The homeobox transcription factor DUXBL controls exit from totipotency

In mice, exit from the totipotent 2-cell (2C) embryo stage requires silencing of the 2C-associated transcriptional program. However, the molecular mechanisms involved in this process remain poorly understood. Here, we demonstrate that the 2C-specific transcription factor DUX mediates an essential negative feedback loop by inducing the expression of DUXBL to promote this silencing.We show that DUXBL gains accessibility to DUX-bound regions specifically upon DUX expression.Furthermore, we determine that DUXBL interacts with TRIM24 and TRIM33, members of the tripartite motif superfamily involved in gene silencing and co-localizes with them in nuclear foci upon DUX expression. Importantly, DUXBL overexpression impairs 2C-associated transcription, whereas Duxbl inactivation in ESC increases DUX-dependent induction of the 2C-transcriptional program. Consequently, DUXBL deficiency in embryos results in sustained expression of 2C-associated transcripts, leading to early developmental arrest.Our study identifies DUXBL as an essential regulator of totipotency exit enabling the first divergence of cell fates. Overall design: Three to twelve week-old C57BL6/J female mice were superovulated by intraperitoneal injection of pregnant mare serum gonadotropin (7.5 IU). 48 h later, human chorionic gonadotropin (hCG, 7.5 IU) was delivered i.p. followed by mating with C57BL6/J males. Next morning, zygotes were isolated from ampulla of the oviducts and brought into culture in EmbryoMax® KSOM Mouse Embryo Media (Millipore-Sigma, MR-106-D) at 37 °C , 5 % CO2, 5% O2. Mouse embryos were injected at the 1-cell stage (24 h post hCG) with 200 ng/ul of either Duxbl-V5-2A-NLS-mCherry- or NLS-mCherry-encoding mRNAs. Embryos were then cultured either until the late 1-cell stage (28-30 h post hCG), until the early 2-cell stage (32 h post hCG) or until the late 2-cell stage (48 h post hCG) and harvested for RNA-seq.