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Lineage regulators TFAP2C and NR5A2 function as bipotency activators in totipotent embryos

Nature Structural & Molecular Biology (2024)Cite this article

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Abstract

During the first lineage segregation, a mammalian totipotent embryo differentiates into the inner cell mass (ICM) and trophectoderm (TE). However, how transcription factors (TFs) regulate this earliest cell-fate decision in vivo remains elusive, with their regulomes primarily inferred from cultured cells. Here, we investigated the TF regulomes during the first lineage specification in early mouse embryos, spanning the pre-initiation, initiation, commitment, and maintenance phases. Unexpectedly, we found that TFAP2C, a trophoblast regulator, bound and activated both early TE and inner cell mass (ICM) genes at the totipotent (two- to eight-cell) stages (‘bipotency activation’). Tfap2c deficiency caused downregulation of early ICM genes, including Nanog, Nr5a2, and Tdgf1, and early TE genes, including Tfeb and Itgb5, in eight-cell embryos. Transcription defects in both ICM and TE lineages were also found in blastocysts, accompanied by increased apoptosis and reduced cell numbers in ICMs. Upon trophoblast commitment, TFAP2C left early ICM genes but acquired binding to late TE genes in blastocysts, where it co-bound with CDX2, and later to extra-embryonic ectoderm (ExE) genes, where it cooperatively co-occupied with the former ICM regulator SOX2. Finally, ‘bipotency activation’ in totipotent embryos also applied to a pluripotency regulator NR5A2, which similarly bound and activated both ICM and TE lineage genes at the eight-cell stage. These data reveal a unique transcription circuity of totipotency underpinned by highly adaptable lineage regulators.

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Fig. 1: The dynamics of TFAP2C chromatin binding in mouse early embryos.
Fig. 2: TFAP2C regulates cell-polarity genes in 8C embryos.
Fig. 3: TFAP2C regulates expression of ICM and TE genes in 8C embryos.
Fig. 4: TFAP2C binding transition during the first lineage specification.
Fig. 5: TFAP2C binding transition from TE to ExE.
Fig. 6: NR5A2 regulates ICM and TE genes in 8C embryos.
Fig. 7: A model for bipotency activation by lineage regulators during the first cell-fate specification.

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Data availability

All datasets generated in this study have been deposited as a super series at the Gene Expression Omnibus (GEO) under accession number GSE216256. Accession codes of the published data in GEO used in this study are as follows: RNA-seq and ATAC-seq of the mouse early embryos, GSE66390; RNA-seq and ATAC-seq of epiblasts, GSE125318; TFAP2C ChIP of mouse TSCs, GSE28455; ATAC, H3K27ac ChIP–seq of mouse TSCs, GSE110950; scRNA of the mouse embryos, GSE45719; scRNA-seq of E4.5 epiblast and PrE, GSE159030; RNA-seq in the Tfap2 knockdown embryos, GSE124755. NR5A2 CUT&RUN and RNA-seq in the Nr5a2 knockdown embryos, GSE229740. Source data are provided with this paper.

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Acknowledgements

We are grateful to members of the Xie 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 Q. Sun (Chinese Academic of Sciences) for providing the Stra8-cre mice. We thank J. Knott (Michigan State University) for insightful discussion. We are grateful to L. Yan, Y. Ren, and N. Wang from Third Hospital of Peking University for help with E4.5 TE collection. This work was funded by the National Key R&D Program of China (2021YFA1100102), the National Natural Science Foundation of China (31988101 and 31725018), the National Key R&D Program of China (2019YFA0508900), and the Tsinghua-Peking Center for Life Sciences. W.X. is a recipient of an HHMI International Research Scholar award and a New Cornerstone Investigator. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.

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  1. These authors contributed equally: Lijia Li, Fangnong Lai.

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

    Lijia Li, Fangnong Lai, Ling Liu, Xukun Lu, Xiaoyu Hu, Bofeng Liu, Qiang Fan, Feng Kong, Qianhua Xu & Wei Xie

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

    Lijia Li, Fangnong Lai, Ling Liu, Xukun Lu, Xiaoyu Hu, Bofeng Liu, Qiang Fan, Feng Kong, Qianhua Xu & Wei Xie

  3. College of Animal Science and Technology College, Beijing University of Agriculture, Beijing, China

    Zili Lin

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Contributions

L. Li, F.L., and W.X. conceived and designed the project. L. Li performed the CUT&RUN, ATAC-seq, and RNA-seq experiments. F.L. and L. Liu performed embryo collection and immunostaining with the help of X.L., Z.L., F.K., and Q.X. Q.F., F.L., and L. Li derived the Tfap2c mzKO mouse ES cells. L. Li performed the western blotting experiments. L. Li constructed the Tfap2c-knockout mouse ES cell line with the help from X.H. L. Li performed the bioinformatics analysis with the help from B.L. L. Li and W.X. prepared most figures and wrote the manuscript, with help from all authors. L. Li and F.L. contributed equally. L. Liu, X.L., X.H., and B.L. contributed equally.

