Abstract
SPAN-Xb is a spermatid-specific protein encoded by the SPAN-XB gene on chromosome Xq27.1. It is a novel Cancer-Testis antigen in multiple myeloma. We recently demonstrated that SPAN-Xb expression in myeloma cells is regulated through promoter methylation and could be upregulated by IL-7 and GM-CSF. In this present study, we set out to investigate the mechanism of SPAN-XB expression and the promoter association with the methyl-CpG binding protein, MeCP2. Elucidation of these interactions is likely to shed light on potential therapeutic strategies to upregulate antigen levels that could be used to improve the outcome of SPAN-Xb-based tumor vaccines.
We previously showed that the putative SPAN-XB promoter resides within exon 1 and contains 433 bp, starting from position −546 to position −104 upstream of the translational start site. Using a panel of truncated promoter constructs generated from the 3′ and 5′ ends of the promoter gene, we localized the core sequence of SPAN-XB promoter to the 73 base pairs at the 3-end of the promoter, a region that lacks CpG dinucleotides within the full length promoter. There are 11 CpG dinucleotides within the putative SPAN-XB promoter. We previously found that DNA methylation provides the primary regulatory mechanism for SPAN-XB gene expression.
We also identified that hypomethylation at positions −310, −307, −299 and −221 strongly predicted for SPAN-XB expression, suggesting the involvement of these four CpG dinucleotides in the regulation of SPAN-XB gene expression through DNA methylation. In the present study, using reporter gene expression assays, we found that the core promoter function is significantly modulated by these adjacent CpG sequences so that mutation of the CpG dinucleotides outside the core sequence resulted in changes in the promoter function. We also previously demonstrated by bisulfite conversion and sequence analysis the association between methylation at specific CpG dinucleotides with repression of SPAN-XB gene. Here, we extended our study to determine whether or not the methylated cytosine binding protein, MeCP2, interacts with the SPAN-XB promoter gene in myeloma cells. Chromatin immunoprecipitation assays revealed a specific association of the MeCP2 with SPAN-XB promoter, and MeCP2 binding strongly correlated with repression of the SPAN-XB gene in myeloma cell lines and CD138-enriched fresh myeloma cells. Reactivation of the SPAN-XB gene by 5-azacytidine treatment resulted in the loss of MeCP2 from this site. We, therefore, conclude that SPAN-XB promoter consists of a core sequence and a regulatory element; the core element resides within the 73 base pairs at the 3′ end of the full length promoter. SPAN-XB gene expression by the core sequence is regulated in myeloma cells by specific CpG nucleotides and associated with MeCP2 binding to the promoter sequence.
Core promoter sequence of SEMG I spans between the two putative GATA-1 binding domains and is responsive to IL-4 and IL-6 in myeloma cells
