Figure 3: Molecular features of ES cells expressing Zscan4–ERT2 and a proposed mode of Zscan4 actions.

(a) Constitutive presence of Zscan4–ERT2 and forced expression of Zscan4 made telomeres longer than controls in both F1-hybrid (129/C57BL/6)-derived ES cells (V6.5) and C57BL/6-derived ES cells (MC2). Telomere lengths (average±s.e.m.; n=3) are shown. (b) Constitutive presence of Zscan4–ERT2 and forced expression of Zscan4 maintained better karyotype in long-term culture of ES cells. (c) Comparisons of global expression profiles between V6.5 ZERT2 #18 ES cells and V6.5 #2 control ES cells (left scatter plot) and between MC1 Em⁺ (Zscan4⁺) ES cells and Em⁻ (Zscan4⁻) ES cells (right scatter plot). Red and green dots represent genes that show expression differences with statistical significance (false discovery rate <0.05 and fold change >2). Venn diagram showing the overlap between genes that are more highly expressed in V6.5 ZERT2 #18 cells than V6.5 #2 cells, and genes that are more highly expressed in Em⁺ (Zscan4⁺) cells than Em⁻ (Zscan4⁻) cells (centre). (d) A proposed model. Upper panel: ES cells oscillate between Zscan4⁻ and Zscan4⁺ states, resulting in the normal ES cells with only 1–5% of Zscan4⁺ cells. While in Zscan4⁻ state, the potency of ES cells gradually decreases, which is restored to some extent by transient activation of Zscan4 (Zscan4⁺ state). However, the potency of ES cells still decreases during the long-term culture. Lower Panel: when the Zscan4 is more frequently activated by expressing an exogenous Zscan4, ES cells are rejuvenated more frequently, resulting in the longer maintenance of the high developmental potency.