Figure 4. NFATc1 inhibits the MyoD N-terminal activation domain.

C3H10T1/2 cells were transfected with the indicated GAL4(DBD) fusion protein expression plasmids and the UAS reporter plasmid pG₅E1b-luciferase. Full length MyoD, NFATc1 (c1), NFATc3 (c3), E12-VP16 or parental expression vector (dashes) were also co-transfected as indicated. (A) MyoD dimerization with GAL4- E12(bHLH) caused potent activation (lane 6), which was significantly inhibited by NFATc1 (lane 7). (B) E12-VP16-GAL4-MyoD(bHLH) dimers strongly activated the pG₅E1b-luciferase reporter (lane 6) and this was not inhibited by NFATc1 (lane 7). (C) Fusion of full length MyoD or the N terminal fragment of MyoD [MyoD(N)] to the GAL4 DBD resulted in potent activation of the UAS reporter due to the strong activation domains of MyoD. Co-expression of NFATc1 significantly inhibited the MyoD activation domain (compare lanes 3, 4 and lanes 5, 6). Results are reported as the mean fold activation plus SEM from 4 independent transfections; n.s., not significant. Models depicting the mammalian two hybrid (A, B) and one-hybrid (C) assays are shown to the right of their respective graphs. See also Figure S4 and Figure S5.