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    The family of MAGE genes is well known due to the majority of MAGE genes expressing specifically in tumor tissues while restrictedly in normal tissues. MAGE-D4 is one of the MAGE family and considered as a promising target for glioma immunotherapy because of its overexpression in glioma and restricted expression in normal tissues. Whereas the mechanism of MAGE-D4 heterogeneous expression in glioma has not yet been elucidated. In this study, the transcriptional regulation mechanism of MAGE-D4 in glioma is focused from the perspectives of promoter methylation and SP1. Dual-luciferase reporter assay was performed to identify the core promoter of MAGE-D4 gene. Mass spectrometry was applied to quantify the methylation status of MAGE-D4 promoter in 50 glioma and 9 normal brain tissues. The influence of methylation and SP1 on MAGE-D4 transcriptional activity was evaluated by dual-luciferase reporter assay, qRT-PCR, western blot and ChIP-qPCR. Decitabine, an epigenetic drug, was used to treat the glioma cells. Then the treated cells were evaluated the influence of demethylation on SP1 binding to MAGE-D4 promoter. The -358 to +172 bp region was identified as the core promoter of MAGE-D4 gene which demonstrated hypomethylated and negative correlation between methylation level and MAGE-D4 mRNA expression in glioma tissues. For single CpG unit analysis, 8 CpG units (CpG unit 1, 2, 3, 4, 5, 6, 9 and 12) in MAGE-D4 core promoter showed hypomethylated in glioma and the methylation level of CpG unit 6 was positively associated with the prognosis of glioma patients. Furthermore, the methylation level of CpG unit 1 and 6 was negative negatively correlated with MAGE-D4 mRNA expression. Then, the results demonstrated that the promoter activity of MAGE-D4 was decreased by methylation in glioma cell lines. In addition, SP1 can binds directly to the MAGE-D4 promoter leading to up-regulation of MAGE-D4 mRNA through activation of its promoter. Finally, demethylation of MAGE-D4 promoter could benefit the SP1 binding and resulting co-activation of MAGE-D4 promoter by demethylation and SP1 in glioma cell lines. These findings indicate that the synergies of promoter hypomethylation and SP1 up-regulated MAGE-D4 transcription in glioma, which implies a potential approach to resolve the heterogeneous expression of MAGE-D4 in order to establish foundation for the MAGE-D4 based glioma therapy. AJTR Copyright © 2021.


    Chang Liu, Yingying Ge, Bin Luo, Xiaoxun Xie, Ning Shen, Weixia Nong, Shuiqing Bi, Lina Lin, Xing Wei, Song Wu, Shaowen Xiao, Qingmei Zhang. Synergistic regulation of methylation and SP1 on MAGE-D4 transcription in glioma. American journal of translational research. 2021;13(4):2241-2255

    PMID: 34017386

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