Epigenetic Control of the S100A6 (Calcyclin) Gene Expression Wiesława Leśniak, Łukasz P. Słomnicki, Jacek Kuźnicki  Journal of Investigative Dermatology  Volume 127, Issue 10, Pages 2307-2314 (October 2007) DOI: 10.1038/sj.jid.5700879 Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 The effect of 5-azacytidine and TSA on S100A6 and S100A8 mRNA level in C6 rat glioma cells. S100A6 (a, b), S100A8 (c). (a) Northern blot; (b) methylene blue staining of 28S RNA; (c) RT-PCR. RNA from untreated control cells (1), from cells treated with 10μm 5-azacytidine for 12 days (2) or with 0.5μg/ml TSA for 24hours (3). Journal of Investigative Dermatology 2007 127, 2307-2314DOI: (10.1038/sj.jid.5700879) Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Schematic representation of CpG dinucleotide distribution in the S100A6 gene and its 5′- and 3′-regions. The exons are represented by gray and introns by white rectangles, respectively. Base numbering is acc. to the UCSC Genome Browser (www.genome.ucsc.edu). The transcription start site is given acc. to Ferrari et al., 1987, and corresponds to base no. 151775092. The regions for which CpG methylation analysis has been performed are indicated below and correspond to the promoter/first exon, second exon/second intron and the 3′-proximal and distal regions, respectively. The CpG island is indicated by a thick black horizontal line. Journal of Investigative Dermatology 2007 127, 2307-2314DOI: (10.1038/sj.jid.5700879) Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Cytosine methylation analysis of the S100A6 gene and its 5′- and 3′-regions. (a) RT-PCR showing S100A6 mRNA level in the examined cell lines. (b) Schematic representation of cytosine residue methylation in the examined regions. Each bar represents one CpG pair: empty bar, cytosine residue unmethylated in all clones examined; filled bar, cytosine residue methylated in all clones examined; partially filled bar, cytosine residue methylated in some of the clones; the extent of filling is proportional to the amount of methylated residues. The number of clones analyzed is given below. *Sequenced as a PCR fragment; nd, not determined. Base numbering is acc. to the UCSC Genome Browser (www.genome.ucsc.edu); only the last four digits are shown for the analyzed fragments. 1, Hep-2 cells; 2, human lymphocytes; 3, HEK293 cells. Journal of Investigative Dermatology 2007 127, 2307-2314DOI: (10.1038/sj.jid.5700879) Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 ChIP assay. (a) A representative experiment showing results of PCR performed on DNA samples precipitated with antibodies against modified histone H3: Ac H3 – anti-acetyl-histone H3, Met Lys9 H3 – anti-trimethyl-histone H3 (Lys 9), Met Lys27 H3 – anti-trimethyl-histone H3 (Lys 27). (b) Quantitative analysis of ChIP experiments. Filled and empty bars represent the results obtained in Hep-2 and HEK 293 cells, respectively, for the S100A6 gene promoter. The result of ChIP performed with anti-acetyl-histone H3 antibody for the glyceraldehyde-3-phosphate dehydrogenase gene promoter is shown for comparison. Densitometric analysis was performed using the Gene Tools software. The signal obtained for a given antigen was normalized against the control and given as a percent of the input signal. Statistical analysis was performed employing the Student's t-test. All data are mean±SEM of at least three independent experiments. **P≤0.01, ***P≤0.001. Journal of Investigative Dermatology 2007 127, 2307-2314DOI: (10.1038/sj.jid.5700879) Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions