 inhibited FGF4-induced differentiation but did not alter FGF4 driven proliferation. This uncoupling of differentiation and proliferation was also observed when oncogenic Ras isoforms were over expressed in ES cells. Knockdown of Mp1 redirected FGF4 signaling from differentiation towards pluripotency and upregulated the pluripotency-related genes Esrrb, Rex1, Tcl1 and Sox2. We also found that human germ cell tumors express low amounts of Mp1 in the invasive embryonic carcinoma and seminoma histologies and higher amounts of Mp1 in the non-invasive carcinoma in situ precursor and differentiated components. Knockdown of Mp1 in invasive germ cell tumor cells resulted in resistance to differentiation, thereby showing a functional role for Mp1 both in normal differentiation of ES cells as well as in germ cell cancer.

ORGANISM(S): Mus musculus

PROVIDER: GSE32595 | GEO | 2011/11/15

SECONDARY ACCESSION(S): PRJNA147071

REPOSITORIES: GEO

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A genome wide RNAi screen in mouse embryonic stem cells identifies Mp1 as a key mediator of differentiation

Project description:Despite intense investigation of intrinsic and extrinsic factors that regulate pluripotency, the process of initial fate commitment of embryonic stem (ES) cells is still poorly understood. Here, we used a genome wide shRNA screen in mouse ES cells to identify genes that are essential for initiation of differentiation. Knockdown of the scaffolding protein Mek binding protein 1 (Mp1, also known as Lamtor3, Map2k1ip1) stimulated self-renewal of ES cells, blocked differentiation and promoted proliferation. Fibroblast growth factor 4 (FGF4) signaling is required for initial fate commitment of ES cells. Knockdown of Mp1 inhibited FGF4-induced differentiation but did not alter FGF4 driven proliferation. This uncoupling of differentiation and proliferation was also observed when oncogenic Ras isoforms were over expressed in ES cells. Knockdown of Mp1 redirected FGF4 signaling from differentiation towards pluripotency and upregulated the pluripotency-related genes Esrrb, Rex1, Tcl1 and Sox2. We also found that human germ cell tumors express low amounts of Mp1 in the invasive embryonic carcinoma and seminoma histologies and higher amounts of Mp1 in the non-invasive carcinoma in situ precursor and differentiated components. Knockdown of Mp1 in invasive germ cell tumor cells resulted in resistance to differentiation, thereby showing a functional role for Mp1 both in normal differentiation of ES cells as well as in germ cell cancer. 8 samples were analyzed, representing four different experimental conditions of which each condition was analyzed by two different shRNAs to prevent off target artifacts.

2011-11-15 | E-GEOD-32595 | biostudies-arrayexpress

The FGF4-dependent phosphoproteome in mouse embryonic stem cells

Project description:Protein kinase signalling is a major mechanism by which embryonic stem cell pluripotency and differentiation is controlled. However, the pathways and components that regulate embryonic stem cell identity have not been systematically defined. Here, we employ FGF4 signalling as a model system to investigate phosphoproteome dynamics in differentiating mouse embryonic stem cells. We report identification and quantitation of more than 10,000 phosphopeptides, of which hundreds of phosphophoylation sites are regulated more than 2-fold by acute FGF4 stimulation. We hypothesise that phosphorylation sites in this dataset are relevant for regulating the transition of mouse embryonic stem cells from pluripotency towards lineage specific differentiation.

2020-02-24 | PXD012069 | Pride

Titration of Sall4 by the NuRD complex facilitates neural differentiation of ES cells

Project description:Sall4 is a transcription factor essential for early mammalian development. Though it is reported to play an important role in embryonic stem (ES) cell self-renewal, whether it is an essential pluripotency factor has been disputed. Though Sall4 is known to associate with the Nucleosome Remodeling and Deacetylase (NuRD) complex, the nature of this interaction is unclear as NuRD and Sall4 serve opposing functions in ES cells. Here we use defined culture conditions and single-cell gene expression analyses to show that Sall4 prevents activation of the neural gene expression programme in ES cells but is dispensable for maintaining the pluripotency gene regulatory network. We further show using genome-wide analyses that while Sall4 interacts with NuRD, it neither recruits NuRD to chromatin nor influences transcription via NuRD. Rather we propose a model where, by titrating Sall4 protein, NuRD limits the differentiation-inhibiting activity of Sall4 in ES cells to enable lineage commitment.

