Synergistic regulation of methylation and SP1 on MAGE-D4 transcription in glioma

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

Am J Transl Res. 2021 Apr 15;13(4):2241-2255. eCollection 2021.

Authors

Chang Liu¹, Yingying Ge^{2

3}, Bin Luo^{2

3}, Xiaoxun Xie^{2

3}, Ning Shen⁴, Weixia Nong^{2

3}, Shuiqing Bi¹, Lina Lin², Xing Wei², Song Wu¹, Shaowen Xiao¹, Qingmei Zhang^{2

3}

Affiliations

¹ Department of Neurosurgery, The First Affiliated Hospital of Guangxi Medical University Nanning, Guangxi, P. R. China.
² Department of Histology and Embryology, School of Pre-Clinical Medicine, Guangxi Medical University Nanning, Guangxi, P. R. China.
³ Central Laboratory, School of Pre-Clinical Medicine, Guangxi Medical University Nanning, Guangxi, P. R. China.
⁴ Department of Oral and Maxillofacial Surgery, The People's Hospital of Guangxi Zhuang Autonomous Region Nanning, Guangxi, P. R. China.

PMID: 34017386
PMCID: PMC8129322

Abstract

Background: 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.

Methods: 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.

Results: 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.

Conclusion: 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.

Keywords: Glioma; MAGE-D4; SP1; methylation.