Figure 6.

Transcriptional activities of WT and variant GCM2 proteins with a synthetic (3X-gbs) and natural (PTH) GCM2 responsive promoter. (A) A synthetic promoter–reporter construct containing three GCM response elements in tandem upstream of a luciferase reporter gene (3xgbs-lux) was transfected into HEK293 cells with vector, WT or variants (V382M, I383M, T386S, Y384S or Y282D). After 48 h, cells were harvested and luciferase activity of extracts measured. Values are mean ± s.e.m. of six estimations. (B) Top panel: representation of the human PTH promoter. Two putative GCM binding sites (−1002/−995) (site A) and (−390/−383) (site B) have been identified (41). Lower panel: the PTH promoter was transfected into HEK293 cells with vector, WT or variants (V382M, I383M, T386S, Y384S or Y282D). After 48 h, cells were harvested and luciferase activity of extracts measured. Values are mean ± s.e.m. of six estimations. (C) All activating variants bind DNA equally. Biotinylated oligonucleotide pull-down assays were conducted on extracts of HEK293 cells that had been transfected 48 h before harvesting with either empty vector alone or Flag-GCM2 WT or variants. The oligonucleotides used represented the WT and variant GCM consensus elements.