Dissecting human endoderm development through a genome-wide CRISPR screen in human embryonic stem cells
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE95729 | GEO | 2019/09/06
REPOSITORIES: GEO
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Project description:Mitogen-activated protein kinase kinase 4 (MKK4) is a dual-specificity kinase activated by environmental stress, cytokines, and peptide growth factors that reportedly can promote or inhibit tumor cell growth and metastasis. Somatic mutations in the gene encoding MKK4 (MAP2K4) have been identified in various human cancers, but the consequences of these mutations on MKK4 function and the biology of tumor cells that have them have not been elucidated. Here we report that, of the eleven mutations within the MAP2K kinase domain described thus far, one had gain-of-function (Q142L) and six had loss-of-function. Three of the loss-of-function mutations are nonsense mutations that produced C-terminally-truncated proteins (I295fs*23, R304*, and W310*) that were highly ubiqitinated and rapidly degraded when introduced into cells, and three are missense mutations in the ATP-binding pocket (N234I), activation loop (S251N), or C-lobe (P326L). We modeled the consequences of MAP2K4 loss-of-function mutations on cells by introducing MKK4 short-hairpin RNA constructs and found that MKK4 depletion enhanced the ability of a weakly tumorigenic murine cancer cell to metastasize when injected into syngeneic mice but had no effect on primary tumor formation. MKK4-depleted cells exhibited an increased capacity to migrate across PET filters and to invade through matrigel but no change in anchorage-dependent or -independent proliferation. Transcriptional profiling of these cells revealed gene expression changes that promote epithelial-to-mesenchymal transition and angiogenesis. We conclude that MKK4 inactivation promoted metastasis but not primary tumor formation. Collectively, these findings implicate loss-of-function MAP2K4 somatic mutations in tumor metastasis and provide one of the few examples of a somatic mutation in cancer cells that exerts a metastasis-specific effect. Keywords: Two-group comparison. 393P cells were infected with retroviruses expressing a MKK4-specific shRNA construct (shZ) or scrambled control (scr). 393P-shZ and 393P-scr were transcriptionally profiled in triplicate cultures.
2011-09-30 | E-GEOD-19091 | biostudies-arrayexpress
Project description:Mitogen-activated protein kinase kinase 4 (MKK4) is a dual-specificity kinase activated by environmental stress, cytokines, and peptide growth factors that reportedly can promote or inhibit tumor cell growth and metastasis. Somatic mutations in the gene encoding MKK4 (MAP2K4) have been identified in various human cancers, but the consequences of these mutations on MKK4 function and the biology of tumor cells that have them have not been elucidated. Here we report that, of the eleven mutations within the MAP2K kinase domain described thus far, one had gain-of-function (Q142L) and six had loss-of-function. Three of the loss-of-function mutations are nonsense mutations that produced C-terminally-truncated proteins (I295fs*23, R304*, and W310*) that were highly ubiqitinated and rapidly degraded when introduced into cells, and three are missense mutations in the ATP-binding pocket (N234I), activation loop (S251N), or C-lobe (P326L). We modeled the consequences of MAP2K4 loss-of-function mutations on cells by introducing MKK4 short-hairpin RNA constructs and found that MKK4 depletion enhanced the ability of a weakly tumorigenic murine cancer cell to metastasize when injected into syngeneic mice but had no effect on primary tumor formation. MKK4-depleted cells exhibited an increased capacity to migrate across PET filters and to invade through matrigel but no change in anchorage-dependent or -independent proliferation. Transcriptional profiling of these cells revealed gene expression changes that promote epithelial-to-mesenchymal transition and angiogenesis. We conclude that MKK4 inactivation promoted metastasis but not primary tumor formation. Collectively, these findings implicate loss-of-function MAP2K4 somatic mutations in tumor metastasis and provide one of the few examples of a somatic mutation in cancer cells that exerts a metastasis-specific effect. Keywords: Two-group comparison.
