FIG. 1.

Schematic of the genetic constructs used in these experiments. Consecutive flag-tag sequences were cloned upstream of the murine Atoh1 gene, and this construct was placed under control of a cytomegalovirus promoter (cmv.Atoh1) to serve as a positive control. The test construct consisted of this same flag-tagged Atoh1 construct ligated to a mutated ER LBD, which had been mutated to selectively bind 4-hydoxytamoxifen rather than endogenous estrogen, and a DsRed sequence to be used as a florescent marker to detect expression of the fusion protein. The three domains were separated by two linker sequences designed to code for triplet proline sequences in order to provide spatial separation and to reduce steric hindrance between moieties of the fusion protein. A negative control construct consisted of the DsRed sequence ligated to the ER LBD separated by one of the linker sequences described above. ATOH1, helix-loop-helix transcription factor atonal-1; CMV, cytomegalovirus; ER LBD, estrogen receptor ligand binding domain.