Zscan4 Inhibits Maintenance DNA Methylation to Facilitate Telomere Elongation in Mouse Embryonic Stem Cells

Abstract

Proper telomere length is essential for embryonic stem cell (ESC) self-renewal and pluripotency. Mouse ESCs (mESCs) sporadically convert to a transient totipotent state similar to that of two-cell (2C) embryos to recover shortened telomeres. Zscan4, which exhibits a burst of expression in 2C-like mESCs, is required for telomere extension in these cells. However, the mechanism by which Zscan4 extends telomeres remains elusive. Here, we show that Zscan4 facilitates telomere elongation by inducing global DNA demethylation through downregulation of Uhrf1 and Dnmt1, major components of the maintenance DNA methylation machinery. Mechanistically, Zscan4 recruits Uhrf1 and Dnmt1 and promotes their degradation, which depends on the E3 ubiquitin ligase activity of Uhrf1. Blocking DNA demethylation prevents telomere elongation associated with Zscan4 expression, suggesting that DNA demethylation mediates the effect of Zscan4. Our results define a molecular pathway that contributes to the maintenance of telomere length homeostasis in mESCs.

Keywords: DNA methylation; Dnmt1; ESCs; Uhrf1; Zscan4; telomere.

Figure 1. 2C-Like mESCs Show Global DNA Hypomethylation Due to Uhrf1 and Dnmt1 Degradation

(A,B) IF analysis of stable mESC clones transfected with the Zscan4-GFP reporter, which shows that GFP⁺ cells (indicated by arrowheads) are severely hypomethylated. Representative images (A) and quantification of the data (B) are shown. Scale bar in (A), 10 μm. The total numbers of cells counted in (B) are indicated, and the results are shown as percentages. (C) Dot blot analysis of DNA from FACS-sorted GFP⁺ and GFP^- populations, which confirms global hypomethylation in GFP⁺ cells. The same membrane was stained with SYTOX Green to verify equal DNA loading. (D) Quantification of the data in (C) by measuring the signal intensities. Presented are relative 5mC levels (mean + s.d.). **** P<0.001. (E) Southern blot analysis of the major and minor satellite repeats after digestion of genomic DNA with methylation-sensitive restriction enzymes (MaeII for major satellite; HpaII for minor satellite), which shows DNA hypomethylation in GFP⁺ cells. (F) Western blot analysis of FACS-sorted GFP⁺ and GFP^- cells, which shows severe downregulation of Uhrf1 and Dnmt1 in the GFP⁺ population. (G,H) IF analysis of stable mESC clones transfected with the Zscan4-GFP reporter, which confirms that Uhrf1 and Dnmt1 levels are extremely low in GFP⁺ cells (G) but largely maintained with MG132 treatment (H) (10 μM MG132, 13 hr for Uhrf1 detection, and 6 hr for Dnmt1 detection). (I) Quantification of the data in (G,H). The total numbers of cells counted are indicated, and the results are shown as percentages. (J) Western blot analysis of GFP⁺ and GFP^- cells sorted after 12 hr of treatment with MG132 or DMSO, which shows that MG132 prevents Dnmt1 and Uhrf1 degradation in GFP⁺ cells. See also Figures S1-S3.

Figure 2. Zscan4 Overexpression Leads to Uhrf1 and Dnmt1 Downregulation and DNA Demethylation

(A) Western blot analysis of stable clones expressing Myc-Zscan4c in Flag-Dnmt1 KI/+ mESCs, which show severe downregulation of Uhrf1 and Dnmt1 (examined by both Dnmt1 and Flag antibodies). Mock, stable Flag-Dnmt1 KI/+ mESC clones transfected with empty Myc vector. (B) Dot blot analysis confirming global DNA hypomethylation in cells overexpressing Myc-Zscan4c. (C) IF analysis of co-cultured Myc-Zscan4c⁺ cells (indicated by arrowheads) and Mock (Myc^-) cells, which shows severely lower levels of Uhrf1, Flag-Dnmt1, and 5mC in Myc-Zscan4c⁺ cells. Scale bars, 10 μm. (D) Western blot analysis showing that Zscan4 shRNA (shZscan4) efficiently depletes Myc-Zscan4c in an overexpression clone and, as a consequence, Uhrf1 and Dnmt1 levels are greatly restored. Untrans, untransfected Flag-Dnmt1 KI/+ mESCs; shControl, control shRNA. (E) Dot blot analysis showing the restoration of 5mC levels in shZscan4-mediated knockdown clones. See also Figure S4.

