Endocrine Abstracts

GATA3 is a member of the family of zinc finger transcription factors that recognise (A/T)GATA(A/G) motifs in DNA, and mutations that lead to haploinsufficiency cause the hypoparathyroidism, deafness and renal dysplasia syndrome (HDR). GATA3 has two zinc fingers, of which the carboxy-terminal finger (ZnF2) is essential for DNA binding, whereas the amino-terminal finger (ZnF1) stabilizes this binding and physically binds with other multi-type zinc finger proteins, which are referred to as Friends of GATA (FOG). GATA3-ZnF1 has been shown to interact with FOG2 but the residues of GATA3-ZnF1 that are involved remain to be elucidated. We have further investigated this by engineering mutations, using site-directed mutagenesis, in 7 of the 27 amino acid residues that constitute the GATA3-ZnF1. These GATA-ZnF1 mutants (Cys264Arg, Glu263Val, GlyAla268/269GlnThr, Pro273Thr, Arg276Gln, Asp278Gly, Asp278Tyr) were then further assessed by electrophoretic mobility shift assays (EMSAs), and by yeast two-hybrid analysis to study protein-protein interactions. EMSAs revealed normal DNA binding by all these mutants. However, interactions between the GATA3-ZnF1 mutants Glu263Val and GlyAla268/269GlnThr and the 4 of 8 studied FOG2 zinc fingers was abolished, whilst it was preserved in the GATA3-ZnF1 mutants Pro273Thr and Arg276Gln; and the GATA3-ZnF1 Asp278Tyr and Asp278Gly mutants interacted with FOG2 ZnFs 1, 5 and 6 but not 8. These results are consistent with the findings from the proposed 3-dimensional model that has been reported for GATA1-ZnF1. Thus, in this model, the GATA3 Glu263 and GlyAla268/269 residues would reside on the FOG2 protein interaction face, with Asp278 in close proximity; however, Pro273 and Arg276 residues would reside on the DNA binding interface. Thus, our results have identified the role of GATA3-ZnF1 residues in the interactions with FOG2, and this will help to elucidate further the roles of these transcription factors in regulating the development of the parathyroids, otic vesicles and kidneys.