The Regulation Of PGC7 With Its Interaction Protein RBBP4 And HDAC1 In Dlk1-Dio3 Imprinted Region

PGC7 also known as Dppa3 or Stella,is a maternal effector that maintains imprinting during early embryonic development.And It is also a small protein that promotes the maintenance of cell pluripotency and the reprogramming process by regulating the imprinted genes.This protein is only 150 amino acids in mice and is a non-folded protein that functions in combination with a variety of functional proteins.HDAC1/2-RBBP4/7 deacetylase complex consists of deacetylase HDAC1/2 and histone chaperone RBBP4/7.This complex can bind to other proteins to form a larger protein complex,including NuRD protein complexes that promotes transcriptional repression by histone deacetylation and nucleosome remodeling and PRC2 complex,which promotes repression of homeotic genes during development.The Dlk1-Dio3 imprinted region is an ideal model for studying mammalian imprinting.Some genes in the Dlk1-Dio3 imprinting region are involved in the cell biology of stem cell levels and the pathogenesis of various diseases,and even the correct expression of non-coding RNA in this region can determine the full differentiation potential of induced pluripotent stem cells.This study found that RBBP4 is a potential PGC7 interacting protein.Firstly,the interaction and intracellular localization of the two were identified and the major methylation regulatory regions of the Dlk1-Dio3 gene cluster were detected by ChIP-qPCR.The mouse F9 cell line with knockdown of RBBP4 gene was further constructed.It was found by real-time PCR that the expression of RBBP4 knockdown resulted in a significant decrease in mRNA expression levels of the imprinted genes Dlk1,Meg3,Peg10 and Rasgrf.DNA methylation sequencing of bisulfite DNA revealed that DNA methylation levels of Dlk1-DMR and Meg3-DMR increased after RBBP4 knockdown Immunofluorescence staining showed no significant change in overall DNA methylation level and decreased H4 acetylation level after RBBP4 knockdown.In order to investigate whether cell pluripotency is affected during the regulation of Dlk1-Dio3 imprinting region,real-time PCR detection of imprinted genes Dlk1,Meg3 and cellular pluripotency-related genes Oct4,Nanog,Sox2 and Pgc7 found that the expression of Dlk1 and Meg3 were significantly increased after retinoic acid treatment,while the expression of pluripotency factor was significantly decreased.Moreover,real-time quantitative PCR detected a decrease in the expression levels of pluripotency genes Sox2,Oct4 and Pgc7 after RBBP4 knockdown.At the same time,alkaline phosphatase staining and CCK cell proliferation assay confirmed that RBBP4 knockdown slowed cell proliferation and accelerated cell differentiation.The RBBP4 protein is a histone chaperone involved in the formation of the HDAC1/2-RBBP4/7 deacetylase complex.We constructed a eukaryotic expression vector for other members of the HDAC1/2-RBBP4/7 complex,and verified the interaction of HDAC1,HDAC2,and RBBP7 with PGC7 by Co-IP.The results show that PGC7 also interacts with HDAC1 and HDAC2.And the interaction between HDAC1 and HDAC2,HDAC1 and RBBP4 proteins disappeared after the addition of PGC7 protein.Due to the effect of PGC7 protein on the HDAC1/2-RBBP4/7 complex,it is speculated that PGC7 may recruit HDAC1 to play a role in the methylation regulatory region.The chromatin immunoprecipitation was used to determine that PGC7 and HDAC1 were co-enriched in the Dlk1-Dio3 imprinting region IG-DMR,and the enrichment of HDAC1 was reduced after interfering with PCG7.It is speculated that PGC7 and HDAC1 may have synergistic regulation in this region.Taken together,this study demonstrates that PGC7 protein interacts with HDAC1,HDCA2 and RBBP4 proteins in the HDAC1/2-RBBP4/7 complex,respectively,and affects the interaction between the complexes.Moreover,this study further explored the regulation that PGC7 with its interaction protein RBBP4 and HDAC1 in Dio3-Dlk1 imprinted region and its relationship with pluripotency maintenance.To provide a basis for further explanation of the relationship between imprinting disorder,histone deacetylation-DNA methylation,imprinting regulation-cell pluripotency.