DUX4 is a multifunctional factor priming human embryonic genome activation

Abstract

Double homeobox 4 (DUX4) is expressed at the early pre-implantation stage in human embryos. Here we show that induced human DUX4 expression substantially alters the chromatin accessibility of non-coding DNA and activates thousands of newly identified transcribed enhancer-like regions, preferentially located within ERVL-MaLR repeat elements. CRISPR activation of transcribed enhancers by C-terminal DUX4 motifs results in the increased expression of target embryonic genome activation (EGA) genes ZSCAN4 and KHDC1P1. We show that DUX4 is markedly enriched in human zygotes, followed by intense nuclear DUX4 localization preceding and coinciding with minor EGA. DUX4 knockdown in human zygotes led to changes in the EGA transcriptome but did not terminate the embryos. We also show that the DUX4 protein interacts with the Mediator complex via the C-terminal KIX binding motif. Our findings contribute to the understanding of DUX4 as a regulator of the non-coding genome.

Keywords: biology of human development; developmental biology; molecular biology.

Graphical abstract

Figure 1

DUX4 activates thousands of newly identified bidirectionally transcribed enhancer-like regions that are enriched for ERVL-MaLR repeats (A) Schematic of the experimental outline. hESCs carrying an inducible DUX4-TetOn construct were doxycycline (dox) induced for 4 h. ATAC-seq and NET-CAGE were performed to identify accessible and transcribed cis-regulatory elements, respectively. (B) Venn diagram showing the number of ATAC-seq peaks in control and DUX4-activated hESC. (C) Bar plot showing the distribution of ATAC–seq peaks in control and DUX4-activated cells across the genome. (D) Bar plot showing the log2 ratio of ATAC–seq peaks overlapping ERVL-MaLR regions over randomly selected background regions (See STAR Methods). (E and F) Global differential expression analysis of DUX4-expressing (dox +) and control (dox -) hESCs for promoters (E) and putative enhancers (F). Log2 mean (counts per million, CPM) of four DUX4-expressing (dox +) and four control (dox -) replicates has been shown. Orange and purple dots indicate significantly upregulated (FDR < 0.05) promoters (E) and putative enhancers (F), respectively. Black dots indicate promoters for known 4-cell stage embryo genome activation genes. White dots indicated enhancers validated using the CRISPR activation assay. Yellow dots indicate significantly downregulated (FDR < 0.05) promoters (E) and putative enhancers (F), respectively. Grey dots indicate non-significantly differentially expressed promoters (E) and putative enhancers (F). (G) Genome browser view showing the putative enhancer-like region for ZSCAN4. The promoter for ZSCAN4 is 20.5 kb downstream of the putative enhancer. ATAC-seq signal indicates the accessibility of chromatin and NET-CAGE signal shows bidirectional transcription start sites of enhancer RNAs in dox (+) hESCs. NET-CAGE reads in red, plus strand; NET-CAGE reads in blue, minus strand. The putative enhancer also overlaps ERVL-MaLR repeat element. See also Figure S5. (H) Bar plot showing the log2 ratio of promoters and putative enhancers overlapping ERVL-MaLR regions over randomly selected background regions. (I and J) Schematic of CRISPR dCas9 activator constructs fused with DUX4 C-terminal end (I) or VP192 (J) used in combination with guide RNA pools to activate putative enhancers. Graphs show ZSCAN4 expression level relative to non-transfected cells (n = 6 from independent cell cultures (I); n = 3 from independent cell cultures (J)). Guide RNA construct for TdT were used as negative control. Data are shown as mean ± SD and p-values were calculated using two-tailed Student’s t-test. See also Figures S1–S5.

