We study embryonic stem cells (ESCs). These cells are pluripotent, meaning they can change into all the cell types of our bodies (differentiation). ESCs can also divide to produce cells identical to themselves, a process termed self-renewal. The simultaneous possession of these properties is what makes ESCs useful.
Pluripotent cell identity is controlled by the action of transcription factors (TFs) that together form cell-specific gene regulatory networks. In ESCs the gene regulatory network is centred on the TFs OCT4, SOX2 and NANOG. We identified NANOG in a genetic screen, searching for molecules that could allow ESCs to self-renew in the absence of the otherwise obligatory cytokine, LIF. Because of this phenotype, we named it Nanog, after Tir nan Og, the mythological Celtic land of the ever-young, where visitors remain unaged. We use Nanog as a tool to investigate how pluripotent cells are regulated.
We study the control of pluripotent stem cell identity, focussing on three strands: (i) how transcription factors (TFs) interact with partner proteins and chromatin to direct efficient self-renewal, (ii) how changes in TF interactions drive commitment to differentiate, (iii) how the pluripotency TF network is reconfigured to enable entry to the germline.
TF interactions regulating ESC identity
TFs control cell identity by changing the activity of the transcriptional machinery. To understand how TFs do this, it is important to identify the proteins that TFs interact with and to then examine the mechanisms that enable these partners to deliver TF function. To examine interacting proteins we use mass spectrometry to identify partner proteins (Gagliardi et al., The EMBO J., 2013).
Repeating this with mutant proteins has enabled us to focus in on a group of 6 NANOG interacting proteins required for NANOG function. We are using biochemical and advanced microscopic approaches to determine how these partners interact with NANOG both in vitro and in cells.
Chromatin mechanisms regulating pluripotent identity
To assess how TFs act on chromatin to alter cell identity, we use detailed temporal analysis of RNA expression together with ChIP assays. We couple ChIP assays with FACS sorting of fluorescent reporters to dissect distinct sub-populations of pluripotent cells. This tells us about TF interdependencies at individual loci before, and at the earliest stages of commitment to differentiation (Festuccia et al. The EMBO J., 2018).
Coupling ChIP with a technique for genome-wide analysis of enhancer activity (ChIP-STARR-seq) enables us to find novel active enhancers, genome-wide in distinct pluripotent populations (Barakat et al. Cell Stem Cell, 2018).
The role of pluripotency TFs in entry to the germline
We have recently shown that OTX2 plays an important role in restricting entry of cells into the germline, that the Otx2 gene is a repressed target of cytokine signals required for germline entry, and that in the absence of OTX2, germline differentiation exhibits aspects of a default differentiation (Zhang et al, Nature, 2018). These results suggest that OTX2 acts like a traffic warden, to restrict access to the germline and to usher cells towards the soma.
While Otx2+/+ cells do not enter the germline in the absence of cytokines, Otx2-null cells do. However, the efficiency with which Otx2-null cells enter the germline increase from about 25% to about 80% in the presence of cytokines. This suggests that additional cytokine-induced mechanisms contribute to germline entry. We aim to identify these mechanisms by single cell RNA-seq analysis of populations of ESCs at early points during PGC differentiation.
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