Figure 3. Zscan4 Depletion Prevents Uhrf1 and Dnmt1 Downregulation and DNA Demethylation in 2C-Like mESCs

(A,B) Western blot and IF analyses of Zscan4-GFP reporter mESC lines stably transfected with Zscan4 shRNA (shZscan4) or control shRNA (shControl), which show that shZscan4 efficiently depletes endogenous Zscan4. Two shControl and four shZscan4 clones are shown in (A). (C-E) DNA methylation analyses by dot blot (C), Southern blot (D), and IF (E) showing that depletion of endogenous Zscan4 prevents DNA hypomethylation in GFP⁺ (2C-like) cells. (F,G) IF analysis showing that Zscan4 depletion prevents downregulation of Uhrf1 (F) and Dnmt1 (G) in GFP⁺ (2C-like) cells (indicated by arrowheads). Scale bars, 10 μm.

Figure 4. Blocking DNA Demethylation Prevents Zscan4-Mediated Telomere Elongation

(A) Western blot analysis of stable mESC clones expressing Myc-Zscan4c alone or both Myc-Zscan4c and Flag-Dnmt1-PCNA, which shows the expression of the transfected proteins. Mock, stable mESC clones transfected with empty Myc vector. (B) Dot blot analysis showing that hypomethylation associated with Myc-Zscan4c expression is blocked with co-expression of Flag-Dnmt1-PCNA. The same membrane was stained with SYTOX Green to verify equal DNA loading. (C,D) Telomere length analyses by Q-FISH (C) and Flow-FISH (D) showing that telomere elongation induced by Myc-Zscan4c is prevented with co-expression of Flag-Dnmt1-PCNA. The relative telomere length (mean ± s.d.) for each sample is indicated. Green lines in (C) indicate median relative telomere length. TFU, telomere fluorescence units. (E,F) CO–FISH analysis after labeling leading strand telomeres showing increased TSCE frequency with Zscan4c expression, which is prevented with co-expression of the Dnmt1-PCNA fusion protein. Chromosomes were stained with DAPI (blue), and telomeres were marked with a Cy3-cojugated telomere probe (red). In the absence of recombination between telomeric repeats, only one telomere at each chromosome end is labeled. A T-SCE event gives rise to three telomeric signals per chromosome (indicated by arrowheads). Representative CO-FISH images (E) and quantification (mean + s.d.) of T-SCE frequency in different groups (F) are shown. See also Figures S5 and S6.

Figure 5. Zscan4 Recruits the Dnmt1-Uhrf1 Complex by Directly Interacting with Uhrf1

(A) Co-IP experiments with Myc- and Flag-tagged proteins expressed in HEK293 cells, which demonstrate that Zscan4c interacts with both Dnmt1 and Uhrf1. (B) Co-IP experiments with Myc-Zscan4c and Flag-Uhrf1 expressed in wild-type (WT) or Dnmt1^-/- mESCs, which show that Dnmt1 deficiency does not affect Zscan4c-Uhrf1 interaction. (C) Co-IP experiments with Flag-Zscan4c and Myc-Dnmt1 expressed in WT or Uhrf1^-/- mESCs, which show that Uhrf1 is required for Zscan4c-Dnmt1 interaction. (D) Mutagenesis and co-IP experiments showing that deletion of the Zscan4c ZNF domain (ΔZNF1-4) or the ZNF4 motif (ΔZNF4) abolishes its interaction with Uhrf1. (E) GST pull down assay showing that GST-Zscan4c, but not GST-ΔZNF1-4 or GST-ΔZNF4, interacts with His-Uhrf1. Shown below is a gel of the purified GST- Zscan4c fusion proteins stained by Coomassie Brilliant Blue. (F) Co-IP experiments with Myc- and HA-tagged Zscan4c proteins showing that Zscan4c self-associates and that deletion of the SCAN domain abolishes selfassociation. See also Figure S7.

Figure 6. Both Uhrf1-Binding and Self-Association abilities of Zscan4 Are Required for Its Effects on DNA Methylation and Telomere Length

(A,B) Western blot and dot blot analyses of mESC stable clones expressing Myc-Zscan4c or deletion mutants, which demonstrate that the ΔZNF4, ΔZNF1-4, and ΔSCAN mutants fail to induce Uhrf1 and Dnmt1 downregulation (A) and hypomethylation (B). Mock, mESC stable clones transfected with empty Myc vector. (C,D) Telomere length analyses by Q-FISH (C) and Flow-FISH (D) showing that WT Zscan4c, but not the ΔZNF4 and ΔSCAN mutants, induces telomere elongation. The relative telomere length (mean ± s.d.) for each sample is indicated. (E,F) CO-FISH analysis showing that WT Zscan4c, but not the ΔZNF4 and ΔSCAN mutants, leads to increased T-SCE events (indicated by arrowheads) compared to Mock control cells. Representative CO–FISH images (E) and quantification (mean + s.d.) of TSCE frequencies (F) in different groups are shown.

Figure 7. Zscan4 Enhances Uhrf1 Self-Ubiquitination and Degradation

(A) Western blot analysis of Dnmt1 and Uhrf1 in mESC stable clones expressing Myc-Zscan4c or mutants (ΔZNF4 or ΔSCAN) at different time points after blocking proteinsynthesis with 100 μg/ml cycloheximide (CHX) to determine Dnmt1 and Uhrf1 stability. (B) Quantification of data in (A). The intensities of the Uhrf1 and Dnmt1 bands were measured and normalized to those of β-actin. Shown are changes in Uhrf1 and Dnmt1 levels over time relative to their levels before CHX treatment. (C) The half-lives of Uhrf1 and Dnmt1 proteins (mean + s.d.) in mESCs expressing WT or mutant Zscan4c. ** p<0.01; *** p<0.001. (D) Ubiquitination assay showing that Myc-Zscan4c, but not the ΔSCAN or ΔZNF4 mutant, enhances ubiquitination of Flag-tagged WT Uhrf1, but not the E3 ligaseinactive mutant (H730A). (E,F) IF analysis of Uhrf1^-/-mESC stable clones expressing Flag-tagged WT Uhrf1 or the E3 ligase-inactive mutants, which shows downregulation of Flag-Uhrf1, but not the E3 ligase-inactive mutants, in Zscan4⁺/2C-like cells. Representative images (E) and quantification of the data (F) are shown. (G,H) IF analysis of Uhrf1^-/- mESCs or Dnmt1^-/- mESCs (transfected with the Zscan4-GFP reporter) showing that Uhrf1 deficiency prevents Dnmt1 downregulation and Dnmt1 deficiency has no effect on Uhrf1 downregulation in Zscan4⁺/2C-like cells. Representative images (G) and quantification of the data (H) are shown. (I) Proposed mechanism by which Zscan4 induces global DNA demethylation and telomere elongation. As mESCs proliferate, they undergo telomere shortening, which ultimately triggers the transition to the 2C-like totipotent state, resulting in a burst of Zscan4 expression. Zscan4, via its ZNF4 motif, recruits the Uhrf1-Dnmt1 complex and, through self-association mediated by its SCAN domain, brings two or more Uhrf1-Dnmt1 heterodimers to close proximity, facilitating Uhrf1-mediated ubiquitination of Uhrf1 itself and Dnmt1, likely through cross actions between Uhrf1 molecules. Subsequent Uhrf1 and Dnmt1 degradation leads to global DNA demethylation, which facilitates telomere recombination and elongation. After telomere lengths are recovered, the cells transition back to the pluripotent state.