DUX-family transcription factors regulate zygotic genome activation in placental mammals

Abstract

In animal embryos, transcription is mostly silent for several cell divisions, until the release of the first major wave of embryonic transcripts through so-called zygotic genome activation (ZGA)1. Maternally provided ZGA-triggering factors have been identified in Drosophila melanogaster and Danio rerio2,3, but their mammalian homologs are still undefined. Here, we provide evidence that the DUX family of transcription factors4,5 is essential to this process in mice and potentially in humans. First, human DUX4 and mouse Dux are both expressed before ZGA in their respective species. Second, both orthologous proteins bind the promoters of ZGA-associated genes and activate their transcription. Third, Dux knockout in mouse embryonic stem cells (mESCs) prevents the cells from cycling through a 2-cell-like state. Finally, zygotic depletion of Dux leads to impaired early embryonic development and defective ZGA. We conclude that DUX-family proteins are key inducers of zygotic genome activation in placental mammals.

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Figure 1: DUX4 promotes transcription of genes expressed during early ZGA.
Figure 2: DUX4 binds TSSs of genes expressed during early ZGA and activates their expression in hESCs.
Figure 3: Dux is necessary for formation of 2C-like mESCs.
Figure 4: TRIM28 regulates formation of 2C-like mESCs by repressing Dux expression.
Figure 5: Dux is necessary for mouse early embryonic development.

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Acknowledgements

We thank T. Macfarlan (NIH) and M.E. Torres-Padilla (Helmholtz Zentrum Munchen) for sharing reagents and for helpful discussions, and we thank the Transgenic and Gene Expression Core Facility (EPFL) and S. Offner for technical assistance. This work was financed through grants from the Swiss National Science Foundation, the Gebert-Rüf Foundation, an INGENIUM grant (FP7 MC-ITN INGENIUM 290123), and the European Research Council (ERC 268721 and ERC 694658) to D.T.

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A.D.I. and D.T. conceived the project, designed the experiments, analyzed the data, and wrote the manuscript; A.D.I., A.C., and S.V. carried out the experiments; E.P. and J.D. performed bioinformatic and statistical analyses.

Corresponding author

Correspondence to Didier Trono.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Identification of genes specifically expressed at each stage of early embryo development.

(A) Pattern of genes with similar expression behavior during human embryo development. The blue line represents the average expression of the genes in each subset, while the gray lines represent each single gene. (B) Fraction of genes belonging to each pattern in (A) whose expression is not changing (gray), upregulated (cyan) or downregulated (orange) when DUX4 is ectopically expressed in human primary myoblast. (C) Fraction of genes belonging to each cluster illustrated in Figure 1B, the expression of which is unchanged (gray), increased (cyan) or decreased (orange) when DUX4 is ectopically expressed in human primary myoblasts. In B and C a fold change bigger than 2 and a p-value lower than 0.05 after Benjamini-Hochberg correction were selected.

Supplementary Figure 2 DUX4 ectopically expressed in hESCs and human primary myoblasts binds similar genomic sequences.

DUX4 was ectopically expressed in hESCs and ChIP-seq for the transcription factor was performed. (A) The Venn diagram represents the ChIP-seq peak overlap between our analysis in hESCs and the publicly available one in human primary myoblasts. (B) Percentage of individual integrants from families of transposable elements bound by DUX4. (C) Comparative expression during early human embryonic development of DUX4 (red), its putative target genes (blue, average), and the transposable elements (TEs) bound by DUX4 in hESCs and displaying increased expression at the peak of DUX4 transcription (green, average of all the TE integrants from each family). Oo, oocyte; Zy, zygote; 2C, 4C, 8C, corresponding n-cell stage; Mo, morula; Bl, blastocyst. (D) DUX4 binding motif identified with Homer. (E) Comparison of the fraction of all DUX4 peaks and the ones selected for being in a 5 kb window around the TSS of ZGA genes (from figure 2A) containing the DUX4 binding motif identified. (F) Comparison of the frequency of DUX4 binding motif identified in all DUX4 peaks or in the ones selected for being in a 5 kb window around the TSS of ZGA genes (from figure 2A).

Supplementary Figure 3 DUX4 binds TSSs of genes expressed during early ZGA when it is overexpressed in human primary myoblasts.

(A) Average coverage of ChIP-seq signal of DUX4 (blue) when overexpressed in human primary myoblasts in a window of 5 kb from the annotated TSS of genes belonging to the 2-4C and 2-8C clusters in figure 1B. Total input is represented in gray (line, average; shade, standard error of the mean). (B) Fraction of genes belonging to each cluster in figure 1B that have a DUX4 peak within a 5 kb window from their annotated TSS. Fisher’s exact test was performed to compare maternal vs 2-4C or 2-8C (p= 1.27e-47 and p= 1.49e-09 respectively). (C) Average coverage of ChIP-seq signal of DUX4 (blue) when overexpressed in human primary myoblasts in a window of 5 kb from TFE of RNAs found specifically upregulated at oocyte-to-4C and 4C-to-8C transitions. Total input is represented in gray (line, average; shade, standard error of the mean). (D) Fraction of TFE from oocyte-to-4C and 4C-to-8C transitions that have a DUX4 peak overlapping with their 5'. Fisher’s exact test was performed to compare 4C-to-8C vs. oocyte-to-4C TFEs (p= 4.48e-17).

Supplementary Figure 4 DUX4 binds TSSs of genes expressed during early ZGA.

UCSC genome browser screenshots on genes belonging to the 2-4C (TRIM43 and TRIM49) and the 2-8C (ZSCAN4 and MBD3L2) subset of genes. Starting from the top are displayed tracks for: RNA expression in early embryos (2C, 4C and 8C); DUX4 interaction with the locus in hESCs and human primary myoblasts; RefSeq track for gene annotation.

Supplementary Figure 5 Genomic and protein sequence organization of mDux.

(A) UCSC genome browser screenshot representing the genomic location of the D4Z4 repeats where the mouse orthologues of DUX4 are located. Tracks for gene annotation from two different Ensembl releases (84 and 75) and RefSeq are displayed. (B) Alignment of DUX4, Dux and Gm4981 protein sequences. Residues are colored according to their physicochemical properties (Clustal Omega code). The black boxes define conserved putative homeodomains and C-terminus.

Supplementary Figure 6 Genes highly expressed at the middle 2C stage are significantly represented in 2C-like mESCs.

(A) Pattern of genes with similar expression behavior during murine embryo development. The blue line represents the average expression of the genes in each subset, while the gray lines represent each single gene. (B) Fraction of genes belonging to mid 2C pattern (A) or rest of the genes, the expression of which is unchanged (gray), increased (cyan) or decreased (orange) in 2C-like cells compared to the rest of mESCs (selected from figure 3B). (C) Comparative expression of genes (Zscan4, Cml2, Dux, Zfp352) and transposable elements (MERVL) activated at ZGA, and two control housekeeping gene (Actb and Gapdh) in mESCs carrying an integrated MERVL-GFP reporter and sorted for GFP expression. Expression was normalized to Actb.

Supplementary Figure 7 CRISPR–Cas9 deletion of the Dux macrosatellite repeat in mESCs.

(A) UCSC genome browser screenshot representing the locus depleted in mESCs carrying a MERVL-GFP reporter, using a combination of two different sgRNAs cutting upstream and downstream of the murine D4Z4 repeat (2 vertical black lines). Starting from the top are displayed tracks for: gene annotation from RefSeq, mobile elements from repeat masker, and mappability of the locus by alignability of 100mers. Lower arrows show the primers used for the genotyping of the WT and Dux KO alleles. Dashed lines represent the PCR product amplified by the primers. (B) PCR products using the WT (upper blot) or KO (lower blot) primer combinations on three different WT (1-3) or Dux KO (4-6) clones, the bulk mESC population before (7) and after (8) Cas9/sgRNA transfection, and water (9). (C) Fraction of genes overexpressed in 2C-like cells compared to the remaining population of mESCs (from figure 3B), the expression of which is unchanged (gray), decreased (orange) or increased (cyan) upon Dux KO in mESCs (fold change bigger than 2 and p-value lower than 0.05 after Benjamini-Hochberg correction).

Supplementary Figure 8 Ectopic expression of Dux rescues the formation of 2C-like cells.

(A) Fraction of genes overexpressed in 2C-like cells compared to the remaining population of mESCs (from figure 3B), the expression of which is unchanged (gray), increased (orange) or decreased (cyan) upon Dux overexpression in Dux KO mESCs (fold change bigger than 2 and p-value lower than 0.05 after Benjamini-Hochberg correction). Comparative expression of indicated RNAs in (B) WT or (C) Dux KO MERVL-GFP mESCs transfected with plasmids encoding for LacZ, DUX4, Dux or Gm4981. RNA expression data is normalized to Actb. Shown are the mean ± SD for three independent clones. *** p ≤ 0.001, t-test. Immunofluorescence staining against (D) NANOG or (E) SOX2 in Dux KO mESC clones ectopically expressing Dux.

Supplementary Figure 9 RNA sequencing analyses show similar patterns of gene expression when Dux is expressed in different contexts.

