The Forkhead Transcription Factor Foxi1 Is a Master Regulator of Vacuolar H+-ATPase Proton Pump Subunits in the Inner Ear, Kidney and Epididymis (original) (raw)
Figure 5
Foxi1 interact and activate ATP6V0A4 promoter reporter construct at putative forkhead binding sites Fk1-3.
(A) Core TTT-triplets of potential forkhead sites were mutated to GGG according to schematic picture. (B) Mutations reduce Foxi1-depedent reporter activation. While Fk3 mutations seem to abolish this activation the effects on Fk1-2 are intermediate and Fk4 seems to contribute very little to Foxi1-dependent reporter gene activation. (C) EMSA using in vitro transcribed/translated Foxi1 demonstrates interactions with [32P] labeled oligonucleotides harboring the −561/−547 (Fk1-3) and −358/−352 (Fk4) regions. The comparatively weak interaction demonstrated for Fk4 correlates well with the modest effects on reporter gene activation seen in Fig. 5 B. (D–F) EMSA showing the −561/−547 interaction is competed for with either wt or mutated oligonucleotides specific for the Fk1, 2 and 3 interactions. As can be deduced, there is a higher sensitivity in EMSA competition experiments using wt probes as compared with mutated probes – typical of a sequence specific significant interaction.