Supplementary MaterialsSupplementary Information 41467_2019_9041_MOESM1_ESM. under different environmental constraints is only partially comprehended. Here, we show that this transcription factor Nanog deploys multiple unique mechanisms to enhance embryonic stem cell self-renewal. In the presence of LIF, which fosters self-renewal, Nanog rewires the pluripotency network by promoting chromatin convenience and binding of other pluripotency factors to thousands of enhancers. In the absence of LIF, Nanog blocks differentiation by sustaining H3K27me3, a repressive histone mark, at developmental regulators. Among those, we show that this repression of plays a preponderant role. Our results underscore the versatility of grasp transcription factors, such as Nanog, to globally influence gene regulation during developmental processes. Introduction Gene regulatory networks driven by grasp transcription factors (TFs) play pivotal functions over a large spectrum of biological processes, from adaptive cell responses1 to cell fate specification during development2. The key properties of TF networks, shared among cell types, developmental contexts and organisms3, are exemplified by the pluripotency network, which plays a dominant role during early mammalian embryogenesis4. The robustness of this network allows to capture the ex vivo of transient biological identity of the pluripotent epiblast through the derivation of self-renewing Embryonic Stem (ES) cells5, which have enabled identification of important TFs NBQX manufacturer (e.g., Oct4, Sox2, Nanog and Esrrb). The study of processes driving the balance between ES cell self-renewal and differentiation has provided us with a canonical picture of how TF networks operate, establishing self-sustaining regulatory loops and acting together through multiple promoters and enhancers6C9. For instance, Oct4, without which pluripotent cells cannot be managed10, acts with the TF Sox2 to recognise and bind chimeric motifs11 found at a large number of regulatory elements driving ES cell-specific transcription. Oct4 and Sox2 also tend to bind with other TFs, including Nanog and Esrrb, at multiple enhancers across the genome, to combinatorially coregulate a large number of targets. This simultaneous and concerted action over hundreds of common targets ensures considerable redundancy, and, therefore, strong genome-wide responses. How these TFs synergise at or compete for common regulatory elements, and how by these means they individually contribute to the networks activity, is usually however not well comprehended. Moreover, several TFs of the pluripotency network are directly connected to cell signalling, enabling ES cells to establish appropriate responses that are instructed extrinsically. A prominent example is usually provided by the LIF cytokine, which promotes self-renewal by activating several pluripotency TFs such as Esrrb12,13. NBQX manufacturer Hence, a key function of the pluripotency network is usually to integrate signalling cues to appropriately respond to changes in the environment, conferring the responsiveness of ES cells NBQX manufacturer and their capacity to readily differentiate. In this regard, it is noteworthy that was first identified as a factor capable of bypassing the requirements for LIF: in the presence of ectopic Nanog expression, ES cell self-renewal is usually strongly enhanced and completely impartial of LIF14. In the current model, Nanog achieves LIF-independent self-renewal by activating LIF-responsive genes, in particular transcription The SunTag system was developed as a versatile tool to either visualise specific molecules in live cells or to perform epigenome editing of endogenous loci when coupled to an enzymatically inert dCas922. It entails the expression of diffusible antibodies (scFv) that interact with high affinity with 10 copies of the GCN4 epitope linked to an enzymatically inert Cas9 (dCas9). These scFv antibodies are fused to GFP and the potent activator VP64, such that upon expression of a gRNA targeting a given genomic region, several VP64 molecules are NTRK2 brought about with high efficiency and specificity. To provide increased flexibility to the system, and facilitate the generation of cell NBQX manufacturer lines transporting an inducible CRISPR-ON system, we engineered a single vector expressing the two SunTag moieties under the control of a Tetracycline Responsive Element. Moreover, dCas9 is usually NBQX manufacturer linked to BFP and HpH through P2A and IRES sequences, respectively (Supplementary Fig.?1A). Hence, upon induction of the system with Doxycycline (Dox), the cells are expected to become green, blue and Hygromycin-resistant, providing a high tractability. This vector was launched in ES cells together with the rtTA activator: two clones (C1 and C2) showing a high percentage of green/blue cells.