OBJECTIVE: To localize and define the region of nucleus export signal (NES) on BRD7, and determine the role of this region in nucleus export of the external protein. METHODS: Based on an in vitro expressed model of green fluorescence protein (GFP), we performed DNA walking analysis to set BRD7 into several sections according to the structural characteristics of BRD7, investigated the effect of different sections of BRD7 on nucleus export of GFP, defined the region of nucleus export signal sequence of BRD7, and further ascertained the content of amino acids in BRD7 and potential localization of BRD7 NES by bioinformatics. RESULTS: B7C1 fragments ranged from aa219 to aa450 in BRD7 were found to target the external protein GFP into the cytoplasm detected by GFP direct fluorescence, which could be inhibited by NES inhibitor Leptomycin B (LMB). This region was rich in hydrophobic amino acid residues but no typical NES with characteristics of leucine-rich sequence by bioinformatics. CONCLUSION The region from aa219 to aa450 is primarily defined as an atypical NES in BRD7.
Animals ; Base Sequence ; COS Cells ; Cell Nucleus ; metabolism ; Chlorocebus aethiops ; Chromosomal Proteins, Non-Histone ; genetics ; metabolism ; Cytoplasm ; metabolism ; Escherichia coli ; genetics ; metabolism ; Green Fluorescent Proteins ; genetics ; Humans ; Molecular Sequence Data ; Nuclear Export Signals ; Recombinant Proteins ; genetics ; metabolism

Background:The musk glands of adult male Chinese forest musk deer(Moschus berezovskii Flerov,1929)(FMD),which are considered as special skin glands,secrete a mixture of sebum,lipids,and proteins into the musk pod.Together,these components form musk,which plays an important role in attracting females during the breeding season.However,the relationship between the musk glands and skin of Chinese FMD remains undiscovered.Here,the musk gland and skin of Chinese FMD were examined using histological analysis and RNA sequencing(RNA-seq),and the expression of key regulatory genes was evaluated to determine whether the musk gland is derived from the skin.Methods:A comparative analysis of musk gland anatomy between juvenile and adult Chinese FMD was conducted.Then,based on the anatomical structure of the musk gland,skin tissues from the abdomen and back as well as musk gland tissues were obtained from three juvenile FMD.These tissues were used for RNA-seq,hematoxylin-eosin(HE)staining,immunohistochemistry(IHC),western blot(WB),and quantitative real-time polymerase chain reaction(qRT-PCR)experiments.Results:Anatomical analysis showed that only adult male FMD had a complete glandular organ and musk pod,while juvenile FMD did not have any well-developed musk pods.Transcriptomic data revealed that 88.24%of genes were co-expressed in the skin and musk gland tissues.Kyoto Encyclopedia of Genes and Genomes(KEGG)signaling pathway analysis found that the genes co-expressed in the abdomen skin,back skin,and musk gland were enriched in biological development,endocrine system,lipid metabolism,and other pathways.Gene Ontology(GO)enrichment analysis indicated that the genes expressed in these tissues were enriched in biological processes such as multicellular development and cell division.Moreover,the Metascape predictive analysis tool demonstrated that genes expressed in musk glands were skin tissue-specific.qRT-PCR and WB revealed that sex-determining region Y-box protein 9(Sox9),Caveolin-1(Cav-1),and androgen receptor(AR)were expressed in all three tissues,although the expression levels differed among the tissues.According to the IHC results,Sox9 and AR were expressed in the nuclei of sebaceous gland,hair follicle,and musk gland cells,whereas Cav-1 was expressed in the cell membrane.Conclusions:The musk gland of Chinese FMD may be a derivative of skin tissue,and Sox9,Cav-1,and AR may play significant roles in musk gland development.