Figure 4.

HES6 binds the N box to repress transcription and does not antagonize HES1 in myoblasts. Transient transfection of C2C12 myoblasts with HES6, HES6-Δ, HES6-VP16, and HES1 expression vectors, alone or in combination. (a) Transcriptional repression by HES6 in C2C12 cells. The control template (pActinLUC) did not contain N boxes, whereas the test template (p6NactinLUC) contained six copies of a canonical N box sequence. Results are expressed as a percentage of expression relative to cells transfected with the reporter and empty vector alone. (b) The HES6–VP16 activation domain fusion protein binds N boxes and activates the N box–containing reporter. Joining the VP16 activation domain to HES6 transformed HES6 from a repressor into an activator, and HES6–VP16 activated transcription specifically from the N box–containing promoter, whereas the full-length VP16 protein had no effect. Results are expressed as fold induction relative to cells transfected with the reporter alone. Vector, empty expression vector backbone. (c) HES6 cooperates with HES1 to maximally repress N box–dependent transcription in muscle cells. The reporter construct was p6NactinLUC. Results are expressed as a percentage of expression relative to cells transfected with the reporter and empty vector alone.