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. 2009 Oct 30;285(1):242–254. doi: 10.1074/jbc.M109.068668

FIGURE 6.

FIGURE 6.

Role of transcriptional repressor HES1 in up-regulation of MAP2 promoter activity by mBRAF. A, mBRAF increases MAP2 promoter activity in melanoma cells. Luciferase activity in cells co-transfected, in triplicate wells, with phMAP2.2 and vector control or mBRAF plasmid was measured, and representative data of three independent experiments are shown as the -fold increase (mean ± S.E.; *, p < 0.001) compared with the activity in empty vector-transfected cells. HeLa cells were used as a non-melanoma control. B, transfection with increasing amounts of mBRAF plasmid increases MAP2 promoter activity. Primary (WM35) and metastatic (451Lu) melanoma were co-transfected with phMAP2.2 promoter luciferase plasmid alone or with increasing amounts (25, 125, and 625 ng) of mBRAF plasmid in triplicate wells, luciferase activity was measured, and -fold increase in promoter activity (mean ± S.E.) in cells transfected with mBRAF over empty vector-transfected cells is shown. C, presence of HES1 binding box is sufficient for up-regulation of MAP2 promoter activity by mBRAF. Shown is a schematic diagram of various human genomic DNA fragments consisting of the MAP2 5′ regulatory region (phMAP2.1–2.4) showing E and N boxes and an N box mutant (ΔN2). Nucleotides are numbered using the transcription start site as +1. 451Lu melanoma cells were transfected with MAP2 promoter constructs and mBRAF expression plasmid, and luciferase activity was measured. Promoterless pGL3 and empty vector (pEFplink) were used as controls. Results are represented as relative luciferase units (RLU) (mean ± S.E.; *, p < 0.01). D, mutation of the HES1-binding N box abolishes BRAF-induced up-regulation of MAP2 promoter activity. The HES1-binding N2 box (GCCGCC) was mutated (mutated nucleotides are shown underlined) as described (12), and wild type and ΔN2 mutant promoter plasmids were co-transfected with mBRAF plasmid. Luciferase activity was measured, and results are shown as -fold change in activity as in A. *, p < 0.05. E, HES1 represses BRAF-induced MAP2 promoter up-regulation. 451Lu melanoma cells were co-transfected with MAP2.4 promoter plasmid, mBRAF expression plasmid, and increasing amounts (10, 20, 30, 40, and 50 ng) of mouse Hes1 expression plasmid pCI-Hes1. Forty-eight hours after transfection, luciferase activity was measured, and data (mean ± S.E.; *, p < 0.001) are represented as percentage change by normalizing with empty vector control.