Expression of the CT Antigen, SPAN-Xb, Is Regulated through Promoter Methylation.
Authors: Wang Zhiqing , Zhang Yana , Zhang Jian , Lim Seah H. 
Published: April 2020

Abstract
SPAN-Xb is a spermatid protein that we have recently identified as a novel Cancer-Testis (CT) antigen in hematologic malignancies. We have also shown that SPAN-Xb expression in tumor cell lines could be upregulated by 5-azacytidine, GM-CSF and IL-7. The ability of 5-azacytidine to increase SPAN-Xb expression suggests that SPAN-Xb gene expression, like the other CT antigens, may be regulated through promoter methylation. On the other hand, the ability of GM-CSF and IL-7 to increase SPAN-Xb expression remained to be determined. In this study, we set out to determine whether or not promoter methylation regulates SPAN-Xb gene expression and the effects of GM-CSF and IL-7 on SPAN-Xb expression is due to their action on the promoter activity.
We first isolated and cloned the SPAN-Xb promoter gene into the CAT (chloramphenicol acetyl transferase) reporter system, pCAT*3-Enhancer vector. In vitro methylation was achieved using SssI methylase and the recombinant vectors were transfected into the myeloma cell line, RPMI 8226 cells. CAT activity was assayed in the lysate of the transfectants 48–72 hours after gene transfer. We observed that CAT activitiy in transfectants containining demethylated recombinant pCAT*3-SPAN-Xb promoter vector. In contrast, CAT activity was abrogated once the recombinant vector was methylated in vitro, supporting the role of DNA methylation in the regulation of SPAN-Xb gene expression. CAT activity in the transfectants containing the demethylated vector could be further increased by GM-CSF and IL-7, suggesting that the increase in SPAN-Xb expression we have observed in cells treated with GM-CSF and IL-7 may be the actions of these cytokines on the SPAN-Xb promoter. These cytokines alone, however, were unable to induce CAT activity since transfectants containing the methylated promoter sequence remained negative for the CAT activity even with the addition of GM-CSF or IL-7.
To further evaluate the role of DNA methylation on the expression of SPAN-Xb, we carried out the bisulfite conversion assays using genomic DNA from tumor cell lines, normal testis, blood, kidney, pancreas and spleen. Following bisulfite conversion, the modified genomic DNA was subjected to PCR amplification, cloning and sequence analysis. Five clones from each tissues were randomly picked for sequence analysis. A total of 11 CpG islands were identified within the promoter sequence. They were put together into 7 groups according to their positions in the sequence: Group I: −502; Group II: −474; Group III: −450; Group IV: −341; Group V: −311 to −300; Group VI: −226 to −222; Group VII: −184 to −181. Following sequence analysis, we observed that SPAN-Xb expressor (normal testis) was consistently demethylated within Groups V and VI CpG islands. In contrast, SPAN-Xb-negative tissues were consistently methylated at these two CpG islands, localizing the promoter activity of the sequence to these two areas of the promoter. The methylation status at the other CpG island did not predict SPAN-Xb expression.
We therefore conclude that: 1. SPAN-Xb expression is regulated by promoter methylation; 2. GM-CSF and IL-7 increase SPAN-Xb expression through their action on the SPAN-Xb promoter, and; 3. The CpG islands between −311 and −300 and −226 and −222 are the regions within the SPAN-Xb promoter sequence that control gene expression.