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Heterochromatin establishment during early mammalian development is regulated by pericentromeric RNA and characterized by non-repressive H3K9me3

Abstract

Following fertilization in mammals, the gametes are reprogrammed to create a totipotent zygote, a process that involves de novo establishment of chromatin domains. A major feature occurring during preimplantation development is the dramatic remodelling of constitutive heterochromatin, although the functional relevance of this is unknown. Here, we show that heterochromatin establishment relies on the stepwise expression and regulated activity of SUV39H enzymes. Enforcing precocious acquisition of constitutive heterochromatin results in compromised development and epigenetic reprogramming, which demonstrates that heterochromatin remodelling is essential for natural reprogramming at fertilization. We find that de novo H3K9 trimethylation (H3K9me3) in the paternal pronucleus after fertilization is catalysed by SUV39H2 and that pericentromeric RNAs inhibit SUV39H2 activity and reduce H3K9me3. De novo H3K9me3 is initially non-repressive for gene expression, but instead bookmarks promoters for compaction. Overall, we uncover the functional importance for the restricted transmission of constitutive heterochromatin during reprogramming and a non-repressive role for H3K9me3.

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Fig. 1: De novo H3K9me3 occurs in the paternal pronucleus immediately after fertilization.
Fig. 2: Pericentromeric RNA modulation of the KMT activity of SUV39H2 attenuates H3K9me3 levels in the early preimplantation mouse embryo.
Fig. 3: H3K9me3 is compatible with gene expression during early preimplantation development.
Fig. 4: SUV39H1 induces constitutive heterochromatin.
Fig. 5: SUV39H1 compromises development.
Fig. 6: SUV39H1 does not prevent zygotic genome activation.
Fig. 7: SUV39H1 inhibits epigenetic reprogramming.

Data availability

Sequencing data generated during this study have been deposited into the GEO under accession codes GSE126021 (single-embryo RNA-seq for Suv39h1wt from the two-cell stage to the morula stage), GSE126185 (RNA-seq for pooled Suv39h1-overexpressing embryos at the two-cell stage), GSE126492 (RNA-seq for pooled embryos with Suv39h2 RNAi at the two-cell stage) and GSE138686 (NicE-seq for embryos with Suv39h2 RNAi at the eight-cell stage). Previously published mouse embryo datasets re-analysed here are available under accession codes GSE45719 and GSE38495 (single-cell RNA-seq), GSE66390 (ATAC-seq) and GSE98149 (H3K9me3 ChIP-seq). All other data supporting the findings of this study are available from the corresponding author upon reasonable request. Source data are provided with this paper.

Code availability

All next-generation sequencing data were analysed with standard programs and packages, as detailed in the Methods. Code is available upon request.

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Acknowledgements

We thank A. J. Bannister for providing the Suv39h1 cDNA and S. Daujat for providing the H3K64me3 antibody, K. Swist for help with expression of recombinant SUV39H enzymes and analysis of data shown in Fig. 2c., A. J. Bannister and members of the IES for helpful discussions. We thank A. Ettinger for help with the blind counting and microscopy. C.G. and T.J. were supported by funds from the Max Planck Society. A.E. was partially funded through a MENRT fellowship. M.-E.T.-P. acknowledges funding from EpiGeneSys NoE, ERC-Stg ‘NuclearPotency’, EMBO Young Investigator Programme, the Schlumberger Foundation for Research and Education and the Helmholtz Association and by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) project-ID 213249687–SFB 1064. N.B. and V.B. were funded through the Laboratoire d’Excellence REVIVE (Investissement d’Avenir, ANR-10-LABX-73) and the INRA animal facilities (IERP, Jouy-en-Josas, France). J.M.V. acknowledges funding from EpiGeneSys NoE and the Max Planck Society and S.G. from the Ministry of Science and Technology of China (grant number 2016YFA0100400).

