scholarly journals Loss of PRC2 subunits primes lineage choice during exit of pluripotency

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Chet H. Loh ◽  
Siebe van Genesen ◽  
Matteo Perino ◽  
Magnus R. Bark ◽  
Gert Jan C. Veenstra
Keyword(s):  
Gene Activation ◽  
Embryonic Stem ◽  
Embryoid Bodies ◽  
Specific Gene ◽  
Cell Fates ◽  
Lineage Specification ◽  
Polycomb Repressive Complex 2 ◽  
Expression Of Genes ◽  
Coordinated Expression ◽  
Lineage Specific Gene

AbstractPolycomb Repressive Complex 2 (PRC2) is crucial for the coordinated expression of genes during early embryonic development, catalyzing histone H3 lysine 27 trimethylation. Two distinct PRC2 complexes, PRC2.1 and PRC2.2, contain respectively MTF2 and JARID2 in embryonic stem cells (ESCs). In this study, we explored their roles in lineage specification and commitment, using single-cell transcriptomics and mouse embryoid bodies derived from Mtf2 and Jarid2 null ESCs. We observe that the loss of Mtf2 results in enhanced and faster differentiation towards cell fates from all germ layers, while the Jarid2 null cells are predominantly directed towards early differentiating precursors, with reduced efficiency towards mesendodermal lineages. These effects are caused by derepression of developmental regulators that are poised for activation in pluripotent cells and gain H3K4me3 at their promoters in the absence of PRC2 repression. Upon lineage commitment, the differentiation trajectories are relatively similar to those of wild-type cells. Together, our results uncover a major role for MTF2-containing PRC2.1 in balancing poised lineage-specific gene activation, whereas the contribution of JARID2-containing PRC2 is more selective in nature compared to MTF2. These data explain how PRC2 imposes thresholds for lineage choice during the exit of pluripotency.

scholarly journals Loss of PRC2 subunits primes lineage choice during exit of pluripotency

2020 ◽  
Author(s):  
Chet H Loh ◽  
Matteo Perino ◽  
Magnus R Bark ◽  
Gert Jan C Veenstra
Keyword(s):  
Es Cells ◽  
Gene Activation ◽  
Embryonic Stem ◽  
Embryoid Bodies ◽  
Specific Gene ◽  
List Type ◽  
Cell Fates ◽  
Germ Layers ◽  
Polycomb Repressive Complex 2 ◽  
Lineage Priming

AbstractPolycomb Repressive Complex 2 (PRC2) is crucial for the coordinated expression of genes during early embryonic development, catalyzing histone H3 lysine 27 trimethylation. There are two distinct PRC2 complexes, PRC2.1 and PRC2.2, which contain respectively MTF2 and JARID2 in ES cells. Very little is known about the roles of these auxiliary PRC2 subunits during the exit of pluripotency. In this study, we explored their roles in lineage specification and commitment, using single-cell transcriptomics and mouse embryoid bodies derived from Mtf2 and Jarid2 null embryonic stem cells (ESCs). We observed that the loss of Mtf2 resulted in enhanced and faster differentiation towards cell fates from all germ layers, while the Jarid2 null cells were predominantly directed towards early differentiating precursors and neuro-ectodermal fates. Interestingly, we found that these effects are caused by derepression of developmental regulators that were poised for activation in pluripotent cells and gained H3K4me3 at their promoters in the absence of PRC2 repression. Upon lineage commitment, the differentiation trajectories were relatively similar to those of wild type cells. Together, our results uncovered a major role for MTF2-containing PRC2.1 in balancing poised lineage-specific gene activation, providing a threshold for lineage choice during the exit of pluripotency.HighlightsEnhanced and faster differentiation into all three germ layers in Mtf2 null embryoid bodiesJarid2 null cells enriched for early differentiating precursors and neuro-ectodermal cell fatesMTF2 is critical for the balance of activation and repression of key developmental regulatorsPRC2 coordinates lineage choice and execution of the lineage-specific program by thresholding of lineage-priming

scholarly journals H3K9me3-heterochromatin loss at protein-coding genes enables developmental lineage specification

