UHRF2 Interacts With H3K9AC Through ITS PHD Domain And Decreases H3K9AC In HEPG2 Cancer Cells

ObjectiveTo investigate the association of UHRF2 with acetylated H3 (H3K9ac and H3K14ac) and find the required domain for UHRF2 interacting with acetylated H3, further to study the effect and mechanism on how UHRF2 regulates H3K9ac and H3K14ac in normal cells and cancer cells.Methods1. HEK293, LO2 and HepG2 were transfected with pCMV-FLAG-UHRF2 to observe the colocation between UHRF2 and H3, H3K9ac and H3K14ac by immunofluorescence assay and laser scanning confocal microscopy. Then the interaction between UHRF2 and H3K9ac, H3K14ac in HEK293, LO2 and HepG2 cells were detected by immunoprecipitation.2. Identification of deletion mutants of UHRF2 plasmids by restriction enzyme digestion. HEK293, LO2 and HepG2 cells were transfected with deletion mutants of UHRF2 to test the functional binding region for UHRF2 interacting with H3K9ac and H3K14ac.3. To detect endogenous expression level of UHRF2, H3K9ac and H3K14ac in HEK293, LO2, HepG2 cells, adjacent non-tumor tissues and HCC tissues by western blotting and immunohistochemistry.4. Overexpression of UHRF2 to study the effect of UHRF2 on H3K9ac and H3K14ac in HEK293, LO2, HepG2 cells by Western blotting analysis. Then, performed immunohistochemistry to further test whether the UHRF2 had effect on the expression of H3K9ac and H3K14ac in HCC tissues.Results1. Laser scanning confocal microscopy showed that UHRF2 were colocalized with H3, H3K9ac and H3K14ac in HEK293, LO2 and HepG2 cells. The immunoprecipitation study demonstrated that UHRF2 interacted with H3K9ac in all of these three cell types.2. The immunoprecipitation study showed that PHD domain was the key domain for UHRF2 to interact with H3K9ac in HEK293, LO2 and HepG2 cells. Moreover, in HepG2 cells except PHD domain, YDG domain was also the key domain for UHRF2 to interact with H3K9ac3. Western blot analysis demonstrated that the level of H3K9ac and H3K14ac were higher in HepG2 cells than in HEK293 and LO2 cells. The immunohistochemistry study showed that UHRF2, H3K9ac and H3K14ac were positive staining in HCC tissues but negative staining in adjacent non-tumor tissues.4. Western blotting analysis indicated that overexpression of UHRF2 increased the level of H3K9ac in HEK293 and LO2 cells but decreased the level H3K9ac in HepG2 cells. And the immunohistochemistry study represented that the expression of H3K9ac was high in low level of UHRF2 HCC tissues but low in high level of UHRF2 HCC tissues.ConclusionTogether, these results suggest that UHRF2 interacts with H3K9 acetylation through its PHD domain and decreases H3K9 acetylation in HepG2 cancer cells。 Further research on its molecular mechanism, namely how UHRF2 regulates H3K9ac and H3K14ac is being addressed。...