Nr0b1 is a negative regulator of Zscan4c in mouse embryonic stem cells

Fujii, Setsuko; Nishikawa‐Torikai, Satomi; Futatsugi, Yoko; Toyooka, Yayoi; Yamane, Mariko; Ohtsuka, Satoshi; Niwa, Hitoshi

doi:10.1038/srep09146

Cited by 38 publications

(38 citation statements)

References 44 publications

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“…It has been shown that Nr0b1 is downregulated in RA conditions (Hosler et al 1993) (see also Fig. 2C), and the downregulation of Nr0b1 results in a substantial increase of Zscan4 c expression as well as in the upregulation of endoderm-related genes in ESCs (Fujii et al 2015). Thus, Nr0b1 (−/−) cells described in the previous report (Fujii et al 2015) are possibly similar to the secondary colonies described in our study.…”

Section: Discussionmentioning

confidence: 84%

“…2C), and the downregulation of Nr0b1 results in a substantial increase of Zscan4 c expression as well as in the upregulation of endoderm-related genes in ESCs (Fujii et al 2015). Thus, Nr0b1 (−/−) cells described in the previous report (Fujii et al 2015) are possibly similar to the secondary colonies described in our study. However, it is not clear whether this is the only or the main mechanism that increases the proportion of Zscan4 (+) cells in secondary colonies.…”

Section: Discussionmentioning

confidence: 99%

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Emergence of undifferentiated colonies from mouse embryonic stem cells undergoing differentiation by retinoic acid treatment

Sharova

Sharov

Piao

et al. 2016

In Vitro Cell.Dev.Biol.-Animal

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Retinoic acid (RA) is one of the most potent inducers of differentiation of mouse embryonic stem cells (ESCs). However, previous studies show that RA treatment of cells cultured in the presence of a leukemia inhibitory factor (LIF) also result in the upregulation of a gene called Zscan4, whose transient expression is a marker for undifferentiated ESCs. We explored the balance between these two seemingly antagonistic effects of RA. ESCs indeed differentiated in the presence of LIF after RA treatment, but colonies of undifferentiated ESCs eventually emerged from these differentiated cells — even in the presence of RA. These colonies, named secondary colonies, consist of three cell types: typical undifferentiated ESCs expressing pluripotency genes such as Pou5f1, Sox2, and Nanog; cells expressing Zscan4; and endodermal-like cells located at the periphery of the colony. The capacity to form secondary colonies was confirmed for all eight tested ESC lines. Cells from the secondary colonies — after transfer to the standard ESC medium — retained pluripotency, judged by their strong alkaline phosphatase (ALP) staining, typical colony morphology, gene expression profile, stable karyotype, capacity to differentiate into all three germ layers in embryoid body formation assays, and successful contribution to chimeras after injection into blastocysts. Based on flow cytometry analysis (FACS), the proportion of Zscan4-positive cells in secondary colonies was higher than in standard ESC colonies, which may explain the capacity of ESCs to resist the differentiating effects of RA and instead form secondary colonies of undifferentiated ESCs. This hypothesis is supported by cell-lineage tracing analysis, which showed that most cells in the secondary colonies were descendents of cells transiently expressing Zscan4.

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Section: Discussionmentioning

confidence: 84%

Section: Discussionmentioning

confidence: 99%

Emergence of undifferentiated colonies from mouse embryonic stem cells undergoing differentiation by retinoic acid treatment

Sharova

Sharov

Piao

et al. 2016

In Vitro Cell.Dev.Biol.-Animal

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“…However, Dax1-deficient ES cells contributed to chimeric embryos, indicating sustained pluripotency [16]. These cells also overexpressed Zscan4c, suggesting that Dax1 is a negative regulator of Zscan4c [16].…”

Section: Gata4 and Gata6 Expression Was Reportedly Induced In Cre-loxmentioning

confidence: 99%

“…conditional knockout ES cells [16]. However, Dax1-deficient ES cells contributed to chimeric embryos, indicating sustained pluripotency [16].…”

Section: Gata4 and Gata6 Expression Was Reportedly Induced In Cre-loxmentioning

confidence: 99%

Esrrb directly binds to Gata6 promoter and regulates its expression with Dax1 and Ncoa3

