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Mapping putative enhancers in mouse oocytes and early embryos reveals TCF3/12 as key folliculogenesis regulators

Nature Cell Biology volume 26, pages 962–974 (2024)Cite this article

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Abstract

Dynamic epigenomic reprogramming occurs during mammalian oocyte maturation and early development. However, the underlying transcription circuitry remains poorly characterized. By mapping cis-regulatory elements using H3K27ac, we identified putative enhancers in mouse oocytes and early embryos distinct from those in adult tissues, enabling global transitions of regulatory landscapes around fertilization and implantation. Gene deserts harbour prevalent putative enhancers in fully grown oocytes linked to oocyte-specific genes and repeat activation. Embryo-specific enhancers are primed before zygotic genome activation and are restricted by oocyte-inherited H3K27me3. Putative enhancers in oocytes often manifest H3K4me3, bidirectional transcription, Pol II binding and can drive transcription in STARR-seq and a reporter assay. Finally, motif analysis of these elements identified crucial regulators of oogenesis, TCF3 and TCF12, the deficiency of which impairs activation of key oocyte genes and folliculogenesis. These data reveal distinctive regulatory landscapes and their interacting transcription factors that underpin the development of mammalian oocytes and early embryos.

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Fig. 1: H3K27ac landscape in mouse gametes, early embryos and tissues.
Fig. 2: Reprogramming of H3K27ac in mouse gametes and early embryos.
Fig. 3: Reprogramming of H3K27ac and H3K27me3 during ZGA.
Fig. 4: Validation of enhancers in FGOs.
Fig. 5: Identification of candidate TF regulators from putative enhancer maps during oogenesis and pre-implantation embryos.
Fig. 6: TCF3 and TCF12 are key folliculogenesis regulators.
Fig. 7: Dynamic enhancer rewiring in mouse oocytes and early embryos.

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Data availability

The generated and analysed data are available in the Gene Expression Omnibus with accession number GSE217970. Source data are provided with this paper.

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Acknowledgements

We are grateful to members of the Xie laboratory and the Li laboratory for the discussion and comments during the preparation of the manuscript and the Animal Center and Biocomputing Facility at Tsinghua University for their support. We thank X. Hu, L. Li, L. Wang, X. Lu, Q. Xu, Z. Du, Y. Zhang, G. Yu, H. Zheng, L. Liu and Y. Li for the help of various experiments and bioinformatics analyses. We are indebted to F. Lan, D. Fang and A. Stark for offering reagents or insightful discussion. We are grateful to Y. Zhuang for the Tcf3flox/flox and Tcf12flox/flox transgenic mice and Y. Su for the Gdf9-Cre mice. This work was funded by the National Natural Science Foundation of China (31988101 to W. Xie), the National Key R&D Program of China (2021YFA1100102 and 2019YFA0508900 to W. Xie, 2022YFC2703000 to J.L. and 2023YFA1800300 to X.W.), the National Natural Science Foundation of China (31830047 and 31725018 to W. Xie) and the Tsinghua-Peking Center for Life Sciences (W. Xie). B.L. is supported by Tsinghua Shuimu Scholar and Center for Life Sciences postdoctoral fellowship. W. Xie is a recipient of an HHMI International Research Scholar award and is a New Cornerstone Investigator.

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  1. These authors contributed equally: Bofeng Liu, Yuanlin He, Xiaotong Wu, Zili Lin, Jing Ma.

Authors and Affiliations

  1. Center for Stem Cell Biology and Regenerative Medicine, MOE Key Laboratory of Bioinformatics, New Cornerstone Science Laboratory, School of Life Sciences, Tsinghua University, Beijing, China

    Bofeng Liu, Zili Lin, Jing Ma, Yunlong Xiang, Feng Kong, Fangnong Lai, Bingjie Zhang, Qiujun Wang, Jingyi Wu, Weikun Xia & Wei Xie

  2. Tsinghua-Peking Center for Life Sciences, Beijing, China

    Bofeng Liu, Xiaotong Wu, Zili Lin, Jing Ma, Yunlong Xiang, Feng Kong, Fangnong Lai, Bingjie Zhang, Qiujun Wang, Jingyi Wu, Weikun Xia, Weimin Shen & Wei Xie

