Subcellular Localization Of Chicken BRD2 Gene Spliceosomes And Screening And Functional Analysis Of Their Interaction Proteins

Bromodomain-containing protein2(BRD2)is a member of the bromodomain and extra-terminal domain protein family,which contains two bromodomains and an exoterminal domain in tandem.BRD2 can specifically bind histone acetylated lysine,and participate in gene transcriptional regulation,chromatin remodeling,and cell proliferation and apoptosis.At present,domestic and foreign studies on BRD2 gene are mainly focused on humans and mouses,while there are relatively few reports on chicken BRD2 gene.Especially,the expression characteristics,subcellular localization and the cellular proteins interacting with chicken BRD2 gene and its splice are still unclear.Therefore,in this study,the chicken BRD2 gene and its spliceosome were cloned,bioinformatics and tissue expression characteristics were analyzed.The recombinant eukaryotic expression vector of chicken BRD2 gene and its spliceosome was constructed and transfected into cells to observe its subcellular localization in cells.Furthermore,cell proteins interacting with chicken BRD2 and its spliceosomal proteins in chicken embryo fibroblasts(DF-1)were identified by immunoprecipitation combined with mass spectrometry and bioinformatics analysis was conducted.Then,one of the cell proteins was selected for interaction verification.The main research results are as follows:1.The ORF of chicken BRD2 gene and its spliceosome was successfully amplified,cloned and sequenced.Sequence analysis showed that the full length of chicken BRD2 gene and its splicosome ORF were 2340bp(BRD2),2283bp(BRD2-X1),2301bp(BRD2-X2)and 1983bp(BRD2-X3),encoding 779,767,761 and 661 amino acids,respectively.The secondary structure of chicken BRD2 and its spliceosome proteins is mainly composed of random coil and α-helix.Fluorescence quantitative PCR results showed that BRD2 gene and its spliceosome were expressed in different tissues at different stages of chicken development,and the mRNA expression levels were different among different tissues at the same day of age,among which BRD2 and BRD2-X2 were up-regulated and the expression levels were the highest in lung.However,BRD2-X1 and BRD2-X3 were down-regulated and had the lowest expression levels in the lung.2.the recombinant eukaryotic expression vectors pEGFP-BRD2,pEGFP-BRD2-x1,pEGFP-BRD2-X2,pEGFP-BRD2-X3 of chicken BRD2 gene and its splice were successfully constructed,and the fusion protein was correctly expressed in cells by Western blotting.The results of subcellular localization showed that chicken BRD2 gene fusion protein GFP-BRD2 and its splice fusion proteins GFP-BRD2-X1,GFPBRD2-X2 and GFP-BRD2-X3 were all located in the nucleus,but GFP-BRD2 and GFP-BRD2-X1 were evenly distributed in the nucleus,while GFP-BRD2-X2 and GFPBRD2-X3 were unevenly distributed in the nucleus3.There are 225,185,137 and 34 cell proteins were screened to interact with chicken BRD2,BRD2-X1,BRD2-X2 and BRD2-X3 proteins,respectively.GO functional annotations showed that the BRD2 interacting proteins were mainly involved in gene transcriptional regulation,cell cycle,development,metabolism and biological regulation.The proteins interacting with BRD2-X1 are mainly involved in gene transcriptional regulation,transcription and translation,metabolism and biological regulation,immunity and viral infection.The proteins interacting with BRD2-X2 are mainly involved in gene transcriptional regulation,metabolism and biological regulation,as well as immune and viral infection processes.The BRD2-X3 interacting proteins are mainly involved in the immune and viral infection processes.The proteins interacting with BRD2 and its spliceosomal proteins all have the molecular functions of nucleic acid binding,protein binding and enzyme activity regulation,and are mainly localized in the nucleus and partly in the cytoplasm.KEGG signaling pathway showed that the proteins interacting with chicken BRD2 and its spliceosome were mainly involved in the process of genetic information,translation,immune system and cell development.4.The constructed eukaryotic expression vectors pEGFP-BRD2 and p CMV-MycMATR3 were co-transfected into DF-1 cells,and the co-localization of chicken BRD2 protein and MATR3 protein in the cells was observed by fluorescence colocalization.The interaction between Brd2 protein and MATR3 protein was verified by immunoprecipitation method.The results showed that GFP-BRD2 and MYC-MATR3,the fusion proteins of chicken BRD2 protein and MATR3 protein,were distributed in the nucleus and had obvious colocalization in the nucleus.The results of immunoprecipitation test showed that there was an interaction between chicken BRD2 protein and MATR3 protein.These results indicated that chicken BRD2 protein and MATR3 protein interact in the nucleus.