Science ◽  
2019 ◽  
Vol 363 (6424) ◽  
pp. 294-297 ◽  
Author(s):  
Dario Nicetto ◽  
Greg Donahue ◽  
Tanya Jain ◽  
Tao Peng ◽  
Simone Sidoli ◽  
...  
Keyword(s):  
Embryonic Stem ◽  
Cell Number ◽  
Specific Gene ◽  
Cell Fates ◽  
Lineage Specification ◽  
Protein Coding ◽  
Protein Coding Genes ◽  
Differentiated Cells ◽  
Cell Type Specific ◽  
Embryologic Development

Gene silencing by chromatin compaction is integral to establishing and maintaining cell fates. Trimethylated histone 3 lysine 9 (H3K9me3)–marked heterochromatin is reduced in embryonic stem cells compared to differentiated cells. However, the establishment and dynamics of closed regions of chromatin at protein-coding genes, in embryologic development, remain elusive. We developed an antibody-independent method to isolate and map compacted heterochromatin from low–cell number samples. We discovered high levels of compacted heterochromatin, H3K9me3-decorated, at protein-coding genes in early, uncommitted cells at the germ-layer stage, undergoing profound rearrangements and reduction upon differentiation, concomitant with cell type–specific gene expression. Perturbation of the three H3K9me3-related methyltransferases revealed a pivotal role for H3K9me3 heterochromatin during lineage commitment at the onset of organogenesis and for lineage fidelity maintenance.

Tie-1-directed expression of Cre recombinase in endothelial cells of embryoid bodies and transgenic mice

2001 ◽  
Vol 114 (4) ◽  
pp. 671-676 ◽  
Author(s):  
E. Gustafsson ◽  
C. Brakebusch ◽  
K. Hietanen ◽  
R. Fassler
Keyword(s):  
Endothelial Cells ◽  
Stem Cell ◽  
Embryonic Stem Cell ◽  
Embryonic Stem ◽  
Cre Recombinase ◽  
Embryoid Bodies ◽  
Lymphoid Cells ◽  
Adult Brain ◽  
Specific Gene

Tissue-specific gene inactivation using the Cre-loxP system has become an important tool to unravel functions of genes when the conventional null mutation is lethal. We report here the generation of a transgenic mouse line expressing Cre recombinase in endothelial cells. In order to avoid the production and screening of multiple transgenic lines we used embryonic stem cell and embryoid body technology to identify recombinant embryonic stem cell clones with high, endothelial-specific Cre activity. One embryonic stem cell clone that showed high Cre activity in endothelial cells was used to generate germline chimeras. The in vivo efficiency and specificity of the transgenic Cre was analysed by intercrossing the tie-1-Cre line with the ROSA26R reporter mice. At initial stages of vascular formation (E8-9), LacZ staining was detected in almost all cells of the forming vasculature. Between E10 and birth, LacZ activity was detected in most endothelial cells within the embryo and of extra-embryonic tissues such as yolk sac and chorioallantoic placenta. Ectopic expression of Cre was observed in approximately 12–20% of the adult erythroid, myeloid and lymphoid cells and in subregions of the adult brain. These results show that the tie-1-Cre transgenic strain can efficiently direct deletion of floxed genes in endothelial cells in vivo.

scholarly journals Bach1 regulates self-renewal and impedes mesendodermal differentiation of human embryonic stem cells

2019 ◽  
Vol 5 (3) ◽  
pp. eaau7887 ◽  
Author(s):  
Xiangxiang Wei ◽  
Jieyu Guo ◽  
Qinhan Li ◽  
Qianqian Jia ◽  
Qing Jing ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Human Embryonic Stem Cells ◽  
Embryonic Stem ◽  
Gene Promoters ◽  
Lineage Specification ◽  
Self Renewal ◽  
Polycomb Repressive Complex 2 ◽  
Direct Target ◽  
Processing Protease