  3. State Key Laboratory of Reproductive Medicine and Offspring Health, Nanjing Medical University, Nanjing, China

    Yuanlin He, Yuexin Qiu & Jing Li

  4. Innovation Center of Suzhou Nanjing Medical University, Suzhou, China

    Yuanlin He & Jing Li

  5. Laboratory of Molecular Developmental Biology, State Key Laboratory of Membrane Biology, Tsinghua University, Beijing, China

    Xiaotong Wu & Weimin Shen

  6. Institute of Epigenetics and Stem Cells (IES), Helmholtz Zentrum München, Munich, Germany

    Mrinmoy Pal & Maria-Elena Torres-Padilla

  7. Center for Stem Cell Biology and Regenerative Medicine, School of Medicine, Tsinghua University, Beijing, China

    Peizhe Wang, Jia Ming & Jie Na

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  1. Bofeng Liu
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  2. Yuanlin He
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W. Xie and J.L. conceived and designed the project. Z.L., F.K., P.W., J. Ming, F.L. and J. Ma collected and prepared WT and Eed KO embryos and oocytes. Y.H. and Y.Q. collected Tcf3/12 DKO, Tcf3 mKO and Tcf12 mKO oocytes. J. Ma, B.L., Y.X. and B.Z. conducted STAR ChIP-seq in mouse oocytes, embryos and mES cells. B.L. conducted ATAC-seq in mouse oocytes. X.W. modified and conducted STARR-seq in mouse oocytes with the help of W.S. Y.H. conducted single-oocyte RNA-seq. X.W. and Y.Q. conducted immunostaining in WT oocytes and embryos with the help of Z.L. and P.W. X.W. conducted plumbagin treatment, EU staining and reporter assay in FGOs with the help of F.K., F.L. and Z.L. F.K., Z.L. and F.L. conducted microinjection in oocytes. Y.Q. conducted qPCR, western blot, IHC, H&E staining in Tcf3/12 DKO oocytes and IP in HEK293T cells. Y.Q. analysed fertility and follicle numbers of Tcf3/12 DKO, Tcf3 mKO and Tcf12 mKO mice. Q.W. prepared Dnmt3a/b−/− mES cells. M.P. and M.-E.T.-P. conducted lamin B1 DamID. W. Xia helped with various experiments. B.L. conducted the bioinformatics analysis with the help of J.W., B.Z. and Y.X. B.L., X.W. and W. Xie prepared most figures and wrote the manuscript with help of J.L., Y.Q., M.-E.T.-P., M.P., J.N. and all other authors.

Corresponding authors

Correspondence to Jing Li or Wei Xie.

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Nature Cell Biology thanks Chih-Jen Lin and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Validation of H3K27ac ChIP-seq data in mouse oocytes and early embryos.

a, Top, immunostaining showing H3K27ac signals in GO-P7 (n=9), GO-P10 (n=8), FGO-NSN (non-surrounded nucleolus) (n=10), FGO-SN (surrounded nucleolus) (n=4), MII oocyte (n=8), one-cell PN5 (n=16), early two-cell (n=19), late two-cell (n=9), 8-cell embryos (n=6), and blastocyst (n=5). One representative image from 3 independent experiments is shown. Scale bar, 20 μm. Bottom, boxplot showing the ratio of H3K27ac to DAPI intensity in immunostaining. The median is indicated by the center line. The bottom, top edges, and whiskers represent the 10th and 90th percentiles and 1.5 times the interquartile range (IQR), respectively. b, The UCSC browser view showing H3K27ac signals at each stage with two biological replicates. c, Heatmap showing the pairwise Pearson correlation between each sample for their H3K27ac levels in oocytes, early embryos, and tissues. Source numerical data and unprocessed blots are available in source data.

Extended Data Fig. 2 H3K27ac, gene expression, and chromatin accessibility in mouse oocytes and early embryos.

a, The UCSC browser view and heatmaps showing H3K27ac signals and RNA expression at representative genes. Oocyte-, pre-implantation- and post-implantation-specific putative enhancers are shaded green, red, and blue, respectively. b, Heatmaps showing the stage-specific distal H3K27ac signals and the mapped chromatin accessibility signals. DHS, DNase I hypersensitive site. c, Line charts showing the cumulative distribution of the distances between promoters of active and inactive genes (top 5,000) and nearest distal H3K27ac sites (top 10,000).

Extended Data Fig. 3 Gene expression in mouse oocytes, early embryos, and tissues.

Heatmaps showing the stage-enriched gene expression in mouse oocytes and early embryos. Mouse adult tissue gene expression97 is also mapped. Example genes are listed on the right.

