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Structure-function relationship of the novel ES cell transcriptional determinant Nanog

Reference	BB/C508834/1
Principal Investigator / Supervisor	Professor Ian Chambers
Co-Investigators / Co-Supervisors	Professor Paul Barlow, Professor Malcolm Walkinshaw
Institution	University of Edinburgh
Department	Inst of Stem Cell Research
Funding type	Research
Value (£)	359,328
Status	Completed
Type	Research Grant
Start date	01/01/2005
End date	31/12/2007
Duration	36 months

Abstract

ES cells possess the capacity to undergo essentially unlimited symmetrical cell divisions, a process referred to as self-renewal as well as to differentiate into all germ layers of the embryo. In order to sustain self-renewing divisions it is necessary to supplement cell cultures with LIF and either serum or BMP. We have recently used a functional screen in ES cells to isolate a cDNA capable of conferring LIF independent self-renewal on transfected ES cells. This cDNA encodes Nanog, a variant homeodomain protein. We have since shown that ES cells overexpressing Nanog no longer require serum or BMP to maintain self-renewal. These features define Nanog as a unique component of the machinery that drives ES cell self-renewal. Here we propose determination of Nanogs high-resolution 3D structure by NMR and/or X-ray crystallography. A large number of fragments in addition to the whole protein will be studied to maximise the likelihood of rapidly being able to collect good quality experimental data. This emerging structural analysis will inform and direct functional analysis of Nanog mutants and allow formulation of hypotheses to explain function-structure relations. These can be tested by further rounds of mutagenesis and functional assay validated by structural methods. Using this strategy, the structural basis of Nanog's ability to carry out the following will be investigated: Interact with DNA, by electrophoretic mobility shift assays; Direct ES cell self renewal in the absence and in the presence of LIF/serum/BMP, by stable transfection of episomal expression constructs; Affect expression from Nanog responsive promoters, by transient expression of reporter constructs driven by multimerised Nanog binding sites; Bind to novel partner proteins, by 2-hybrid studies and determining the sites of interaction on Nanog and on the partner protein required for interaction. We also intend to test the ability of particular mutants to isolate a subset of target genes by chromatin immunoprecipitation. In addition we will perform a random mutagenesis screen to identify residues important for self-renewal which cannot be predicted from the protein structure or sequence comparisons. In summary, the intention is to exploit the complementary expertise available at the University of Edinburgh to quickly build up a thorough knowledge of the relationship between sequence, structure and function for this relatively new protein.

Summary

unavailable

Committee	Closed Committee - Biochemistry & Cell Biology (BCB)
Research Topics	X – not assigned to a current Research Topic
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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