2016-08-02 | PXD003614 | Pride

Heparan Sulfation Dependent FGF Signalling Maintains ES Cells Primed for Differentiation in a Heterogeneous State

Project description:Embryonic stem (ES) cells continuously decide whether to maintain pluripotency or differentiate. While exogenous LIF and BMP4 perpetuate a pluripotent state, less is known about factors initiating differentiation. We show that heparan sulfate (HS) proteoglycans are critical co-receptors for signals inducing ES cell differentiation. Genetic targeting of NDST1 and 2, two enzymes required for N-sulfation of proteoglycans, blocked differentiation. This phenotype was rescued by HS presented in trans or by soluble heparin. NaClO3-, which reduces sulfation of proteoglycans, potently blocked differentiation of wild type cells. Mechanistically, N-sulfation was identified to be critical for functional autocrine FGF4 signalling. Micro array analysis identified the pluripotency maintaining transcription factors Nanog, KLF2/4/8, Tbx3 and Tcf3 to be negatively regulated, whereas markers of differentiation such as Gbx2, Dnmt3b, FGF5 and Brachyury were induced by sulfation-dependent-FGFR signalling. We show that several of these genes are heterogeneously expressed in ES cells and targeting of heparan sulfation or FGFR-signalling facilitated a homogenous Nanog/KLF4/Tbx3 positive ES cell state. This finding suggests that the recently discovered heterogeneous state of ES cells is regulated by HS-dependent FGFR signalling. Similarly, culturing blastocysts with NaClO3- eliminated GATA6 positive primitive endoderm progenitors generating a homogenous Nanog positive inner cell mass. Functionally, reduction of sulfation robustly improved de novo ES cell derivation efficiency. We conclude that N-sulfated HS is required for FGF4 signalling to maintain ES cells primed for differentiation in a heterogeneous state. Inhibiting this pathway facilitates a more naÃ¯ve ground state. Four groups with three biological replicates and a technical duplicate in each

2010-04-09 | E-GEOD-15974 | biostudies-arrayexpress

Molecular Insights into Non-teratoma-forming Induced Pluripotent Stem Cells

Project description:Human induced pluripotent stem (iPS) cells are capable of differentiating into derivatives of the three embryonic germ layers both in vitro and in vivo. To date the the molecular differences between teratoma-forming cells and non-teratoma-forming cells has not been analysed. A cell line, B1, bears typical ES cell-like morphology, expression of pluripotency-associated genes, and in vitro pluripotency capacity, but fails to form teratomas after subcutaneously injected into immune-deficient mice based on histological analysis. Besides histological analysis, we characterized the tumors derived from line B1, and teratomas derived from bona fida iPS and ES (line H1) cells respectively, using microarray-based gene expression analysis. The expression levels of pluripotency-associated markers in B1 cells were comparable to that in iPS and ES cells, while the complexity of tissue expression commitment was decreased upon spontaneous differentiation of B1 cells as compared to iPS and ES cells. Total RNA obtained from HFF1 (human foreskin fibroblast) cells, line B1, iPS-A4, iPS-B4 and ES (line H1) cells, and their derived tumors in immune-deficient mice.

