Identification And Functional Characterization Of PGC7 Interacting Proteins

PGC7(Primordial germ cell 7)is a maternal factor essential for early development,which specifically expressed in primordial germ cells,oocytes,preimplantation embryos,and pluripotent cells.Now,multiple studies have shown that PGC7 plays critical roles in imprinting maintenance,transcriptional repression,chromatin condensation,and cell division and the maintenance of cell pluripotentiality.However,the molecular mechanism that PGC7 regulates these biological processes are not clear.Based on the comprehensive prediction analysis,we found that PGC7 is an intrinsically disordered protein.As PGC7 is not equipped with known enzymatic domains,it is most likely that its interacting proteins function to regulate above-mentioned biological processes.Thus,we investigated the functions of PGC7 and its interacting proteins start with analyzing PGC7 protein interactome.The main works and results were as follows:1.The functional intrinsic disorder analysis of PGC7 protein.We employed a broad set of computational tools for intrinsic disorder analysis to gain information on the structural peculiarities and intrinsic disorder predispositions of PGC7.The result indicated the PGC7 was a highly disordered protein(the contents of the predicted disordered residues > 30%).2.To identify the PGC7-interacting proteins in HEK293 T cells,two cell samples were generated in this study.HEK293 T cells expressing BirA and biotinylated PGC7 protein were used for the enrichment and purification of PGC7-interacting proteins.The cells only expressing BirA were established to serve as a control.After verification of biotinylation of PGC7,the cellular localization of biotinylated and unmodified PGC7 were compared by immunofluorescence analysis.The result demonstrated that biotinylated PGC7 protein had similar cellular localization to the unmodified PGC7.Subsequently,PGC7 protein complexes were isolated by streptavidin-mediated affinity purification and identified by mass-spectrometric analysis.In total,291 potential PGC7-interacting proteins were identified,of which 290 are new candidates of PGC7-interacting partners found in this study.Bioinformatics analysis showed that PGC7-interacting proteins were enriched in GO molecular functions,such as nucleotide binding,RNA binding,structural molecule activity and structural constituent of ribosome.KEGG pathway analysis confirmed that PGC7-interacting proteins were enriched in ribosome and oocyte meiosis pathways.3.Protein-protein interaction network analysis.In this study,we constructed theinteraction network of PGC7-interacting proteins by using STRING database and Cytoscape v3.4.0 softwave.We further analyzed the interaction network for densely connected regions using the MCODE plugin tool in Cytoscape.14 highly connected clusters were identified from the interaction network,and the functions of these clusters were involved in transcription,RNA processing,protein transportation and cell cycle.4.According to the results of bioinformatics analysis,28 PGC7-interacting protein eukaryotic expression vectors were constructed in this study.The interaction of PGC7 with 23PGC7-interacting proteins,including UHRF1,MAGED2,KPNB1,GNB2L1,FBL,STAT1,CDK2,HNRNPR,YBX1,HSPA2,CDK5,AKAP8 L,PPP2R1A,NOP2,MCM6,RPL14,YWHAG,U2AF2,HP1BP3,DMAP1,NUPL1,KPNA1 and RBBP4,were confirmed by coimmunoprecipitation(co-IP).5.Studies of the interaction between PGC7 and HP1BP3.We constructed a series of eukaryotic expression vectors that expressing two PGC7 truncation mutants and three HP1BP3 truncation mutants.The C-terminal of PGC7(76-150 aa)interacts with HP1BP3 globular domains(158-412 aa)was confirmed by co-IP.Further investigation demonstrated that overexpression of PGC7 disrupts the interaction of HP1BP3 and HP1.6.PGC7 and HP1BP3 co-regulate the expression and methylation of imprinted genes.We constructed a Hp1bp3 gene knockout F9 cell line in this study.Real-time PCR analysis showed that the expression of several imprinted genes was significantly changed in Hp1bp3 gene knockout F9 cell line,such as H19,Rasgrf1,Gtl2 and Peg1 were downregulated,and Peg10 was upregulated.We found that HP1BP3 overexpressed in Hp1bp3 gene knockout F9 cell line returned the expression of H19,Rasgrf1,Gtl2 and Peg10 to normal levels.PGC7 overexpressed in Hp1bp3 gene knockout F9 cell line significantly upregulated the expression of Gtl2.Bisulfite sequencing analysis showed that HP1BP3 knockout significantly upregulated the methylation level of Gtl2 promoter region.HP1BP3 or PGC7 overexpressed in Hp1bp3 gene knockout F9 cell line downregulated the methylation level of Gtl2 promoter region.Further ChIP-qPCR analysis demonstrated that PGC7 and HP1BP3 co-regulate the expression and methylation of Gtl2 by preventing DNMT3 A binding.7.Function studies of DMAP1 protein.Based on the predicted protein folding information,we constructed three DMAP1 truncation mutant eukaryotic expression vectors.The C-terminal of PGC7(76-150 aa)interacts with DMAP1 Coiled-coil domain(235-412 aa)was confirmed by co-IP.Further investigation demonstrated that DMAP1 mediates the interaction between PGC7 and DNMT1.In addition,we constructed Dmap1 monoallelic gene knockout F9 cell lines.The immunofluorescence staining analysis showed that DMAP1 knockdown had no effect on global DNA methylation status.Real-time PCR analysis showedthat the expression of H19 and Peg1 were significantly downregulated,and the expression of Peg10 was significantly upregulated in DMAP1 knockdown F9 cell.However,bisulfite sequencing analysis demonstrated that DMAP1 knockdown had no effect on the DNA methylation status of imprinted genes,such as,Peg1,Peg10,H19 and Rasgrf1.Conclusively,the identification of these potential interactors of PGC7 expands our knowledge on the PGC7 interactome.Additionally,the knowledge of these new PGC7-interacting proteins and the function studies of DMAP1 and HP1BP3 both provide a valuable resource for understanding the diverse functions of these proteins.