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Contributions

A.B. and M.-E.T.-P. conceived the project and wrote the manuscript. A.B., V.B., C.G., H.G.C. and A.E. performed and designed experiments with the supervision of N.B., T.J., S.P. and M.-E.T.-P. E.R.R.-M., Q.R., D.R.-T., K.K., S.L.G., V.S.U., X.L. and C.W., performed and designed computational analysis with the supervision of S.G., S.P., J.M.V. and M.-E.T.-P.

Corresponding author

Correspondence to Maria-Elena Torres-Padilla.

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Extended data

Extended Data Fig. 1 De novo H3K9me3 activity occurs immediately after fertilization.

a, b. Wild-type and Suv39h1/2 double knock-out MEF cells immunostained with a anti-SUV39H1 and b anti-SUV39H2 b antibody. Shown is a representative single confocal section from 2 independent experiments. Scale bar 20 µm. c. Zygotes microinjected with HA-Suv39h1 mRNA as described in Fig. 4a were fixed and immunostained with anti-HA and anti-SUV39H2 antibodies after 8 h. A representative single confocal section of the paternal pronucleus in 7 zygotes from 2 independent experiments is shown. The anti-SUV39H2 antibody does not recognise SUV39H1 protein. Scale bar 10 µm. d. SUV39H2 immunostaining in the two pronculei in the early zygote (19 h post-hCG). Shown is a representative embryo of 11 from 3 independent experiments. e. Knockdown efficiency of dsRNA targeting Suv39h2 was assessed by RT-qPCR of Suv39h2 mRNA at 30 h post-hCG in single embryos, the same timing as the immunofluorescence experiments shown in Fig. 1g, h. The data represents the average fold change of Suv39h2 levels normalized to average levels of Actin-b and Gapdh in each embryo. N = the indicated number of embryos across 3 independent experiments. The box plots depict the median and interquartile range. The two sided Mann-Whitney U-test was used to compare experimental groups. f. Representative maximum intensity projections of 2-cell stage embryos upon Suv39h2 RNAi using dsRNA, immunostained with anti-H3K9me3 antibodies. N = total number of individual embryos analysed in each group across 3 independent experiments for Suv39h2 and 2 for control knockdown. Scale bar 10 µm. g. Quantification of average fluorescence intensity for H3K9me3 in 2-cell stage nuclei. Data were normalized to the average H3K9me3 signal in non-injected embryos and are presented as mean ± S.E.M (n = as in f). Statistical analysis was performed using the two-sided Mann-Whitney U test for comparing nonparametric distributions. Statistical source data are shown in Source Data Extended Data Fig. 1. Source data

Extended Data Fig. 2 RNA modulation of the KMT activity of SUV39H2 attenuates H3K9me3 levels in the early preimplantation mouse embryo.

a. Representative full z-series projections of confocal stacks of the indicated number of embryos manipulated as described in Fig. 2d, injected instead with single-stranded forward or reverse major satellite transcripts from 2 independent experiments. Paternal and maternal pronuclei are indicated. Scale bar 10 µm. b. Quantification of the levels of H3K9me3 staining in the maternal and paternal pronuclei across embryos represented in a. Maternal and paternal H3K9me3 levels were normalized to average levels in non-injected embryo. The graph depicts the mean ± S.E.M. (n = the number of embryos analysed as indicated in a across 2 independent experiments). Statistical analysis was performed using the two-sided Mann-Whitney U test for comparing nonparametric distributions. Statistical source data are shown in Source Data Extended Data Fig. 2. Source data

Extended Data Fig. 3 H3K9me3 is compatible with gene expression during early preimplantation development.

