scholarly journals GFAT2 and AMDHD2 act in tandem to control the hexosamine biosynthetic pathway

2021 ◽  
Author(s):  
Virginia Kroef ◽  
Sabine Ruegenberg ◽  
Moritz Horn ◽  
Kira Allmeroth ◽  
Lena Ebert ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Biosynthetic Pathway ◽  
Embryonic Stem ◽  
Chemical Mutagenesis ◽  
Lower Sensitivity ◽  
Loss Of Function ◽  
Critical Function ◽  
Hexosamine Biosynthetic Pathway ◽  
Rate Limiting

AbstractThe hexosamine biosynthetic pathway (HBP) produces the essential metabolite UDP-GlcNAc and plays a key role in metabolism, cancer, and aging. The HBP is controlled by its rate-limiting enzyme glutamine fructose-6-phosphate amidotransferase (GFAT) that is directly inhibited by UDP-GlcNAc in a feedback loop. HBP regulation by GFAT is well studied but other HBP regulators have remained obscure. Elevated UDP-GlcNAc levels counteract the glycosylation toxin tunicamycin (TM) and thus we screened for TM resistance in haploid mouse embryonic stem cells (mESCs) using random chemical mutagenesis to pinpoint new HBP regulators. We identified the N-acetylglucosamine deacetylase AMDHD2 that catalyzes a reverse reaction in the HBP and its loss strongly elevated UDP-GlcNAc. To better understand AMDHD2, we solved the crystal structure and found that loss-of-function is caused by protein destabilization or interference with its catalytic activity. Finally, we show that mESCs express AMDHD2 together with GFAT2 instead of the more common paralog GFAT1. Compared with GFAT1, GFAT2 had a much lower sensitivity to UDP-GlcNAc inhibition, explaining how AMDHD2 loss-of-function resulted in HBP activation. This HBP configuration in which AMDHD2 serves to balance GFAT2 activity was also observed in other mESCs and, consistently, the GFAT2/GFAT1 ratio decreased with differentiation of mouse and human embryonic stem cells. Together, our data reveal a critical function of AMDHD2 in limiting UDP-GlcNAc production in cells that use GFAT2 for metabolite entry into the HBP.

scholarly journals Transcriptional programs regulating neuronal differentiation are disrupted in DLG2 knockout human embryonic stem cells and enriched for schizophrenia and related disorders risk variants

2022 ◽  
Vol 13 (1) ◽  
Author(s):  
Bret Sanders ◽  
Daniel D’Andrea ◽  
Mark O. Collins ◽  
Elliott Rees ◽  
Tom G. J. Steward ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Neuronal Differentiation ◽  
Human Embryonic Stem Cells ◽  
De Novo ◽  
Embryonic Stem ◽  
Neuropsychiatric Disorders ◽  
Loss Of Function ◽  
Excitatory Neurons

AbstractCoordinated programs of gene expression drive brain development. It is unclear which transcriptional programs, in which cell-types, are affected in neuropsychiatric disorders such as schizophrenia. Here we integrate human genetics with transcriptomic data from differentiation of human embryonic stem cells into cortical excitatory neurons. We identify transcriptional programs expressed during early neurogenesis in vitro and in human foetal cortex that are down-regulated in DLG2−/− lines. Down-regulation impacted neuronal differentiation and maturation, impairing migration, morphology and action potential generation. Genetic variation in these programs is associated with neuropsychiatric disorders and cognitive function, with associated variants predominantly concentrated in loss-of-function intolerant genes. Neurogenic programs also overlap schizophrenia GWAS enrichment previously identified in mature excitatory neurons, suggesting that pathways active during prenatal cortical development may also be associated with mature neuronal dysfunction. Our data from human embryonic stem cells, when combined with analysis of available foetal cortical gene expression data, de novo rare variants and GWAS statistics for neuropsychiatric disorders and cognition, reveal a convergence on transcriptional programs regulating excitatory cortical neurogenesis.

