Zygotic genome activation (ZGA) activates the quiescent genome to enable the maternal-to-zygotic transition1,2. However, the identity of transcription factors that underlie mammalian ZGA in vivo remains elusive. Here we show that OBOX, a PRD-like homeobox domain transcription factor family (OBOX1–OBOX8)3,4,5, are key regulators of mouse ZGA. Mice deficient for maternally transcribed Obox1/2/5/7 and zygotically expressed Obox3/4 had a two-cell to four-cell arrest, accompanied by impaired ZGA. The Obox knockout defects could be rescued by restoring either maternal and zygotic OBOX, which suggests that maternal and zygotic OBOX redundantly support embryonic development. Chromatin-binding analysis showed that Obox knockout preferentially affected OBOX-binding targets. Mechanistically, OBOX facilitated the ‘preconfiguration’ of RNA polymerase II, as the polymerase relocated from the initial one-cell binding targets to ZGA gene promoters and distal enhancers. Impaired polymerase II preconfiguration in Obox mutants was accompanied by defective ZGA and chromatin accessibility transition, as well as aberrant activation of one-cell polymerase II targets. Finally, ectopic expression of OBOX activated ZGA genes and MERVL repeats in mouse embryonic stem cells. These data thus demonstrate that OBOX regulates mouse ZGA and early embryogenesis.
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All data are available within the article and its Supplementary Tables. All data have been deposited at GEO with accession no. GSE215813. Accession codes of the published data in GEO used in this study are as follows: RNA-seq of oocytes and early embryos and L2C H3K4me3, GSE71434; Ribo-lite data of oocytes and early embryos, GSE165782; RiboTag data of oocytes, GSE135525; total RNA-seq of oocytes and early embryos, GSE169632; 1C ATAC-seq, GSE169632; E2C and L2C ATAC-seq, GSE92605; Pol II stacc–seq of early embryos, GSE135457; L2C H3K27ac, GSE72784; RNA-seq of Dux-overexpressed and control mES cells, GSE85632; RNA-seq of Dux KO embryos, GSE121746 and GSE134832; RNA-seq of 2C-like cells and control mES cells, GSE75751; RNA-seq of Nr5a2 knockdown and control 2C embryos, GSE178661. Source data are provided with this paper.
Software used to analyse these data are listed in Methods and are all publicly available.
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We thank members of the Schultz and Xie laboratories for discussion and comments during the OBOX study and preparation of the manuscript, and the Animal Center and Biocomputing Facility at Tsinghua University for their support. We thank H. Wang for advice on embryo culture. This work was funded by the National Natural Science Foundation of China (grant 31988101 to W.X.), the National Key R&D Program of China (nos. 2021YFA1100102 and 2019YFA0508900 to W.X.), the National Natural Science Foundation of China (nos. 31830047 and 31725018 to W.X.) and the Tsinghua-Peking Center for Life Sciences (W.X.). This work was also supported in part by NIH grant HD022681 (R.M.S.) and the Intramural Research Program of the NIH, National Institutes of Environmental Health Sciences grant 1ZIAES102985 (C.J.W.). S.J. and F.C. are supported by postdoctoral fellowships from the Tsinghua-Peking Center for Life Sciences. W.X. is a recipient of an HHMI International Research Scholar award and is a New Cornerstone Investigator.
The authors declare no competing interests.