Uranishi

Akagi

Koide

et al. 2016

Biochemical and Biophysical Research Communications

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Estrogen-related receptor beta (Esrrb) is expressed in embryonic stem (ES) cells and is involved in self-renewal ability and pluripotency. Previously, we found that Dax1 is associated with Esrrb and represses its transcriptional activity. Further, the disruption of the Dax1-Esrrb interaction increases the expression of the extra-embryonic endoderm marker Gata6 in ES cells. Here, we investigated the influences of Esrrb and Dax1 on Gata6 expression. Esrrb overexpression in ES cells induced endogenous Gata6 mRNA and Gata6 promoter activity. In addition, the Gata6 promoter was found to contain the Esrrb recognition motifs ERRE1 and ERRE2, and the latter was the responsive element of Esrrb. Associations between ERRE2 and Esrrb were then confirmed by biotin DNA pulldown and chromatin immunoprecipitation assays. Subsequently, we showed that Esrrb activity at the Gata6 promoter was repressed by Dax1, and although Dax1 did not bind to ERRE2, it was associated with Esrrb, which directly binds to ERRE2. In addition, the transcriptional activity of Esrrb was enhanced by nuclear receptor co-activator 3 (Ncoa3), which has recently been shown to be a binding partner of Esrrb.Finally, we showed that Dax1 was associated with Ncoa3 and repressed its transcriptional activity. Taken together, the present study indicates that the Gata6 pCAGIP-Myc-Dax1, and pCAGIP-Myc-Dax1LTm, were generated as described previously [9,21]. The Ncoa3 coding region and its truncated mutants were amplified using polymerase chain reaction (PCR) before being cloned into expression vectors.The Gata6 gene promoter region (-0.9 kb) was amplified using PCR and was cloned into pGL4.10 (Promega, Madison, WI, USA) to produce the plasmid pGL4.10-Gata6P 0.9k. To construct the Esrrb-responsive element (ERRE)-mutant pGL4.10-Gata6P 0.9k plasmids, mutant ERRE1 or ERRE2 elements were generated using PCR with specific primers. PCR products were then cloned into pGL4.10 to produce pGL4.10-Gata6P 0.9k-ERRE1 mut and -ERRE2 mut vectors. All primer sequences are listed in Supplemental Table S1. 9Plasmids were introduced into cultured cells using Lipofectamine 2000 (Life Technologies, Grand Island, NY, USA). Two days after transfection, cells were treated with 1 μg/mL puromycin for 5-7 days, and RNA samples for RT-PCR were isolated and examined as described previously [21]. The cell extracts for the luciferase assays were prepared 48 h after transfection, and luciferase activity was measured using a luciferase assay kit (Promega) and an AB-2200 luminometer (ATTO, Tokyo, Japan). Biotin-labeled DNA pull-down and chromatin immunoprecipitation assaysBiotin-labeled DNA pull-down assays were performed as described previously [21]. Briefly, biotin-labeled oligonucleotides were incubated with A3-1 ES extracts or HEK293 cell extracts that had been transfected with pCAGIP-Myc-Dax1 and/or pCAGIP-Esrrb in the presence of streptavidin-agarose (Novagen, Darmstadt, Germany).Subsequently, non-labeled oligonucleotide (either wild-type or mutant) was added for competition assays at a ...

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“…Although Nanog-deficient ES cells retain the capacity for self-renewal (11), deletion of the Nanog gene causes early embryonic lethality, whereas forced expression of Nanog in ES cells accelerates their self-renewal in a LIF-independent manner (12,13). Furthermore, other transcriptional regulators, including ESRRB (14 -16), DAX1 (17)(18)(19), SALL4 (20 -22), ZIC3 (23), KLF4 (24), MYC (25,26), and MAX (27), have been identified as key regulators of the self-renewal capacity and pluripotency of ES cells.…”

mentioning

confidence: 99%

ETS-related Transcription Factors ETV4 and ETV5 Are Involved in Proliferation and Induction of Differentiation-associated Genes in Embryonic Stem (ES) Cells

Akagi

Kuure

Uranishi

et al. 2015

Journal of Biological Chemistry

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Background: Characteristics of ES cells are controlled by gene regulatory networks, and ETV4 and -5 participate in the network. Results: Proliferation, induction of ectodermal marker genes, and expression of stem cell-related genes were impaired in Etv4/5 double KO ES cells. Conclusion: ETV4 and -5 crucially define properties of ES cells. Significance: Gene regulatory networks are dysregulated in the double KO ES cells.

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Nr0b1 is a negative regulator of Zscan4c in mouse embryonic stem cells

Cited by 38 publications

References 44 publications

Emergence of undifferentiated colonies from mouse embryonic stem cells undergoing differentiation by retinoic acid treatment

Emergence of undifferentiated colonies from mouse embryonic stem cells undergoing differentiation by retinoic acid treatment

Esrrb directly binds to Gata6 promoter and regulates its expression with Dax1 and Ncoa3

ETS-related Transcription Factors ETV4 and ETV5 Are Involved in Proliferation and Induction of Differentiation-associated Genes in Embryonic Stem (ES) Cells

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