a. Enrichment heatmap (log 2 observed/expected TE density) in regions with differing paternal H3K9me3 levels. Columns correspond to 10 kb binned genomic regions sorted from left to right by increasing H3K9me3 levels. Rows correspond to individual TE types hierarchically clustered using the centroid linkage method. Clusters are coarsely annotated with the dominant TE families. H3K9me3 Chipseq data from12 was reanalyzed for panels a and b. b. Examples of paternal H3K9me3 levels in a 40 kb region around selected TE classes (red line, 5% trimmed mean; grey, randomized signal). c-d. Violin plots showing relative expression values of the indicated repetitive elements (c) or genes (d) in knockdown of Suv39h2 against control (lacZ) knockdown embryos at the 2-cell stage (46 h post-hCG). The mean expression value of n = 10 embryos (control) and n = 13 embryos (Suv39h2 knockdown) from 3 independent experiments is depicted as a red point and the individual expression in each embryo as black dots. Statistical analysis was performed using the two-sided Mann-Whitney U test. See Supplementary Table 4 for a list of genes (sheet1), REs (sheet2), and for the genes Taqman® assay IDs and reason(s) for inclusion. e. Shown is the mean (± S.E.M.) proportion of embryos that reached the blastocyst stage 3 days after microinjection (n = the total number of embryos analysed is indicated from 5 independent experiments). Statistical analysis was performed using the N-1 Chi-squared test for comparing independent proportions. On the right are shown representative brightfield images of embryos. Images were taken at 120 h post-hCG injection. Scale bar 100 µm. f. Comparison of the genomic distribution of open chromatin identified by NicE-seq compared to ATAC-seq in 8-cell stage mouse preimplantation embryos. Statistical source data are shown in Source Data Extended Data Fig. 3. Source data

Extended Data Fig. 4 SUV39H1 induces constitutive heterochromatin.

a. Zygotes were microinjected with HA-Suv39h1 mRNA, cultured for 24 or 48 h, fixed and immunostained with an anti-HA antibody. A representative full z-series projection from at least 3 independent experiments is shown. Scale bar 10 µm. b. Mean levels of H3K9me3 (±S.E.M) in 2-cell embryos (42–44 h post-hCG). Confocal stacks were reconstructed with IMARIS and nuclei were segmented using the DAPI channel. Fluorescence intensity was quantified in each nucleus after background subtraction. N = numbers as in Fig. 4d. Statistical analysis was performed using the two-sided Mann-Whitney U test. c. Zygotes were microinjected with Ehmt2 (G9a) or Setdb2 mRNA, fixed at the 2-cell stage (42–44 h post-hCG) and immunostained with anti-H3K9me3 and anti-HA antibodies. A representative full z-series projection is shown. Mean fluorescence levels (± S.E.M.) are shown on the right (n = the total number of embryos analysed as indicated on the left panels across 2 independent experiments). Statistical analysis was performed using the Mann-Whitney U test. Scale bar 20 µm. d. As in c but with Setdb1 mRNA. Mean fluorescence levels (± S.E.M.) are shown on the right (n = the total number of embryos analysed as indicated on the left panels across 2 independent experiments). Statistical analysis was performed using the two-sided T test. Scale bar 20 µm. e. Mean levels (± S.E.M) of H3K64me3 in 2-cell stage embryos (42–44 h post-hCG injection) after Suv39h1wt or Suv39h1mut expression. N = total number of embryos analysed is show in Fig. 4h across 5 independent experiments for Suv39h1wt and 4 for Suv39h1mut. Statistical analysis was performed using the two-sided Mann-Whitney U test. f. Summary of the stages of development for which embryos arrested as described in Fig. 5b. N = numbers as in Fig. 5b. Statistical source data are shown in Source Data Extended Data Fig. 4. Source data

Extended Data Fig. 5 SUV39H1 compromises development.