Extended Data Fig. 4 Repeat enrichment in H3K27ac peaks.

Heatmaps showing enrichment of all repeat subfamilies at distal H3K27ac peaks compared to that in random peaks in oocytes, early embryos, and mESCs. The H3K27ac enrichment was calculated as a log2 ratio for the numbers of observed peaks overlapped with repeats divided by those for random peaks (shuffled peaks with lengths matched).

Extended Data Fig. 5 Dynamics of putative enhancers during mouse oogenesis.

a, Heatmaps showing the expression for oocyte stage-specific and common genes with promoter H3K27ac signals mapped. b, The UCSC browser views and heatmaps showing H3K27ac signals and RNA expression, respectively, in GO-P7, GO-P10, FGO, and mESCs (ENCODE) with replicates at representative genes. c, Bar charts showing the expression levels of genes (left, n=5,505) and the numbers of active repeats (right; GO-P7, n=17,082; GO-P10, n=34,430; FGO, n=70,975; 8C, n=33,384; Epi, n=34,265; mESC, n=14,026) in gene-poor regions at each stage. d, The UCSC browser views showing H3K27ac signals at representative repeats in gene-poor regions in GO-P7, GO-P10, and FGOs.

Extended Data Fig. 6 The transition of putative enhancers during fertilization and ZGA.

a, Heatmap and bubble plot comparing distal H3K27ac signals and motif enrichment (-log10 p-value, hypergeometric test with Bonferroni correction, one-sided from HOMER57, Methods), respectively, at putative enhancers in FGO, one-cell, early two-cell, and late two-cell stage embryos. b, Scatter plots comparing allelic H3K27ac and Lamin B1 DamID signals22 in the one-cell, late two-cell, 8-cell embryos, and mESCs. M, maternal. P, paternal. Pearson correlations of each stage are also shown. c, Lamin B1 DamID and DamID-only control signal profiles in FGOs22 and GO-P10 for chromosome 1 (n=4 biological replicates).

Extended Data Fig. 7 Analyses of H3K4me3-marked enhancers in oocytes and embryos.

a, The UCSC browser view showing H3K4me3 signals in control and Dnmt3a/b knockout FGOs40, and H3K27ac and DNA methylation signals in wild-type FGOs. H3K4me3-gain regions upon Dnmt3a/b knockout are shaded. b, Top, line charts showing H3K4me3 signals at putative enhancers (orange) and promoters (green) in wild-type (left) and Dnmt3a/b KO (right) mESCs. The dashed lines indicate the peaks of H3K4me3 signals at putative enhancer regions. Bottom, the UCSC browser views showing H3K4me3 signals at promoters and putative enhancers (annotated ENCODE dCRE) at representative genes in wild-type and Dnmt3a/b KO mESCs. Putative enhancer and promoter regions are shaded orange and green, respectively. c, Bar chart showing the percentages of H3K27ac/H3K4me3 co-marked and H3K27ac only marked enhancers bound by distal Pol II34 at each stage. d, Line charts showing the cumulative distribution of the distance between transcription start sites (TSSs) of active gene and nearest distal putative enhancers marked by either H3K27ac only (blue) or both H3K27ac and H3K4me3 (red) in FGO (left) and the 8-cell embryos (right). e, Bar chart showing the percentages of H3K27ac only (blue) and H3K27ac/H3K4me3 (red) peaks that also overlap ENCODE dCREs. f, Bar chart showing the percentages of CAGE-defined enhancer sites that also overlap H3K27ac sites, H3K4me3 sites, or both. Random sites with identical lengths and numbers were similarly analysed as controls.

Extended Data Fig. 8 Analyses of enhancer activities in oocytes.

a, Representative images of H3K27ac immunostaining (green) and EU staining (red) in DMSO or plumbagin-treated FGO-NSN and FGO-SN. DNA was stained with DAPI (blue). Scale bar, 20μm. b, Bar charts showing the relative intensities of H3K27ac or EU in DMSO (blue) or plumbagin (red) treated FGO-NSN and FGO-SN. The dots indicate the relative intensities of individual oocytes. The numbers of oocytes used: 7, 13, 7, and 5 for H3K27ac (top), and 11, 17, 8, and 9 for EU staining (bottom). P-values (t-test, two-sided) are also provided. c, Line charts showing H3K4me3 signals at CAGE-enhancers and H3K4me3-marked distal H3K27ac peaks in Mll2 control and KO oocytes40. Pie charts showing the percentages of CAGE-enhancers and H3K4me3 at distal H3K27ac peaks in Mll2 KO oocytes compared with wild-type. d. The UCSC browser views showing CAGE in GO-P14 (ref. 46), H3K27ac, and H3K4me3 in FGOs. Putative enhancer regions defined by CAGE (red arrows) or distal H3K27ac (blue shades) are indicated. Source numerical data and unprocessed blots are available in source data.

