Endocrine Abstracts

GATA3 mutations cause the congenital autosomal dominant hypoparathyroidism, deafness and renal dysplasia (HDR) syndrome. GATA3 belongs to a family of dual zinc-finger nuclear transcription factors that recognise the consensus (A/T)GATA(A/G) motif and are involved in vertebrate embryonic development. To date, the mechanisms by which GATA3 mutations lead to haploinsufficiency of the GATA3 protein, which comprises 444 amino acids, have been shown to include loss of DNA binding, reduced affinity of DNA binding, and loss of interaction with co-factor Friend of GATA type 2 (FOG2). We hypothesized that, in addition to these mechanisms, interference with correct nuclear localisation of GATA3 by mutations in the critical amino acid residues of the nuclear localisation signal (NLS) may also lead to haploinsufficiency. The NLS of the 50 kDa GATA3 has not been previously defined. To identify these residues, enhanced green fluorescent protein-tagged truncation and point GATA3 mutants were generated, transiently expressed in COS-7 cells, and assessed for their sub-cellular localisation by fluorescence cell imaging. Western blot analyses of nuclear and cytoplasmic fractions from the transfected cells were also performed. The results showed that the NLS of GATA3 does not conform to either a classical mono- or bi-partite signal, but rather contains multiple cooperative NLS elements residing around the N-terminal zinc finger (ZnF1), where residues 249 to 311 were sufficient for nuclear targeting. Mutating all basic residues within ZnF1 resulted in mislocalisation of the mutant GATA3 protein. The lack of a single NLS site that completely accounts for nuclear targeting means that naturally occurring missense mutations in GATA3 protein are unlikely to disrupt the function of the protein by mislocalising the mutant protein to the cytoplasm. Furthermore, the basic residues within ZnF1 are highly conserved among GATA family members suggesting that these residues could be part of the NLS common to all GATA family members.