2012-04-29 | E-GEOD-31970 | biostudies-arrayexpress

Molecular Insights into Non-teratoma-forming Induced Pluripotent Stem Cells

2012-04-30 | GSE31970 | GEO

Heparan Sulfation Dependent FGF Signalling Maintains ES Cells Primed for Differentiation in a Heterogeneous State

2009-11-13 | GSE15974 | GEO

Prediction and Testing of Novel Transcriptional Networks Regulating Embryonic Stem Cell Self-Renewal and Commitment

Project description:We have used mouse embryonic stem cells (ESCs) as a model to study the signaling mechanisms that regulate self-renewal and commitment to differentiation. We hypothesized that genes critical to stem cell fate would be dynamically regulated at the initiation of commitment. Time course microarray analysis following initiation of commitment led us to propose a model of ESC maintenance in which highly regulated transcription factors and chromatin remodeling genes (down-regulated in our time course) maintain repression of genes responsible for cell differentiation, morphogenesis and development (up-regulated in our time course). Microarrays of Oct4, Nanog and Sox2 shRNA knockdown cell lines confirmed predicted regulation of target genes. shRNA knockdowns of candidate genes were tested in a novel high throughput screen of self-renewal, confirming their role in ESC pluripotency. We have identified genes that are critical for self-renewal and those that initiate commitment and developed draft transcriptional networks that control self-renewal and early development. Keywords: genetic modification Gene expression in Oct4 knockdown, Sox2 knockdown and their empty vector contol ES cells was analyzed.

2010-06-11 | E-GEOD-7520 | biostudies-arrayexpress

Transcription profiling of mouse ES cells overexpressing Akt response to LIF

Project description:Embryonic stem (ES) cells can self-renew indefinitely without losing their differentiation ability to any cell types. Phosphoinositide-3 kinase (PI3K)/Akt signaling plays a pivotal role in various stem cell systems, including the formation of embryonic germ (EG) cells from primordial germ cells and self-renewal of neural stem cells. Here, we show that myristoylated, active form of Akt (myr-Akt) maintained the undifferentiated phenotypes in mouse ES cells without the addition of leukemia inhibitory factor (LIF). The effects of myr-Akt were reversible, because LIF dependence and pluripotent differentiation activity were restored by the deletion of myr-Akt. In addition, myr-Akt-Mer fusion protein, whose enzymatic activity is controlled by 4-hydroxy-tamoxifen, also maintained the pluripotency of not only mouse but also cynomolgus monkey ES cells. These results clearly demonstrate that Akt signaling sufficiently maintains pluripotency in mouse and primate ES cells, and support the notion that PI3K/Akt signaling axis regulates 'stemness' in a broad spectrum of stem cell systems.

2008-06-12 | E-GEOD-2516 | biostudies-arrayexpress

Mediator Med23-deficiency Enhances Neural Differentiation of Embryonic Stem Cells through Modulating BMP Signaling

Project description:Unraveling the mechanisms underlying early neural differentiation of ESCs is crucial to the cell-based therapies of neurodegenerate diseases. Neural fate acquisition is proposed to be controlled by a âdefaultâ mechanism, for which the molecular regulation is not well understood. In this study, we investigated the functional roles of Mediator Med23 in pluripotency and lineage commitment of embryonic stem cells (ESCs). Unexpectedly we found that, despite the largely unchanged pluripotency and self-renewal of ESCs, Med23-depletion rendered the cells prone to neural differentiation in different differentiation assays. Knockdown of other Mediator subunit, Med1 or Med15, did not alter the neural differentiation of ESCs; and Med15 knockdown selectively inhibited endoderm differentiation, suggesting the specificity of cell fate control by distinctive Mediator subunits. Gene profiling revealed that Med23-depletion attenuated the BMP signaling in ESCs. Mechanistically, MED23 modulated Bmp4 expression by controlling the activity of ETS1 that is involved in the Bmp4 promoter-enhancer communication. Interestingly, Med23 knockdown in zebrafish embryos also enhanced the neural development at early embryogenesis, which could be reversible by coinjection of bmp4 mRNA. Taken together, our study reveals an intrinsic, restrictive role of MED23 in early neural development, thus providing new molecular insights for neural fate determination. We used microarrays to detail the global gene expression after MED23 knockout We examined the gene expression level in WT and MED23 knockout ES cells in steady culture condition.

2015-03-05 | E-GEOD-64500 | biostudies-arrayexpress

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