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Correspondence to Wei Xie.

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Extended data

Extended Data Fig. 1 Validation of low-input TFAP2C CUT&RUN in TSCs and ESCs.

a, The boxplots showing the Spearman correlation between the expression of lineage regulator genes and ICM-TE specific genes across single cells at each stage8. Centre line, median; box, 25th and 75th percentiles; whiskers, 1.5 × interquartile range. Early ICM gene, n = 224; late ICM gene, n = 136; early TE genes, n = 191; late TE genes, n = 149; all genes, n = 22,654. b, Scatter plots comparing the expression of Tfap2c with representative lineage regulator genes across single cells in 8C, 16C embryos, and blastocysts8. The Spearman correlation coefficients are also shown. c, The UCSC browser views showing TFAP2C CUT&RUN using TSCs with cell numbers ranging from 50,000 to 500, and TFAP2C ChIP-seq47 signals in TSCs. d, Sequence logos and ranks of TF motif enrichment for TFAP2C peaks identified from TFAP2C CUT&RUN and ChIP-seq in TSCs. The top four TFs motifs are shown. e, The Western blot showing TFAP2C and GAPDH expression in wild-type (WT) and Tfap2c−/− mESCs. f, The UCSC browser views showing TFAP2C CUT&RUN using WT mESCs with cell numbers ranging from 2,000 to 500, and 2,000 Tfap2c−/− mESCs.

Source data

Extended Data Fig. 2 Validation of TFAP2C CUT&RUN in mouse embryos.

a, Immunostaining of TFAP2C (red) and DAPI (blue) in mouse 2-cell, 4-cell, 8-cell embryos, E3.5 and E4.5 blastocysts (two biological replicates). Dotted lines mark inner cells. Scale bar: 20 μm. b, Immunostaining showing TFAP2C, SOX2, and CDX2 protein levels and DAPI in E3.5 (n = 7) and E4.5 (n = 7) blastocysts. Scale bar: 20 μm. Quantification of TFAP2C signal intensity (relative to DAPI signal) in ICM/Epi cells (SOX2 positive or CDX2 negative) and TE cells (SOX2 negative or CDX2 positive) and P values (t-test, two-sided) are shown. Each dot represents a single blastomere. c, The UCSC browser views showing H3K27ac and H3K27me3 enrichment near Tfap2c, and heat maps showing the expression levels of Tfap2c. d, Scatter plots comparing the TFAP2C CUT&RUN signals (5-kb window for the entire genome) between two biological replicates. The Pearson correlation coefficients are also shown. e, Bar charts showing the numbers of strong peaks identified from TFAP2C CUT&RUN in 2C, 4C, and 8C embryos, E3.5 ICM, E4.5 TE, E6.5 ExE, and TSCs (two biological replicates at each stage). f, Bar charts showing the percentages of TFAP2C strong peak distribution in the promoter (TSS ± 2.5 kb), intragenic, and intergenic regions.

Source data

Extended Data Fig. 3 Characterization of TFAP2C binding dynamics in mouse early development.

a, Scatter plots showing pairwise correlation of TFAP2C CUT&RUN signals at its promoter or distal binding peaks across different stages and TSCs, for two replicates. Pearson correlation coefficients are also shown. b, Left, heat maps showing the TFAP2C binding, motif density, H3K27ac, ATAC-seq signals, and CpG density at stage-specific and shared TFAP2C binding peaks. Right, the functional enrichment of nearby genes for TFAP2C binding peaks by the GREAT analysis are shown. c, Bar charts showing the TFAP2C peak distribution in promoter (TSS ± 2.5 kb), intragenic and intergenic regions in each cluster.

Extended Data Fig. 4 TFAP2C binds both accessible and inaccessible regions at early stages.

a, Bar charts showing percentages of TFAP2C binding peaks overlapped with ATAC-seq peaks at the promoters and distal regions. Random peaks of the same length of each peak are shuffled and generated. b, The UCSC browser views and heat maps showing TFAP2C binding and ATAC-seq enrichment, and RNA expression of representative genes. TFAP2C binding in accessible regions and inaccessible regions is shaded. c, Scatter plots comparing the TFAP2C CUT&RUN signals (5-kb window for the entire genome) and ATAC-seq signals at each stage. The Pearson correlation coefficients are also shown. d, Immunofluorescence of TFAP2C (red) and DAPI (blue) in mouse control and Tfap2c KO 8-cell embryos (two biological replicates). Scale bar: 20 μm. e, Bright field views of control and Tfap2c KO embryos at E7.5 and E10.5 (two biological replicates). Scale bar: 200 μm. f, Control and Tfap2c mzKO blastocysts, and their outgrowths grown in culture for 6 days (three biological replicates). Scale bar: 50 μm. g, Morphology of 2i mESCs derived from control and Tfap2c KO blastocysts (three biological replicates). Scale bar: 100 μm. h, Western blot showing TFAP2C, NANOG, SOX2, OCT4, and TUBULIN proteins in control and Tfap2c KO mESCs (2 clones).

Source data

Extended Data Fig. 5 Transcriptome analyses of Tfap2c mzKO embryos.

a, Volcano plot showing the gene expression fold changes (Tfap2c mzKO vs. control embryos) and the P values from edgeR. 2, 2, 3, 5, 2, 5, and 5 biological replicates are used for 2C, 4C, 8C, E3.5 ICM, E4.5 TE, E6.5 Epi, and ExE, respectively. Up-regulated (log2(Fold change) > 2, P value < 0.01) and down-regulated (log2(Fold change) < 2, P value < 0.01) genes are colored in red and blue, respectively. The GO terms and example genes are also shown.

Extended Data Fig. 6 TFAP2C regulates both early ICM and early TE genes at the 8C stage.

a, The PCA analysis based on RNA-seq of wild-type embryos18, control and Tfap2c KO embryos. b, Venn diagram shows the overlap between down- or up-regulated genes in Tfap2c KO 8C embryos (three biological replicates) and TFAP2C target genes with inaccessible (left) or accessible (right) TFAP2C peaks at their promoters. P values, one-sided Fisher exact test. c, The cumulative distributions of down-regulated, up-regulated, and all genes with defined distances (x-axis) between their TSSs and nearest distal 8-cell TFAP2C accessible or inaccessible peaks, with P values (Wilcoxon test, two-sided) indicated. d, Left, the boxplots showing the average enrichment of TFAP2C signals at the promoters of various lineage genes at the 8C stage. Centre line, median; box, 25th and 75th percentiles; whiskers, 1.5 × interquartile range. Early ICM gene, n = 224; early TE genes, n = 191; PrE specific genes73, n = 979; Ectoderm (Ect) specific genes, n = 84; Mesoderm specific genes, n = 222; Endoderm (End) specific genes, n = 585; all genes, n = 22654. Right, the cumulative distributions of various lineage genes with defined distances (x-axis) between their TSSs and nearest distal TFAP2C binding peaks. e, The cumulative distributions of early-late ICM or TE genes with defined distances (x-axis) between their TSSs and nearest distal TFAP2C binding peaks. f, Box plots showing expression of representative genes in control and Tfap2c knockdown 8C embryos (four biological replicates)17, with P values (t-test, two-sided) indicated. g, The cumulative distributions of down-regulated or up-regulated ICM-specific, TE-specific, and other genes in Tfap2c KO 8C embryos with defined distances (x-axis) between their TSSs and nearest distal 8C TFAP2C binding peaks, with P values (Wilcoxon test, two-sided) indicated.

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Extended Data Fig. 7 TFAP2C depletion activates cell apoptosis in ICMs.

a, Immunostaining showing NANOG protein levels and DAPI in control and Tfap2c mzKO E3.5 blastocysts (two biological replicates). Scale bar: 20 μm. b, Pie charts showing the percentages of ICM- and TE-specific genes among down-regulated genes in Tfap2c mzKO ICM and TE. c, The cumulative distributions of down-regulated or up-regulated, and all genes in Tfap2c KO ICM embryos with defined distances (x-axis) between their TSSs and nearest distal ICM TFAP2C binding peaks, with P values (Wilcoxon test, two-sided) indicated. d, Left, TUNEL assay and immunostaining showing CDX2 protein levels and DAPI in control and Tfap2c mzKO early blastocysts. Scale bar: 20 μm. Right, bar charts show the percentages of control (n = 12) and Tfap2c mzKO (n = 14) early blastocysts with different TUNEL foci incidence (CDX2 + , TE; CDX2-, ICM, indicated by dashed circles). e, Box plots showing expression of representative genes in control and Tfap2c mzKO embryos. f, TF motifs identified from 8C-specific (n = 2060), ICM-specific (n = 299), TE-specific (with CDX2 binding, n = 1227; without CDX2 binding, n = 843), and 8C-ICM-TE shared (with CDX2 binding, n = 1860; without CDX2 binding, n = 913) distal TFAP2C peaks. Sizes of circles indicate the relative motif enrichment. Expression levels of TFs in 8C embryos, ICM, and TE are color-coded for 8C-specific, ICM-specific, and TE-specific peaks, respectively. Expression of TFs is not shown for 8C-ICM-TE shared peaks. g, Line plots showing average motif densities for selected TFs (10 bp resolution) within 2 Kb around peak centers identified from TE-specific and 8C-ICM-TE shared distal TFAP2C peaks, with or without CDX2 binding in TE.

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Extended Data Fig. 8 Co-localization of CDX2 and TFAP2C in TE.

a, Sequence logos and ranks of TF motifs enriched in CDX2 peaks (n = 19,620) identified from CDX2 CUT&RUN at E4.5 TE. The top five TFs motifs are shown. b, The UCSC browser views showing CDX2 binding and TFAP2C binding signals in E4.5 TE (two biological replicates). c, The UCSC browser views showing enrichment of TFAP2C binding, CDX2 binding in the control and Tfap2c mzKO TE. Shades indicate TE-specific TFAP2C binding site (red), 8C-ICM-TE shared TFAP2C binding site (green) and CDX2-only binding site (blue). The arrows indicate reduction of CDX2 binding at TE-specific TFAP2C peaks in TE. d, Left, heat maps showing TFAP2C binding, CDX2 binding at the 8C-specific, ICM-specific, TE-specific, and 8C-ICM-TE shared TFAP2C peaks, and TE CDX2-only peaks. Right, heat maps and average plots show enrichment of CDX2 CUT&RUN (replicate 2) signals in control and Tfap2c mzKO E4.5 TE. e, Immunostaining showing TFAP2C, CDX2 protein levels and DAPI in control and Tfap2c mzKO E4.5 blastocysts (three biological replicates). Scale bar: 20 μm.

Extended Data Fig. 9 Co-localization of SOX2 and TFAP2C in ExE.

a, The UCSC browser views showing SOX2 binding and TFAP2C binding signals in ExE (two biological replicates). b, Sequence logos and ranks of TF motifs enriched in SOX2 peaks (n = 69,743) identified from SOX2 CUT&RUN at E6.5 ExE. The top four TF motifs are shown. c, Scatterplot comparing global binding (5 Kb bin) of SOX2 and TFAP2C in ExE for two replicates. Pearson correlation coefficients are also shown. d, Heat maps showing enrichment of SOX2, TFAP2C binding signals, and motif densities (numbers of motif per bp) at SOX2-only, TFAP2C-only, and SOX2-TFAP2C co-bound peaks in ExE (left). The functional enrichment of nearby genes around binding peaks by the GREAT analysis is also shown (right). e, Box plots showing expression of Sox2 in control and Tfap2c mzKO E6.5 ExE (five biological replicates). f, Immunostaining showing SOX2, OCT4 protein levels and DAPI in control and Tfap2c mzKO E6.5 embryos (two biological replicates). Scale bar: 50 μm.

Extended Data Fig. 10 Comparisons of TFAP2C and NR5A2 binding.

a, The boxplots showing the average enrichment of NR5A2 signals at the promoters of early-late ICM or TE genes at each stage. P values, two-sided Wilcoxon rank-sum test. Centre line, median; box, 25th and 75th percentiles; whiskers, 1.5 × interquartile range (same for b). Early ICM gene, n = 224; late ICM gene, n = 136; early TE genes, n = 191; late TE genes, n = 149; all genes, n = 22654. b, Boxplots showing the average enrichment of NR5A2 binding signals25 at the promoter (in WT embryos) of down-regulated or up-regulated genes identified in Nr5a2 knockdown (KD) 8C embryos25 for ICM-specific, TE-specific, and other genes, with P values (t-test, two-sided) indicated. All genes were similarly analyzed and are shown as controls. Down ICM genes, n = 180; up ICM genes, n = 164; down TE genes, n = 168; up TE genes, n = 69; down other genes, n = 628; up other genes, n = 573. c, Left, Heat maps showing TFAP2C binding, NR5A2 binding25, ATAC-seq signals18, and motif density at TFAP2C-only, NR5A2-only, TFAP2C-NR5A2 shared binding peaks in 2C embryos, separated by promoter and distal regions. d, Heat maps showing TFAP2C binding, NR5A2 binding25, ATAC-seq signal, and motif density at TFAP2C-only, NR5A2-only, TFAP2C-NR5A2 shared binding peaks in 8C embryos, separated by promoter and distal regions. The percentages of open (+) or not-open (-) binding sites for each cluster are also shown. e, Venn diagram showing the overlap between down- (top) or up-regulated (bottom) genes in Tfap2c KO and Nr5a2 KD25 8C embryos. The functional enrichment of shared downregulated genes is also shown. P values, one-sided Fisher exact test.

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Supplementary information

Reporting Summary

Supplementary Table 1

Tfap2c mzKO RNA expression table

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Li, L., Lai, F., Liu, L. et al. Lineage regulators TFAP2C and NR5A2 function as bipotency activators in totipotent embryos. Nat Struct Mol Biol (2024). https://doi.org/10.1038/s41594-023-01199-x

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