The transcription factor BTB and CNC homology 1 (Bach1) is expressed in the embryos of mice, but whether Bach1 regulates the self-renewal and early differentiation of human embryonic stem cells (hESCs) is unknown. We report that the deubiquitinase ubiquitin-specific processing protease 7 (Usp7) is a direct target of Bach1, that Bach1 interacts with Nanog, Sox2, and Oct4, and that Bach1 facilitates their deubiquitination and stabilization via the recruitment of Usp7, thereby maintaining stem cell identity and self-renewal. Bach1 also interacts with polycomb repressive complex 2 (PRC2) and represses mesendodermal gene expression by recruiting PRC2 to the genes’ promoters. The loss of Bach1 in hESCs promotes differentiation toward the mesendodermal germ layers by reducing the occupancy of EZH2 and H3K27me3 in mesendodermal gene promoters and by activating the Wnt/β-catenin and Nodal/Smad2/3 signaling pathways. Our study shows that Bach1 is a key determinant of pluripotency, self-renewal, and lineage specification in hESCs.

scholarly journals Intronic enhancers regulate the expression of genes involved in tissue-specific functions and homeostasis

2020 ◽  
Author(s):  
Beatrice Borsari ◽  
Pablo Villegas-Mirón ◽  
Hafid Laayouni ◽  
Alba Segarra-Casas ◽  
Jaume Bertranpetit ◽  
...  
Keyword(s):  
Gene Expression ◽  
Embryonic Stem ◽  
Gene Expression Pattern ◽  
Specific Gene ◽  
Control Of Gene Expression ◽  
Tissue Specific ◽  
Tissue Specific Gene ◽  
Specific Gene Expression ◽  
Expression Of Genes ◽  
Genomic Location

AbstractTissue function and homeostasis reflect the gene expression signature by which the combination of ubiquitous and tissue-specific genes contribute to the tissue maintenance and stimuli-responsive function. Enhancers are central to control this tissue-specific gene expression pattern. Here, we explore the correlation between the genomic location of enhancers and their role in tissue-specific gene expression. We found that enhancers showing tissue-specific activity are highly enriched in intronic regions and regulate the expression of genes involved in tissue-specific functions, while housekeeping genes are more often controlled by intergenic enhancers. Notably, an intergenic-to-intronic active enhancers continuum is observed in the transition from developmental to adult stages: the most differentiated tissues present higher rates of intronic enhancers, while the lowest rates are observed in embryonic stem cells. Altogether, our results suggest that the genomic location of active enhancers is key for the tissue-specific control of gene expression.

Characterization of hematopoietic lineage-specific gene expression by ES cell in vitro differentiation induction system

Blood ◽  
2000 ◽  
Vol 95 (3) ◽  
pp. 870-878 ◽  
Author(s):  
Takumi Era ◽  
Toshiaki Takagi ◽  
Tomomi Takahashi ◽  
Jean-Christophe Bories ◽  
Toru Nakano
Keyword(s):  
Gene Expression ◽  
Cell Lines ◽  
Embryonic Stem ◽  
Experimental System ◽  
Specific Gene ◽  
Specific Gene Expression ◽  
Differentiation Induction ◽  
Lineage Specific Gene ◽  
Megakaryocytic Cell

