Abstract TET1, TET2 and TET3 are DNA demethylases with important roles in development and differentiation. To assess the contributions of TET proteins to cell function during early development, single and compound knockouts of Tet genes in mouse pluripotent embryonic stem cells (ESCs) were generated. Here, we show that TET proteins are not required to transit between naïve, formative and primed pluripotency states. Moreover, ESCs with double knockouts of Tet1 and Tet2 or triple knockouts of Tet1, Tet2 and Tet3 are phenotypically indistinguishable. TET1,2,3-deficient ESCs exhibit differentiation defects and fail to activate somatic gene expression, retaining expression of pluripotency transcription factors. Therefore, TET1 and TET2, but not TET3 act redundantly to facilitate somatic differentiation. Importantly however, TET-deficient ESCs can differentiate into primordial germ cell-like cells (PGCLCs), and do so at high efficiency in the presence or absence of PGC-promoting cytokines. Moreover, acquisition of a PGCLC transcriptional programme occurs more rapidly in TET-deficient cells. These results establish that TET proteins act at the juncture between somatic and germline fates: without TET proteins, epiblast cell differentiation defaults to the germline.

Poor survival and lack of treatment response in glioblastoma (GBM) is attributed to the persistence of glioma stem cells (GSCs). To identify novel therapeutic approaches, we performed CRISPR/Cas9 knockout screens and discovered TGFβ activated kinase (TAK1) as a selective survival factor in a significant fraction of GSCs. Loss of TAK1 kinase activity results in RIPK1-dependent apoptosis via Caspase-8/FADD complex activation, dependent on autocrine TNFα ligand production and constitutive TNFR signaling. We identify a transcriptional signature associated with immune activation and the mesenchymal GBM subtype to be a characteristic of cancer cells sensitive to TAK1 perturbation and employ this signature to accurately predict sensitivity to the TAK1 kinase inhibitor HS-276. In addition, exposure to pro-inflammatory cytokines IFNγ and TNFα can sensitize resistant GSCs to TAK1 inhibition. Our findings reveal dependency on TAK1 kinase activity as a novel vulnerability in immune-activated cancers, including mesenchymal GBMs that can be exploited therapeutically.

Posttranslational modifications of histones (PTMs) are associated with specific chromatin and gene expression states1,2. Although studies in Drosophila melanogaster have revealed phenotypic associations between chromatin-modifying enzymes and their histone substrates, comparable studies in mammalian models do not exist3-5. Here, we use CRISPR base editing in mouse embryonic stem cells (mESCs) to address the regulatory role of lysine 27 of histone H3 (H3K27), a substrate for Polycomb repressive complex 2 (PRC2)-mediated methylation and CBP/EP300-mediated acetylation6,7. By generating pan-H3K27R (pK27R) mutant mESCs, where all 28 alleles of H3.1, H3.2 and H3.3 have been mutated, we demonstrate similarity in transcription patterns of genes and differentiation to PRC2-null mutants. Moreover, H3K27 acetylation is not essential for gene derepression linked to loss of H3K27 methylation, or de novo activation of genes during cell-fate transition to epiblast-like cells (EpiLCs). In conclusion, our results show that H3K27 is an essential substrate for PRC2 in mESCs, whereas other PTMs in addition to H3K27 acetylation are likely involved in mediating CBP/EP300 function. Our work demonstrates the feasibility of large-scale multicopy gene editing to interrogate histone PTM function in mammalian cells.

Deciphering the mechanisms that control the pluripotent ground state is key for understanding embryonic development. Nonetheless, the epigenetic regulation of ground-state mouse embryonic stem cells (mESCs) is not fully understood. Here, we identify the epigenetic protein MPP8 as being essential for ground-state pluripotency. Its depletion leads to cell cycle arrest and spontaneous differentiation. MPP8 has been suggested to repress LINE1 elements by recruiting the human silencing hub (HUSH) complex to H3K9me3-rich regions. Unexpectedly, we find that LINE1 elements are efficiently repressed by MPP8 lacking the chromodomain, while the unannotated C-terminus is essential for its function. Moreover, we show that SETDB1 recruits MPP8 to its genomic target loci, whereas transcriptional repression of LINE1 elements is maintained without retaining H3K9me3 levels. Taken together, our findings demonstrate that MPP8 protects the DNA-hypomethylated pluripotent ground state through its association with the HUSH core complex, however, independently of detectable chromatin binding and maintenance of H3K9me3.

