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Transient transcription in the early embryo sets an epigenetic state that programs postnatal growth

Nature Genetics volume 49, pages 110–118 (2017)Cite this article

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Abstract

The potential for early embryonic events to program epigenetic states that influence adult physiology remains an important question in health and development. Using the imprinted Zdbf2 locus as a paradigm for the early programming of phenotypes, we demonstrate here that chromatin changes that occur in the pluripotent embryo can be dispensable for embryogenesis but instead signal essential regulatory information in the adult. The Liz (long isoform of Zdbf2) transcript is transiently expressed in early embryos and embryonic stem cells (ESCs). This transcription locally promotes de novo DNA methylation upstream of the Zdbf2 promoter, which antagonizes Polycomb-mediated repression of Zdbf2. Strikingly, mouse embryos deficient for Liz develop normally but fail to activate Zdbf2 in the postnatal brain and show indelible growth reduction, implying a crucial role for a Liz-dependent epigenetic switch. This work provides evidence that transcription during an early embryonic timeframe can program a stable epigenetic state with later physiological consequences.

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Figure 1: A cellular model of the dynamic regulation of the Gpr1/Zdbf2 locus.
Figure 2: Liz transcription promotes sDMR methylation and Zdbf2 expression.
Figure 3: Zdbf2 is repressed by PRC2.
Figure 4: DNA methylation is required to derepress Zdbf2.
Figure 5: Liz RNA regulates sDMR and Zdbf2 in vivo.
Figure 6: Liz paternal-knockout mice exhibit a lifelong growth defect.
Figure 7: Model of Liz-dependent epigenetic programming of Zdbf2 expression and growth potential on the paternal allele.

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Acknowledgements

We would like to thank members of the Bourc'his laboratory for insightful experimental and conceptual input; E. Heard, R. Margueron, J. Barau and R. Duffié for critical advice on the manuscript; M. Weber for the DNMT3-mutant embryos; and C. Jouhanneau and the Institut Curie Animal Facility for excellent mouse husbandry. High-throughput sequencing was performed by the ICGex NGS platform of the Institut Curie supported by grants from the ANR 'Investissements d'Avenir' program (ANR-10-EQPX-03, Equipex and ANR-10-INBS-09-08, France Génomique Consortium) and from Cancéropôle Île-de-France. The laboratory of D.B. is part of the Laboratoire d'Excellence (LABEX) entitled DEEP (11-LBX0044). This research was supported by grants from the ERC (ERC-Cog EpiRepro) and the ANR (ABS4NGS-ANR-11-BINF-0001). M.V.C.G. was supported by ARC and EMBO (LTF 457-2013) postdoctoral fellowships. J.G. was a recipient of a PhD fellowship from DIM Biotherapy, Île-de-France. M.W. was the recipient of a PhD fellowship from the École Polytechnique.

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Author notes

  1. Máté Borsos

    Present address: Present address: Institut de Génétique et Biologie Moléculaire et Cellulaire, CNRS, INSERM, Strasbourg University, Illkirch, France.,

  2. Maxim V C Greenberg and Juliane Glaser: These authors contributed equally to this work.

Authors and Affiliations

  1. Institut Curie, PSL Research University, INSERM, CNRS, Paris, France

    Maxim V C Greenberg, Juliane Glaser, Máté Borsos, Marius Walter, Aurélie Teissandier & Déborah Bourc'his

  2. Institut Curie, CNRS, Paris, France

    Fatima El Marjou

  3. École des Mines, Paris, France

    Aurélie Teissandier

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Contributions

Conceptualization: M.V.C.G., J.G. and D.B. Methodology: M.V.C.G., J.G. and D.B. Formal analysis: A.T. Investigation: M.V.C.G., J.G., M.B., M.W. and F.E.M. Writing, original draft: M.V.C.G., J.G. and D.B. Writing, final draft: M.V.C.G., J.G. and D.B. Supervision: D.B. Funding acquisition, D.B.

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Correspondence to Déborah Bourc'his.

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Integrated supplementary information

Supplementary Figure 1 Liz/Zdbf2 dynamics in vivo and in vitro.

