Cryo-electron microscopy studies of pluripotency factor Nanog
Item statusRestricted Access
Embargo end date14/06/2024
Embryonic stem (ES) cells are derived from the inner cell mass of the blastocyst in the early mammalian embryo. These cells are characterised by the ability to self-renew and to give rise to cell types from all lineages, known as pluripotency. Culture of ES cells in vitro requires addition of signalling molecules such as LIF to block differentiation signals and maintain self-renewal. At the transcriptional level, self-renewal is governed by the ‘network of pluripotency’; a group of transcription factors which includes Nanog, Sox2 and Oct4. Nanog is a homeodomain protein which, when over-expressed, can drive cytokine-independent self-renewal of undifferentiated ES cells. Dimerisation of Nanog through the tryptophan repeat (WR) region, a low complexity domain including 10 tryptophan residues, has been shown to be vital for this function. Although Nanog is well characterised from a biological perspective, detailed structural information has previously been limited to the homeodomain only. To provide a structural basis for how oligomerisation of Nanog drives self-renewal, cryo-electron microscopy and single particle analysis were utilised to determine a 3D structure of Nanog. By performing size exclusion chromatography and analytical ultracentrifugation, it was discovered that Nanog forms large oligomers of 860 kDa which corresponds to a complex of 24 Nanog protomers. Negative stain electron microscopy showed that the oligomers are globular with a size of 220 x 200 Å. Cryo-electron microscopy and single particle analysis approaches produced a model of the Nanog oligomers showing a triangular shape, however, inherent flexibility within the assembly limited the resolution to approximately 10 Å. Attempts to limit this flexibility for further structural studies included binding of Nanog to DNA and to partner protein Sox2. Negative stain microscopy of Nanog bound to a DNA element derived from the Tcf3 promoter showed a strikingly different, larger complex compared to Nanog alone. Preliminary biophysical and negative stain studies suggested that the Nanog-Sox2 complex may exist in a variety of oligomeric species. Altogether, this thesis provides insight on how Nanog behaves structurally and biophysically. Further work will be required to limit flexibility within these Nanog homo-oligomers to allow determination of a high resolution structure.