For stem cell populations to remain effective they need to balance the paradoxical capabilities for both self-renewal and differentiation. These two properties define pluripotent Embryonic Stem (ES) cells and make them useful, so uncovering the molecular basis of these properties will reveal what being a stem cell means.
Our goal is to determine how ES cells balance self-renewal and differentiation. To achieve this it is important to (i) identify the molecules that direct self-renewal, (ii) determine how these molecules function and (iii) define how these molecules interact with their target proteins / genes, to deliver that function.
We cloned the pluripotent cell specific transcription factor Nanog using a genetic screen for enhanced self-renewal. As Nanog allows ES cells to self-renew under conditions in which they would normally differentiate, we named it after Tir nan Og, the mythological Celtic land of the ever-young. We are using Nanog as a molecular route into understanding of pluripotent cell regulation.
We have found that undifferentiated ES cells are not all the same. In fact undifferentiated ES cells fluctuate between states of high Nanog expression, associated with a high probability of self-renewal, and low Nanog, associated with a pre-disposition towards differentiation. Therefore loss of Nanog can be dissociated from commitment to differentiation.
A colony of ES cells showing heterogenous Nanog protein (green) in a more uniform Oct4 background (red).
We hypothesize that this heterogeneity underpins the functional heterogeneity seen in populations of ES cells in culture and that this is important in obtaining what we refer to as the Goldilocks balance between self renewal and differentiation. Rather than being essential for ES cell self-renewal, Nanog acts like a dimmer switch to modulate ES cell self-renewal efficiency (see figure below).
Nanog heterogeneity is affected by another pluripotency transcription factor, Oct4, but not in the way that might at first be expected. Oct4 positively regulates Nanog, so it was a surprise to find that Oct4+/- ES cells showed homogenous high Nanog expression. In work published in May 2013 in Cell Stem Cell we show that Oct4+/- cells have increased expression of Wnt pathway ligands and increased sensitivity to LIF. In collaboration with Huck-Hui Ng's lab at the A-star Genome Institute of Singapore we show by Chip-Seq analysis that, paradoxically, Oct4+/- ES cells have enhanced binding of Oct4 to the chromatin at key nodes in the pluripotency gene regulatory network. This reinforced positive feedback may explain the high homogenous expression of Esrrb, Klf4 and Nanog and the resulting robust self renewal of Oct4+/- ES cells.
To understand how Nanog works, we need to know how it interacts with its partner proteins, including itself. We have shown that Nanog exists in equilibrium between monomeric and dimeric forms (Kd of 3 µM). The dimerisation region is a sequence in which every fifth amino acid is tryptophan, and is essential for Nanog to confer cytokine independent self-renewal.
To identify partner proteins important in mediating Nanog function, we have collaborated with Raymond Poot's lab at Erasmus Medical Center, Rotterdam, to develop an affinity purification method coupled to mass spectrometric identification of proteins. We have used this approach re-iteratively to identify an extended network of interacting proteins centred on Oct4. In work published in the EMBO Journal in August 2013 we have extended this approach to Nanog (see Figure). This allowed us to define the residues mediating interaction between Nanog and Sox2 proteins.
As well as identifying partner proteins for Nanog, it is important to understand how Nanog mediates changes in gene expression. We have used Nanog-null ES cells carrying Tamoxifen-inducible Nanog protein to do this. Using bioinformatics tools (www.geneprof.org) developed by Simon Tomlinson's group, we have identified the genes that directly respond to Nanog protein. Surprisingly, in contrast to the many thousands of genes which bind Nanog in global chromatin immunoprecipitation studies, less than 100 genes change expression upon Nanog activation.
The transcription factor showing the strongest upregulation by Nanog is Essrb. In work published in October 2012 in Cell Stem Cell we show that Esrrb delivers many of the functional outputs of Nanog. Of particular note is our demonstration that cytokine-independent self renewal requires Esrrb, neatly closing a circle that began with our cloning of Nanog in 2003 on the basis of its ability to confer cytokine independent self-renewal.
Interestingly, Nanog does not simply work as a transcriptional activator. In work published in December 2012 in the EMBO Journal we show that Nanog protein represses Nanog gene activation and that this autorepression contributes to heterogeneous Nanog expression.
In collaboration with Val Wilson's group we have investigated the loss of pluripotency in embryos. In work published in July 2012 in Development we find that pluripotency disappears at the onset of somite formation, when Nanog expression is switched off and as Oct4 levels decline. Excitingly, we are able to reverse this loss of pluripotency by re-expression of Oct4.
Although Nanog -/- ES cells maintain somatic pluripotency, Nanog is required for primordial germ cell development between E11.5 and E12.5. This indicates that Nanog functions in vivo at two developmental time points (E3.5 and E11.5) that coincide with periods of major epigenetic reprogramming that include reactivation of both X chromosomes in females. We shall therefore extend on analyses of the pluripotency transcription factors to examine the fundamental similarities in transcription factor function occuring at both of these times.
We are investigating the differences between pluripotent stem cells expressing different levels of pluripotency transcription factors and are particularly interested in the mechanisms by which cells switch between alternate states. We are also investigating the molecular relationship between ES cells and the developmentally more advanced pluripotent cells of the post implantation embryo and their in vitro derivatives, epiblast stem cells (EpiSCs).
Do you want to join our group?
We always welcome speculative approaches from people with a strong work ethic and with a genuine interest in the fundamental controls of pluripotent stem cell function. Please address informal enquiries to ichambers [at] ed [dot] ac [dot] uk.