2011-10-01 | GSE19091 | GEO
Project description:A conserved molecular pathway has emerged controlling endoderm formation in Xenopus zebrafish and mice. Key genes in this pathway include Nodal ligands and transcription factors of the Mix-like paired homeodomain class, Gata4-6 zinc finger factors and Sox17 HMG domain proteins. While a linear epistatic pathway has been proposed, the precise hierarchical relationships between these factors and their downstream targets are largely unresolved. Here we used a combination of microarray analysis and loss-of-function experiments to examine the global regulatory network controlling Xenopus endoderm formation. We identified over 300 transcripts enriched in the gastrula endoderm, including most of the known endoderm regulators as well as over a hundred uncharacterized genes. Surprisingly only 10% of the endoderm transcriptome is regulated as predicted by the current linear model. We find that Nodals, Mixer and Sox17 have both shared and distinct sets of downstream targets and that a number of unexpected autoregulatory loops exist between Sox17 and Gata4-6, Sox17 and Bix1, 2, 4 and between Sox17 and Xnr4. We find that Mixer does not function primarily via Sox17 as previously proposed. This data provides a new insight into the complexity of endoderm formation and will serve as valuable resource for establishing a complete endoderm gene regulatory network. Experiment Overall Design: Define a set of transcripts with enriched expression in the gastrula endoderm of the Xenopus laevis embryo and determine how these are regulated by nodal signaling, Mixer and Sox17 using loss-of-function experiments. For more specific details see Sinner et al., (2006) Global analysis of the transcriptional network controlling Xenopus endoderm formation.
2007-10-05 | E-GEOD-4448 | biostudies-arrayexpress
Project description:The mitogen-activated protein kinase kinase (MEK) kinase 1 (MEKK1), also known as MAP3K1, mediates activin B (ActB) signals required for eyelid epithelium morphogenesis during mouse fetal development. The present study investigates the role of MEKK1 in epithelial wound healing, another activin-regulated biological process. In a skin wound model, injury markedly stimulates MEKK1 expression and activity, which are in turn required for the expression of genes involved in extracellular matrix (ECM) homeostasis. MEKK1 ablation or down-regulation by interfering RNA significantly delays skin wound closure and impairs activation of Jun NH2-terminal kinases, induction of plasminogen activator inhibitor (PAI)-1, and restoration of cell-cell junctions of the wounded epidermis. Conversely, expression of wild-type MEKK1 accelerates reepithelialization of full-thickness skin and corneal debridement wounds by mechanisms involving epithelial cell migration, a cell function that is partially abolished by neutralizing antibodies for PAI-1 and metalloproteinase III. Our data suggest that MEKK1 transmits wound signals, leading to the transcriptional activation of genes involved in ECM homeostasis, epithelial cell migration, and wound reepithelialization.
2022-07-13 | GSE201823 | GEO
Project description:Agrobacterium tumefaciens is a special plant pathogen causing crown gall disease. This pathogen is well known for the technology Agrobacterium-mediated transformation. As a pathogen, Agrobacterium triggers plant immunity, and this affects transformation. But the signaling components and pathways in plant immunity to Agrobacterium remain elusive. We demonstrate two Arabidopsis MAPKKs MKK4/MKK5 and their downstream MAPKs MPK3/MPK6 play a major role in both Agrobacterium-triggered immunity and Agrobacterium-mediated transformation. Agrobacteria induce MPK3/MPK6 activity and plant defense responsive genes expression in a very early stage. This process is dependent on MKK4/MKK5 function. Loss of function of MKK4 and MKK5 or their downstream MPK3 and MPK6 abolishes plant immunity to agrobacteria, and increases the transformation frequency, while activation of MKK4 and MKK5 enhances the plant immunity and represses the transformation. Global transcriptome indicates agrobacteria induce various plant defense pathways, including ROS production, ethylene and SA-mediated defense responses, and MKK4/MKK5 is essential for these pathways induction. Activation of MKK4 and MKK5 promotes ROS production and cell death in agrobacteria infection process. Ethylene and SA act bypass of MKK4/MKK5 signaling to regulate transformation. Based on these results, we propose MKK4/5-MPK3/6 cascade is an essential signaling pathway to regulate Agrobacterium-mediated transformation by modulating Agrobacterium-triggered plant immunity.
2021-10-20 | GSE179628 | GEO
Project description:The transcription factors Mixer and Sox17beta have well characterized roles in endoderm specification during Xenopus embryogenesis. In order to more thoroughly understand the mechanisms by which these endodermal regulators act, we expressed Mixer and Sox17beta in naïve ectodermal tissue and, using oligonucleotide-based microarrays, compared their genomic transcriptional profile to that of unaffected tissue. Using this novel approach, we identified 71 transcripts that are upregulated by Mixer or Sox17beta, 63 of which have previously uncharacterized roles in endoderm development. Furthermore, an in situ hybridization screen using antisense probes for several of these clones identified six targets of Mixer and/or Sox17beta that are expressed in the endoderm during gastrula stages, providing new and regional markers of the endoderm. Our results contribute further insight into the functions of Mixer and Sox17beta and bring us closer to understanding at the molecular level the pathways that regulate endoderm development.