The Polycomb repressive complex 2 (PRC2) catalyzes H3K27 methylation across the genome, which impacts transcriptional regulation and is critical for establishment of cell identity. Because of its essential function during development and in cancer, understanding the delineation of genome-wide H3K27 methylation patterns has been the focus of intense investigation. PRC2 methylation activity is abundant and dispersed throughout the genome, but the highest activity is specifically directed to a subset of target sites that are stably occupied by the complex and highly enriched for H3K27me3. Here, we show, by systematically knocking out single and multiple non-core subunits of the PRC2 complex in mouse embryonic stem cells, that they each contribute to directing PRC2 activity to target sites. Furthermore, combined knockout of six non-core subunits reveals that, while dispensable for global H3K27 methylation levels, the non-core PRC2 subunits are collectively required for focusing H3K27me3 activity to specific sites in the genome.

Tet1, Tet2, and Tet3 encode DNA demethylases that play critical roles during stem cell differentiation and reprogramming to pluripotency. Although all three genes are transcribed in pluripotent cells, little is known about the expression of the corresponding proteins. Here, we tagged all the endogenous Tet family alleles using CRISPR/Cas9, and characterised TET protein expression in distinct pluripotent cell culture conditions. Whereas TET1 is abundantly expressed in both naïve and primed pluripotent cells, TET2 expression is restricted to the naïve state. Moreover, TET2 is expressed heterogeneously in embryonic stem cells (ESCs) cultured in serum/leukemia inhibitory factor, with expression correlating with naïve pluripotency markers. FACS-sorting of ESCs carrying a Tet2 Flag-IRES-EGFP reporter demonstrated that TET2-negative cells have lost the ability to form undifferentiated ESC colonies. We further show that TET2 binds to the transcription factor NANOG. We hypothesize that TET2 and NANOG co-localise on chromatin to regulate enhancers associated with naïve pluripotency genes.

Pluripotent cells reorganise themselves into an epithelium before they initiate differentiation, but it is not clear how these two events are mechanistically linked. Here we use quantitative imaging approaches to measure cellular rearrangements that accompany exit from naive pluripotency. We show that competence to epithelialise, like competence to differentiate, is a regulated process. The pro-differentiation transcription factor Tcf15 prospectively identifies cells that are competent to epithelialise. We identify early upregulation of the laminin receptor integrin alpha3 prior to differentiation and show that Tcf15 helps to regulate this change. Finally, we show that Tcf15 identifies and is required for efficient differentiation of a primed subpopulation of pluripotent cells. We conclude that competence to epithelialise is actively regulated and linked to differentiation-competence through the transcription factor Tcf15.

Deletion of Sox2 from mouse embryonic stem cells (ESCs) causes trophectodermal differentiation. While this can be prevented by enforced expression of the related SOXB1 proteins, SOX1 or SOX3, the roles of SOXB1 proteins in epiblast stem cell (EpiSC) pluripotency are unknown. Here, we show that Sox2 can be deleted from EpiSCs with impunity. This is due to a shift in the balance of SoxB1 expression in EpiSCs, which have decreased Sox2 and increased Sox3 compared to ESCs. Consistent with functional redundancy, Sox3 can also be deleted from EpiSCs without eliminating self-renewal. However, deletion of both Sox2 and Sox3 prevents self-renewal. The overall SOXB1 levels in ESCs affect differentiation choices: neural differentiation of Sox2 heterozygous ESCs is compromised, while increased SOXB1 levels divert the ESC to EpiSC transition towards neural differentiation. Therefore, optimal SOXB1 levels are critical for each pluripotent state and for cell fate decisions during exit from naïve pluripotency.