(A) Principal component analysis and (B) heatmap showing the clustering of all the RNA-sequencing analysis of WT and Dux KO mESCs used in this manuscript. Pearson distances and complete agglomeration method were used to compute the heatmap’s dendrogram.

Supplementary Figure 10 DUX4 and Dux have partial interspecies activity on ZGA genes.

(A) GFP expression of 293T carrying several copies of integrated MT2/gag sequence regulating a GFP reporter and transfected with plasmids encoding for LacZ, DUX4, Dux or Gm4981. (B) Comparative expression in 293T of four genes activated at ZGA (ZSCAN4, ZSCAN5B, MBD3L2 and DUXA) and two control housekeeping gene (ACTB and TBP) 24 hours after transfection with plasmids expressing LacZ, DUX4, Dux or Gm4981. Expression was normalized to ACTB. (C) Graphical representation of the MERVL regulatory sequence and truncated mutants used in luciferase reporter assays, with canonical (in gag) and altered (♦; in the MT2_mm LTR) DUX4 binding sequences indicated. Luciferase activity was quantified 24 hours after transfection of 293T (D) and mESCs (E) with pGL4 plasmids containing a promoterless reporter driven by MT2/gag, the truncated mutants or a human PGK promoter together with plasmids expressing LacZ, DUX4, Dux or Gm4981. Ratios between the pGL4 plasmids containing sequences upstream of the luciferase reporter and the empty pGL4 plasmid are represented. Shown are the mean ± standard deviation for three experiments performed on different days. ** p ≤ 0.01, *** p ≤ 0.001, unpaired t-test.

Supplementary Figure 11 TRIM28 regulates formation of 2C-like mESCs by repressing Dux expression.

(A) Fraction of genes overexpressed in 2C-like cells compared to the remaining population of mESCs (from figure 3B), the expression of which is unchanged (gray), increased (orange) or decreased (cyan) upon Trim28 KD in WT (first two bars) or Dux KO (last two bars) in mESCs (fold change bigger than 2 and p-value lower than 0.05 after Benjamini-Hochberg correction). (B) Comparative expression of indicated RNAs in control or Dux KO MERVL-GFP mESCs transduced with lentiviral vectors targeting a control or Trim28. RNA expression data is normalized to Actb. (C) RNA sequencing of control and Trim28 KD on WT mESC clones. The dot plot represents the average gene expression of three independent clones in the two different conditions. The blue dot represents Trim28, pink dots different Dux transcripts, green dots the genes specifically expressed in 2C-like mESCs (selected from figure 3B), grey dots the rest of the genes. (D) Comparative expression of indicated RNAs in control or Trim28 KO mESCs transduced with control or Dux KD (sh2 and sh5) lentiviral vectors targeting a control or Dux. RNA expression data is normalized to Actb. (E) Fold enrichment of Trim28 ChIP-qPCR in mESCs of IAPEz (positive control) and Dux over MERVL (negative control). (F) Fold enrichment of H3K9me3 ChIP-qPCR in mESCs transduced with control or Trim28 KD lentiviral vector of IAPEz (positive control) and Dux over MERVL (negative control). Shown in (B) are the mean ± SD for three independent clones. Shown in (D) and (F) are the mean ± standard deviation for three experiments performed on different days. Shown in (E) are the mean ± standard deviation for two experiments performed on different days. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, t-test.

Supplementary Figure 12 Ex vivo analysis of Dux KO in preimplantation embryos.

(A) Average percent of embryos reaching the different embryonic stages or failing differentiation (dead) over 4 days. Shown in the bar plot are the mean ± standard deviation for three experiments following between 16 and 23 embryos for each condition performed on different days. * p ≤ 0.05, ** p ≤ 0.01, two-way ANOVA. Statistical tests were made between control and Dux sgRNA-injected embryos. (B) Brightfield images of day 4 embryos injected with control or Dux-specific sgRNAs (* healthy blastocyst). (C) PCR products using the Dux KO specific primer combinations (Supplementary figure 7) on: a pool of day 4 embryos from Figure 5B injected with the control (1) or Dux-specific (2) sgRNAs, WT (3) and Dux KO (4) mESC clones, and water (5).

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Supplementary Figures 1–12 (PDF 2735 kb)

Supplementary Table 1

Genes differentially expressed in human primary myoblasts upon ectopic expression of DUX4 (XLSX 38 kb)

Supplementary Table 2

Primer list (XLSX 28 kb)

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De Iaco, A., Planet, E., Coluccio, A. et al. DUX-family transcription factors regulate zygotic genome activation in placental mammals. Nat Genet 49, 941–945 (2017). https://doi.org/10.1038/ng.3858

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