Notch1 Pathway Activation Results from the Epigenetic Abrogation of Notch-Related MicroRNAs in Mycosis Fungoides  Fernando Gallardo, Juan Sandoval, Angel Díaz-Lagares, Ricard Garcia, Teresa D'Altri, Jessica González, Victor Alegre, Octavio Servitje, Ana-Belén Crujeiras, Ólafur-Andri Stefánsson, Blanca Espinet, Maria-Inmaculada Hernández, Beatriz Bellosillo, Manel Esteller, Ramon-Maria Pujol, Anna Bigas, Lluis Espinosa  Journal of Investigative Dermatology  Volume 135, Issue 12, Pages 3144-3152 (December 2015) DOI: 10.1038/jid.2015.328 Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Notch1 and Notch ligand gene expression analyses. (a) Double immunofluorescence expression showing nuclear localization of Notch1 detected with anti-cleaved Notch1 (α-N1Icv, Cell Signaling at 1:200 in green) and anti-CD3 (Novocastra NCL-L-CD3-PS1, 1:400 in red) on mycosis funogides tumor stage (MFt) samples. Nuclear control with 4',6-diamidino-2-phenylindole (DAPI) in blue. (b) T-cell population showing positive immunofluorescence for the Notch ligand Jagged1. (i/ii) Insets representing in detail representative immunofluorescence results. (c) Upregulated expression levels of Jagged1 and Hes1 in MFt patients compared with inflammatory dermatoses (IDs) are represented. Values were determined in triplicates by quantitative reverse-transcription PCR and are expressed as mean±SEM. *Statistical significance with P<0.05 using a nonparametric Mann–Whitney test. Journal of Investigative Dermatology 2015 135, 3144-3152DOI: (10.1038/jid.2015.328) Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Hierarchical clustering analysis and DNA methylation profiles of mycosis funogides tumor stage (MFt) patients and inflammatory dermatoses (IDs) donors using Notch1-related microRNAs (miRNAs). (a) Unsupervised hierarchical clustering and heatmap associated with the methylation profile (according to the color scale shown) of the sample specimens based on the β-values of the 221 microRNA CpGs. Two different types of samples are represented: inflammatory disease (IDs) and mycosis funogides tumor stage (MFt). (b) Supervised analysis differentiating MFt from IDs. Each column represents an individual patient and each row an individual CpG. Journal of Investigative Dermatology 2015 135, 3144-3152DOI: (10.1038/jid.2015.328) Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Representation of specific promoter differentially methylated microRNAs. Schematic overview of three selected differentially methylated miRNAs comparing CpGs DNA methylation levels between mycosis fungoides tumor stage (MFt) patients (violet) and inflammatory dermatosis donors (light blue) throughout the promoter region. miR-200c and miR-124-2 show gain of methylation in MFt patients, whereas miR-495 shows loss of methylation. TSS corresponds to transcription start site. *CpGs that show statistical significance using a nonparametric Mann–Whitney U-test. Journal of Investigative Dermatology 2015 135, 3144-3152DOI: (10.1038/jid.2015.328) Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 DNA methylation–associated silencing of miR-200c in cutaneous T-cell lymphoma (CTCL) cell lines. (a) DNA methylation levels of miR-200c promoter determined by pyrosequencing. Data represent the mean of the methylation level of two consecutive promoter CpGs. (b) Restored expression of DNA methylated miR-200c in CTCL cell lines after treatment with the DNA demethylating agent 5-aza-2′-deoxycytidine (AZA). Values were determined in triplicates by quantitative reverse-transcription PCR and are expressed as mean±SEM (n=3). *Statistical significance (P<0.05) with respect to untreated cells (control) using a nonparametric Mann–Whitney test. Journal of Investigative Dermatology 2015 135, 3144-3152DOI: (10.1038/jid.2015.328) Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Epigenetic abrogation of miR-200c drives Jagged1 overexpression and Notch1 pathway activation in mycosis funogides tumor stage (MFt). (a) Comparative pattern of expression using relative fold change values between Jagged1 and miR-200c. Gene and microRNA (miRNA) expression values in each of the 11 MFt patients were normalized using the average value of controls (inflammatory dermatosis donors). Relative fold change was calculated by means of the ratio between Jagged1 and miR-200c. Discontinuous horizontal line with the Jagged1/miR-200c ratio value=1 corresponds to controls. (b) Western blot analysis with the indicated antibodies from different cell lines infected with the control or the lentiviral vector encoding for miR-200c. It is noteworthy that the presence of the full-length Jagged1 at 150kDa and several additional bands corresponding to functional Jagged1 fragments that are detected with two different antibodies and are downregulated by miR-200c, coinciding with a reduction in the levels of active Notch1 (ICN1). In contrast P-STAT3 levels remained constant. Journal of Investigative Dermatology 2015 135, 3144-3152DOI: (10.1038/jid.2015.328) Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions