Research ArticleGENETICS

Zscan4 binds nucleosomal microsatellite DNA and protects mouse two-cell embryos from DNA damage

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Science Advances  20 Mar 2020:
Vol. 6, no. 12, eaaz9115
DOI: 10.1126/sciadv.aaz9115
  • Fig. 1 Analysis of Zscan4 genomic occupancy in 2C-like cells.

    (A) Schematic of the workflow describing the two mESC reporter lines and FACS strategy for ChIP-seq experiments. Reporter lines contain a transgene with a 3.6-kb region upstream from the Zscan4 open reading frame driving either GFP (Zprom::GFP, left) or a GFP-Zscan4 transgene (Zprom::GFP-Zscan4; right). LIF, leukemia inhibitory factor. (B) Heat map representation of GFP-Zscan4, endogenous Zscan4, Dux (13), and H3K4me3 (14) ChIP-seq signal enrichments over indicated number of Zscan4 sites, active TSSs, and Dux sites (±5 kb from the center). Heat maps are sorted by the strength of GFP-Zscan4 ChIP-seq signals. The relative signal intensity is indicated in a color scale. Dux and H3K4me3 datasets are from (13, 14). (C) The top sequence motif recovered from the top 3000 peaks in Zscan4 ChIP-seq is highly similar to the GFP-Zscan4 motif and partially overlaps with the previously published SELEX motif (15). Logos for the consensus motifs were generated using SeqPos.

  • Fig. 2 Zscan4 binds to DNA in a sequence-specific manner.

    (A) Heat maps display density of repetitive elements and sequence features over Zscan4, active TSSs, and Dux sites. (B) Degree of enrichment/depletion of Zscan4 occupancy at indicated sequence repeats, with the strongest enrichment observed at (TG)n and (CA)n repeats and their variations. (C) Violin plot of the size distribution of the predicted Z-DNA prone regions, for Zscan-bound (right) and Zscan-free (left) instances. (D) Titration curve quantifying the binding affinity of purified Zscan4(ZnF) to its consensus TG repeat sequence in vitro. Kd is the mean value of three independent experiments. (E and F) EMSAs showing interaction of increasing amounts of Zscan4(ZnF) as indicated with a Cy5-labeled oligo containing an Oct4 consensus sequence, a (GC)n sequence (E), or a (TG)n repeat (F).

  • Fig. 3 Zscan4 associates with nucleosome-rich regions in 2C-like cells.

    (A) Heat map of ATAC-seq signal from 2C-like cells FACS-sorted from the Zprom::GFP line. Signals were centered and sorted as in Fig. 1B. (B) Representative browser tracks illustrating ChIP-seq profiles from H3K4me3 (blue), Dux (red), endogenous and transgenic Zscan4 (green), and ATAC-seq (black). Z-DNA motif enrichment is shown at the bottom. Z-DNA motif predictions were downloaded from the non-B DB database (41). Colored rectangles highlight examples of Zscan4 binding sites (green), a Dux binding site (red), and an active TSS (blue). (C) ChIP-qPCR analysis in 2C-like cells (GFP+) FACS-sorted from the Zprom::GFP line measuring H3 occupancy, at a representative panel of Zscan4 binding sites and open chromatin regions, as determined by ATAC-seq. Error bars denote SD from three replicates. Primer sequences are provided in table S1. (D) Average ATAC-seq signal from reads > 147 bp, indicating nucleosome positioning at TSSs, Dux, and Zscan4 sites. Signal enrichment at the center of TSS and Dux sites indicates open chromatin with positional nucleosomes on either side, while a dip in signal at the center of Zscan4 binding site suggests nucleosomal protection.

  • Fig. 4 Zscan4 binds to nucleosomes in vitro.

    (A and B) EMSAs showing the interaction of increasing amounts of Zscan4(ZnF), as indicated, with 147 bp of Cy5-labeled DNA sequence containing (TG)n consensus sequence (left) and with Cy5-labeled NCP, assembled with the same 147 bp of DNA (right). Detection with Cy5 fluorophore is shown in (A) and detection with SYBR Gold for visualization of nucleic acids is shown in (B). (C) Titration curve quantifying the binding affinity of purified Zscan4(ZnF) under identical conditions to the naked 147-bp DNA and NCP in vitro. Kd is the mean value of three independent experiments.

  • Fig. 5 Zscan4 stabilizes nucleosomal DNA under torsional strain.

    (A) Top: Schematic illustrating the relationship between curaxin and SSRP1. Curaxin intercalation into DNA results in nucleosome destabilization, negative supercoiling, and conversion of susceptible microsatellite regions to Z-DNA, which is recognized by SSRP1 (12). Bottom: Heat maps of SSRP1 ChIP-seq in mESCs treated with DMSO or indicated concentrations of curaxin. ChIP-seq signal enrichments were sorted and centered as in Fig. 1B. (B and C) EMSAs monitoring interaction between NCP and Zscan4(ZnF) in the presence of curaxin. Schematic above each panel summarizes the reaction performed. Gels were visualized using the fluorescent Cy5 signal present on the octamer (bottom panels) and SYBR Gold for visualization of nucleic acids (top panels). (D) Titration curve displaying the relationship between increasing concentrations of curaxin and percentage of free DNA released upon nucleosome destabilization, in the presence and absence of Zscan4(ZnF).

  • Fig. 6 Zscan4 depletion leads to transcriptionally dependent elevation of DNA damage in 2C embryos.

    (A) Representative immunofluorescence images of γH2A.X and Zscan4 staining in mouse 2C embryos in G2 stage (31 hpf), derived from zygotes injected with control or Zscan4 siRNA and treated with DMSO or triptolide (2 hours prior fixation in the presence of EU) as indicated. (B and C) Quantification of Zscan4 staining (B) or γH2A.X foci (C) in siControl (green) and siZscan4 (pink) 2C embryos (31 hpf) treated with DMSO (23 siControl and 12 siZscan4 embryos) or triptolide (21 siControl and 15 siZscan4 embryos) as shown. P values were determined by Wilcoxon test. (D) Proposed model of transcriptionally dependent regulation of genome stability by Zscan4 in early development. See the main text for details.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/6/12/eaaz9115/DC1

    Fig. S1. Zscan4 reporter validation.

    Fig. S2. Enrichment of Zscan4 at repetitive elements.

    Fig. S3. Nucleosomal binding of Zscan4 in 2C-like cells.

    Fig. S4. Nucleosomal binding of Zscan4 in vitro.

    Fig. S5. SSRP1 binding at repetitive elements.

    Fig. S6. Transcription at Zscan4 sites in 2C embryo.

    Table S1. List of primers for ChIP-qPCR in Fig. 3C.

    Reference (42)

  • Supplementary Materials

    This PDF file includes:

    • Fig. S1. Zscan4 reporter validation.
    • Fig. S2. Enrichment of Zscan4 at repetitive elements.
    • Fig. S3. Nucleosomal binding of Zscan4 in 2C-like cells.
    • Fig. S4. Nucleosomal binding of Zscan4 in vitro.
    • Fig. S5. SSRP1 binding at repetitive elements.
    • Fig. S6. Transcription at Zscan4 sites in 2C embryo.
    • Table S1. List of primers for ChIP-qPCR in Fig. 3C.
    • Reference (42)

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