Mechanistic differences in DNA binding behaviour of Isl1/Lhx3 homeodomains in transcriptional complexes

Smith NC, Stokes PH and Matthews JM

University of Sydney, Sydney, NSW, Australia.

LIM-homeodomain (LIM-HD) transcription factors act as key developmental regulators, both through their ability to bind DNA through homeodomain:DNA interactions, and through their ability to form higher order complexes through protein:protein interactions. The LIM-HD proteins Islet-1 (Isl1) and LIM homeobox protein 3 (Lhx3) have been implicated in the development of a broad range of tissues, most comprehensively in cell fate determination in the developing central nervous system. These proteins, along with the protein cofactor LIM-domain binding protein 1 (Ldb1) interact to form cell-specific transcriptional complexes. The presence or absence of Isl1 in complex with Lhx3 and Ldb1 is sufficient to redirect the complex to target different genes. This then directs the differentiaton of neural precursors into motor neurons or interneurons. Recapitulating this system in vitro, we have shown that Isl1 and Lhx3 bind differently in isolation than they do in combination, displaying a change in specificity that is not explained by simple cooperative binding of the homeodomains. Through the use of comparative electrophoretic mobility shift assays (EMSAs), multi angle laser light scattering (MALLS) and small angle X-ray scattering (SAXS) experiments, we we have studied the binding behaviours of these homeodomains in different combinations with each other and with or without additional protein:protein interaction domains. Through this, we have gained new understanding of the mechanisms by which these complexes can discern targets in the cell. Our results suggest that while the stoichiometry of binding is as expected, only one homeodomain is responsible for the affinity of binding to different targets, while the other appears to be important in stabilising binding to specific target sequences. Taken together, these data suggest a novel molecular mechanism for the combinatorial action of these transcription factors that act to influence cell fate during development.