Expression of FoxO3a is regulated by DNA methylation.

<p>(<b>A</b>) WT cells were treated with trichostatin A (TSA), 5-aza-deoxycytidine (5-Aza), or a combination of TSA and 5-Aza for 48 h, and FoxO3a mRNA levels were determined by quantitative real-time PCR. Fold change in mRNA levels was calculated after normalization against β-tubulin mRNA (internal control). **<i>P</i><0.01 and *<i>P</i><0.05 (Student's <i>t</i>-test). (<b>B</b>) DNA methylation status of <i>FoxO3</i> CpG islands in p18KO, p18Rev, and WT cells was analyzed by bisulfite sequencing. The methylation status in the gray boxed region was determined for ten clones obtained from each cell line. Individual clones are indicated by lines with circles. Methylated and non-methylated cytosines are indicated by closed and open circles, respectively. (<b>C</b>) A series of fragments from the intronic region of the mouse FoxO3a gene were subcloned upstream of a luciferase reporter gene. Each construct was transfected into MEFs, and luciferase activity was measured and normalized against pRLTK activity. Normalized luciferase activity is expressed as means±SD (<i>n</i> = 3). ***<i>P</i><0.001 and **<i>P</i><0.01 (Student's <i>t</i>-test).