Figure 2

DUX4 expression dynamics and localization of the DUX4 protein in human zygotes and early embryos (A) Bar plot showing the log2 transcripts per million (TPM) of DUX4 mRNA reads in human MII oocytes (n = 20), zygotes (n = 59), 2-cell (n = 4), 4-cell (n = 15), and 8-cell (n = 14) embryos. Source data Tohonen et al., (2015). A pseudo count of 1 was added. (B and C) Human diploid zygotes (n = 3), 2-cell (n = 3), 4-cell (n = 4), and 8-cell (n = 2) embryos were immunostained with monoclonal DUX4 antibody (green) (B), and nuclei were counterstained with DAPI (magenta) (C). Orthogonal views along the depicted lines are shown (B, C). On the lower right corner (C) a magnification of the indicated region is shown. Scale bars 130 μm. (D) A box plot showing quantification of the DUX4 staining intensity in the nucleus in 3D normalized to the intensity in the cytoplasm. The samples are as described in (B and C). In each box the median is indicated, the edges are the 25^th and 75^thpercentiles, the whiskers extend to the data points not considered outliers. See also Figure S6 and Videos S1, S2, S3, and S4.

Figure 3

DUX4 knockdown in human zygotes leads to minor changes in the embryonic transcriptome (A) Schematic of 5’ STRT RNA sequencing of human embryos microinjected with either control or DUX4 targeting siRNA. (B) Representative confocal images of human embryos immunostained with monoclonal DUX4 antibody (green) 24 h after microinjection with either control (n = 4 zygotes) or DUX4 targeting (n = 5 zygotes) siRNA. Nuclei counterstained with DAPI (blue). Overlay of single DUX4 and DAPI z-planes is shown on the left together with an inset of a nucleus, and the corresponding z-planes and the bright field plane are shown on the right. Scale bars 50 μm. (C) A scatter plot showing the comparison of median log10 gene expression levels in the siControl blastomeres (n = 12 cells from two embryos) versus siDUX4 blastomeres (n = 15 cells from three embryos). Red and blue dots represent significantly upregulated and downregulated genes, respectively. Significance was calculated using Wilcoxon test, FDR < 0.05. Grey dots represent non-significantly differentially expressed genes. (D) Boxplots showing the expression levels in individual siControl and siDUX4 cells for selected oocyte-specific genes (left) and 8-cell stage genes seen during embryonic genome activation (right). All genes are statistically significant by Wilcoxon test, FDR < 0.05. In each box, the median line is indicated, the edges are the 25^th and 75^th percentiles, the whiskers extend to the data points not considered outliers. (E) Heatmaps showing Z score normalized RNA-seq expression levels (Yan et al., 2013) (GEO: GSE36552) for significantly upregulated (left) and downregulated (right) genes from (C). Upregulated genes (left) are mainly expressed in oocyte, zygote and 2-cell stages while downregulated genes (right) are expressed from 8-cell stage onwards. See also Figure S6.

Figure 4

DUX4 C-terminal KIX binding domain interacts with MED15 (A) DUX4 protein-protein interactome enriched for MED protein complex is shown. BioID -interactions are shown with red lines and AP-MS -interactions are shown with blue lines. If a protein appeared in both data sets it is shown with black line and outlined in black. Known prey-prey interactions are shown in grey (iREF). (B) Domain structure of full-length DUX4 is shown: N-terminal homeodomains HD1 and HD2, and C-terminal region. Conservation of residues in primates versus human sequences (green curve) C-terminal to residue G153 and sequence alignment of three conserved regions with a disorder value lower than 0.5 (red curve). Residue numbering from UniProt: Q9UBX2. Two helical regions are predicted within the C-terminal region, the first one (cyan helices) and the second one (salmon helix) both containing the amphipathic “ΦXXΦΦ” motif (Φ, bulky hydrophobic amino acid; X, any amino acid) found in several transcription factors reported to interact with KIX (Goto et al., 2002; Radhakrishnan et al., 1997; Wang et al., 2012). The position of the 9aaTAD (blue letters) and KBM (KIX binding motif; red letters) sequences are indicated by black bars. (C) Modelled interactions of the human KIX domain (PDB: 2LXT) with DUX4 9aaTAD (cyan) and KBM (salmon). (D) Replicate wells of HEK-GripTite 293 MSR cells were transfected with either wildtype DUX4 (DUX4 WT) or KIX-binding motif mutant DUX4 (DUX4-KBM mut) both tagged with HA, and with either LACZ or MED15 both tagged with V5. The cell lysates were precipitated for HA-tag. Immunoblots are shown for V5 (above) and HA (below) antibodies. See also Figures S7–S9 and Video S5.