Figure 2

Transcriptional activity of mutants mHNF1(V133M), mHNF1(T196A), mHNF1(E235E), mHNF1(R271W) and mHNF1(P378R). Cos7 (A-D) and Min6 (E-G) cells were cotransfected with 250 of the reporter constructs pGL3-GLUT2, β28-Luc, pGL3-HNF4AP2 or pGL3-RIP; 200 ng pCMVβ; and the indicated amounts of wild-type or mutant HNF1A expression vectors, which were defined to obtain a sufficiently strong response with the wild-type (at least a two-fold activation) but avoid saturation of the system. Cells were harvested 24 h after transfection and assayed for luciferase and β-galactosidase activities. Luciferase activity was normalized by the β-galactosidase activity of the internal transfection efficiency control pCMVβ. For a typical experiment, β-galactosidase activity was 1.8 ± 0.1 (mean ± SEM) optical density (OD)₄₂₀ units/[min • mg total protein]. Normalized luciferase values obtained from cells cotransfected with HNF1A expression vectors are shown as fold activation relative to the controls lacking any HNF1A expression vector, which were set as 1. (H) Cos7 cells were cotransfected with 200 ng pGL3-GLUT2, 200 ng pCMVβ and 100 ng wild-type pcDNA3-mHNF1A expression vector alone or together with 100 ng of each mutant expression vector, as indicated. Normalized luciferase values obtained from cells transfected with wild-type HNF-1α alone are set as 100%. All experiments were performed in duplicate and repeated at least 3× with different DNA preparations. The Student t test was used to compare the mean relative fold activation values between groups. Error bars indicate SEM. ^*P ≤ 0.05; ^**P ≤ 0.005. Note that human residue Pro379 corresponds to mouse Pro378.