(a) Schematic of Liz/Zdbf2 regulation during early development. Summary from Duffié et al.18. Liz transcript initiates from the unmethylated paternal gDMR in the preimplantation embryo. After implantation, Liz expression is turned off and the gDMR is methylated on both alleles. From this stage on, DNA methylation is present at the sDMR and Zdbf2 is expressed from its canonical promoter on the same allele from which Liz was previously expressed, i.e., the paternal allele. Filled circles represent methylated CpGs, and unfilled circles represent unmethylated CpGs. (b) BS–pyro data measured in Figure 1d and split into individual CpGs at the gDMR (left; 10 CpGs) and the sDMR (right; 7 CpGs). The CpGs within the clones analyzed in Figure 1c are depicted in red. Data are shown as means ± s.e.m. from two cell culture replicates. (c) RT–qPCR analysis of Liz expression in E3.5 blastocysts and in day 2 EpiLCs. Values relative to the Hprt1 normalizer are similar. Data are shown as means ± s.e.m. from two blastocyst pools and four cell culture replicates.

Supplementary Figure 2 Characterization of the Gpr1–/– cell line.

(a) Schematic of the homozygous Gpr1-mutant cell line generated on the E14 background. The size and genomic coordinates (in brackets) of the deletion carried by each allele are indicated. The positions of the CRISPR sgRNAs (L, left; R, right) around Gpr1 exon 1 are depicted in red. (b) RT–qPCR analysis shows marked depletion of Gpr1 (left) with little effect on Liz (right) expression upon EpiLC differentiation of Gpr1–/– cells in comparison to E14 wild-type cells. The spliced form of Liz was measured here; similar results were obtained with the unspliced form (data not shown). (c) BS–Pyro analysis of the gDMR (left) and sDMR (right) indicates normal kinetics of DNA methylation in these regions upon differentiation in the absence of Gpr1. Data are shown as means ± s.e.m. from two cell culture replicates.

Supplementary Figure 3 Characterization of the ΔLiz and Liz::2×STOP mutant cell lines.

(a) Schematic of the homozygous ∆Liz cell line generated on the E14 background. The size and genomic coordinates (in brackets) of the deletion carried by each allele are indicated. The positions of the TALEN guides (L, left; R, right) around Liz exon 1 are displayed in red. (b) NanoString custom set data representing expression of active Dnmt genes in wild-type and ΔLiz cells across differentiation. (c) NanoString custom set data representing expression of selected pluripotency genes in wild-type and ΔLiz cells across differentiation. (d) Schematic of the 2× polyadenylation cassette inserted downstream of the first exon of Liz by homology-dependent repair. The selectable marker was removed by transient expression of Cre. (e) RT–qPCR measures at the first exon of Liz across differentiation. Transcription initiation is not affected by the presence of the 2×STOP cassette. Data are shown as means ± s.e.m. from three cell culture replicates for b and c and from two replicates for e.

Supplementary Figure 4 Characterization of H3K36me3 regulation and role at the Liz/Zdbf2 locus.

(a) H3K36me3 ChIP–qPCR in wild-type and ΔLiz cell lines at day 2 of EpiLC differentiation. In the absence of Liz, H3K36me3 is depleted along the path of Liz transcription (Liz intron 2, intergenic CGI, sDMR, Zdbf2 promoter, and Zdbf2 gene body). Data are presented as the percentage of input; control loci are indicated on the right. (b) Schematic of the homozygous Setd2–/– cell line generated on the E14 background. The size and genomic coordinates (in brackets) of the deletion carried by each allele are indicated. The positions of the CRISPR sgRNAs (L, left; R, right) around Setd2 exon 3 are displayed in red. (c) Immunoblot showing that global H3K36me3 levels are drastically reduced in Setd2–/– as compared to wild-type ESCs. (d) RT–qPCR analysis of spliced Liz shows normal kinetics of expression upon differentiation of Setd2–/– EpiLCs. (e) ChIP–qPCR analysis indicates a marked depletion of H3K36me3 levels along the path of Liz in Setd2–/– as compared to wild-type ESCs. Data are presented as the percentage of input; control loci are indicated on the right. (f) BS–pyro analysis of the gDMR (top) and sDMR (bottom) in the Setd2–/– line across EpiLC differentiation. DNA methylation is not affected at the final time point, despite a slight delay at day 2 in the sDMR region. *P ≤ 0.05. (g) BS–pyro analysis of the gDMR (top) and sDMR (bottom) from limb buds of E11.5 embryos. We analyzed Dnmt3a–/– and Dnmt3b–/– embryos along with their respective wild-type littermates. The gDMR is around 20% less methylated in Dnmt3a–/– and Dnmt3b–/– embryos, indicating partial redundancy of DNMT3A and DNMT3B. In contrast, the sDMR is normally methylated in both Dnmt3a–/– and Dnmt3b–/– embryos, highlighting complete redundancy of these two enzymes at this locus. *P ≤ 0.05. Data are shown as means ± s.e.m. from two cell culture replicates for a and d–f and from two mouse embryos for each genotype in g.

