AP-2a Targets Methylation Figure 4. AP-2a interacts with HDAC1/2, and HDAC activity is required for targeted methylation in HNSCC cells. a) ChIP analysis was performed using AP-2a, SP1, HDAC1, and HDAC2 antibodies in SCC22B cells with and without AP-2a downregulation. ChIP eluate was then utilized for quantitative PCR using CDH1, RASGRF2, MLH1, RASSF1A, and PTEN promoter primers. b) Western blot analysis demonstrating increased acetylation following Trichostatin A (TSA) treatment. c) Bisulfite sequencing analysis was performed in SCC22B cells before and after TSA treatment. Solid circles represent methylated CpGs; whereas, open circles represent unmethylated CpG sites. d) Graphical representation of the average methylation percentage in the 3 target genes analyzed. * P-value = 0.0452; ** P-value = 3.7761026. doi:10.1371/journal.pone.0006931.g004 class I and II HDACs were inhibited via Trichostatin A (TSA). Efficacy of TSA treatment was verified via western analysis for acetyl histone H4K5 (Figure 4b). Following TSA treatment, target methylation was then compared with that of the SCC22B cells with intact HDAC activity. Both PTEN and MLH1 demonstrated a significant decrease in methylation subsequent to TSA treatment; however, RASSF1A remained unchanged (Figure 4c; ** P-value = 3.7761026 and * 0.0452, respectively; graphical quantitation shown in Figure 4d). This correlates with previous findings that TSA alone is insufficient to change the histone acetylation status of RASSF1A [23]. However, no obvious change in target gene expression resulted from the increased acetylation (data not shown). This may be due to the brief time course (24 hours), which may not allow enough time for the methylation changes to reflect their impact on expression [24]. AP-2a downregulation correlates with decreased mitosis Because AP-2a appears to target methylation and silencing of certain tumor suppressor genes in this HNSCC cell line, downregulation of AP-2a (i.e. relief of tumor suppressor silencing) should correlate with decreased proliferation. We performed cell cycle analysis by flow cytometry on the HNSCC cells SCC22B with endogenous AP-2a, AP-2a overexpression (Figure 5a), and downregulation of AP-2a. The pattern in AP-2a overexpressing cells looked similar to endogenous AP-2a expression; however, HNSCC cells with AP-2a downregulation exhibited significantly less cells in mitosis (Figure 5b; P-value = 0.0003). Lack of AP-2a inhibits remethylation of PTEN following 5- aza-29 deoxycytidine treatment In order to exclude the possibility that AP-2a is affecting DNA methylation at the global level, an methylation sensitive digest was performed on DNAs from SCC22B cells with and without AP-2a downregulation. This revealed no substantial difference in DNA methylation levels between the two cell types (Figure 5c), suggesting the DNA methylation changes from AP-2a are gene- specific. To determine whether AP-2a acts to maintain or induce this specific target DNA methylation, the HNSCC cells SCC22B (+/2 AP-2a shRNA) were treated with a demethylating drug (5-aza-29deoxycytidine) and then permitted to recover methylation by continued culturing in the absence of the drug. Cells collected at 7 days post-treatment showed no PTEN methylation in both cell types (Figure 5d). However, after 10 days PTEN methylation was returning in the SCC22B cells with AP-2a expression, whereas, PTEN remained unmethylated in the cells lacking AP-2a ex- pression (Figure 5d). Therefore, this is suggestive of a mechanism by which AP-2a induces target methylation. Discussion It has previously been shown that AP-2a is expressed at relatively low levels in normal keratinocytes and is significantly elevated in cancerous and proliferating keratinocytes in squamous cell carcinoma of the skin [7]. Forced expression of AP-2a in keratinocytes causes increased expression of the oncogene PLoS ONE | www.plosone.org 5 September 2009 | Volume 4 | Issue 9 | e6931