Our result reveals that Znf179 can inter act with the endogenous Plzf protein. Mapping the sites of interaction between Znf179 and Plzf To determine the region in Plzf that was required for its interaction with Znf179, various deletion constructs of Plzf were generated and cotransfected with EGFP Znf179 into COS 1 cells. Cell lysates were immunoprecipitated with anti Flag antibody, followed by Western Nutlin-3a mechanism blot analysis with anti Znf179 antibody. As shown in Figure 3A, two fragments of Plzf interacted with Znf179, which was consistent with the findings in yeast two hybrid assay. In contrast, the N terminal fragment and the last seven zinc fingers of Plzf did not interact with Znf179. We also generated the N and C terminal fragments of Znf179 and found that the C terminal but not N terminal fragment resulted in the recruitment of Znf179 protein from the nucleoplasm to the Plzf localized nuclear bodies.

Taken together, these results indicate that these two proteins indeed interact with each other in vivo and the sub cellular localization of Znf179 is influenced by the expression of Plzf. Overexpression of Znf179 does not affect Plzf mediated transcriptional repression Plzf can function as a transcriptional repressor. To examine whether Znf179 affected the transcriptional re pression activity of Plzf through protein protein inter action, we used a Gal4 based transactivation assay. The constructs consisting of Plzf or Znf179, fused with the DNA binding domain GSK-3 of the yeast Gal4 transcrip tion factor, were cotransfected with the Gal4 response element containing luciferase reporter.

In agreement with its transcriptional repressor function, our results showed that Gal4 DBD Plzf inhibited the Gal4 luciferase reporter activity. However, we did not observe a significant difference of Gal4 luciferase reporter acti vities in cells cotransfected with Gal4 DBD Plzf and ei ther a control vector or Znf179 expression plasmid. We also found that although Gal4 DBD Znf179 did not dis play autonomous transcriptional regulatory activity, the Gal4 luciferase reporter activity was inhibited by coex pression of Plzf, suggesting that Gal4 DBD Znf179 might recruit Plzf to the Gal4 reporter gene and was required for the interaction of Znf179 with Plzf.

Effect of Plzf co expression on subcellular localization of Znf179 To further determine the sub cellular localization of Znf179 and the interaction of Znf179 and Plzf, HeLa cells were transiently transfected with individual constructs or co transfected with combinations of the HA tagged Plzf and EGFP tagged Znf179 constructs and subsequently stained with an anti HA antibody followed by an im munofluorescence analysis. As shown in Figure 4, Plzf was mostly localized in nuclei and concentrated in nuclear bodies as previous studies reported, while Znf179 was predominantly localized in nuclei with faint sellckchem cytoplas mic staining.