Supplementary Figure 5 Role of Polycomb at the Liz/Zdbf2 locus.

(a) Screenshots of H3K27me3 ChIP–seq tracks in wild-type and ΔLiz ESCs grown in 2i+vitC medium. While H3K27me3 is enriched over the Liz/Zdbf2 sDMR in comparison to surrounding regions in wild type (blue), the absolute levels are lower than at typical Polycomb targets (Pax5, Pax9 and Gata6), which are enriched in CGIs. In contrast, H3K27me3 enrichment at the sDMR increases to similar levels as seen for Polycomb targets in ΔLiz ESCs (red). This suggests that basal Liz transcription before induction of EpiLC differentiation already competes with Polycomb occupancy. The positions of CGIs are highlighted in green. The y axis for the H3K27me3 tracks indicates units of reads per million mapped reads (rpm). (b) H3K27me3 and H3K4me3 ChIP–qPCR analysis of control loci for Figure 3b (left) and Figure 3e (right). Data are shown as means ± s.e.m. from three (left) and two (right) cell culture replicates. (c) LUMA quantification of global CpG methylation. Even with prolonged culture in 2i+vitC medium, global methylation levels remain high in Eed–/– ESCs at day 0, in single-knockout Eed–/– or double-knockout ΔLiz; Eed–/– cells, relative to their wild-type counterparts, E14 and J1, respectively. Data are shown as means ± s.e.m. from two cell culture replicates. (d) BS–pyro analysis of CpG methylation of the sDMR in Eed-mutant cell lines upon EpiLC differentiation. As with the global genome in c, DNA methylation remains high at the sDMR in Eed-mutant ESCs in 2i+vitC medium (day 0). At day 4 of EpiLC differentiation, global genomic methylation levels were identical for the four cell lines. The time course is interrupted at day 4, as Eed-mutant cells die after this time point. Data are shown as means ± s.e.m. from two to four cell culture replicates. (e) Schematic of the homozygous Eed–/– cell line generated on the ΔLiz background. The size and genomic coordinates (in brackets) of the deletion carried by each allele are indicated. The positions of the sgRNAs (L, left; R, right) around Eed exon 6 are displayed in red. (f) Immunoblotting for EED and H3K27me3 in Eed–/– cell lines. There was no detectable protein and corresponding histone marks in either homozygous line, indicating proper null mutations.

Supplementary Figure 6 Characterization of DNMT triple-knockout EpiLC differentiation.

(a) NanoString custom set data representing expression of selected pluripotency genes in DNMT triple-knockout cells across EpiLC differentiation. Despite lack of DNA methylation, these cells exhibit similar dynamics as wild-type cells (J1). Data are shown as means ± s.e.m. from three cell culture replicates. (b) Representative images of wild-type and DNMT triple-knockout EpiLCs at day 5. There are few, if any, differences at the morphological level. (c) BS–pyro analysis of the gDMR and sDMR confirms no DNA methylation in DNMT triple-knockout cells upon EpiLC differentiation. Data are shown as means ± s.e.m. from three cell culture replicates. (d) H3K27me3 ChIP–qPCR analysis of control loci for Figure 4c. Data are shown as means ± s.e.m. from two cell culture replicates.

