Fig. 5

Fig. 5

MITF and TFAP2A Affect IRF4 Expression by Binding Regulatory Elements in Intron 4

(A) Schematic view presents the IRF4 gene. The sequence shows a comparison of MITF- and TFAP2A-binding sites in intron 4 of IRF4 among humans (H sap [Homo sapiens]), gorilla (G gor [Gorilla gorilla]), chimpanzee (P tro [Pan troglodytes]), and mouse (M mus [Mus musculus]). The location of the rs12203592 polymorphism is indicated.

(B) ChIP analysis of a GST-tagged MITF protein (a-GST AB) was performed in 501mel cells. Primers specific for HSPA1B (negative control), TYR (positive control), and intron 4 of IRF4 are indicated. The GST-tagged Mitf only precipitated TYR and IRF4 sequences.

(C) ChIP analysis of TFAP2A in 501mel cells is presented. PCR products specific for TGFA (positive control), HSPA1B (negative control), and intron 4 of IRF4 are shown. TFAP2A only precipitated TGFA and INT4 sequences.

(D) Gel shift analysis shows the binding of TFAP2A to the ancestral sequence (INT4-WT) in intron 4 of IRF4 (coordinates chr6: 396309–396333 in build GRCh37.p5 of the human genome), but not to the rs12203592-T sequence (INT4-RS) or to a completely mutated sequence (INT4-mut).

(E) Luciferase reporter assays were performed in 501mel melanoma cells using intron 4 of IRF4 as a reporter. The ancestral IRF4 intron 4 sequence (intron 4, blue), the rs12203592-T polymorphic sequence (intron 4-rs), a sequence where all MITF-binding sites were mutated (intron 4-3xE), and a sequence containing mutated MITF sites and the rs12203592-T polymorphism (intron 4-3xE+rs) were tested. The luciferase reporters were cotransfected with the wild-type MITF and TFAP2A proteins and with a dominant-negative version of MITF (MITF^mi). Statistical analysis was done using unpaired t test. Data are represented as mean ± SEM.

(F) Cells homozygous for the ancestral allele (CC) express significantly higher levels of IRF4 than cells homozygous for the rs12203592-T polymorphism (TT). Fold expression data are represented relative to pooled mean TT expression ± SEM. Statistical analysis was performed using ANOVA (p < 0.001).

(G) A western blot shows expression of the IRF4 protein in melanocytes from CC and TT individuals. Intensity quantification is relative to GAPDH-loading control. Note the decrease of basal IRF4 protein in the TT melanocyte cell lines.

See also Figures S3, S4, and S5.