2007-06-28 | E-GEOD-3334 | biostudies-arrayexpress
Project description:Faithful embryogenesis requires the precise coordination between embryonic and extraembryonic tissues. Although embryonic and extraembryonic stem cells have been derived from several mammalian species including humans, they are cultured in different conditions, which makes it difficult to study their intercommunication. Here, by simultaneously activating FGF, TGF-β and WNT pathways, we derived stable pluripotent stem cells (PSCs), trophoblast stem cells (TSCs) and extraembryonic endoderm stem cells (XENs) from mouse blastocysts under the same condition (FTW). Co-culture of PSCs and XENs in the same environment uncovered, among other interactions, a previously unrecognized control of proliferation of epiblast cells by extraembryonic endoderm cells. FTW condition also supported de novo derivation of XENs from cynomolgus monkey and human blastocysts, and enabled setting up co-culture of human iPSCs and XENs. Crosspieces comparison revealed conserved and divergent processes and genes regulating XENs and ligand-receptor interactions between pluripotent and extraembryonic endoderm cells. Our study establishes a unique stem cell co-culture strategy to examine embryonic and extraembryonic lineage crosstalk during early mammalian development, and opens the door for developing more faithful in vitro models and differentiation protocols.
2023-04-13 | GSE178459 | GEO
Project description:Faithful embryogenesis requires the precise coordination between embryonic and extraembryonic tissues. Although embryonic and extraembryonic stem cells have been derived from several mammalian species including humans, they are cultured in different conditions, which makes it difficult to study their intercommunication. Here, by simultaneously activating FGF, TGF-β and WNT pathways, we derived stable pluripotent stem cells (PSCs), trophoblast stem cells (TSCs) and extraembryonic endoderm stem cells (XENs) from mouse blastocysts under the same condition (FTW). Co-culture of PSCs and XENs in the same environment uncovered, among other interactions, a previously unrecognized control of proliferation of epiblast cells by extraembryonic endoderm cells. FTW condition also supported de novo derivation of XENs from cynomolgus monkey and human blastocysts, and enabled setting up co-culture of human iPSCs and XENs. Crosspieces comparison revealed conserved and divergent processes and genes regulating XENs and ligand-receptor interactions between pluripotent and extraembryonic endoderm cells. Our study establishes a unique stem cell co-culture strategy to examine embryonic and extraembryonic lineage crosstalk during early mammalian development, and opens the door for developing more faithful in vitro models and differentiation protocols.
2023-12-19 | GSE233614 | GEO
Project description:This study examined the importance of intracellular kinase MEKK1 in breast tumor-induced on gene expression in non-tumor mammary cells. The hypothesis tested in this study was that the kinase MEKK1 regulates breast tumor cells-induced expression of fibroblast genes that change the tumor microenvironment to promote breast tumor progression to metastasis. Results reveal that MEKK1 regulates expression of chemokines and other genes in normal breast stroma cells that contribute to the development of a tumor microenvironment that promotes metastasis.
2021-02-20 | GSE167130 | GEO
Project description:A conserved molecular pathway has emerged controlling endoderm formation in Xenopus zebrafish and mice. Key genes in this pathway include Nodal ligands and transcription factors of the Mix-like paired homeodomain class, Gata4-6 zinc finger factors and Sox17 HMG domain proteins. While a linear epistatic pathway has been proposed, the precise hierarchical relationships between these factors and their downstream targets are largely unresolved. Here we used a combination of microarray analysis and loss-of-function experiments to examine the global regulatory network controlling Xenopus endoderm formation. We identified over 300 transcripts enriched in the gastrula endoderm, including most of the known endoderm regulators as well as over a hundred uncharacterized genes. Surprisingly only 10% of the endoderm transcriptome is regulated as predicted by the current linear model. We find that Nodals, Mixer and Sox17 have both shared and distinct sets of downstream targets and that a number of unexpected autoregulatory loops exist between Sox17 and Gata4-6, Sox17 and Bix1, 2, 4 and between Sox17 and Xnr4. We find that Mixer does not function primarily via Sox17 as previously proposed. This data provides a new insight into the complexity of endoderm formation and will serve as valuable resource for establishing a complete endoderm gene regulatory network. Keywords: Embryonic, Development. Endoderm, Xenopus, nodal, Sox17, Mixer, microarray, gene regulatory network
2006-03-11 | GSE4448 | GEO