a. Violin plots depicting absolute expression levels (log2 scale) in single Suv39h1wt, Suv39h1mut-expressing embryos and non-injected controls at the morula stage (78 h post-hCG). Expression values in individual embryos are indicated by black points and the mean by a red point. Genes with a significant difference (P < 0.05; two-sided Mann-Whitney U test) between the Suv39h1wt group and both control groups are shown on the left. N = numbers and stages of the embryos acquired from 3 independent experiments are shown in Supplementary Table 5 and a list of genes analysed are shown in Supplementary Table 6. b, c. Embryos injected with mRNA encodinb Suv39h1mut (control) or (c) Suv39h1wt were cultured to the blastocyst stage, fixed and immunostained with the indicated antibodies. Shown are representative full z-series projections. N = number of embryos analyzed from 2 independent experiments is indicated. Scale bar 20 µm. Arrows in Nanog panel in c indicate cells ectopically expressing low levels of NANOG. The graphs below show the levels of NANOG and CDX2 in individual nuclei, for the representative embryos above. A clear segregation of CDX2 or NANOG positive populations is apparent in the controls, but is less pronounced in the Suv39h1wt-injected embryos. The red box indicates a consistent population of double CDX2/NANOG positive cells present in Suv39h1wt embryos only. d. The absolute difference in intensity (ΔCDX2/NANOG) for each nucleus (plotted points) from the embryos classified as blastocysts (>32 cells) plotted against the sum of the NANOG and CDX2 signal (n = 7 embryos for Suv39h1mut and 5 for Suv39h1wt across 2 independent experiments). Fewer nuclei have high ΔCDX2/NANOG and more nuclei have high sum CDX2/NANOG scores in the Suv39h1wt embryos, suggesting a defect in the resolution of the outer and inner lineages. Statistical source data are shown in Source Data Extended Data Fig. 5. Source data

Extended Data Fig. 6 SUV39H1 inhibits epigenetic reprogramming.

a. Violin plots depicting absolute expression levels in log2 scale in single Suv39h1wt, Suv39h1mut-expressing embryos and non-injected controls at the late 2-cell stage (46 h post-hCG). N = 9, 9 and 11 single embryos respectively across 3 independent experiments. Expression values in individual embryos are indicated by black points and the mean by a red point. The only gene or RE displaying significant difference using the two-sided Mann-Whitney U test between groups is Suv39h1 itself. See Supplementary Table 4 for a list of genes (sheet1), REs (sheet2), and for the genes Taqman® assay IDs and reason(s) for inclusion. b. The mean number of nucleolar-like bodies (NLBs) (± S.E.M.) inside each 2-cell stage nucleus from confocal stacks of DAPI stained embryos was counted manually (n = the number of embryos analysed is indicated across 4 independent experiments). Nuclear transfer embryos were fixed at 25 and 33 h post-activation with similar results obtained at the two time points. Statistical testing was performed using the two-sided Mann-Whitney U test for comparing nonparametric distributions. c. Shown is a summary of the developmental progression of nuclear transfer embryos microinjected with the indicated mRNAs according to the workflow in Fig. 7a. Developmental progression was monitored daily and the proportion of embryos that reached each stage of development was calculated. The data represents the mean (±S.E.M) (n = the total number of embryos analysed as indicated in Fig. 7c across 3 independent experiments except for H2B-cherry alone, which was done twice. d. The mean proportion of embryos injected with Suv39h1mut or GfpmRNA developing to the blastocyst stage is shown. N = the number of embryos tested across 2 independent experiments is indicated on the graph. Statistical analysis was performed using the N-1 Chi-squared test for comparing independent proportions. Statistical source data are shown in Source Data Extended Data Fig. 6. Source data

Supplementary information

41556_2020_536_MOESM1_ESM.pdf

Reporting Summary

Supplementary Tables

Supplementary Table 1: P values of H3K9me3 enrichment on repetitive elements on the paternal chromatin. P values were calculated using Mann–Whitney U-test on the distribution of all average paternal H3K9me3 values in the binned non-smoothed genome versus the distribution of values in bins that contained a copy of the given TE class and adjusted using the Bonferroni procedure. Relates to Extended Data Fig. 3a,b. Supplementary Table 2: list of pooled 5 × 2-cell stage samples sequenced for RNA-seq analysis after Suv39h2 knockdown, with sequencing and mapping information. Relates to Fig. 3c,d. Supplementary Table 3: RNA-seq was performed on six pools for RNAi Suv39h2 and 5 pools for RNAi lacZ-injected embryos at the late two-cell stage (46 h post-hCG), Pools consisted of five two-cell stage embryos collected from three independent experiments. Differential expression analysis was performed using DESeq2. List of differentially expressed genes between dsRNA for Suv39h2 and lacZ groups. The left shows all genes sorted by adjusted P value and the right shows all repetitive elements sorted by adjusted P value. Relates to Fig. 3c,d. Supplementary Table 4: list of Taqman assays used for gene expression analysis by qPCR in two-cell stage embryos in Extended Data Figs. 3c,d and 6a. The function of each gene during preimplantation development is indicated. Assays targeting repetitive elements were custom-made (sequences available upon request). Supplementary Table 5: number and stage of embryos analysed for gene expression patterns in Extended Data Fig. 5a. Supplementary Table 6: list of Taqman assays used for gene expression analysis at the morula stage in Extended Data Fig. 5a. Supplementary Table 7: list of differentially expressed genes between Suv39h1wt and Suv39h1mut groups by total RNA-seq in two pools of 20 embryos per sample; each pool collected from three independent experiments. DEQSeq2 was used for differential expression analysis. The upper part shows significant genes, (adjusted P value < 0.05) sorted by fold change in expression and the lower part shows all genes, sorted by adjusted P value (relates to Fig. 6a). Supplementary Table 8: list of differentially expressed repetitive elements between Suv39h1wt and Suv39h1mut groups by total RNA-seq in two pools of 20 embryos per sample; each pool collected from three independent experiments. DEQSeq2 was used for differential expression analysis. The upper part shows significant repetitive elements, (adjusted P value < 0.05) sorted by fold-change in expression and the lower part shows all repeats, sorted by adjusted P value (relates to Fig. 6b). Supplementary Table 9: list of single embryo samples sequenced for RNA-seq analysis after Suv39h1wt or Suv39h1mut gain-of-function, with time of collection, sequencing and mapping information. Relates to Fig. 6c–e. Supplementary Table 10: list of differentially expressed genes (adjusted P value < 0.05) between Suv39h1wt and Suv39h1mut groups at 65 h and 90 h by RNA-seq in single embryos. DESeq2 was used for differential expression analysis. Embryos at all time points were collected from four independent experiments including the three sample groups. At 48 h and 75 h time points, no statistically significant differentially expressed genes were found between Suv39h1wt and Suv39h1mut injected embryos. Relates to Fig. 6c–e.

Supplementary Video 1

A representative Gfp-injected zygote (Fig. 4d) from six independent experiments fixed at the two-cell stage and immunostained with the indicated antibodies. Confocal stacks were reconstructed in 3D using Imaris software. The scale bar is 10 µm. (N = 9).

Supplementary Video 2

A representative Suv39h1wt-injected zygote (Fig. 4d) from six independent experiments fixed at the two-cell stage and immunostained with the indicated antibodies. Confocal stacks were reconstructed in 3D using IMARIS software. The scale bar is 10 µm. (N = 20).

Supplementary Video 3

A representative Suv39h1mut-injected zygote from two independent experiments fixed at the two-cell stage and immunostained with the indicated antibodies. Confocal stacks were reconstructed in 3D using Imaris software. The scale bar is 10 µm. (N = 12).

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Burton, A., Brochard, V., Galan, C. et al. Heterochromatin establishment during early mammalian development is regulated by pericentromeric RNA and characterized by non-repressive H3K9me3. Nat Cell Biol 22, 767–778 (2020). https://doi.org/10.1038/s41556-020-0536-6

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