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Z-DNA formation regulates the totipotent-like state and primes Zscan4-dependent chromatin compartmentalization

Nature Structural & Molecular Biology (2026)Cite this article

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Abstract

The progression from the one-cell to the two-cell stage constitutes a remarkable transition, accompanied by the activation of a specific set of embryonic genes, epigenome reprogramming and nuclear architecture reorganization. Some of these characteristics are recapitulated in vitro with the spontaneous emergence of two-cell-like cells from mouse embryonic stem cells, which exhibit a transcriptomic signature resembling the two-cell stage, including the expression of genes such as Dux, Zscan4 and the repetitive element MERVL, as well as a more relaxed chromatin state. Here we show that interchromosomal and intrachromosomal interactions driven by Zscan4 chromatin factors form during this transition and segregate into a distinct genomic compartment, the Z compartment, independently of cohesin and CCCTC-binding factor. Mechanistically, the formation of Z-DNA, an alternative DNA conformation regulated by polyamine levels, appears to promote the emergence of totipotent-like cells and the establishment of the Z compartment. This compartment is characterized by a decrease in active histone marks and a reduced expression of genes associated with differentiation and late developmental processes. Overall, these findings suggest that Z-DNA formation may have a dual role, first in initiating zygotic genome activation (ZGA) and later in guiding genome compartmentalization to safeguard the totipotent-like state by restricting the expression of non-ZGA genes within a permissive chromatin environment.

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Fig. 1: Identification of specific genomic interactions in 2CLCs.
Fig. 2: Identification and characterization of the Z compartment in 2CLCs.
Fig. 3: Zscan4 is required for the specific genomic interactions characteristic of 2CLCs.
Fig. 4: Curaxin-induced Z-DNA formation increases the emergence of both murine 2CLCs and human 8CLCs.
Fig. 5: Zscan4-driven interactions within the Z compartment do not require cohesin.
Fig. 6: Polyamine synthesis inhibition disrupts Zscan4-dependent compartmentalization in 2CLCs.
Fig. 7: Epigenetic and transcriptomic dynamics during the pluripotent-to-totipotent-like conversion.

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Data availability

All raw and processed sequencing data generated in this study were deposited to the Gene Expression Omnibus under accession number GSE291992. Source data are provided with this paper.

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Acknowledgements

We thank R. Koszul for assistance with Hi-C experiments and for providing us with the necessary equipment. We are grateful to E. Nora (University of California, San Francisco) for the AID cell lines. We acknowledge J.-S. Seeler for helpful discussions and insightful comments. We thank S. Schmutz, P.-H. Commere and the Flow Cytometry Platform (Institut Pasteur) for cell sorting and the Biomics Platform (Institut Pasteur) for Hi-C and scRNA-seq sequencing. Sequencing for methyl-seq/RNA-seq was performed by the GenomEast Platform, a member of the France Génomique consortium (ANR-10-INBS-0009). This work was supported by grants from the European Research Council (AdG SUMiDENTITY), Agence Nationale de la Recherche (ANR-19-CE12-0011-01) and Sjöberg Foundation to A.D. The work conducted by A.T. was supported by a grant from European Research Council (COG SynarchiC).

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Author notes

  1. Laure Asselin & Jack-Christophe Cossec

    Present address: Université Paris Cité, CNRS UMR7216 Epigenetics and Cell Fate, Dynamics of Biological Identities Team, Department of Cell Biology and Infection, Institut Pasteur, Paris, France

Authors and Affiliations

  1. Institut Pasteur, Université Paris Cité, Nuclear Organization and Oncogenesis Unit, Paris, France

    Shireen Shajahan, Yann Loe-Mie, Laure Asselin, Marion Salmon-Legagneur, Tatiana Traboulsi, Anne Dejean & Jack-Christophe Cossec

