MYO5A Gene Is a Target of MITF in Melanocytes Cleidson P. Alves, Satoru Yokoyama, Lucas Goedert, Carmen L.S. Pontes, Josane F. Sousa, David E. Fisher, Enilza M. Espreafico  Journal of Investigative Dermatology  Volume 137, Issue 4, Pages 985-989 (April 2017) DOI: 10.1016/j.jid.2016.11.026 Copyright © 2016 The Authors Terms and Conditions

Figure 1 Expression of myosin-Va is dependent on MITF. Expression levels of myosin-Va and MITF 3 days after transfection of siMITF or siControl in primary melanocytes assessed by western blot (a) and qRT-PCR (b). Western-blot image is representative of three independent experiments. *P < 0.05. (c) Primary melanocytes from three different donors were stimulated with α-MSH or forskolin after 18 hours of starvation. Total RNA was collected at the indicated points and submitted to qRT-PCR. (d) Three days after transfection with siControl or siMITF, primary melanocytes were treated with α-MSH. Total RNA was collected at the indicated points. For all qPCR experiments, β-actin was used as control for normalization. Values represent mean ± SD from three experiments performed in triplicates. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MITF, microphthalmia-associated transcription factor; α-MSH, α-melanocyte-stimulating hormone; qRT-PCR, quantitative real-time reverse transcriptase-PCR; SD, standard deviation; TRPM1, transient receptor potential cation channel subfamily M member 1. Journal of Investigative Dermatology 2017 137, 985-989DOI: (10.1016/j.jid.2016.11.026) Copyright © 2016 The Authors Terms and Conditions

Figure 2 MITF binds to an E-boxes-containing region within the intron-1 of MYO5A and activates MYO5A gene expression. (a) Genomic structure and the MITF target sequences in the intron-1 of MYO5A gene. Four E-boxes identified by us are present in the “R1” region. The bar below the E-boxes region represents the sequence identified by ChIP-Seq by Strub et al. (2011). Arrows indicate the map location of the pair of primers for the R1 region and arrowheads indicate the map location of the control primers (Ctrl), both pairs used to probe the chromatin immunoprecipitated samples. (b) Binding of MITF to the endogenous MYO5A gene region in forskolin 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-specific antibody or control antibody. PCR primers spanning either the MYO5A “R1” region (MYO5A (+)) or the control region (MYO5A (–)), as well as a known MITF target region upstream of the TYR gene (TYR), were employed. The data show promoter occupancy relative to control antibody and represent mean ± SD of three independent experiments. P < 0.05. (c) Diagram of the construct covering 1.2 kb of the promoter region of MYO5A gene and the “R1” region containing the MITF target sequences. (d) UACC62 and HEK293 cells were transiently transfected with the reporter plasmids, in addition to overexpression vector for MITF or empty vector. After 48 hours, firefly luciferase activity was measured and normalized to Renilla luciferase (transfection control). For the experiments with siRNA, siMITF or siControl were transfected 2 days before the transfections of the constructs. The data show fold of stimulation and represent mean ± SD of three (UACC62) or two (HEK293) experiments performed in triplicate. ATG, translation start codon; siRNA, small interfering RNA; TSS, transcription start site. Journal of Investigative Dermatology 2017 137, 985-989DOI: (10.1016/j.jid.2016.11.026) Copyright © 2016 The Authors Terms and Conditions