Fig. 5

FOXN1 genomic context, potential methylation substrate C20 and CpG methylation status on hTEC. Panel (a) shows the genomic localization of C20 region on FOXN1 gene and (b) (upper panel) presents the relative GC content, CpG island prediction, repetitive region (simple), histone modifications and transcriptional factor binding sites on C20 region. Histone modifications and transcriptional factor binding sites were evaluated by ENCODE consortium laboratories using chromatin immunoprecipitation followed by DNA sequencing [39]. The CpG island was predicted using MethPrimer web-based software [68] and the repetitive region was obtained in the RepeatMasker database [74]. Lower panel in (b) shows the C20 nucleotide sequence. Above is the normal genome sequence and below is the bisulfite converted sequence. In red is presented the CpG residues. The arrows (> and <) indicate the forward and reverse specific bisulfate primers used for sequencing. The CpG island is underlined in black and the yellow label marks the repetitive region. CpG methylation status on C20 region was obtained for FOXN1expressing human skin (c) and FOXN1 non-expressing human leukocytes (d) from the ENCODE database on DNA Methylation by Reduced Representation Bisulfite Sequencing data provided by ENCODE/HudsonAlpha laboratory. FOXN1 gene graphic representation and DNA elements regulation were adapted from UCSC genome browser from the human genome assembly hg19 using “ENCODE regulation” track features [39, 67]. The graph in (e) represents the percentage of CpG methylation, for each residue, and in hTEC cell line clone sequences, while the diagrams in (f) show the 13 CpG-s presented as lollipop diagrams, where the methylated residue is shown in black circles, while the non-methylated residue is in open circles. Percent values were obtained after multiple sequence alignment using ClustalW algorithm followed for methylated CpG quantification using BiQ analyzer software