Fig. 4.

MITF directly regulates BCL2A1 in the melanocytic lineage. Expression of antiapoptotic BCL-2 family members, MITF, and MITF-regulated targets in (A) the NCI-60 tumor panel and (B) an independent dataset of 954 cancer cell lines (GlaxoSmithKline). (C) The cAMP agonist forskolin (20 µM) induces MITF, TRPM1 (a known MITF target), and BCL2A1 mRNA. A representative of three independent experiments using different donors is shown. (D) Knockdown of MITF by siRNA suppresses BCL2A1 expression in melanoma cells and primary melanocytes of different donors. Indicated RNA was quantified 72 h after siRNA transfection. (E) Knockdown of MITF by two independent lentiviral-expressed shMITFs suppresses BCL2A1 in UACC-62 melanoma cells. (F) Genomic structure of BCL2A1 promoter with conserved E-box at −7 kb site and within 5′UTR (gray boxes), showing exons 1 and 2 and location of primers used for chromatin precipitation. Below, alignment of the BCL2A1 promoter among mammalian species at the −7 kb site, based on Feb 2009 Build. (G) Chromatin immunopreciptation of indicated genomic region with no antibody, anti-MITF, or rabbit IgG. Precipitated DNA was amplified using primers surrounding the −7 kb or 5′UTR E-boxes. Results are normalized to input DNA. ***P < 0.001 compared with rabbit IgG control; **P < 0.01. (H) BCL2A1 promoters were cloned upstream of the luciferase gene as indicated. UACC-62 cells were transfected with the indicated promoters and two distinct shRNA hairpins targeting MITF (#2 and #4). Forty-eight hours later, luciferase activity was determined. Results reported are averages of at least three independent experiments, performed in duplicate and normalized for transfection efficiency using Renilla luciferase. All data are normalized to the wild-type BCL2A1 promoter transfected with the control. ***P < 0.001 compared with control.