Endocrine Abstracts

Glial-cell missing b (GCMB) is a parathyroid-specific gene whose loss results in parathyroid agenesis while haploinsufficiency of GATA3, which belongs to a family (GATA1-6) of dual zinc-finger transcription factors causes the hypoparathyroidism-deafness-renal dysplasia (HDR) syndrome. We investigated the possibility that GATA3 regulates GCMB expression. Our bioinformatics analysis revealed 5 putative GATA3 binding sites in the 1.3 kb region upstream of exon 1 of GCMB and this included a double GATA motif at position −1180 to −1166 bp. These motifs were functionally assessed by electrophoretic mobility shift assays (EMSAs); luciferase reporter assays; and chromatin immunoprecipitation (ChIP) assays. EMSAs using nuclear extracts containing GATA3 protein in binding reactions with double stranded DNA oligonucleotides containing GATA elements of the GCMB promoter revealed specific binding by GATA3 to the double GATA motif. Luciferase reporter constructs of the GCMB region containing the 5 putative GATA motifs, and deletion constructs that resulted in a sequential loss of each motif were cloned into the pGL3 vector upstream of the firefly luciferase gene and transfected into COS7 cells with or without with a GATA3 expressing construct. The presence of GATA3 caused a 2 to 6 fold increase in the reporter constructs that contained the double GATA motif at position −1180 to −1166 bp. Engineered mutations of this double GATA motif abolished transactivation by GATA3, thereby demonstrating that this double GATA motif needs to be intact for transcriptional activity. To assess the in vivo utilization of these upstream GCMB-associated GATA motifs, parathyroid adenoma nuclear extracts were used in ChIP assays together with real-time PCR assays. This demonstrated GATA3 specific binding to the double GATA motif within the GCMB promoter. Thus, these results are the first to report that GCMB is a downstream target gene for GATA3 in a likely transcriptional cascade that regulates parathyroid gland development.