Investigating the role of protein-protein and protein-DNA interactions in the function of Isl1

LIM-homeodomain (LIM-HD) transcription factors act as key developmental regulators, through their ability to both bind DNA through homeodomain-DNA interactions, and to form larger complexes through protein-protein interactions. Many interactions that have been characterised are formed using their LIM domains, but likely also involve other regions, which have not yet been described for many LIM-HD proteins. The LIM-HD protein Isl1 has been implicated in the development of many tissues. However, relatively little detail is known about how Isl1 functions in these systems and the pathways in which it acts. The first part of this thesis aimed to identify and characterise novel binding partners for Isl1. An earlier project isolated ~180 potential binding partners through use of yeast two-hybrid mating screens; throughout this thesis further methodology was developed to identify additional proteins in a medium throughput manner. Validation protocols were then applied to determine which interactors were likely to represent biologically relevant interaction partners for Isl1. The second part of this thesis focussed on the mechanisms by which Isl1 and Lhx3 direct cell fate determination in the developing central nervous system. These proteins, along with Ldb1, interact via LIM:LID interactions to form cell-specific transcriptional complexes that target genes different to those targeted by either LIM-HD protein alone. It was not known if the homeodomains target these different sites solely because of the LIM:LID interactions or if the homeodomains themselves bind cooperatively to DNA. The DNA-binding behaviour of various iterations of the Lhx3/Isl1/Ldb1 complex are described, and structural characterisation of the Isl1/Lhx3 DNA-binding unit has been pursued. These data provide new insights into the mechanisms by which Isl1 and Lhx3 work together in regulating gene expression