Figure 6. Mixl1 functions as a sequence-specific transcriptional activator in differentiating i-Mix ES cells.

(A) General experimental protocol for transcription assays using i-Mixl1 ES cells. Undifferentiated i-Mixl1 ES cell monolayer cultures were transiently transfected with reporter plasmids in the LIF-containing medium at day 0. At 6 hr (day 0.25) post transfection, the LIF-containing medium in half of the cultures was replaced by LIF-free medium and the cells were cultured in the absence (−) or presence (+) of DOX (0.4 μg/ml). Cell lysates were prepared 48 hr post transfection for luciferase assays. (B) i-Mixl1 ES cells were transfected with the reporter plasmid (pGL2pro or pGL2pro-6xMBS) and then cultured in the LIF-containing or LIF-free medium in the presence or absence of DOX. Induction of Mixl1 (+DOX) resulted in the activation of the reporter luciferase gene linked to the multimerized MBS (pGLpro-6xMBS) in the presence or absence of LIF. (C) The reporter plasmids for analyzing the role of the TAAT/ATTA half sites in Mixl1-mediated transcriptional activation. Each plasmid except for pGL2pro contained six copies of the MBS or mutant MBS (MBS M1, MBS M2, or MBS M3). The TAAT/ATTA half sites are marked by inverted arrows and mutations are underlined. (D) i-Mixl1 ES cells were transfected with the individual reporter plasmids and then cultured in LIF-free medium in the presence or absence of DOX. Induction of Mixl1 (+DOX) resulted in the activation of the reporter luciferase gene linked to the multimerized MBS (pGL2pro-6xMBS) but not any of the mutant MBS (pGL2pro-6xMBS M1, -6xMBS M2, and -6xMBS M3).