Figure 4.

The BRAF/BRN2 mediated induction of MITF expression requires PAX3. (a) Sequence of the M‐MITF promoter (−53/−23) with BRN2 (−36/−51) and PAX3 (−25/−39) binding sites with respective mutations. (b) In vitro DNA binding. Cell extracts from Hela cells overexpressing PAX3 or BRN2 were incubated with the −77 to −20 region of the MITF promoter and bound proteins were analysed by Western blotting. (c) Luciferase assay for the WT or mutated M‐MITF promoter (−333/+120) in A375 cells transfected with ^V600EBRAF, PAX3, or BRN2 expression plasmids. (d) Luciferase assay for the M‐MITF promoter (−333/+120) in A375 cells transfected with control, PAX3 or BRN2 siRNA and plasmids expressing either BRN2 or PAX3. (e) ChIP qRT‐PCR for BRN2 in WM164 cells transfected with control or PAX3 siRNA. (f) ChIP qRT‐PCR for PAX3 in WM164 cells transfected with control or BRN2 siRNA. (g) Western blot of GST pull down of endogenous BRN2. Extracts from A375 cells were incubated with immobilized GST or GSTPAX3, expression of which was analysed by Coomassie staining. (h) Co‐immunoprecipitation of PAX3 with BRN2 from WM164 cells or from ectopically PAX3 overexpressing WM266‐4 cells. (i) Proposed mechanism of regulation of MITF by BRAF. Data presented as the mean± SEM are from at least three biological repeats