The Role Of Poly Comb-like Protein PCL2 In The Regulation Of Centrosome Duplication

Centrosome,a cytoplasmic membrane-free organelle composed of a pair of barrel-shaped centrioles and pericentriolar material,is the major microtubule-organizing center in animal cells.Centrosomes regulate the life activities of many animal cells by forming the microtubule skeleton of cell cilia and spindles,such as maintenance of cell morphology,establishment of cell polarity,cell division,and cell migration.At present,centrosome abnormalities have been reported in a variety of tumors and neurodegenerative diseases,and their abnormalities can be used as clinical markers for tumor detection.However,the duplication process of centrosome is complicated,and the research on its duplication mechanism is still insufficient.In theory,a clear study of the duplication process of the centrosome will provide potential treatment options for clinically abnormal diseases of the centrosomePCL2 is a polycomb-like protein originally found in Drosophila.It has 593 amino acids and contains Tudor,PHD 1/2,and EH domains.With the deepening of epigenetics research,PCL2 is gradually recognized as a subunit of the epigenetic complex PRC2,which can recruit the PRC2 complex to hypomethylated CpG islands to perform the one/two/triple methyl group of the 27 lysine of histone H3.The trimethylation of 27 lysine of H3 is then recognized by PRC1,which ubiquitinates the 119 lysine of histone H2A,thereby suppressing gene transcription.In addition,PCL2 can also regulate the stability of P53.Current research on it is mainly focused on the field of apparent regulation of the genome in the nucleus,and there are few reports on other aspects of research.This article deepens its understanding by studying its role and regulation mechanism in centrosome duplication.In summary,this study firstly identified that by bingding with Sas-6 PCL2 is recruited to centrosome via CCNA2 and regulates the duplication of centrosomes by modulating the protein stability of Plk4.Object:To clarify the role of PCL2 in the process of centrosome duplication and to explore the regulation mechanism of PCL2 in centrosome duplication.Methods:1.Synchronize the cells,and detect the changes of PCL2 protein level and localization in each cell cycle by Western blotting and immunofluorescence.2.Use GeneMANIA to predict whether PCL2 interacts with centrosome duplication-related proteins.3.Detect whether PCL2 co-localizes with centrosomal proteins y-tublin,Sas-6 and Cep97 by immunofluorescence,and Co-IP to detect whether PCL2 directly interacts with centroso mL proteins.4.Use immunofluorescence to test whether PCL2 and CCNA2 co-localize.After knocking down CCNA2,the immunofluorescence was used to test whether PCL2 aggregated to the centrosome,and the Co-IP was used to test whether PCL2 directly interacted with CCNA2.5.Overexpression PCL2,using immunofluorescence to detect changes in the number of centrosomes.6.Overexpression and knockdown PCL2.Western blotting and q-PCR were used to detect changes in Plk4 protein level and mRNA level.Immunofluorescence and Co-IP were used to detect whether Plk4 and PCL2 co-localized and interacted.Results:1.Western blotting results of synchronized cells show that PCL2 protein level is changed as the cycle process,and its protein expression level is highest in the S phase of cell cycle.Immunofluorescence results show that PCL2 aggregates in the cell as cell cycle changes.It indicates that PCL2 is involved cell duplication.2.GeneMANIA analysis found that PCL2 was co-localized with PLK4,CCNA2,and CEP 135,indicating that PCL2 may be involved in the regulation of centrosomes duplication.3.Immunofluorescence results after synchronization of cells showed that PCL2 and y-tubulin co-localized from S phase to G2/M phase and co-localized with Sas-6 and Cep97.Co-IP results showed that there was a direct interaction between PCL2 and Sas-6.4.The immunofluorescence and Co-IP results of PCL2 and CCNA2 showed that there was co-localization and interaction between PCL2 and CCNA2.After knocking down of CCNA2,the immunofluorescence results showed that PCL2 no longer aggregated to the centrosome,indicating that CCNA2 recruited PCL2 to the centrosome.5.After PCL2 was overexpressed,immunofluorescence results showed that centrosomes could not be separated normally and centrosome number was abnormal.6.After overexpression or knockdown of PCL2,Western blotting and q-PCR results showed that Plk4 protein level was changed significantly,but mRNA level did not vary significantly.Immunofluorescence and Co-IP results show that PCL2 and Plk4 are co-localized and directly interact.Conclusions:1.PCL2 may be involved in the regulation of cell division.2.PCL2 may participate in the regulation of centrosome duplication.3.PCL2 co-localizes with centrosome and directly interacts with centrosomal protein Sas-6.4.PCL2 was recruited to the centrosome by CCNA2.5.Overexpression of PCL2 induces centrosomal abnormalities6.PCL2 regulates centrosome duplication via modulating the protein level of Plk4.