Figure 4. DICER is an MITF target gene.
(A) Primary human melanocytes were transfected with siMITF or scrambled siRNA. At 48 hrs post-transfection, cells were serum-depleted for 12 hours, followed by forsk stimulation. Total RNA from stimulated melanocytes was used for qRT-PCR analysis. Graphs present MITF (upper) or DICER (lower) mRNA levels upon stimulation, in either siMITF-transfected cells (dash bars) or cells transfected with control siRNA (full bars). Results are normalized to Actin and are relative to non stimulated (−) values. The data show fold of induction relative to siCont and represent mean ±SD of 5 replicates.
(B) Primary human melanocytes were infected with either MITF wild-type (wt), MITF dominant-negative (dn) or empty vector adenovirus (MOI:200). 72 hours post infection, cells were serum-depleted for 12 hours, stimulated with forsk for the indicated time, and harvested for immunoblot analysis. Tubulin was used as loading control.
(C) Diagram of the human DICER gene promoter region showing two putative MITF binding sites (filled gray boxes) at positions 2323 and 928 nucleotides upstream of the transcription start site.
(D) Binding of MITF to the endogenous DICER promoter region in forsk activated and resting (−) primary human melanocytes. Chromatin immunoprecipitations were performed using log-phase primary human melanocytes. Protein:chromatin-crosslinked complexes were immunoprecipitated with either MITF antibody or control antibody. PCR primers spanning either DICER promoter (“pro”) or downstream (“down”) regions were employed. Tyrosinase promoter (“Tyr pro”) primers were used as a positive control. The data show promoter occupancy relative to control Ab and represent mean ±SD of 3 independent experiments.
(E) UACC62 melanoma cells were transiently transfected with DICER promoter reporter plasmids, in addition to siMITF, control siRNA, over expression vector for MITF, or empty vector. The same set of experiments was performed on DICER promoter wt, mutated E-boxes or empty reporter plasmids as indicated. After 48h, firefly luciferase was measured and normalized to renilla luciferase (transfection control). The data show fold of stimulation and represent mean ±SD of three experiments performed in triplicate.