MITF binds to the promoter of innate immune target genes and acts as a transcriptional repressor.

<p><b>(A, B)</b> Relative gene expression in melan-a cells 48 hours after transfection with siRNA. In <b>(A)</b>, cells were transfected with siRNAs targeting <i>Mitf</i> (<i>siMitf</i> and <i>siMitf-OM</i>) or an analogous scrambled control siRNA (<i>siMitf-scram</i>). In <b>(B)</b>, cells were transfected with one of two siRNAs targeting <i>Irf4</i> (<i>siIrf4_a</i>, <i>siIrf4_b</i>) or <i>siNC1</i>. The bars represent the mean ± standard deviation, and the asterisks indicate gene expression changes with a <i>q</i>-value of <0.05 using the two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli, with <i>Q</i> = 5%. Data presented in A and B are representative of two independent knockdown experiments performed in melan-a cells at two different cell passages. <b>(C)</b> Venn diagrams demonstrating the intersection of gene lists. The left diagram represents the genes exhibiting a 1.5-fold or greater difference in expression in <i>Mitf</i>^{<i>mi-vga9/+</i>} over wild-type McSCs (<i>Mitf</i>^{<i>mi-vga9/+</i>}DEGs, top circle) overlapped with the list of genes associated with MITF ChIP-seq peaks reported by Webster et al. 2014 (MITF ChIP-seq genes, bottom circle) [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2003648#pbio.2003648.ref041" target="_blank">41</a>] and are considered potential direct targets of MITF. These 108 direct targets are provided in <b><a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2003648#pbio.2003648.s004" target="_blank">S4 Data</a></b>. Direct target genes were further identified by comparing them with a list of known pigmentation genes in the upper right diagram and with the list of 55 innate immune-related genes up-regulated in <i>Mitf</i>^{<i>mi-vga9/+</i>} McSCs (as defined in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2003648#pbio.2003648.g003" target="_blank">Fig 3A</a>) in the lower right diagram. <b>(D)</b> MITF ChIP-qPCR performed in melan-a cells and assayed for enrichment at the indicated gene loci (<i>Dct</i>, <i>Ifih1</i>, <i>Ifit3</i>, <i>Stat1</i>). A centromeric DNA sequence was amplified as a negative control (neg con). Error bars indicate mean ± standard deviation of two independent ChIP pulldowns. The raw data used to generate the graphs in (A), (B), and (D) are available in <b><a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2003648#pbio.2003648.s001" target="_blank">S1 Data</a></b>. <i>Actb</i>, actin beta; ChIP, chromatin immunoprecipitation; ChIP-qPCR, chromatin immunoprecipitation quantitative polymerase chain reaction; ChIP-seq, chromatin immunoprecipitation sequencing; DEG, differentially expressed gene; IgG, immunoglobulin G; McSC, melanocyte stem cell; MITF, melanogenesis associated transcription factor; neg con, negative control; <i>siNC1</i>, nontargeting control siRNA; siRNA, small interfering RNA.