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Extended data figures and tables
a, The UCSC genome browser snapshots showing Obox location and expression. b, Heatmap showing Obox mRNA levels in oocytes and embryos. c, CPE and PAS locations in maternal Obox 3’UTRs. d, Line plots showing poly(A) tail lengths25 of maternal Obox during oocyte maturation. e, Bar chart showing Obox3 mRNA levels in WT (2-4 biological replicates;10 oocytes or embryos for each group). f, OBOX3 immunofluorescence in 2C embryos. M2C, mid-2-cell; M-L2C, mid-to-late 2-cell (3 biological replicates). Scale bar, 20 μm. Arrow, nuclear OBOX3.
a-b, Line plots showing Obox mRNA and translation levels during oocyte maturation (2 biological replicates) and early embryo development (2 biological replicates) based on datasets from the previous publications30,31. NA, data not available. c, OBOX antibody epitope locations. d, Immunofluorescence showing OBOX signals detected by OBOX antibodies upon Flag-OBOX-GFP overexpression in mESCs (2 biological replicates). Scale bar, 10 μm. e–h, OBOX immunofluorescence in mouse oocytes and embryos (3 biological replicates). BL, blastocyst. Scale bar, 20 μm.
a, Schematic of individual Obox knockdown. IVM, in vitro maturation. PA, parthenogenetic activation. IVC, in vitro culture. h, hour. b, Bar chart showing the Obox knockdown efficiency in embryos (2 biological replicates; 10 embryos for each group). The control RNA levels were normalized to 1. Arrow, targeted Obox. c, Embryo morphology upon individual Obox knockdown at the blastocyst stage (2 biological replicates). Scale bars, 100 μm. d, Developmental rate upon individual Obox knockdown (2 biological replicates).
a, Whole-genome sequencing (WGS) and RNA-seq showing Obox genes and expression. Yellow shade, the deleted Obox. #1/#2/#3, three Obox mzKO mice. b, RNA-seq showing Obox levels (2 or 3 biological replicates). KO, the knocked out Obox genes. c, OBOX staining in WT and Obox mzKO embryos (2 biological replicates). Scale bar, 20 μm. d, Tubulin and OBOX staining in WT and Obox−/− oocytes (3 biological replicates). Scale bars, 5 μm (top) and 20 μm (bottom). e, Bar chart showing offspring numbers with different crossing strategies. 37, 23, and 16 cages for WT × WT, heterozygote × heterozygote, and homozygote × homozygote, respectively. ns, not significant (P-value = 0.69, two-sided t-test). Data are presented as mean values ± SD. f, Fertility test of mzKO (four female mice per group). g, HE staining (3 biological replicates). Scale bar, 0.25 mm. h, Bright-field images and bar charts showing oocyte morphology and maturation percentages upon OBOX depletion (2 biological replications). GVBD, germinal vesicle breakdown; PB1, the first polar body. Scale bar, 75 μm. i, Bar chart showing the numbers of ovulated oocytes per mouse. n, number of mice used. P-value = 0.84, two-sided t-test. Data are presented as mean values ± SD. j, Volcano plot showing gene expression changes between Obox−/− and WT oocytes (2 biological replicates). Dashed line, adjusted P-value threshold 0.05. k, Embryo morphology and developmental rate in vitro (5 biological replicates). Scale bar, 75 μm.
a, Expression of stage-specific genes in WT, Obox mutant, and rescued embryos. BL*2C, Obox mzKO embryos arrested at 2C when WT developed to blastocyst. b, Schematic of OBOX3 rescue in Obox mzKO embryos with embryo morphology and developmental rates shown (3 biological replicates). Scale bar, 100 μm. c-d, OBOX4 expression (c), embryo morphology, and developmental rate (d) with or without Obox4 rescue (3 biological replicates). Scale bar, 75 μm. e, RNA-seq showing Obox levels in WT and maternal Obox knockout embryos. Check and cross, the presence or absence of Obox mRNAs. f, OBOX3 immunofluorescence in WT and Obox mKO embryos (3 biological replicates). Scale bar, 20 μm. g-h, Embryo morphology, developmental rate (g), and expression of stage-specific genes (h) for WT and Obox mKO embryos in vivo at the blastocyst stage (2 biological replicates). Scale bar, 100 μm. i, Obox expression levels in Obox mutant embryos.
a, Hierarchical clustering based on RNA-seq (2 biological replicates for E2C and 3 for L2C). b, Volcano plot showing gene expression changes upon Obox depletion (2 biological replicates for E2C and 3 for L2C). Dashed line, adjusted P-value threshold 0.05. GO terms are shown. c, Balloon plot showing gene expression changes (mzKO/WT) for MERVL and ZGA genes at 2C (2 biological replicates for E2C and 3 for L2C). d, Scatter plot showing gene expression fold-changes upon Obox depletion (2 biological replicates for E2C and 3 for L2C). FC, fold-change. Yellow lines, local regression fitting. e, Violin plot showing maternal and ZGA gene expression changes from oocytes to E2C or L2C in WT and Obox mzKO embryos (2 biological replicates for MII, E2C and 3 for L2C). Centre line, median; box, 25th and 75th percentiles; whiskers, 1.5 × IQR.
a, Stage-specific gene expression upon Obox overexpression in WT embryos. b, Luciferase reporter assay showing OBOX gene activation abilities in HEK293 cells (2 biological replicates). ΔHD, homeobox domain deletion. c, Heatmap showing OBOX binding at L2C. OBOX motif densities and H3K27ac42 are shown. d, Scatter plot comparing OBOX binding at L2C. e, Bar chart showing the genomic distribution of OBOX binding at L2C. f, Heatmap showing OBOX binding on MERVL at L2C. OBOX motif is shown. n, peak number. g, Motif identified in OBOX binding sites in embryos. Percentages and P-values are shown. h, OBOX motif reporter assay in WT mouse embryos (2 biological replicates). Exposure time is shown. i, Luciferase reporter intensities in HEK293 cells (2 biological replicates). j, OBOX motif reporter assay in WT and Obox mzKO embryos (3 biological replicates). + and −, presence and absence of Obox1/5 mRNAs or extended motif, respectively. Scale bar, 75 μm. k, Bar chart showing OBOX motif occurrence at the stage-specific gene promoters. l-m, Box plots showing OBOX binding enrichment at major ZGA gene promoters (l) and distal regions (m) in WT L2C. 234, 272, 232, 169, and 201 genes have 0, 1, 2, 3, and >3 OBOX motifs on promoters, respectively. 9,855, 18,416, 7,142, 2,135, and 1,257 distal OBOX1 binding peaks have 0, 1, 2, 3, and >3 OBOX motifs, respectively. 5,918, 15,350, 4,795, 1,000, and 261 distal OBOX3 binding peaks have 0, 1, 2, 3, and >3 OBOX motifs, respectively. P-values, two-sided Wilcoxon rank-sum test. Centre line, median; box, 25th and 75th percentiles; whiskers, 1.5 × IQR. n, Percentages of ZGA genes that showed gene expression changes upon Obox depletion at L2C (3 biological replicates).
Extended Data Fig. 8 Depletion of OBOX led to Pol II preconfiguration defects and ectopic activation of 1C Pol II targets.
a, Pol II binding, CG density, and OBOX motif enrichment at 1C-specific, shared, and L2C-specific Pol II peaks in WT and Obox mzKO embryos. Red and blue arrows indicate L2C-specific Pol II binding and enrichment of the OBOX motif, respectively. b, Top, OBOX binding at example genes in WT embryos. OBOX motif and CG density are shown. Middle, Pol II binding and ATAC enrichment in WT and Obox mzKO embryos (2 biological replicates). P (+/−), promoter with or without the OBOX motif; D (+), distal enhancer with the OBOX motif. Bottom, bar charts showing gene expression (2 biological replicates for MII and 3 for L2C). Error bars, mean ± SE. c, Hierarchical clustering based on Pol II Stacc-seq (2 biological replicates). d, Percentages of Pol II or ATAC peaks with OBOX motif at the promoters or distal regions at L2C. e, Box plot showing RNA levels of ectopically activated genes, major ZGA genes, and maternal genes. n, gene number. Centre line, median; box, 25th and 75th percentiles; whiskers, 1.5 × IQR. f, Percentages of ectopically activated genes or all genes (control) that are 1C-specific Pol II targets or Polycomb targets (PcG). P-values, two-sided Fisher’s exact test. g, RNA levels in WT oocytes and embryos for ectopically activated genes (left). GO terms and example genes are shown (right). Centre line, median; box, 25th and 75th percentiles; whiskers, 1.5 × IQR. h, Heatmap showing gene expression in ICM (inner cell mass), TE (trophectoderm), and the ratio of TE/ICM in WT embryos for ectopically activated ICM and TE genes in Obox knockout embryos. Gene expression for W and Obox mzKO MII oocytes (2 biological replicates) and embryos (3 biological replicates) is mapped. n indicates gene number. 4C*, the stage when WT developed to 4C and Obox mzKO embryos arrested at 2-4C. i, Bar chart showing gene expression of example ICM and TE genes from h.
a, Obox expression levels upon overexpression in 2i mESCs (4 biological replicates). Error bars, mean ± SE. b, Bar chart showing the activated ZGA gene numbers upon Obox overexpression in 2i mESCs (4 biological replicates). P-values, two-sided Fisher’s exact test. c, Venn diagram showing the overlaps among Obox OE upregulated genes in 2i mESCs and ZGA genes. P-value, two-sided Fisher’s exact test. Green indicates the combined ZGA gene list activated by OBOX3/5. d, Scatter plot showing gene expression fold-changes upon Obox overexpression in 2i mESCs (4 biological replicates). e, OBOX binding at example OBOX-activated ZGA genes and MERVL in embryos. OBOX motif and RNA levels are shown. f, OBOX binding enrichment in embryos at the promoters of differentially expressed genes (DEGs) upon Obox overexpression in 2i mESCs. g, Line charts showing DEG upon Obox overexpression in 2i mESCs (4 biological replicates) for their expression in oocytes and embryos. Error bars, mean ± SE. n, gene number. h, Venn diagram showing the overlap between Obox activated ZGA genes in 2i mESCs (4 biological replicates) and downregulated ZGA genes in Obox mzKO embryos (2 biological replicates for E2C and 3 for L2C)). P-value, two-sided Fisher’s exact test. Green indicates the combined ZGA gene list activated by OBOX3/5 and downregulated in Obox mzKO embryos. i, Volcano plot showing the repeat expression changes upon Obox overexpression in 2i mESCs (4 biological replicates). Dashed line, adjusted P-value threshold 0.05.
a, Bar charts showing Dux, Zscan4, and Dppa expression in 2C embryos (top, 2 biological replicates for E2C and 3 for L2C) and mESCs (bottom, 4-5 biological replicates). b, The UCSC browser snapshots showing OBOX binding at 2C. Pol II, ATAC, and OBOX motif are shown. c, Heatmap showing Obox expression upon Dux overexpression16 in 2i mESCs (2 biological replicates). d, Heatmap showing Obox expression upon Dux knockout20 (2 biological replicates for late 1-cell (L1C) and 3 for L2C). e, Venn diagram showing the overlap of downregulated ZGA genes between Obox knockout and Dux knockout embryos19,20. n, ZGA gene number. f, Venn diagram showing the overlap of OBOX-activated ZGA genes and upregulated ZGA genes in 2CLCs compared to mESCs. 2CLC, 2-cell-like cell. P-value, two-sided Fisher’s exact test. Green indicates the combined ZGA gene list activated by OBOX3/5 and in 2CLCs. g, Heatmap showing Obox expression upon Nr5a2 knockdown21 in embryos. h, Scatter plot comparing the ZGA gene expression changes upon Obox overexpression between WT and Nr5a2 knockout mESCs (2 replicates).
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Ji, S., Chen, F., Stein, P. et al. OBOX regulates mouse zygotic genome activation and early development. Nature 620, 1047–1053 (2023). https://doi.org/10.1038/s41586-023-06428-3