Extended Data Fig. 9 STARR-seq and reporter assay in oocytes.

a, Schematic of STARR-seq in FGOs. 70 candidates and 16 negative controls were manually cloned into the STARR-seq constructs and then the pooled products were injected into the nuclei of FGOs. The RNA was recovered by a method adapted from Smart-seq2 (ref. 54) (Methods) to suit low-input cells, followed by sequencing. b, Scatter plots showing STARR-seq signals (RNA output vs. DNA input) (Methods) in both replicates and strands. Red, enhancer candidates; blue, negative control elements. c, UCSC genome browser showing FGO STARR-seq RNA output and DNA input signals on chromosome 16. d, Heatmaps showing STARR-seq (STARR/input) signals in FGO with two replicates and Pol II signals in GO-P14 and FGO at enhancer candidates and negative control regions. e. Top, fluorescence and bright fields of mouse FGOs in an enhancer reporter assay (Pro, mini promoter). Scale bar, 100 μm. Bottom, boxplot showing the ratio of GFP to mCherry intensity in the enhancer reporter assay. The dashed line indicates the ratio in the empty vector group. The numbers of oocytes used in each group: 17, 14, 17, 18, 10, 18, 15, 17, 13, 10, 12, 19, 11, and 6. The median is indicated by the center line. The bottom, top edges, and whiskers represent the 10th and 90th percentiles and 1.5 times the interquartile range (IQR), respectively. Source numerical data and unprocessed blots are available in source data.

Extended Data Fig. 10 TCF3/12 regulate oocyte transcription and development.

a. Heatmaps showing STARR-seq signals (RNA/DNA input) and TCF3/12 motif densities (counts per kb) for enhancer candidates (including those showing positive and negative STARR-seq signals) and negative controls. b. Bar charts showing TCF3/12 motif enrichment (-log10 p-value, hypergeometric test with Bonferroni correction, one-sided from HOMER57, Methods) in enhancers previously evaluated by STARR-seq and negative controls. Ranks of TCF3/12 motifs among all motifs are also shown. c, qPCR results showing relative RNA levels of Tcf3 and Tcf12 in wild-type and Tcf3 or Tcf12 mKO GO-P5 oocytes (n=3 biological replicates). P-value (t-test, two-sided) is also shown. d, qPCR results showing relative RNA levels of Tcf3 and Tcf12 in WT and Tcf3/12 DKO GO-P5 oocytes (n=3 biological replicates). P-value (t-test, two-sided) is also shown. e, Western blot showing protein levels of TCF3 and TCF12 in WT and Tcf3/12 DKO GO-P5 ovaries (n=3 biological replicates). f, Immunohistochemistry (IHC) showing TCF3 and TCF12 in WT (n=5) and Tcf3/12 DKO (n=5) ovaries. Scale bar, 50 μm. g, RNA expression and ribosome-protected fragment (RPF, indicating translation level) levels of Sp1 from FGOs to late two-cell embryos are shown98. Source numerical data and unprocessed blots are available in source data.

Supplementary information

Reporting Summary

Supplementary Tables 1–3 and 5

Supplementary Table 1. Candidate enhancer regions tested in STARR-seq and corresponding cloning primers. Table 2. Candidate enhancer regions tested in the reporter assay and corresponding cloning primers. Table 3. PCR primers for genotyping and qPCR for Tcf3/12 DKO mice. Table 5. Candidate enhancers identified by CAGE in GO-P14.

Supplementary Table 4

Enhancer candidates identified by distal H3K27ac.

Source data

Source Data Fig. 1

Statistical source data.

Source Data Fig. 2

Unprocessed blots and gels.

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Liu, B., He, Y., Wu, X. et al. Mapping putative enhancers in mouse oocytes and early embryos reveals TCF3/12 as key folliculogenesis regulators. Nat Cell Biol 26, 962–974 (2024). https://doi.org/10.1038/s41556-024-01422-x

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