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. 2010 Apr 8;464(7290):858-63.
doi: 10.1038/nature08882. Epub 2010 Mar 24.

Zscan4 Regulates Telomere Elongation and Genomic Stability in ES Cells

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Free PMC article

Zscan4 Regulates Telomere Elongation and Genomic Stability in ES Cells

Michal Zalzman et al. Nature. .
Free PMC article

Abstract

Exceptional genomic stability is one of the hallmarks of mouse embryonic stem (ES) cells. However, the genes contributing to this stability remain obscure. We previously identified Zscan4 as a specific marker for two-cell embryo and ES cells. Here we show that Zscan4 is involved in telomere maintenance and long-term genomic stability in ES cells. Only 5% of ES cells express Zscan4 at a given time, but nearly all ES cells activate Zscan4 at least once during nine passages. The transient Zscan4-positive state is associated with rapid telomere extension by telomere recombination and upregulation of meiosis-specific homologous recombination genes, which encode proteins that are colocalized with ZSCAN4 on telomeres. Furthermore, Zscan4 knockdown shortens telomeres, increases karyotype abnormalities and spontaneous sister chromatid exchange, and slows down cell proliferation until reaching crisis by passage eight. Together, our data show a unique mode of genome maintenance in ES cells.

Figures

Figure 1
Figure 1. Zscan4 is transiently expressed in ES cells
a, Zscan4 expression visualized by RNA in situ hybridization and GFP-Emerald reporter gene (green). b, FACS analysis of pZscan4-Emerald cells. c, Time-course fluorescent/DIC images of pZscan4-Emerald cells after sorting and culturing only Em+ cells or Em− cells. d, Flow cytometry analysis of cells shown in c. e, Images of pZscan4-CreERT2 cells maintained in tamoxifen and stained with X-gal at each passage. Inserts: a single colony. f, A % fraction of LacZ+ cells measured by flow cytometry analysis using CMFDG fluorescence LacZ assay: tamoxifen was present continuously (Tam+: P1–P9), only for the first three passages (Tam+: P1–P3), or absent (Tam−, control).
Figure 2
Figure 2. Characterization of Zscan4 knockdown cells
a, Confirmation of Zscan4 knockdown and rescue by qPCR analysis using a common primer for all Zscan4 paralogs (top) and Western blot analysis (bottom). Cells were cultured for 3 days in Dox+ or Dox− conditions. shCont (control shRNA in the same parental cells) was used to exclude off-target effects. See Supplementary Fig 7 and 8 for additional controls. b, Reduction of proliferation by Zscan4 knockdown, until cells died abruptly at passage 8 (P+8). Rescue improved proliferation. Assays were done in triplicate in four independent experiments. c, Annexin-V Apoptosis assay performed by flow cytometry. d, Karyotype deterioration seen after Zscan4 knockdown.
Figure 3
Figure 3. Zscan4 regulates telomere length
a, A distribution diagram of relative telomere length, analyzed by Q-FISH and TFL-Telo software (results of 10 pooled nuclei, totaling 1,600 telomeres). b, A distribution of relative telomere length in Zscan4c-overexpressing cells.
Figure 4
Figure 4. Zscan4 promotes T-SCE but inhibits spontaneous SCE in non-telomeric regions
a, T-SCE (arrows) shown by chromosome orientation FISH (CO-FISH): chromosomes stained with DAPI (blue); telomeres marked with a Cy3-cojugated telomere probe (red). Left, tet-Zscan4c cells (Dox+). Right, Zscan4-overexpressing cells (Dox−). b, A summary of total T-SCE events in >20 nuclei/sample based on three independent experiments. c, The number of cells with spontaneous genomic SCEs was decreased by Zscan4 overexpression (*P=0.01) but increased by knockdown (**P=0.005). d, The number of SCEs per metaphase was reduced by Zscan4 overexpression (*P=0.02) but elevated by knockdown (**P=0.0016). Assays were done in three independent experiment with 50 metaphases/sample (total n=150). Error bars indicate s.e.m.
Figure 5
Figure 5. ZSCAN4 forms foci on telomeres along with meiosis specific homologous recombination mediators
a, confocal microscope images with chromosomal DNAs stained with DAPI (blue). Top, colocalization of ZSCAN4 (red, immunostaining) and telomeres (green, T-FISH using Alexa488-conjugated DNA probe). Middle, SPO11 localized on telomeres following Zscan4c induction for 3 days. Bottom, colocalization of ZSCAN4-FLAG foci (green: immunostaining with antibodies against FLAG, fused to ZSCAN4 ORF19) and SPO11 (red). b, qPCR analysis showing upregulation of meiosis-specific homologous recombination genes, following Zscan4c overexpression. c, Western blot confirmed b at protein levels. Lanes 1, Zscan4 Dox+; 2, Zscan4 overexpression Dox−; 3, Empty Dox+; and 4, Empty Dox−. d, A model for Zscan4 action in ES cells.

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