Identification Of Mineral Dust-induced Gene Interaction Partners Through Proteomics

Mineral dust-induced gene (mdig, also named as mina53, MINA, or NO52), was first identified in alveolar macrophages (AMs) of coal miners with chronic lung diseases. Increased expression of mdig has been implicated in the pathogenesis of a number of human cancers. However, how mdig contributes to the cancer cell growth or genes epigenetic regulation remains to be fully understood. Previous studies suggested that mdig protein may be involved in the demethylation of the tri-methyl lysine 9 of histone H3 (H3K9me3) through its JmjC domain. But distinguishing from typical histone demethylases, the mdig proteins lacks chromatin-binding domains, such as WD repeat and/or PHD fingers. Thus, the activities of mdig on chromatin or histone proteins are most likely achieved through its interaction with other chromatin binding partners.Part 1 Identification of mineral dust-induced gene interaction partners through proteomicsObject:In this study, we investigate the interacting partners of mineral dust-induced gene through proteomic, and explore the function of mdig in cell metabolisms and cancer development.Method:The protein complexes are immunoprecipitated by anti-mdig antibody from either the lung cancer cell line A549 or the human bronchial epithelial cell line BEAS-2B cells. After stained by coomassie blue, protein bands can be observed in SDS-PAGE gels. Then the protein complexes was identified by proteomics analysis using both Orbitrap Fusion and Orbitrap XL nano ESI-MS/MS system in four independent experiments. The interaction of mdig with some of detected proteins was further validated by immunoblotting. The pathway analysis was performed by IPA software.Result:On SDS-PAGE gels, three to five unique protein bands were consistently observed in the complexes pulled-down by mdig antibody, but not the control IgG. In addition to the mdig protein, several DNA repair or chromatin binding proteins, including XRCC5, XRCC6, RBBP4, CBX8, PRMT5, and TDRD, were identified in the mdig pull-down complexes. Immunoblotting results confirmed the binding between mdig and these proteins. The banding pattern obtained with SDS-PAGE is very similar for the BEAS-2B and A549 cell mdig IP samples. However, several proteins that interact with mdig were only detected in the BEAS-2B cells, such as CBX3 and CBX5.Conclusion:These data, provide evidence suggesting that mdig accomplishes its functions on chromatin, DNA repair and cell growth through interacting with the partner proteins.Part 2 Mdig protein participates in DNA non-homologous end-joining repairObject:In mammalian cells, DNA double-strand breaks (DSBs) repair pathways are essential for the maintenance of genomic integrity and the prevention of tumorigenesis. Proteomics analysis results indicated the interacting role of mdig with the key subunits of non-homologous end-joining repair, such as XRCC5, XRCC6, DNA-PK, etc. This study aims to explore the regulatory function of mdig in NHEJ pathway, and give us the evidence that mdig achieved its function in DNA damage repair by interacting with XRCC6.Method:A549 cell lines with stable expression of control vector, mdig-GFP, control shRNA, or mdig shRNA were established. All of these cells were exposed to 30 uM phleomycin for 0,0.25,0.5,1,2,4 hours. The proteins level of NHEJ pathway subunits and DNA damage kinases was tested by immunoblotting experiments. The expression of yH2AX and pDNA-PK was measured by immunofluorescent staining. Phleomycin-induced DNA damage was detected by comet assay.Result:Mdig protein can physically bind to NHEJ subunit XRCC6, and affect the expression of pDNA-PK and pATM following phleomycin treatment. Overexpression of mdig sensitizes phleomycin-induced double-strand breaks of DNA in A549 cells. Silencing mdig by shRNA, on the other hand, alleviated DNA DSBs induced by phleomycin.Conclusion:Association of mdig with the complexes of XRCC5, XRCC6, and DNA-PK may weaken the NHEJ repair capacity, and consequently, increase the opportunity of genomic mutation and carcinogenesis in response to toxins that target genomic DNA.