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doi: 10.1038/srep09146.

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

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

Setsuko Fujii et al. Sci Rep..

Abstract

Nuclear receptor subfamily 0, group B, member 1 (Nr0b1, also known as Dax1) is regarded as an important component of the transcription factor network that governs pluripotency in mouse embryonic stem (ES) cells. Here we generated inducible knockout ES cells for Nr0b1 using the Cre-loxP system and analyzed its precise function. We succeeded in establishing the Nr0b1-null ES cells and confirmed their pluripotency by showing their contribution to chimeric embryos. However, they proliferated slowly with over-expression of 2-cell stage specific transcripts including Zscan4c, which is known to be involved in telomere elongation in ES cells. We revealed that over-expression of Zscan4c prevents normal self-renewal by inducing arrest at G2 phase followed by cell death and that Nr0b1 directly represses the Zscan4c promoter. These data indicated that Nr0b1 is not essential to maintain pluripotency but is involved in the proper activation of 2-cell specific transcripts for self-renewal.

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Conflict of interest statement

The authors declare no competing financial interests.

Figures

Figure 1
Figure 1. Generation of inducibleNr0b1-null ES cells.
(a) Schematic representation of the strategy to generate the inducibleNr0b1–null ES cells. (b) PCR genotyping of the ES cells at each step of genetic engineering. P1 and P2 were the results of PCR with primer pairs KO PCR 1 and 2, respectively. (c) Colony formation of the inducibleNr0b1-null ES cells. The stem cell colonies were scored by the compact morphology after Leischman staining. Error bars indicate standard deviation (n = 3). (d) Depletion of NR0B1 protein in theNr0b1-null ES cells determined by western blotting.
Figure 2
Figure 2. Defective proliferation ofNr0b1-null ES cells.
(a) Colony morphologies ofNr0b1fl/Y andNr0b1KO/Y ES cells after the culture for 5 days on feeder cells. (b) Proliferation ratio of wild-type (WT),Nr0b1fl/Y (fl/Y) andNr0b1KO/Y (KO/Y) ES cells. 1 × 104 cells were seeded on feeder cells and the numbers of cells were counted after 1, 3 and 5 days. Error bars indicate standard deviation (n = 3). (c) Quantitative RT-PCR analysis ofNr0b1fl/Y (fl/Y) andNr0b1KO/Y (KO/Y) ES cells for the expressions of pluripotency-associated genes. The level of expression of each transcript inNr0b1fl/Y ES cells was set at 1.0. Error bars indicate standard deviation (n = 3). (d) Cell-cycle profiling of wild-type (WT),Nr0b1fl/Y (fl/Y) andNr0b1KO/Y (KO/Y) ES cells by FACS with propidium iodide (PI) staining. Proportions of the cells at each cell cycle were shown in the graph with error bars indicating standard deviation (n = 3). Asterisk indicates statistic difference (P < 0.05; t-test). (e) Analysis of early apoptosis and dead cells in wild-type (WT),Nr0b1fl/Y (fl/Y) andNr0b1KO/Y (KO/Y) ES cells by FACS with Annexin-V and PI staining. Proportions of the dead cells and the cells at early apoptosis were shown in the graph with error bars indicating standard deviation (n = 3). Asterisk indicates statistic difference (P < 0.05; t-test).
Figure 3
Figure 3. Chimeric embryos withNr0b1-null ES cells.
Chimeric embryos at 13.5 dpc obtained by injection ofNr0b1fl/Y (fl/Y) andNr0b1KO/Y (KO/Y) ES cells carrying constitutively-activeEgfp transgene.
Figure 4
Figure 4. Up-regulation of 2-cell specific transcripts inNr0b1-null ES cells.
