Epigenetic mechanisms are essential to establish and safeguard cellular identities in mammals. They dynamically regulate the expression of genes, transposable elements, and higher-order chromatin structures. Expectedly, these chromatin marks are indispensable for mammalian development and alterations often lead to diseases such as cancer. Molecularly, epigenetic mechanisms rely on factors to establish patterns, interpret them into a transcriptional output, and maintain them across cell divisions. A global picture of these phenomena has started to emerge over the years, yet many of the molecular actors remain to be discovered. In this context, we have developed a reporter system sensitive to epigenetic perturbations to report on repressive pathways based on Dazl, which is normally repressed in mouse ES cells. We used this system for a genome-wide CRISPR knock-out screen, which yielded expected hits (DNMT1, UHRF1, MGA), as well as novel candidates. We prioritized the candidates by secondary screens, and led further experiments on 6 of them: ZBTB14, KDM5C, SPOP, MCM3AP, BEND3, and KMT2D. Our results show that all 6 candidates regulate the expression of germline genes. In addition, we find that removal of ZBTB14, KDM5C, SPOP and MCM3AP led to similar transcriptional responses, including a reactivation of the 2-cell like cell (2CLC) signature. Therefore, our genetic screen has identified new regulators of key cellular states.

In mammals, only the zygote and blastomeres of the early embryo are fully totipotent. This totipotency is mirrored in vitro by mouse "2-cell-like cells" (2CLCs), which appear at low frequency in cultures of Embryonic Stem cells (ESCs). Because totipotency is incompletely understood, we carried out a genomewide CRISPR KO screen in mouse ESCs, searching for mutants that reactivate the expression of Dazl, a robust 2-cell-like marker. Using secondary screens, we identify four mutants that reactivate not just Dazl, but also a broader 2-cell-like signature: the E3 ubiquitin ligase adaptor SPOP, the Zinc Finger transcription factor ZBTB14, MCM3AP, a component of the RNA processing complex TREX-2, and the lysine demethylase KDM5C. Functional experiments show how these factors link to known players of the 2 celllike state. These results extend our knowledge of totipotency, a key phase of organismal life.

In mammals, only the zygote and blastomeres of the early embryo are fully totipotent. This totipotency is mirrored in vitro by mouse "2-cell-like cells" (2CLCs), which appear at low frequency in cultures of Embryonic Stem cells (ESCs). Because totipotency is incompletely understood, we carried out a genome-wide CRISPR KO screen in mouse ESCs, searching for mutants that reactivate the expression of Dazl, a robust 2-cell-like marker. Using secondary screens, we identify four mutants that reactivate not just Dazl, but also a broader 2-cell-like signature: the E3 ubiquitin ligase adaptor SPOP, the Zinc Finger transcription factor ZBTB14, MCM3AP, a component of the RNA processing complex TREX-2, and the lysine demethylase KDM5C. Functional experiments show how these factors link to known players of the 2 cell-like state. These results extend our knowledge of totipotency, a key phase of organismal life.