The continuous generation of mature blood cells from hematopoietic progenitor cells requires a highly complex series of molecular events. To examine lineage-specific gene expression during the differentiation process, we developed a novel method combiningLacZ reporter gene analysis with in vitro hematopoietic differentiation induction from mouse embryonic stem cells. For a model system using this method, we chose the erythroid and megakaryocytic differentiation pathways. Although erythroid and megakaryocytic cells possess distinct functional and morphologic features, these 2 lineages originate from bipotential erythro-megakaryocytic progenitors and share common lineage-restricted transcription factors. A portion of the 5′ flanking region of the human glycoprotein IIb (IIb) integrin gene extending from base −598 to base +33 was examined in detail. As reported previously, this region is sufficient for megakaryocyte-specific gene expression. However, previous reports that used human erythro-megakaryocytic cell lines suggested that one or more negative regulatory regions were necessary for megakaryocyte-specific gene expression. Our data clearly showed that an approximately 200-base enhancer region extending from −598 to −400 was sufficient for megakaryocyte-specific gene expression. This experimental system has advantages over those using erythro-megakaryocytic cell lines because it recapitulates normal hematopoietic cell development and differentiation. Furthermore, this system is more efficient than transgenic analysis and can easily examine gene expression with null mutations of specific genes.

scholarly journals Phosphorylation of Tet3 by cdk5 is critical for robust activation of BRN2 during neuronal differentiation

2019 ◽  
Vol 48 (3) ◽  
pp. 1225-1238 ◽  
Author(s):  
Vinay Kumar Rao ◽  
Adusumalli Swarnaseetha ◽  
Guo-Hong Tham ◽  
Wei-Qi Lin ◽  
Bin-Bin Han ◽  
...  
Keyword(s):  
Neuronal Differentiation ◽  
Knockout Mouse ◽  
Catalytic Domain ◽  
Dynamic Balance ◽  
Embryonic Stem ◽  
Specific Gene ◽  
Dependent Manner ◽  
Wild Type ◽  
Expression Of Genes

Abstract Tet3 regulates the dynamic balance between 5-methylcyotsine (5mC) and 5-hydroxymethylcytosine (5hmC) in DNA during brain development and homeostasis. However, it remains unclear how its functions are modulated in a context-dependent manner during neuronal differentiation. Here, we show that cyclin-dependent kinase 5 (cdk5) phosphorylates Tet3 at the highly conserved serine 1310 and 1379 residues within its catalytic domain, changing its in vitro dioxygenase activity. Interestingly, when stably expressed in Tet1, 2, 3 triple-knockout mouse embryonic stem cells (ESCs), wild-type Tet3 induces higher level of 5hmC and concomitant expression of genes associated with neurogenesis whereas phosphor-mutant (S1310A/S1379A) Tet3 causes elevated 5hmC and expression of genes that are linked to metabolic processes. Consistent with this observation, Tet3-knockout mouse ESCs rescued with wild-type Tet3 have higher level of 5hmC at the promoter of neuron-specific gene BRN2 when compared to cells that expressed phosphor-mutant Tet3. Wild-type and phosphor-mutant Tet3 also exhibit differential binding affinity to histone variant H2A.Z. The differential 5hmC enrichment and H2A.Z occupancy at BRN2 promoter is correlated with higher gene expression and more efficient neuronal differentiation of ESCs that expressed wild-type Tet3. Taken together, our results suggest that cdk5-mediated phosphorylation of Tet3 is required for robust activation of neuronal differentiation program.

scholarly journals Expression of the liver-specific gene Cyp7a1 reveals hepatic differentiation in embryoid bodies derived from mouse embryonic stem cells

2004 ◽  
Vol 9 (12) ◽  
pp. 1297-1308 ◽  
Author(s):  
Kinji Asahina ◽  
Hiroaki Fujimori ◽  
Keiko Shimizu-Saito ◽  
Yuji Kumashiro ◽  
Kentaro Okamura ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Embryonic Stem ◽  
Mouse Embryonic Stem Cells ◽  
Embryoid Bodies ◽  
Specific Gene ◽  
Hepatic Differentiation

scholarly journals GATA-1 Dominantly Activates a Program of Erythroid Gene Expression in Factor-Dependent Myeloid FDCW2 Cells

