Piwil2 Activates STAT3 and Inhibits P53 Figure 2. Piwil2 induces phosporylation of STAT3. A, Western blot analysis showed that phosphorylation level of STAT3 was significantly increased 12 hours after being transient transfected with Piwil2. However, at 24 hours after transfection, STAT3 phosphorylation level had almost returned to normal. Similar while opposite profile was observed followed transfection of Piwil2-siRNA. Nevertheless, stable transfectants of Piwil2 (ST) showed constantly high STAT3 activity. B, pYSTAT3 was subcellular localized in Piwil2 transfected cells by using immunofluorescence. Signal of FITC- labeled secondary antibody was observed significantly increased in cytoplasm 6 hours after transfection, and expanded to nucleus 12 hour after transfection. C, time-course analysis showed that STAT3 phosphorylation was notably sensitive to changes in Piwil2 level and responded rapidly, at least 6 hours before P53 regulation. D, Western blot analysis revealed that Piwil2 induced STAT3 phosphorylation and repressed P53 in HepG2 cells. doi:10.1371/journal.pone.0030999.g002 Furthermore, we examined the level of P53 and pYSTAT3 in another tumor cell line HepG2, revealing that Piwil2 functioned similarly in HepG2 in contrast to HeLa cells (Fig. 2D). To verify this hypothesis, we determined the interaction between STAT3 protein and P53 promoter using chromatin immunoprecipitation (ChIP) assay followed by Real-time quanti- tative PCR. Bioinformatic examination revealed that a potential STAT binding site which contains a consensus sequence TT(N4)AA or TT(N5)AA [19,23] can be located 179 bp upstream of the noncoding first exon of human P53 (NM_000546.4) (Fig. 3A). In additional, overexpression of Piwil2 led to an increased level of interaction between STAT3 and P53 promoter 12 hours after transfection. In contrast, knockdown of Piwil2 suppressed such interaction (Fig. 3B). To investgate whether P53 was silenced at transcription level, methylation state of H3K4 and H3K9 in P53 promoter region were analyzed by ChIP method. The results showed that transient transfection of Piwil2 resulted in approxi- mately a 2-folder increase in the methylation state of H3K9 and significant decrease in the methylation state of H3K4, both of which represented the transcriptional silencing of P53. In contrast, knockdown of Piwil2 resulted in significant decrease in the methylation state of H3K9 and increase in the methylation of H3K4 (Fig. 3C). In order to identify the specific sequence bound by STAT3, the indicated region of p53 gene promoter was mutated and constructed into a pVAX-Luciferase plasmid. Dual-luciferase analysis showed that the translation of mRNAs containing the wildtype but not mutated STAT3 binding site was downregulated by Piwil2 (Fig. 3D). Src Kinase is required for STAT3 phosphorylation by Piwil2 To search for potential upstream factors of STAT3, HeLa cells were treated with tyrosine kinase inhibitors before transfection. Western analysis showed that overexpression of Piwil2 still enhanced STAT3 phosphorylation in HeLa cells pretreated with JAK inhibitor AG490. Thus we can rule out JAK family kinases as a phosphate giver. However, in HeLa cells pretreated with Src inhibitor PP2, STAT3 phosphorylation was blocked after overexpression of Piwil2 (Fig. 4A). This result indicated that Src, rather than JAK, played a role in Piwil2-induced STAT3 phosphorylation, which was further confirmed by western blotting using phosphorylated c-Src antibody. Analysis revealed that Y416- PLoS ONE | www.plosone.org 3 January 2012 | Volume 7 | Issue 1 | e30999