AP-2a Targets Methylation Figure 3. Decreased MSI following AP-2a downregulation in HNSCC cell lines SCC22B and SCC17as. a) 9 MSI markers were used with DNA from SCC22B and SCC17as cells with and without AP-2a downregulation, and the products were analyzed on an 8% PAGE gel. b) ChIP analysis using an AP-2a antibody in SCC22B cells with and without AP-2a downregulation. ChIP eluate was then utilized for quantitative PCR using MLH1 promoter primers. c) Bisulfite sequencing analysis of 3 additional DNA repair genes in WT SCC22B cells. Because no methylation was observed in the WT cells, it was unnecessary to analyze methylation in AP-2a downregulated cells. Solid circles represent methylated CpGs; whereas, open circles represent unmethylated CpG sites. doi:10.1371/journal.pone.0006931.g003 suggests a potential decrease in MSI in these cells correlating with decreased AP-2a (Figure 3a). As expected, the cell lines that exhibited no change in MLH1 methylation (SCC11B, HT29, and HCT116) in the region tested did not reflect any changes in MSI with AP-2a downregulation (data not shown). In order to confirm AP-2a actually binds to the MLH1 promoter, ChIP analysis was performed in SCC22B with and without AP-2a downregulation. This revealed AP-2a does bind to the MLH1 promoter, and this binding is decreased in the AP-2a- downregulated cells (Figure 3b). Methylation screening was performed for additional DNA repair genes (i.e. MSH3, PMS2, and RAD50) that contained both CpG islands and potential AP-2a binding sites within their promoter elements. This bisulfite sequencing analysis revealed relatively no methylation of these genes (Figure 3c). Decreased AP-2a and HDAC1/2 binding to target promoters following AP-2a downregulation In a previous publication, AP-2a binding to target promoters was shown to precede recruitment of HDAC proteins to the region for more stable silencing [15]. Therefore, to address the mechanism by which AP-2a may affect gene methylation and silencing in these HNSCC cells, ChIP analysis was performed on SCC22B cells with and without downregulation of AP-2a. As expected, this semi-quantitative analysis revealed AP-2a and HDAC1/2 binding to all of the target promoters tested, except PTEN, in the wildtype SCC22B cells (Figure 4a). Furthermore, both AP-2a and HDAC binding was reduced in the AP-2a downregulated cells. HDAC1 was found to be more frequently associated with the targets compared to HDAC2, which did not pulldown RASSF1A. Since AP-2a and SP1 bind to the same consensus sequence, SP1 binding was also investigated in order to assess whether increased target gene expression was due to the restored binding of the activator SP1 following removal of AP-2a. With the exception of CDH1, decreased AP-2a binding did not allow increased SP1 binding as would have been expected. HDAC inhibition’s effect on target methylation correlates with AP-2a downregulation In order to investigate whether the mechanism of AP-2a- targeted methylation involved HDAC recruitment and activity, PLoS ONE | www.plosone.org 4 September 2009 | Volume 4 | Issue 9 | e6931