Aims
We study pluripotent Embryonic Stem (ES) cells. Our goal is to define the mechanisms by which key regulatory molecules direct ES cell self-renewal and differentiation, with our principal focus on self-renewal. To achieve this it is important to (i) identify the molecules involved in directing self-renewal, (ii) determine the biological function of these molecules and (iii) define how these molecules interact at an atomic level to fulfil their function.

Background
Embryonic stem (ES) cells present an interesting paradox because they can both self-renew and differentiate into derivative cells of all three primary germ layers. Indeed it is the simultaneous possession of these two properties that defines ES cells and makes them useful.

We isolated the homeodomain-containing transcription factor Nanog through a functional screen for molecules capable of directing ES cell self-renewal in the absence of the otherwise obligatory LIF signal. Since Nanog allows ES cells to self-renew under conditions in which they would normally differentiate, we named the gene after Tir nan Og, the mythological Celtic land of the ever-young.

We have since shown that Nanog exists in equilibrium between momomeric and dimeric forms with a K_d of 3 µM. The dimerisation region has been identified as a sequence in which every fifth amino acid is tryptophan and removal of dimerisation capacity abolishes Nanog function.

We have also shown that Nanog is expressed in a mosaic fashion within the Oct4+ ES cell population. Using a GFP reporter targeted to the Nanog locus, we showed that GFP- cells can give rise to GFP+ cells, suggesting that ES cells expressing no or low Nanog may retain self-renewal capacity. To examine this possibility rigorously we used conditional deletion strategies to remove Nanog from ES cells. Counter to the prevailing view, we showed that Nanog -/- ES cells can self-renewal. However, the efficiency of self-renewal in the absence of Nanog is drastically reduced. Comparative studies show a stepwise reduction in self-renewal efficiency in Nanog overexpressing, Nanog +/+, Nanog +/- and Nanog -/- ES cells. These results indicate that loss of Nanog can be dissociated from commitment to differentiation and that rather than being essential for ES cell self-renewal, Nanog acts like a dimmer switch to modulate ES cell self-renewal efficiency (see Figure).

Although Nanog -/- ES cells maintain somatic pluripotency, primordial germ cell development fails in the absence of Nanog between E11.5 and E12.5. Intriguingly, this is around the time of epigenetic reprogramming and our recent findings indicate that the most dramatic epigenetic function that occurs in mammalian development, the activation of the silent X-chromosome is dictated through binding of the triumvirate of transcription factors, Nanog, Oct4 and Sox2 to the gene encoding the cis acting mediator of X-inactivation, Xist.

Approaches and progress

To investigate how ES cells self-renew, we use biochemical, genetic and imaging techniques as well as genetic screens.

Selected publications

Research papers

• Silva J, Nichols J, Theunissen TW, Guo G, Van Oosten AL, Barrandon O, Wray J, Yamanaka S, Chambers I, Smith A. 2009. Nanog is the gateway to the pluripotent ground. Cell 138(4):722-737.
• Navarro P, Chambers I, Karwacki-Neisius V, Chureau C, Morey C, Rougelle C, Avner P. 2008. Direct molecular coupling of Xist regulation and pluripotency. Science 321:1693-1695. Press release.
• Mullin NP, Yates A, Rowe A, Nijmeijer B, Barlow PN, Walkinshaw MD, Chambers I. 2008. The pluripotency rheostat Nanog functions as a dimer. Biochem J 411:227-231.
• Chambers I, Silva J, Colby D, Nichols J, Robertson M, Nijmeijer B, Vrana J, Jones K, Grotewold L, Smith A. 2007. Nanog safeguards pluripotency and mediates germ cell development. Nature 450:1230-1234.
• Chambers I, Colby D, Robertson M, Nichols J, Lee S, Tweedie S and Smith AG. 2003. Functional expression cloning of Nanog, a pluripotency sustaining factor in mouse embryonic stem cells. Cell 113: 643-655.

Reviews

• Chambers I and Tomlinson S. 2009. The transcriptional foundation of pluripotency. Development 136:2311-2322.
• Chambers I and Smith A. 2004. Self-renewal of teratocarcinoma and embryonic stem cells.
  Oncogene 23(43):7150-7160.

Funding
The Wellcome Trust
Juvenile Diabetes Research Fund (JDRF)
EuroSyStem
Scottish Funding Council