KIT Is a Frequent Target for Epigenetic Silencing in Cutaneous Melanoma Christina Dahl, Cecilie Abildgaard, Rikke Riber-Hansen, Torben Steiniche, Johanne.

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KIT Is a Frequent Target for Epigenetic Silencing in Cutaneous Melanoma Christina Dahl, Cecilie Abildgaard, Rikke Riber-Hansen, Torben Steiniche, Johanne Lade-Keller, Per Guldberg Journal of Investigative Dermatology Volume 135, Issue 2, Pages 516-524 (February 2015) DOI: 10.1038/jid.2014.372 Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Genome-wide screen for aberrant promoter methylation in melanoma cell lines. (a) Plots from the Agilent DNA Analytics software showing the methylation patterns for the PPP1R3C and NID1 promoters in four melanoma cell lines and normal human melanocytes (HEMn). For each sample, Cy5-labeled methylation-enriched DNA and Cy3-labeled input genomic DNA were competitively hybridized to an Agilent Human Promoter Array. Each point represents a probe plotted at its genomic position (hg18 build; x axis) relative to its log2(Cy5/Cy3) (M-value; y axis). The gene is represented by horizontal bars. (b) Analysis of the methylation status of the PPP1R3C and NID1 promoters by MS-MCA. Bisulfite-treated DNA was amplified in the presence of SYBR Green I, and the melting characteristics of the PCR products were determined by continuous fluorescence monitoring during a temperature transition. Shown is the negative derivative of fluorescence over temperature (-dF/dT) versus temperature. Universal methylated DNA (IVM) and whole-genome amplified (WGA) DNA provided positive controls for methylated and unmethylated alleles, respectively. (c) Expression levels of DDIT4L, NID1, PPP1R3C, and RRAD in melanoma cell lines and normal melanocytes determined by qRT-PCR and normalized to PBGD expression. Expression in melanocytes was set at 1. The data represent the mean+SD of three measurements. MS-MCA, methylation-sensitive melting-curve analysis; qRT-PCR, quantitative RT-PCR. Journal of Investigative Dermatology 2015 135, 516-524DOI: (10.1038/jid.2014.372) Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Evaluation of the methylation status of the KIT promoter region in melanoma cell lines and normal melanocytes. (a) MeDIP analysis showing the pattern of CpG methylation at the KIT promoter. See legend to Figure 1a for details. (b) MS-MCA of KIT promoter methylation. (c) Plots from the PyroMark Q24 software showing methylation levels at individual CpG sites (shaded regions) in the KIT promoter determined by bisulfite pyrosequencing. The sequence at the bottom refers to the nucleotide dispensation, and the y axis represents the intensity of the fluorescent signal. (d) Methylation density at the KIT promoter in melanoma cell lines and HEMn. The data shown represent the results obtained for 34 CpG sites analyzed by bisulfite pyrosequencing. (e) KIT expression levels in melanoma cell lines and HEMn determined by qRT-PCR and normalized to PBGD expression. The data represent the mean+half-range of two measurements. (f) Re-expression of KIT induced by 5-aza-2′-deoxycytidine (5-aza-CdR) in melanoma cells carrying a hypermethylated KIT promoter. HEMn, human melanocytes; MeDIP, methylated DNA immunoprecipitation; MS-MCA, methylation-sensitive melting-curve analysis. Journal of Investigative Dermatology 2015 135, 516-524DOI: (10.1038/jid.2014.372) Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Evaluation of KIT promoter methylation in uncultured melanomas. (a) MS-MCA of the KIT promoter in the ESTDAB-023 melanoma cell line and corresponding uncultured tumor. Universal methylated DNA (IVM) and DNA from peripheral blood leukocytes (PBL) served as controls. (b) Bisulfite pyrosequencing showing methylation levels at the KIT promoter in uncultured melanoma metastases. (c) Immunohistochemical analysis of KIT expression in primary melanomas with (Epi56, left) or without (Epi96, right) KIT promoter hypermethylation. MS-MCA, methylation-sensitive melting-curve analysis. Scale bar=50 μm. Journal of Investigative Dermatology 2015 135, 516-524DOI: (10.1038/jid.2014.372) Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Epigenetic downregulation of KIT expression in melanoma cells in response to SCF. (a) FACS analysis showing the expression of KIT in ESTDAB-013. Cells were grown in the presence or absence of SCF (200 μM) for 3 weeks. (b) qRT-PCR analysis of KIT expression in ESTDAB-013 cells grown in the presence or absence of SCF. Expression levels were normalized to PBGD expression, and expression in untreated cells was set at 100%. The data represent the mean+SD of two independent experiments. (c) MS-MCA and (d) bisulfite pyrosequencing showing increased levels of methylation at the KIT promoter in ESTDAB-013 cells after 3 weeks in culture in the presence of SCF. SCF, stem cell factor. Journal of Investigative Dermatology 2015 135, 516-524DOI: (10.1038/jid.2014.372) Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions