Homeodomain-interacting protein kinase 2 regulates DNA damage response through interacting with heterochromatin protein 1γ

Homeodomain-interacting protein kinase 2 (HIPK2) is a potential tumor suppressor that has a crucial role in the DNA damage response (DDR) by regulating cell-cycle checkpoint activation and apoptosis. However, it is unclear whether HIPK2 exerts distinct roles in DNA damage repair. The aim of this study was to identify novel target molecule(s) of HIPK2, which mediates HIPK2-dependent DNA damage repair. HIPK2-knockdown human colon cancer cells (HCT116) or hipk1/hipk2 double-deficient mouse embryonic fibroblasts could not remove histone H2A.X phosphorylated at Ser139 (gamma H2A.X) after irradiation with a sublethal dose (10 J/m(2)) of ultraviolet (UV)-C, resulting in apoptosis. Knockdown of HIPK2 in p53-null HCT116 cells similarly promoted the UV-C-induced gamma H2A.X accumulation and apoptosis. Proteomic analysis of HIPK2-associated proteins using liquid chromatography-tandem mass spectrometry identified heterochromatin protein 1 gamma (HP1 gamma) as a novel target for HIPK2. Immunoprecipitation experiments with HCT116 cells expressing FLAG-tagged HIPK2 and one of the HA-tagged HP1 family members demonstrated that HIPK2 specifically associated with HP1 gamma, but not with HP1 alpha or HP1 beta, through its chromo-shadow domain. Mutation of the HP1box motif (883-PTVSV-887) within HIPK2 abolished the association. HP1 gamma knockdown also enhanced accumulation of gamma H2A.X and apoptosis after sublethal UV-C irradiation. In vitro kinase assay demonstrated an HP1 gamma-phosphorylating activity of HIPK2. Sublethal UV-C irradiation phosphorylated HP1 gamma. This phosphorylation was absent in endogenous HIPK2-silenced cells with HIPK2 3'UTR siRNA. Overexpression of FLAG-HIPK2, but not the HP1box-mutated or kinase-dead HIPK2 mutant, in the HIPK2-silenced cells increased HP1 gamma binding to trimethylated (Lys9) histone H3 (H3K9me3), rescued the UV-C-induced phosphorylation of HP1 gamma, triggered release of HP1 gamma from histone H3K9me3 and suppressed gamma H2A.X accumulation. Our results suggest that HIPK2-dependent phosphorylation of HP1 gamma may participate in the regulation of dynamic interaction between HP1 gamma and histone H3K9me3 to promote DNA damage repair. This HIPK2/HP1 gamma pathway may uncover a new functional aspect of HIPK2 as a tumor suppressor.