scholarly journals Mapping Gene Circuits Essential for Germ Layer Differentiation via Loss-of-Function Screens in Haploid Human Embryonic Stem Cells

2020 ◽  
Vol 27 (4) ◽  
pp. 679-691.e6
Author(s):  
Atilgan Yilmaz ◽  
Carmel Braverman-Gross ◽  
Anna Bialer-Tsypin ◽  
Mordecai Peretz ◽  
Nissim Benvenisty
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Human Embryonic Stem Cells ◽  
Embryonic Stem ◽  
Germ Layer ◽  
Loss Of Function ◽  
Gene Circuits ◽  
Mapping Gene ◽  
Germ Layer Differentiation

scholarly journals TET2 Inhibits Differentiation of Embryonic Stem Cells but Does Not Overcome Methylation-Induced Gene Silencing

2014 ◽  
Vol 2014 ◽  
pp. 1-9 ◽  
Author(s):  
Louis Norman ◽  
Paul Tarrant ◽  
Timothy Chevassut
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Genomic Dna ◽  
Es Cells ◽  
Embryonic Stem ◽  
Gene Promoters ◽  
Loss Of Function ◽  
Promoter Sequences ◽  
Maintenance Methylation

TET2 is a methylcytosine dioxygenase that is frequently mutated in myeloid malignancies, notably myelodysplasia and acute myeloid leukemia. TET2 catalyses the conversion of 5′-methylcytosine to 5′-hydroxymethylcytosine within DNA and has been implicated in the process of genomic demethylation. However, the mechanism by which TET2 loss of function results in hematopoietic dysplasia and leukemogenesis is poorly understood. Here, we show that TET2 is expressed in undifferentiated embryonic stem cells and that its knockdown results in reduction of 5′-hydroxymethylcytosine in genomic DNA. We also present DNA methylation data from bone marrow samples obtained from patients with TET2-mutated myelodysplasia. Based on these findings, we sought to identify the role of TET2 in regulating pluripotency and differentiation. We show that overexpression of TET2 in a stably integrated transgene leads to increased alkaline phosphatase expression in differentiating ES cells and impaired differentiation in methylcellulose culture. We speculate that this effect is due to TET2-mediated expression of stem cell genes in ES cells via hydroxylation of 5′-methylcytosines at key promoter sequences within genomic DNA. This leads to relative hypomethylation of gene promoters as 5′-hydroxymethylcytosine is not a substrate for DNMT1-mediated maintenance methylation. We sought to test this hypothesis by cotransfecting the TET2 gene with methylated reporter genes. The results of these experiments are presented.

scholarly journals Role of TGFβ1 and WNT6 in FGF2 and BMP4-driven endothelial differentiation of murine embryonic stem cells

Angiogenesis ◽  
2021 ◽  
Author(s):  
Anna Gualandris ◽  
Alessio Noghero ◽  
Davide Cora’ ◽  
Elena Astanina ◽  
Marco Arese ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Culture Medium ◽  
Expression Profiles ◽  
Es Cells ◽  
Embryonic Stem ◽  
Endothelial Differentiation ◽  
Loss Of Function ◽  
Pure Cultures

AbstractEmbryonic stem cells (ES) are a valuable source of endothelial cells. By co-culturing ES cells with the stromal PA6 cells, the endothelial commitment can be achieved by adding exogenous FGF2 or BMP4. In this work, the molecular pathways that direct the differentiation of ES cells toward endothelium in response to FGF2 are evaluated and compared to those activated by BMP4. To this purpose the genes expression profiles of both ES/PA6 co-cultures and of pure cultures of PA6 cells were obtained by microarray technique at different time points. The bioinformatics processing of the data indicated TGFβ1 as the most represented upstream regulator in FGF2-induced endothelial commitment while WNT pathway as the most represented in BMP4-activated endothelial differentiation. Loss of function experiments were performed to validate the importance of TGFβ1 and WNT6 respectively in FGF2 and BMP4-induced endothelial differentiation. The loss of TGFβ1 expression significantly impaired the accomplishment of the endothelial commitment unless exogenous recombinant TGFβ1 was added to the culture medium. Similarly, silencing WNT6 expression partially affected the endothelial differentiation of the ES cells upon BMP4 stimulation. Such dysfunction was recovered by the addition of recombinant WNT6 to the culture medium. The ES/PA6 co-culture system recreates an in vitro complete microenvironment in which endothelial commitment is accomplished in response to alternative signals through different mechanisms. Given the importance of WNT and TGFβ1 in mediating the crosstalk between tumor and stromal cells this work adds new insights in the mechanism of tumor angiogenesis and of its possible inhibition.

