Enhanced transcription of the IFN-alpha inducible gene IFITM3 by means of dynamic promoter demethylation in the presence of the TGF-beta inducible small calcium binding protein S100A2

In human melanoma cell lines, the calcium binding protein S100A2 augments the antiproliferative activity of interferon-alpha (IFN) by an unknown mechanism. I show by microarray profiling that recombinant over-expression of S100A2 upregulates the expression of a subset of IFNα response genes beyond the maximal cytokine inducible level including IFITM3, a gene with documented antiproliferative activity. I have chosen IFITM3 as chromosomal IFN response reporter gene in a model system consisting of two human melanoma cell lines ME15 and D10 described previously (Brem, Oraszlan-Szovik et al. 2003). In ME15 cells IFITM3 expression is strictly IFN dependent whilst it is constitutively expressed in the IFN resistant D10 cells. It was shown that S100A2 is sufficient to restore IFN sensitivity in D10 (Foser, Redwanz et al. 2006) and I show by indirect immunofluorescence cytoplasmic localization of S100A2, which eliminates a direct function as a transcriptional enhancer of IFITM3 expression and other antiproliferative genes. I show that treatment of ME15 melanoma cells with the demethylating agent 5-aza-2’deoxycytidine (DAC) results in a significant increase of IFITM3 expression following IFNα stimulation suggesting a DNA methylation mediated mechanism. Based on bisulfite sequencing of the IFITM3 core promoter, I show that D10 cells exhibit hypomethylation and I demonstrate that S100A2 is required for kinetic and reversible IFN induced CpG demethylation in ME15 cells. Since p53 signaling is not functional in D10 cells I propose an indirect mechanism of methylation that involves p53 controlled signaling pathways.