  2. INSERM, U993, Paris, France

    Shireen Shajahan, Yann Loe-Mie, Laure Asselin, Marion Salmon-Legagneur, Tatiana Traboulsi, Anne Dejean & Jack-Christophe Cossec

  3. Sorbonne Université, Collège Doctoral, Complexité du Vivant, Paris, France

    Shireen Shajahan

  4. Institut Pasteur, Université Paris Cité, Bioinformatics and Biostatistics HUB, Paris, France

    Yann Loe-Mie

  5. Université Paris Cité, BioSPC, Paris, France

    Laure Asselin

  6. Institut Pasteur, CNRS UMR 3525, Université Paris Cité, Unité Régulation Spatiale des Génomes, Paris, France

    Agnès Thierry

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Contributions

S.S. and J.-C.C. conceptualized and designed the project. S.S. performed most of the investigations. Y.L.-M. performed most of the bioinformatic analysis. L.A. and M.S.-L. contributed to the Hi-C, DNA-FISH and immunofluorescence experiments. T.T. produced the scRNA-seq data. A.T. provided an optimized Hi-C protocol and assisted S.S. with the initial Hi-C experiments. A.D. contributed to research design and funding. S.S. and J.-C.C. wrote the paper with input from all authors.

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Correspondence to Jack-Christophe Cossec.

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Extended data

Extended Data Fig. 1 Characterization of the genomic architecture in 2CLCs.

(a) Decay of intra-chromosomal contact probability as a function of genomic distance in ESCs (red) and 2CLCs (cyan). (b) Aggregate TAD positions annotated in ESCs and 2CLCS. (c) Aggregate over loop positions in ESCs and 2CLCS. (d) Saddle plots showing chromatin compartmentalization between 50 kb genomic bins ranked according to their eigenvector value (PC1) in ESCs and 2CLCs. (e) Example of Hi-C maps for full chromosomes 4, 6, and 8 (left) in ESCs and 2CLCs, along with a zoomed-in view of one end of their subtelomeric regions, opposite the centromere (right).

Extended Data Fig. 2 Similar genomic interactions observed in both spontaneous and induced 2CLCs.

a) Hi-C matrix for chromosome 5 in 2CLCs, published by Zhu et al.39, with a zoomed-in view highlighting the specific chromatin interactions (arrows) described in Fig. 1a. (b) Screenshot showing the positions of DNA-FISH probes 1 (purple) and 2 (green) on chromosome 5, used to visualize the interaction marked by the dotted square on the Hi-C map. (c) Representative images obtained as a z-stack (z-resolution = 1 µm) of a Zscan4-positive 2CLC, showing the overlap of two pairs of DNA-FISH probes. N = 3 independent experiments. (d) Alignment of computed TADs, represented as dark triangles on the Hi-C map of the full chromosome 5 (top), as well as on a zoomed-in region (bottom) in 2CLCs. TADs that show significantly increased interactions in 2CLCs compared to ESCs are represented as a track below the Hi-C map. Dashed lines indicate TAD-TAD interactions. (e) Representative dot plots from flow cytometry analysis showing an increase in the MERVL-tdTomato-positive population (2CLCs) upon treatment with Pladienolide B (PlaB) (right) compared to untreated ESCs (left). (f) Quantification of the flow cytometry analysis as in “e” (N = 4 independent biological replicates, Mean value), *** p = 0.0009, two-tailed t-test. (g) Hi-C map illustrating similar 3D genome organization in PlaB-induced 2CLCs (top right) and spontaneous 2CLCs (bottom left) in a defined region of chromosome 5.

Source Data

Extended Data Fig. 3 Involvement of Zscan4, but not Dux, in the genomic compartmentalization in 2CLCs.

(a) Saddle plot showing contact probability between 20 kb genomic bins ranked according to the number of Dux binding motifs per bin in 2CLCs (top) and ESCs (bottom). (b) Representative DNA-FISH images of probe 1 (chromosome 4) and probe 2 (chromosome 5) combined with immunofluorescence in ESC cultures. 2CLCs were identified by Zscan4 staining, and DNA was counterstained with DAPI. Scale bar: 5 µm. The zoomed-in image shows overlapping probes in a Zscan4-positive 2CLC. Scale bar: 2 µm. Analysis of n = 300 Zscan4-negative cells and n = 100 Zscan4-positive cells. (c) Representative immunofluorescence images showing 2CLCs detected by an anti-Nelfa antibody (red) and anti-Zscan4 (green). DNA is counterstained with DAPI. Scale bar: 5 µm. N = 3 independent experiments. (d) Zscan4 peak density per kilobase within the Z compartment, plotted according to PC1 quantiles (Hi-C 2CLCs/ESCs; see Supplementary Table 4), reveals that the top 5% of PC1 exhibits the highest Zscan4 density. (e) Saddle plots showing cis- and trans-contact probabilities between 20 kb genomic bins in 2CLCs and ESCs, ranked according to their eigenvector value (PC1) from the differential Hi-C analysis between 2CLCs and ESCs. (f) Screenshot of the Z-compartment, and the trans-interacting regions identified in ESCs by Quinodoz et al.49 across the 19 autosomal chromosomes. (g) Density distributions of the PC1 value (2CLCs versus ESCs) in regions showing trans-contacts in ESCs from Quinodoz et al.49 and in whole genome. The dash line represents the PC1 value that has been selected to determine the Z-compartment (Genomic regions within the top 5% of positive PC1 values). (h) Number of Zscan4 peaks in Z-compartment (red bar) in comparison to randomly shuffled peaks (1000 sets of random peaks were tested, Mean +/- SD, permutation test: p < 1e−3). (i) As in (h) in interacting TADs (iTADs). (j) Pile-up plots of cis-contact probability enrichment at Zscan4 binding sites located (left) within or outside the Z compartment, (right) within or outside iTADs in 2CLCs. (k) Pile-up plots of cis-contacts (left) and trans-contacts (right) showing probability enrichment at Zscan4 binding sites within the Z compartment, either intersecting with iTADs (top) or not (bottom), in 2CLCs. (l) Percentage of base pairs (bp) of Zscan4 peaks per bin of 200 kb across the Z, A and B compartment in 2CLCs. The adjusted p-values were obtained from a two-sided Student’s t-test with a Bonferroni correction. Box plots show the median (center line), 25th and 75th percentiles (box limits), and whiskers extend to 1.5× IQR; outliers are plotted individually.

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Extended Data Fig. 4 Genomic clustering of upregulated genes upon Zscan4 knockdown in 2CLCs.

(a) Quantification of Zscan4 protein upon knockdown with siRNAs (siZscan4) compared to the non-targeting control (siNeg) in ESCs based on immunoblot analysis, N = 4 independent experiments, Mean Value, *** p < 0.001, two-tailed t-test. (b) Unsupervised hierarchical clustering analysis of the top 100 2CLC markers based on their expression in the indicated samples. The color scale bar represents relative gene expression changes between conditions, normalized by the standard deviation. (c) Representative dot plots from flow cytometry analysis after siZscan4 (left) and siNeg (middle) transfection in ESCs. The red square highlights the MERVL-positive 2CLC population. The quantification of N = 6 independent experiments is shown on the right, Mean value. ‘ns’ indicates non-significant (two-tailed t-test). (d) Hi-C map showing 2CLC-specific chromatin interactions on chromosome 4 in control 2CLCs (black arrows) and their reduction in Zscan4-depleted 2CLCs (gray arrows). Resolution: 250 kb. (e) Immunoblots showing induction of Flag-Zscan4 upon doxycycline (Dox) treatment, using an anti-Flag antibody and an anti-Zscan4 antibody. The asterisk represents a nonspecific band. Actin was used as a housekeeping control. N = 2 independent experiments. (f) Pile-up plots of cis-contacts (left) and trans-contacts (right) showing no notable enrichment at Zscan4 binding sites in sorted MERVL-negative ESCs expressing exogenous Zscan4 ( + Dox) or not ( − Dox). (g) Localization of autosomal genomic regions enriched for upregulated and downregulated genes in Zscan4-depleted 2CLCs compared to control 2CLCs. The track showing Zscan4 peaks is also indicated. (h) Number of Zscan4 peaks in downregulated gene-rich regions upon Zscan4 knockdown in 2CLCs (red bar) in comparison to random regions (1000 sets of random regions were tested, Mean +/- SD), permutation test: ns for non-significant. (i) Ontology analysis of the biological processes associated with downregulated genes upon Zscan4 knockdown in 2CLCs compared to control 2CLCs. (j) K-means clustering analysis of genes expressed during early development. The arrow indicates the optimal number of clusters, which is 6. (k) Heatmap showing the transcriptional levels of genes in early embryos, consistent with the k-means clustering analysis in panel ‘j’.

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Extended Data Fig. 5 Transcriptomic analysis of the impact of Z-DNA induction on the emergence of murine 2CLCs and human 8CLCs.

(a) Alignment of Z-DNA motifs and Zscan4 peaks along chromosomes 5 and 8. (b) Saddle plot showing contact probability between 20 kb genomic bins ranked according to the number of Z-DNA motifs per bin in 2CLCs (left) and ESCs (right). (c) Additional immunofluorescence images showing 2CLCs detected by an anti-Nelfa antibody (red) and Z-DNA stained with an anti-Z-DNA antibody (green). DNA is counterstained with DAPI. Scale bar: 5 μm. N = 3 independent experiments. (d) Gene expression levels of the indicated transcripts were quantified by RT-qPCR and normalized to Gapdh in ESCs treated with curaxin (0.1-0.5 μM). Mean + SD, * p < 0.05, ** p < 0.01, *** p < 0.001, two-tailed t-test, N = 3 independent biological replicates. (e) Representative immunofluorescence images showing an increase in the number of 2CLCs stained with an anti-Nelfa antibody (red) in mESCs treated with 0.5 μM curaxin for 48 h, compared to the control condition, and the associated quantification for N = 3 independent experiments, ** p = 0.0071, two-tailed t-test. Scale bar: 50 μm. (f) Heatmap showing the relative expression of the top 100 2CLC markers60 across the six clusters identified from the scRNA-seq analysis of ESCs with or without curaxin treatment (see Fig. 4e). The color scale bar represents relative gene expression changes between conditions, normalized by the standard deviation. (g) UMAP plot as in ‘Fig. 4e’ showing the expression of Tdpoz3 and Pramel19. (h) KEGG Pathway enrichment categories for the top 200 gene markers of clusters 1 + 4 (see Fig. 4e). (i) UMAP plot as in ‘Fig. 4e’ showing the expression of Cdkn1a. (j) Representative example of immunoblots for p53, phospho-p53 (p-p53) as well as Histone H3 and Gapdh, used as the housekeeping controls in ESCs treated or not with curaxin. N = 3 independent experiments. (k) Heatmap showing the relative expression of the top 100 human 8CLC markers60 across the 11 clusters identified from the scRNA-seq analysis of human iPSCs with or without curaxin treatment (see Fig. 4h). The color scale bar represents relative gene expression changes between conditions, normalized by the standard deviation. (l) UMAP plot as in ‘Fig. 4h’ showing the expression of KLF17 and TPRX1. (m) KEGG Pathway enrichment categories for the top 200 gene markers of clusters 2 + 3 + 4 + 7 + 10 (see Fig. 4h). (n) UMAP plot as in ‘Fig. 4h’ showing the expression of CDKN1a. (o) Representative immunoblots for p53, Zscan4, and Gapdh (used as a housekeeping control) in ESCs transfected with non-targeting (NT) or p53-targeting siRNAs, treated or not with curaxin (0.5 μM). (p) Gene expression levels of the indicated transcripts were quantified by RT-qPCR and normalized to Gapdh in ESCs transfected with non-targeting (NT) or p53-targeting siRNAs, treated or not with curaxin (0.5 μM). N = 2.

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Extended Data Fig. 6 Zscan4-driven interactions in the Z compartment are CTCF-independent.

(a) (Left) contact probability matrix for the indicated portion of the chromosome 5 (Chr 5), comparing the curaxin-induced 2CLCs to spontaneous 2CLCs. (Right) Pile up plots of cis- and trans-contact probability enrichment at Zscan4 binding sites in curaxin-induced 2CLCs. (b) Immunoblots for Rad21 and Actin (used as a housekeeping control) in Rad21-degron ESCs treated with auxin and/or curaxin, or not treated. N = 2 independent experiments. (c) Immunoblots for Ctcf and Actin (used as a housekeeping control) in Ctcf-degron ESCs treated with auxin and/or curaxin, or not treated. N = 2 independent experiments. (d) Aggregate TAD positions annotated in untreated, curaxin-treated and curaxin + auxin-treated Ctcf-degron ESCs. (e) Contact probability matrix for the indicated portion of chromosome 5 (Chr5) in untreated (left panel), curaxin-treated (middle panel), and curaxin + auxin-treated (right panel) Ctcf-degron ESCs. (f) Pile-up plots of cis- and trans-contact probability enrichment at Zscan4 binding sites in untreated (left panel), curaxin-treated (middle panel), and curaxin + auxin-treated (right panel) Ctcf-degron ESCs.

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Extended Data Fig. 7 Transcriptomic analysis of 2CLCs upon polyamine inhibition.

(a) A simplified schematic of the polyamine synthesis pathway highlighting the action of DFMO and AMXT-1501 (red crosses) in reducing polyamine concentration in cells. (b) Bar graph showing the gradual depletion of polyamine concentration over time in ESCs treated with 0.25 mM DFMO and 15 μM AMXT-1501. N = 2. (c) Representative dot plots from flow cytometry analysis of control ESCs and ESCs treated for 24 or 48 h with polyamine pathway inhibitors. The red square highlights the MERVL-positive 2CLC population. (d) Ontology analysis of biological processes associated with upregulated genes (left) and downregulated genes (right) in 2CLCs after 48 h of polyamine inhibition (0.25 mM DFMO and 15 μM AMXT-1501). (e) Number of Zscan4 peaks in downregulated gene-rich regions upon polyamine inhibition in 2 CLCs (red bar) in comparison to random regions (1000 sets of random regions were tested, Mean +/- SD, permutation test: p < 1e−3). (f) Localization of autosomal genomic regions enriched for upregulated and downregulated genes in 2 CLCs upon polyamine inhibition compared to control 2 CLCs. Tracks displaying Zscan4 peaks and regions enriched for genes upregulated in Zscan4-depleted 2 CLCs (as in Extended Data Fig. 4e) are also indicated. Overlapping genomic regions enriched for upregulated genes in Zscan4-depleted 2 CLCs and downregulated genes in 2 CLCs upon polyamine inhibition are colored in red.

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Extended Data Fig. 8 Examples of epigenetic and transcriptomic dynamics during the conversion of ESCs to 2 CLCs.

(a) Screenshot of Z-DNA motifs, DNA replication initiation zones, Zscan4 peaks, Z-compartment, and the log2 fold change of histone marks, DNA methylation, and RNA transcription signals between 2 CLCs and ESCs across chromosome 4. (b) Same as ‘a’ for chromosome 2.

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Shajahan, S., Loe-Mie, Y., Asselin, L. et al. Z-DNA formation regulates the totipotent-like state and primes Zscan4-dependent chromatin compartmentalization. Nat Struct Mol Biol (2026). https://doi.org/10.1038/s41594-026-01751-5

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