1998 ◽  
Vol 18 (6) ◽  
pp. 3278-3288 ◽  
Author(s):  
Dhaya Seshasayee ◽  
Peter Gaines ◽  
Don M. Wojchowski
Keyword(s):  
Gene Expression ◽  
Stem Cell ◽  
Cell Cycle Progression ◽  
Receptor Gene ◽  
Globin Gene ◽  
Gene Activation ◽  
Embryonic Stem ◽  
Lineage Specification ◽  
Epo Receptor ◽  
Receptor Gene Expression

ABSTRACT Erythrocyte development has previously been shown to depend upon the expression of the lineage-restricted trans-acting factor GATA-1. Despite predicted roles for this factor during early development, GATA-1-deficient cells in chimeric mice and embryonic stem cell cultures mature to a late proerythroblast stage and express at least certain genes that normally are thought to be regulated by GATA-1 (including erythroid Krüppel-like factor [EKLF] and the erythropoietin [Epo] receptor). Opportunities to test roles for GATA-1 in erythroid gene activation in these systems therefore are limited. In the present study, in an alternate approach to test the function of GATA-1, GATA-1 has been expressed together with the Epo receptor in myeloid FDCW2 cells and the resulting effects on cytokine-dependent proliferation and erythroid gene expression have been assessed. GATA-1 expression at low levels delayed FDCW2ER cell cycle progression at the G1 phase specifically during Epo-induced mitogenesis. Upon expression of GATA-1 at increased levels, proliferation in response to Epo, interleukin-3 (IL-3), and stem cell factor was attenuated and endogenous GATA-1, EKLF and βmaj-globin gene expression was activated. Friend of GATA-1 (FOG) transcript levels also were enhanced, andets-1 and c-mpl but not Epo receptor gene expression was induced. Finally, in FDCW2 cells expressing increased levels of GATA-1 and a carboxyl-terminally truncated Epo receptor, Epo (with respect to IL-3 as a control) was shown to markedly promote globin transcript expression. Thus, novel evidence for select hierarchical roles for GATA-1 and Epo in erythroid lineage specification is provided.

scholarly journals Activation of the amino acid response modulates lineage specification during differentiation of murine embryonic stem cells

2013 ◽  
Vol 305 (3) ◽  
pp. E325-E335 ◽  
Author(s):  
Jixiu Shan ◽  
Takashi Hamazaki ◽  
Tiffany A. Tang ◽  
Naohiro Terada ◽  
Michael S. Kilberg
Keyword(s):  
Stem Cells ◽  
Amino Acid ◽  
Embryonic Stem Cells ◽  
Embryoid Body ◽  
Embryonic Stem ◽  
Embryoid Bodies ◽  
Specific Cell ◽  
Lineage Specification ◽  
Amino Acid Response

In somatic cells, a collection of signaling pathways activated by amino acid limitation have been identified and referred to as the amino acid response (AAR). Despite the importance of possible detrimental effects of nutrient limitation during in vitro culture, the AAR has not been investigated in embryonic stem cells (ESC). AAR activation caused the expected increase in transcription factors that mediate specific AAR pathways, as well as the induction of asparagine synthetase, a terminal AAR target gene. Neither AAR activation nor stable knockdown of activating transcription factor (Atf) 4, a transcriptional mediator of the AAR, adversely affected ESC self-renewal or pluripotency. Low-level induction of the AAR over a 12-day period of embryoid body differentiation did alter lineage specification such that the primitive endodermal, visceral endodermal, and endodermal lineages were favored, whereas mesodermal and certain ectodermal lineages were suppressed. Knockdown of Atf4 further enhanced the AAR-induced increase in endodermal formation, suggesting that this phenomenon is mediated by an Atf4-independent mechanism. Collectively, the results indicate that, during differentiation of mouse embryoid bodies in culture, the availability of nutrients, such as amino acids, can influence the formation of specific cell lineages.

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