A direct link between MITF, innate immunity, and hair graying
(A, B) Relative gene expression in melan-a cells 48 hours after transfection with siRNA. In (A), cells were transfected with siRNAs targeting Mitf (siMitf and siMitf-OM) or an analogous scrambled control siRNA (siMitf-scram). In (B), cells were transfected with one of two siRNAs targeting Irf4 (siIrf4_a, siIrf4_b) or siNC1. The bars represent the mean ± standard deviation, and the asterisks indicate gene expression changes with a q-value of <0.05 using the two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli, with Q = 5%. Data presented in A and B are representative of two independent knockdown experiments performed in melan-a cells at two different cell passages. (C) 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 Mitfmi-vga9/+ over wild-type McSCs (Mitfmi-vga9/+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)  and are considered potential direct targets of MITF. These 108 direct targets are provided in S4 Data. 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 Mitfmi-vga9/+ McSCs (as defined in Fig 3A) in the lower right diagram. (D) MITF ChIP-qPCR performed in melan-a cells and assayed for enrichment at the indicated gene loci (Dct, Ifih1, Ifit3, Stat1). 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 S1 Data. Actb, 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; siNC1, nontargeting control siRNA; siRNA, small interfering RNA.