DUX4 regulates oocyte to embryo transition in human

Abstract

45During the human oocyte-to-embryo transition, the fertilized oocyte undergoes 46 final maturation and the embryo genome is gradually activated during the first 47 three cell divisions. How this transition is coordinated in humans is largely 48 unknown. We show that the double homeodomain transcription factor DUX4 49 contributes to this transition. DUX4 knockdown in human zygotes caused 50 insufficient transcriptome reprogramming as observed three days after 51 fertilization. Induced DUX4 expression in human embryonic stem cells activated 52 transcription of thousands of newly identified bi-directional transcripts, including 53 putative enhancers for embryonic genome activation genes such as LEUTX. DUX4 54 protein interacted with transcriptional modifiers that are known to couple 55 enhancers and promoters. Taken together, our results reveal that DUX4 is a 56 pioneer regulating oocyte-to-embryo transition in human through activation of 57 intergenic genome, especially enhancers, and hence setting the stage for early 58 human embryo development. 59 60 61 62 63 64 65 4 Mammalian pre-implantation development commences with oocyte-to-embryo 66 transition, which involves fundamental changes in the epigenetic landscapes, 67 modulation of cell cycle control, and translation or clearance of selected maternal 68 mRNAs, culminating to embryonic genome activation 1,2 . The pioneer regulators 69 orchestrating the oocyte-to-embryo transition and first embryo genome activation steps 70 in human remain poorly understood. The conserved double homeodomain transcription 71factor DUX4 represents a plausible candidate regulating the oocyte-to-embryo 72 transition in humans, given its capacity to activate germline genes and genomic repeat 73 elements 3-5 . Here we show that DUX4 is able to launch the first reprogramming steps 74 from oocyte to embryo in human by activating thousands of novel enhancers and 75 therein, modulating the transcriptome and chromatin. Human DUX4 knockdown 76 embryos are viable until the third day of development, but their transcriptome is 77 severely altered. Our proteomics approaches suggest that DUX4 binds to Mediator 78 complex and chromatin modifiers through its C-terminal domains, providing a likely 79 explanation to how DUX4 may extensively modulate the genome. This study implies a 80 wider role for DUX4 as a cellular gate keeper acting both as a general genomic modifier 81 of cell fate as well as a specific inducer of first wave embryo genome activation genes. 82 83Results 84 Quantification of DUX4 in human embryos 85The DUX4 induced gene network is highly conserved 6 and recent reports showed that 86 DUX4 is expressed in early human embryos 3,4 . However, details of this dynamic 87 process, including initiation of DUX4 expression, remained ambiguous. Therefore, we 88Given the short-term and precise manifestation of DUX4 mRNA and protein in human 107 zygotes and early cleavage stage embryos, we next asked how DUX4 regulates the first 108 steps of human embryo development. We microinjected either DUX4...

A genome-wide CRISPR/Cas phenotypic screen for modulators of DUX4 cytotoxicity reveals screen complications

“…The cells should be highly proliferative, easily transfected and display a robust DUX4-induced phenotype. Fortunately, DUX4 is a so-called pioneer factor 49,50 , capable of regulating its target genes independent of their chromatin-state.…”

Transient DUX4 expression induces blastomere-like expression program that is marked by SLC34A2

“…Notably, cells at 24 h after 15 and 30 min of induction showed highly similar expression profiles (r = 0.96, Spearman correlation) (Figure S1C). The expression changes of DUX4 target genes present in early embryo (Resnick et al, 2019) were similar between continuously (4 h) treated cells (Vuoristo et al, 2020), and pulsed cells at 24 h post-induction, suggesting that the 15 and 30 min pulse were sufficient to activate DUX4 target genes (Figures 1D and S1D).…”

“…DUX4-TetOn hESCs (Vuoristo et al, 2020) were maintained on hESC-qualified Geltrex (Thermo Fisher Scientific) coated tissue culture dishes in Essential 8 culture medium (Thermo Fisher Scientific) in 5% CO2 at 37°C. The cells were passaged every three to five days after a 3 min incubation with 0.5 mM EDTA (Thermo Fisher Scientific).…”

“…The STRT RNA-seq data of continuous DUX4 induction, treated by doxycycline for 4 h, was obtained from Vuoristo et al (Vuoristo et al, 2020) and reprocessed as described above. The expression of transposable elements was quantified using TEtranscripts (v2.2.1) with 'uniq' mode (Jin et al, 2015).…”

“…DUX4, a double homeobox transcription factor, is a key regulator of human embryonic genome activation (EGA) by inducing cleavage stage-specific genes and repetitive elements (De Iaco et al, 2017;Hendrickson et al, 2017;Vuoristo et al, 2020;Whiddon et al, 2017). It is specifically and transiently expressed in the human cleavage-stage embryo (Töhönen et al, 2017).…”