AP-2a binding sites [17]. Therefore, it is reasonable to hypothesize that in cancer cells AP-2a may bind to specific target genes, silence transcription, and propagate DNA methylation in order to stably silence tumor suppressors and DNA repair genes. The following study investigates 8 target genes in HNSCC for the possibility of AP-2a-induced targeted methylation and subsequent gene silencing. Decreased AP-2a expression was found to correlate with decreased target methylation, increased target gene expression, and decreased microsatellite instability. ChIP analysis revealed an association between AP-2a and HDAC binding to target promoters. Also, the effect of HDAC inhibition on target methylation mirrored that of AP-2a downregulation. Therefore, the mechanism of AP-2a-induced methylation is likely explained by HDAC recruitment. Results AP-2a Targets Methylation methylation was analyzed in 16 potential AP-2a target genes’ promoters (list shown in Table S1). These genes were selected based on previous evidence for methylation in a variety of malignancies and potential regulation by AP-2a (i.e. the presence of consensus AP-2a binding sites in the target’s promoter). A preliminary methylation screen was performed in wildtype SCC22B cells, which yielded methylation in 8/16 of the targets tested: RARB2, DCC, DAPK, PTEN, MLH1, CDH1, P73, and RASSF1A (Figure 1b and Figure S2). Bisulfite sequencing was then performed on these eight genes in SCC22B cells with and without AP-2a downregulation (detailed gene diagrams are displayed in Figure S1). Of these eight genes, five genes (RARB2, PTEN, MLH1, CDH1, and RASSF1A) showed some level of demethylation in the SCC22B cells with AP-2a downregulation compared to the wildtype cells (Figure 1b; graphical representation in Figure 1d). AP-2a downregulation decreases target gene methylation Using a previously generated HNSCC cell line (SCC22B) in which AP-2a is stably downregulated [14](Figure 1a), DNA Decreased target gene methylation correlates with decreased expression In order to assess the significance of the target methylation, western blot analysis was performed on lysates from the wildtype Figure 1. Targeted methylation and repression via AP-2a in HNSCC cell lines SCC22B. a) Western blot analysis to confirm AP-2a downregulation in SCC22B. b) Via bisulfite sequencing analysis, methylation of 5 target genes was compared between SCC22B cells with and without AP-2a expression. Gene drawings indicate the CpG island and bisulfite PCR product in relationship to the transcription start site (arrow). Solid circles represent methylated CpGs; whereas, open circles represent unmethylated CpG sites. The small bars represent the location of the predicted AP-2a site. c) Western blot analysis was performed to analyze methylation’s effect on target expression. d) Graphical representation of the average methylation percentage in the 5 target genes analyzed. * P-value = 0.042; ** P-value = 0.040; *** P-value = 0.014. doi:10.1371/journal.pone.0006931.g001 PLoS ONE | www.plosone.org 2 September 2009 | Volume 4 | Issue 9 | e6931