Use of Chemical Mutagenesis in Mouse Embryonic Stem Cells

2006 ◽  
pp. 397-408
Author(s):  
Martin Hrabé de Angelis ◽  
Sonja Becker ◽  
Johannes Beckers
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Embryonic Stem ◽  
Mouse Embryonic Stem Cells ◽  
Chemical Mutagenesis

scholarly journals Genes essential for embryonic stem cells are associated with neurodevelopmental disorders

2019 ◽  
Author(s):  
Shahar Shohat ◽  
Sagiv Shifman
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Neurodevelopmental Disorders ◽  
Embryonic Stem ◽  
Basic Research ◽  
Essential Genes ◽  
Loss Of Function ◽  
Cellular Functions ◽  
Human Escs ◽  
A Genome

AbstractMouse embryonic stem cells (mESCs) are a key component in generating mouse models for human diseases and performing basic research on pluripotency, but how many genes are essential for mESCs is still unknown. We performed a genome-wide screen for essential genes in mESCs and compared it to human cells. We found that essential genes are enriched for basic cellular functions, are highly expressed in mESCs, and tend to lack paralog genes. Notably, we discovered that genes that are essential specifically in mESCs play a role in pathways associated with their pluripotent state. We show that 25% of human genes that are intolerant to loss-of-function mutations are essential in mouse or human ESCs and that the human phenotypes most significantly associated with essential genes are neurodevelopmental. Our results provide insights into essential genes in the mouse, the pathways which govern pluripotency, and suggest that many genes associated with neurodevelopmental disorders are essential at very early embryonic stages.

Toward the yeastification of mouse genetics: chemical mutagenesis of embryonic stem cells

2000 ◽  
Vol 11 (7) ◽  
pp. 598-602 ◽  
Author(s):  
Yijing Chen ◽  
John Schimenti ◽  
Terry Magnuson
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Embryonic Stem ◽  
Mouse Genetics ◽  
Chemical Mutagenesis

scholarly journals LncRNA Mrhl orchestrates differentiation programs in mouse embryonic stem cells through chromatin mediated regulation

2019 ◽  
Author(s):  
Debosree Pal ◽  
C V Neha ◽  
Utsa Bhaduri ◽  
Zenia ◽  
Subbulakshmi Chidambaram ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Cell Fate ◽  
Embryonic Stem ◽  
Mouse Embryonic Stem Cells ◽  
Loss Of Function ◽  
Genome Wide ◽  
Development And Differentiation ◽  
Lineage Specific Genes

AbstractLong non-coding RNAs (lncRNAs) have been well-established to act as regulators and mediators of development and cell fate specification programs. LncRNA Mrhl (meiotic recombination hotspot locus) has been shown to act in a negative feedback loop with WNT signaling to regulate male germ cell meiotic commitment. In our current study, we have addressed the role of Mrhl in development and differentiation using mouse embryonic stem cells (mESCs) as our model system of study. We found Mrhl to be a nuclear-localized, chromatin-bound lncRNA with moderately stable expression in mESCs. Transcriptome analyses and loss-of-function phenotype studies revealed dysregulation of developmental processes and lineage-specific genes along with aberrance in specification of early lineages during differentiation of mESCs. Genome-wide chromatin occupancy studies suggest regulation of chromatin architecture at key target loci through triplex formation. Our studies thus reveal a role for lncRNA Mrhl in regulating differentiation programs in mESCs in the context of appropriate cues through chromatin-mediated responses.

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