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. 2014 May 5;9(5):e95151. doi: 10.1371/journal.pone.0095151

Figure 4. Human Mutations in Mouse Tbx1.

Figure 4

A: The position of the three known mutations in TBX1 in human patients are shown with respect to its domain structure [19]–[21]. The three mutations lie within the region that was cloned to generate the GST-TBX1 fusion protein. B: After mutagenesis, the wild type (WT) and mutant proteins were induced, purified and used for EMSAs. The protein-DNA complexes are shown before and after IPTG induction. C, D: EMSAs of the WT and three mutated proteins using the radiolabeled TR (C) and ½SPS motifs (D). E: Luciferase assays were performed after co-transfecting the reporter construct (6x TR or 6x ½ SPS) with WT or each mutated full length Tbx1-pCDNA3.1 construct. The F148Y mutation led to a decrease in activation (TR- 17 fold decrease, ♦p<0.0005; ½SPS- 4.5 fold decrease, ♦p<0.0003) when compared to the WT transfection. The H194Q mutation did not lead to a statistically significant change in activation but the trend was in the direction of decreased activity (not significant- n.s). The G310S mutation led to a smaller but still significant decrease in activation (TR- 2.2 fold decrease, ♦p<0.01; ½SPS- 1.3 fold decrease, ♦p<0.05). Equal amounts of WT and mutated Tbx1 was co-transfected with the respective reporter constructs to determine if there was suppression of the mutant phenotype. There was a slight increase in activation when compared to the mutated F148Y alone transfection (TR-4 fold, •p<0.006; ½SPS- 1.9 fold, •p<0.006). Under these new conditions, the F148Y mutated TBX1 with WT protein still showed reduced activation when compared to WT TBX1 (TR- *p<0.0003; ½SPS- *p<0.006). The H194Q+WT combination did not show any significant change (TR- p<0.4; ½ SPS- p<0.1). The G310S+WT combination showed a significant increase in activation (TR- 2 fold, •p<0.001; ½SPS 1.5 fold, •p<0.02) when compared to G310S mutant alone. All data are presented as means ±SD; n≥3. p-values were determined using the Student's t-test. F: Immunoflourescence experiments were performed with antibodies to TBX1 on transfected Jeg3 cells to valdiate that the mutated constructs were localized to the nucleus (green). Nuclear localization was confirmed by observing expression in DAPI stained nuclei shown in blue.