The Role And Mechanism Of Histone Methyltransferase SETDB2 In Mitosis

Objective:Mitosis is the main way for eukaryotic cells to divide and spindle assembly checkpoint is necessary to ensure accurate chromosome separation.It’s reported that histone methyltransferase SETDB2 is involved in chromosome segregation,SETDB2 knockdown leads to abnormal chromosome segregation and delayed mitosis,this function is associated with H3K9me3 distribution and CENP proteins location[1],but the mechanism in mitosis are not clear.Therefore,the study explored the function and mechanism of SETDB2 in mitosis.Methods:1.SETDB2 was knocked down by siRNA,then immunofluorescence assay and flow cytometry were performed to analyze the influence of SETDB2 on mitosis;2.The expression of SETDB2 was restored and the influence of SETDB2 on mitosis was further analyzed by immunofluorescence and flow cytometry;3.The interaction between SETDB2 and BUBR1 was analyzed by co-immunoprecipitation assay;4.Coimmunoprecipitation assay was performed to detect the influence on interaction between CDC20 and BUBR1 and APC3;5.SETDB2 WT and methyltransferase activity defect mutant were restored,then immunofluorescence and co-immunoprecipitation were performed to analyze whether the influence of SETDB2 on mitosis was dependent on its methyltransferase activity;6.Co-immunoprecipitation was used to analyzed the interaction between SETDB2 and α-TUBULIN and β-TUBULIN;7.Immunofluorescence assays was used to detect the subcellular localization of SETDB2 and TUBULIN in interphase and different phases during mitosis.Result:1.SETDB2 knockdown significantly increased the percent of cells with abnormal nuclei and led to abnormal spindle and chromosomal isolation during metaphase and anaphase in mitosis,flow cytometry assay showed that SETDB2 knockdown led to cell arrest in G2/M phase;2.Restored SETDB2 level rescued mitosis defects and cell cycle arrest;3.Co-IP assay showed that SETDB2 interacted with BUBR1;4.Co-IP showed that knocking down of SETDB2 decreased the interaction between CDC20 and BUBR1 and APC3;5.Restoring SETDB2 WT and methyltransferase activity defect mutant both rescued mitosis defects and the interaction between SETDB2 and BUBR1 was independent on its methyltransferase activity;6.Co-IP assay showed the interaction between SETDB2 and a-TUBULIN and β-TUBULIN;7.Immunofluorescence assay showed SETDB2 and TUBULIN co-localized during metaphase and anaphase,while less co-localized during interphase,prophase and telophase.Conclusion:The microtubule binding protein SETDB2 interacts with BUBR1 to promote CDC20-APC3 interaction and guarantee accurate chromosome segregation independent of its methyltransferase activity.