Supplementary Figure 7 Generation of Liz paternal-knockout mice and the regulatory effect on Zdbf2.

(a) Schematic of the Liz deletions generated in vivo. The size and genomic coordinates (in brackets) of the deletion carried by the two independent founders (393 and 403) are indicated. The positions of the sgRNAs (L, left; R, right) around Liz exon 1 are displayed in red. (b) sDMR DNA methylation analysis performed by BS–pyro as in Figure 5b, but with E9.5 progenies from the independent founder line 403. Both founder deletions resulted in the same DNA methylation phenotype. n is the number of embryos analyzed. Data are shown as means ± s.e.m. from n embryos. (c) Bisulfite cloning and sequencing shows nearly complete lack of DNA methylation at the sDMR in Liz paternal-knockout embryos at E9.5 (line 393), in comparison to wild-type littermates. (d) Zdbf2 RT–qPCR analysis performed as in Figure 5c, but with E9.5 progenies from the independent founder line 403. n is the number of embryos analyzed. Data are shown as means ± s.e.m. from n embryos. (e) BS–pyro analysis as performed in Figure 5d, but in 8-month-old mice. The data demonstrate that the epigenetic setting of sDMR methylation persists upon aging of Liz paternal-knockout mice. (f) RT–qPCR analysis of Zdbf2 expression in a bank of tissues from 6-week-old wild-type mice. Zdbf2 expression is mostly limited to brain tissues, with the hypothalamus exhibiting the highest levels. (g) X-gal staining of wild-type (left) and Zdbf2::lacZ (right) placental sections at E18.5. De, deciduum; Sp, spongiotrophoblast; lab, labyrinth. Top: 10× magnification, scale bars = 100 μm; bottom: 40× magnification, scale bars = 25 μm. (h) RT–qPCR analysis as performed in Figure 5g, but in 8-month-old mice. The data demonstrate that the effect on Zdbf2 expression persists in aged Liz paternal-knockout mice. ****P ≤ 0.0001, ***P ≤ 0.001, **P ≤ 0.01, *P ≤ 0.05. Data are shown as means ± s.e.m. from three (f) or two (e,g) mice.

Supplementary Figure 8 Phenotypic characterization of Liz knockout mice.

(a) Genotype ratios observed at birth (heterozygotes and wild type) upon paternal and maternal transmission of the Liz deletion after three backcrosses of the 393 line. Numbers of counted animals of each genotype are given on the histograms. (b) Sex ratios observed at birth among Liz heterozygotes upon paternal (PatKO) and maternal (MatKO) transmission of the deletion after three backcrosses of the 393 line. Numbers of counted animals of each sex are given on the histograms. (c) Individual organ weights plotted as the percentage of total body weight in Liz paternal-knockout mice in comparison to wild-type littermates. Data are shown as means ± s.e.m. from n individuals. (d,e) Growth curves as in Figure 6b and Figure 6c, respectively, but comparing Liz maternal-knockout mice with their wild-type littermates. The Liz deletion behaves as a silent mutation upon maternal transmission: there is no growth-related phenotype in Liz maternal-knockout progenies. Between 15 and 30 mice were analyzed per genotype, depending on age and sex. Data are shown as means ± s.e.m. (f) Scatterplot comparing the body weight of wild-type and Liz paternal-knockout embryos (top) and placentae (bottom) at E18.5. Each point represents one individual. n is the number of embryos or placentae analyzed. (g) Hematoxylin and eosin staining of wild-type and Liz paternal-knockout placentae at E18.5. De, deciduum; Sp, spongiotrophoblast; Lab, labyrinth. Top: 2× magnification, scale bars = 500 μm; bottom: 10× magnification, scale bars = 100 μm.

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Greenberg, M., Glaser, J., Borsos, M. et al. Transient transcription in the early embryo sets an epigenetic state that programs postnatal growth. Nat Genet 49, 110–118 (2017). https://doi.org/10.1038/ng.3718

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