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. 2007 Apr 16;27(11):3911–3919. doi: 10.1128/MCB.01455-06

FIG. 4.

FIG. 4.

Mitf regulates MLC-1a expression through binding to E-box elements in MLC-1a promoter. (A) Four E boxes have been described in the proximal promoter region. E2 and E3 were included in one PCR fragment (E23). The sequence and the position of each of the four E boxes are depicted below. (B) EMSA results for MLC-1a promoter fragments are shown. E23 was used as a radiolabeled probe. fp, free probe; wt, wild-type Mitf; ce, ce/ce Mitf. E23, E1, and E4 were used as cold competitors with labeled E23. One representative experiment out of four is shown. (C) Binding of nuclear extracts of NIH 3T3 cells either overexpressing Mitf-H or transfected with an empty vector (empty). Supershifting of H9C2 nuclear extracts with either polyclonal anti-Mitf antibody directed against the common C terminus (anti Mi), preimmune serum (Pre), or without any sera (No AB) was performed. An arrow indicates the supershifted band. (D) E boxes E2 and E3 in the E23 element were point mutated to gTGtTG and gAGtTG, respectively, and were used for cold competition. Either wild-type E23 oligonucleotide (wt) or E23-mutated cold oligonucleotides (depicted as mut E2, mut E3, and mut E23), as indicated above each lane, competed with the normal E23 probe. (E) Chromatin immunoprecipitation assay in cardiomyocytes. Chromatin was immunoprecipitated with an anti-Mitf antibody directed against the C terminus of Mitf (anti Mi) or preimmune rabbit IgG (Pre) and then PCR amplified using primers for the MLC-1a promoter. (F) Transient transfection of NIH 3T3 cells with the MLC-1a promoter construct. Wild-type Mitf-H or ce/ce mutant plasmid constructs were cotransfected with the MLC-1a promoter reporter construct. The luciferase activity was normalized to that of total protein and divided by the value obtained for the wild type. The results shown represent the mean ± standard error (n = 4). (G) MLC-1a mutations in the E23 element. Either the wild-type MLC-1a promoter (wt) or MLC-1a E23 mutants (mut E2, mut E3, and mut E23) were cotransfected with Mitf expression vector. Luciferase activity was normalized as described above and divided by the value obtained for the E23 mutant promoter. A schematic representation of the different mutations is depicted below. The results shown represent the mean ± standard error (n = 3).