Supervisors: Atlanta Cook, Philipp Voigt

Project Description:

Limb development is a highly orchestrated set of events in animal development requiring the coordinated action of DNA binding proteins to control transcription. This ensures that body plans are correctly executed.  Promyelocytic leukaemia zinc finger protein (PLZF) is a mammalian protein expressed in embryonic stem cells that is implicated in both limb development and fertility. A human patient with a PLZF point mutation, which is likely to interfere with DNA binding, showed limb defects, mental retardation and genital hypoplasia. Furthermore, PLZF knockout mice have altered body plans. Ectopic expression of PLZF fusion proteins in adults is associated with a specific form of leukaemia.

PLZF has an N-terminal dimerisation domain and five zinc finger domains that target it to DNA. A separation-of-function mutation in the dimerisation domain of PLZF has been described in mice, which leads to seven hind-limb toes but no fertility defects. We hypothesise that this mutation disrupts protein-protein interactions between PLZF and an unknown protein partner. This gives us an opportunity to understand interactions required for limb development that are independent of fertility. Using co-precipitation and quantitative mass spectrometry methods we will compare interactomes of wild-type and mutant PLZF to find binding partners that are specifically affected by this mutation.

Based on the mass spectrometry results as well as candidate binding partners from the literature, purified PLZF will be reconstituted with its binding partners. These protein complexes will be characterised using structural and biophysical methods.  This will give an insight into the players required for hind limb development and their protein-protein interactions and provide a foundation for future functional studies in mammalian development.

References:

Hobbs et al (2012) Functional Antagonism between Sall4 and Plzf Defines Germline Progenitors, Cell Stem Cell 10:284 DOI 10.1016/j.stem.2012.02.004

Ching et al (2010) An allele separating skeletal patterning and spermatogonial renewal functions of PLZF, BMC Dev. Biol. 10:33

Ahmad et al (1998) Crystal structure of the BTB domain from PLZF. PNAS 95:12123

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