Fig. 3.

EVI1 does not mediate ATRA regulation of the MDS1/EVI1 promoter, but decreases the ATRA response of its own RARE and enhances that of the RARβ RARE. (A) Four genomic fragments, whose positions are indicated relative to the transcriptional start site of the MDS1/EVI1 gene (assumed to be identical to that of the MDS1 gene; GenBank accession no. NM_004991), were cloned into the luciferase reporter vector pGL3basic. NTERA-2 cells were cotransfected with these reporter constructs, HA-EVI1/pEFzeo or empty vector, and the renilla luciferase vector pRL-SV40. EVI1(+86/+1106)/pGL3 served as a positive control for the ATRA response; pEREluc, which contains two copies of an EVI1 DNA binding site, as a positive control for the effects of EVI1; and empty pGL3basic as a negative control. ATRA or dimethylsulfoxide was added 1 day after transfection, and cells were lysed and luciferase activities determined another ∼ 24 h later. Relative luciferase units (RLU) were derived by normalizing firefly to renilla luciferase activities. Error bars represent the standard deviations between duplicate measurements. (B, C) Luciferase assays after transfection of NTERA-2 cells with the indicated reporter plasmids and the expression vectors HA-EVI1/pEFzeo or HA-MDS1/EVI1/pEFzeo were performed as described in (A). pRARE-tk-luc contains two copies of the RARE of the human RARβ gene promoter and the tk minimal promoter in pGL2; pGL2-tk-luc contains only the tk minimal promoter in pGL2.