The homeobox transcription factor DUXBL controls exit from the totipotent 2C state [RNA-seq]

Upon exit from the totipotent 2-cell (2C) embryo stage, the 2C-associated transcriptional program needs to be efficiently silenced. However, the molecular mechanisms involved in this process remain mostly unknown. Here, we determined that the 2C-specific transcription factor DUX directly induced the expression of DUXBL to promote this silencing. CUT&RUN analysis revealed that DUXBL gained accessibility to DUX-bound regions in DUX-induced ESC while it was unable to bind those regions in uninduced cells. Importantly, DUX expression in Duxbl-knockout ESC caused increased induction of the 2C-transcriptional program, whereas DUXBL overexpression impaired 2C-associated transcription. Mechanistically, we determined that DUXBL interacted with TRIM24 and TRIM33, two members of the tripartite motif superfamily involved in gene silencing, and co-localized with them in nuclear condensates upon DUX expression. Importantly, DUXBL downregulation in mouse zygotes led to a penetrant 2C-stage arrest. Our data revealed an unexpected role for DUXBL in controlling the exit from totipotency. Overall design: RNAseq experiments of WT, DUXBL-KO, TRIM24-KO, and TRIM33-KO embryonic stem cells with and without DOX treatment.

全能性二细胞（2C）胚胎阶段退出后，与2C相关的转录程序需被高效沉默。然而，该过程的分子机制目前仍大多未被阐明。本研究发现，2C特异性转录因子DUX可直接诱导DUXBL的表达，进而介导该沉默过程。CUT&RUN分析显示，在DUX诱导的胚胎干细胞（Embryonic Stem Cell, ESC）中，DUXBL获得了结合DUX靶向结合区域的能力；而在未诱导的细胞中，DUXBL无法结合此类区域。

值得注意的是，在DUXBL基因敲除的胚胎干细胞中诱导DUX表达，会使2C转录程序的诱导水平升高；而过表达DUXBL则会削弱与2C相关的转录过程。机制层面研究显示，DUXBL可与参与基因沉默的三重基序（tripartite motif, TRIM）超家族的两个成员TRIM24、TRIM33相互作用，并在DUX诱导后与二者共定位于核凝聚体中。

此外，在小鼠受精卵中下调DUXBL的表达，会引发显著的2C阶段发育阻滞。本研究数据揭示了DUXBL在调控细胞全能性退出过程中此前未被报道的作用。

实验设计：对经/未经多西环素（Doxycycline, DOX）处理的野生型（Wild Type, WT）、DUXBL基因敲除（DUXBL-KO）、TRIM24基因敲除（TRIM24-KO）及TRIM33基因敲除（TRIM33-KO）胚胎干细胞开展RNA测序（RNA Sequencing, RNAseq）实验。