(a) DNA microarray analyses ofNr0b1fl/Y (fl/Y) ES cells cultured with or without tamoxifen (Tx) for 4 days, andNr0b1KO/Y (KO/Y) ES cells. Scatter plots of log-ratios of relative expression levels were shown for each indicated pairs. The genes shown statistic difference (>2-fold, FDR = 0.05) were highlighted with red or green colors. (b) Highlight of the relative gene expressions of each category from the microarray data sets. (c) Immunostaining ofNr0b1fl/Y (fl/Y) andNr0b1KO/Y (KO/Y) ES cells for Tcstv1. (d) Fluorescent photomicrograph ofNr0b1fl/Y (fl/Y) andNr0b1KO/Y (KO/Y) ES cells carryingpPB-Zscan4c-mCherry. (e) FACS analysis ofNr0b1fl/Y (fl/Y) andNr0b1KO/Y (KO/Y) ES cells carryingpPBCAG-Egfp-IZ andpPB-Zscan4c-mCherry. (f) Fluorescent intencity of mCherry inNr0b1fl/Y (fl/Y) andNr0b1KO/Y (KO/Y) ES cells carryingpPB-Zscan4c-mCherry. The cells undergoing proliferation and cell death were identified in live imaging and the fluorescent intensities were measured. The average intensities in each genotype are indicated with standard error.
Figure 5
Figure 5. Enrichment of G2 phase byZscan4c expression.
(a) Cell-cycle profiles of Zscan4c-mCherry positive and negative cells ofNr0b1fl/Y (fl/Y) andNr0b1KO/Y (KO/Y) ES cells. Each fraction was sorted and analyzed by PI staining. Proportions of the cells at each cell cycle were shown in the box with error values indicating standard deviation (n = 3). (b) Cell-cycle-dependent expression of Zscan4c in ES cells. ES cells at different cell-cycle phases were separaed by FACS based on the Fucci system and the expressions of the indicated genes were quantified by Quantitative RT-PCR. The level of expression of each transcript at G1 phase was set at 1.0. Error bars indicate standard deviation (n = 3). (c) Cell-cycle profiles of ES cells with or without the expression of the Zscan4c transgene. ES cells carrying the doxycline (Dox) inducible Zscan4c transgene were cultured with or without Dox for 2 days and analyzed by PI staining. Proportions of the cells at each cell cycle were shown in the box.
Figure 6
Figure 6. Transcriptional repression ofZscan4c by Nr0b1.
(a) The activity of the Zscan4c promoter inNr0b1fl/Y (fl/Y) andNr0b1KO/Y (KO/Y) ES cells. The relative luciferase activity ofpZscan4c-Fluc was measured by dual-luciferase assay with or without the expression of exogenous Nr0b1. Error bars indicate standard deviation (n = 3). tk (tk-luc) acts as a negative control. (b) Quantitative RT-PCR analysis ofNr0b1fl/Y (fl/Y) ES cells cultured with Tx for the expressions of 2-cell specific transcript genes. The level of expression of each transcript inNr0b1fl/Y ES cells was set at 1.0. Error bars indicate standard deviation (n = 3). The results of two independent experiments were shown.
Figure 7
Figure 7. Phenotypic reversion ofNr0b1-null ES cells by inducible expression of Nr0b1 transgene.
(a) The scheme of the rescue experiments ofNr0b1-null ES cells with tetracycline-inducibleNr0b1 transgene. (b) Proliferation ratio of wild-type (WT),Nr0b1fl/Y (fl/Y),Nr0b1KO/Y (KO/Y) andNr0b1KO/Y with inducibleNr0b1-transgene (clones Tg2 and Tg3) ES cells. 1 × 104 cells were seeded on feeder cells and the numbers of cells were counted after 1, 3 and 5 days. Error bars indicate standard deviation (n = 3). (c) Quantitative RT-PCR analysis of wild-type (WT),Nr0b1fl/Y (fl/Y),Nr0b1KO/Y (KO/Y) and Tg ES cells for the expressions of 2-cell specific transcripts. The level of expression of each transcript inNr0b1fl/Y ES cells was set at 1.0. Error bars indicate standard deviation (n = 3). (d) Immunostaining ofTg2 + Dox andTg2 − Dox ES cells for Tcstv1. Photomicrographs were captured with confocal microscopy. (e) Proportion of Tcstv1-positive cells in Tg ES cells cultured with or without Dox. 10 images captured with confocal microscopy were quantified for each genotype. The average proportions are indicated with standard error. (f) Quantitative RT-PCR analysis ofTg2-Dox ES cells cultured with Dox for the expressions ofNr0b1 transgene and 2-cell specific transcript genes. The level of expression of each transcript inTg2-Dox ES cells was set at 1.0. Error bars indicate standard deviation (n = 3).
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