Fig. 1: Synthetic ZFP189 transcription factors exert opposite transcriptional control at a luciferase target gene in vitro.

A A cartoon representation of the three synthetic ZFP189 transcription factor (TF) proteins. B A cartoon of the ZFP189 DNA response element (RE) luciferase plasmid reporter target gene. TK is thymidine kinase gene promoter. RenSP is the LightSwitch™ luciferase gene. C Transient transfection of the ZFP189 RE luciferase plasmid and individual ZFP189 TFs in N2a cells reveals opposing transcriptional regulation of the luciferase target gene. Specifically, ZFP189^WT down-regulates and ZFP189^VPR up-regulates luciferase-driven relative light units (RLUs) relative to control conditions. One-way ANOVA followed by Bonferroni’s multiple comparisons test relative to mock transfection condition. *p-value < 0.05, ****p-value < 0.0001; n = 3 per condition. D Co-transfecting synthetic ZFP189 TFs of opposing function compete for regulation of the luciferase target gene. ZFP189^NFD, and to a greater degree ZFP189^WT, inhibit ZFP189^VPR-mediated gene activation. One-way ANOVA followed by Bonferroni’s multiple comparisons test. *p-value < 0.05, ****p-value < 0.0001; n = 9 per condition. E Removing ZFP189 REs from the promoter of our luciferase target gene (∆ RE motifs) ameliorates the gene activation of ZFP189^VPR. Data normalized to ZFP189 RE condition, by experiment. Two-tailed, unpaired Student’s t-test; ****p-value < 0.0001. n = 12 per condition. F Removing ZFP189 REs from the promoter of our luciferase target gene releases the gene repression of ZFP189^WT. Data normalized to ZFP189 RE condition, by experiment. Two-tailed, unpaired Student’s t-test; ****p-value < 0.0001. n = 9 per condition. G Removing ZFP189 REs from the promoter of our luciferase target gene does not impact the functions of ZFP189^NFD. Data normalized to ZFP189 RE condition, by experiment. Two-tailed, unpaired Student’s t-test; p-value > 0.05. n = 12 per condition.