SPAN‐XB core promoter sequence is regulated in myeloma cells by specific CpG dinucleotides associated with the MeCP2 protein

Abstract

SPAN-Xb 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 shed light on potential therapeutic strategies to upregulate antigen levels for SPAN-Xbbased tumor vaccines. Using a panel of truncated promoter constructs, we localize the core sequence of SPAN-XB promoter to the 73 bp at the 3 0 end of the promoter, a region within the full length promoter that lacks CpG dinucleotides. Reporter gene expression assays showed that the core promoter function is significantly modulated by the adjacent CpG sequences. Chromatin immunoprecipitation assays revealed a specific association of MeCP2 with the promoter, and MeCP2 binding strongly correlated with repression of SPAN-XB gene. 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 core promoter function in myeloma cells is associated with MeCP2 protein binding and regulated by specific CpG dinucleotide sequences. ' 2006 Wiley-Liss, Inc.Key words: SPAN-Xb; CT antigen; MeCP2 protein SPAN-Xb is a spermatid-specific protein encoded by the SPAN-XB gene on chromosome Xq27.1. SPAN-XB has a genomic organization consisting of 2 exons with a small intron and belongs to the SPAN-X family of genes. 1 SPAN-Xb differs from its other family members (-C, -A1/2 and -D); it contains a 6 amino acid insertion in the middle of the sequence and also contains a potential Nlinked glycosylation site. Also, unlike the other members of the SPAN-X protein that are predominantly nuclear in distribution, immunohistochemistry staining localized SPAN-Xb to the cytoplasm, suggesting that its function is likely to be distinct from those of its other family member proteins. There is no homology between the SPAN-X proteins and any other known proteins. SPAN-Xb was previously shown to be a novel cancer-testis antigen in hematologic malignancies, including multiple myeloma (MM). 2 This is a new class of antigens that consist of a group of normal testicular-specific protein aberrantly expressed in tumor cells. The aberrant expression of SPAN-Xb in tumor cells is associated in vivo with the generation of high titers IgG directed at SPAN-Xb protein, suggesting the immunogenicity of the protein in the cancer-bearing autologous host. The immunogenicity and highly restricted normal tissue expression pattern makes SPANXb an excellent candidate protein to be used for the design of tumor vaccines.One major problem preventing effective tumor immunotherapy is the heterogeneity of tumor antigen expression, even within individual specimens. Furthermore, these antigens are only detected in the tumor cells from approximately 30% of patients, although the expression frequency genera...

The Roles of the Methyl-CpG Binding Proteins in Cancer

“…26 As well as its role in Rett syndrome, a neurodevelopmental disorder, 64 it has also been shown to have a role in tumorigenesis 2,3 as many cancers rely on genes becoming silenced via hypermethylation of their promoter. Recently, it has been shown to target genes in vivo in myeloma, 65 hematological malignancies, 66 and breast, 67,68 colorectal, 69 lung, 70 liver, 71 and prostate 72 cancer. Despite its relevance to cancer, we still have little understanding of how the protein itself is regulated.…”

Cancer‐testis antigens in haematological malignancies

“…Histone deacetylation suppresses gene expression by removing the acetyl groups from histones, which leads to compact and transcriptionally inactive chromatin (Jones et al, 1998;Ng & Bird, 2000). Using antibodies directed at the MeCP2 protein in chromatin immunoprecipitation, it has also been demonstrated that MeCP2 binding to the promoter sequence of SPAN-Xb (Wang et al, 2006b) and SEMG 1 (Zhang et al, 2007) was associated with repression of these genes. Treatment of tumour cells with 5-azacytidine resulted in the dissociation of the MeCP2 from the promoters and subsequent expression of the SPAN-Xb and SEMG 1 genes and proteins.…”

Epigenetic Mechanisms Underlying the Dynamic Expression of Cancer-Testis Genes, PAGE2, -2B and SPANX-B, during Mesenchymal-to-Epithelial Transition

“…Expression of all CT genes studied thus far including PAGE2 and SPANX-B have been associated with the demethylation of CpG residues within regions proximal to the transcription start site [1], [2], [23]–[26]. In this line, both PAGE2 and SPANX-B can be up-regulated by 5-aza 2′-deoxycytidine treatment ( Figure S8 ).…”