Molecular Determinants of Totipotency

Division Of Basic Sciences - Nci

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Abstract

To understand totipotency we used the recently described totipotent-like conversion induced by expression of the homeoprotein DUX. Expression of DUX induces most of features that characterize 2-cell embryos (transcriptional profile, loss of heterochromatin or activation of retroviral dormant sequences). Thus, we generated mESCs carrying a doxycycline (DOX)-inducible DUX cDNA using the KH2 mESC line (hereafter, ESCDux). We verified that DUX is expressed upon addition of DOX along with well-known DUX-dependent downstream ZGA-associated genes. We observed that sustained expression of DUX leads to DNA-damage, revealed by the increased levels of the gH2AX marker, and efficient cell death in a dose dependent manner correlating with the conversion into the 2C-like state. We determined that DNA-damage was taking place at a subset of CTCF-binding sites and hypothesized that CTCF was a barrier to totipotent-like conversion. Interestingly, we observed that CTCF depletion in ESC induced a 2C-like conversion in an spontaneous manner. Finally, we also showed that 2C-like conversion upon CTCF-depletion was dependent on the endogenous activation of the cluster of genes ZSCAN4. Taking all together, we made great progress in this project, and we have this work accepted for publication in Nature Communications. We are also interested in exploring the DUX-dependent induction of DUXBL, a gene that belongs to the DUX family of proteins only found intact in rodents. This gene is part of a triplicated region of the genome spanning more than 300kb with three identical copies of DUXBL. Using published ChIP-seq data for DUX we observed that all three DUXBL copies contain three binding sites for DUX. In fact, induction of DUX in ESCDux cells leads to elevated levels of DUXBL in a dose dependent manner. We performed Cut&Run to identify the putative binding sites in ESCs and found it enriched in different enhancer and promoters. Interestingly, by performing morpholino microinjections in zygotes to downregulate Duxbl, we observed that this protein is essential for development. Indeed, Duxbl-knockdown zygotes arrested at the 2C-stage of development. RNAseq analysis of wild-type and Duxbl-knockdown 2C embryos revealed strong defects in splicing. This project has moved forward during this past FY and these analyses will set the basis to further explore the role of DUXBL during embryonic development.