Semenogelin 1 Expression in Myeloma Cells: Interaction between DNA Methylation, MeCP2 Protein and Specific Cytokines.

Semenogelin (SEMG) 1 is a protein of semen coagulum with limited expression in normal tissues. It plays an important role in sperm clotting and is normally degraded into smaller fragments by prostate-specific antigen. The gene encoding SEMG 1 has been localized to the long arm of chromosome 20, a region of chromosome 20 that is frequently deleted in myeloproliferative diseases and myelodysplastic syndrome. We previously found SEMG 1 to be aberrantly expressed by tumor cells of hematologic malignancies, including multiple myeloma (MM). The aberrant expression of SEMG 1 in tumor cells of hematologic malignancies is associated in vivo with the generation of high titers IgG directed at SEMG 1 protein, suggesting the immunogenicity of the protein in the cancer-bearing patients. The combination of being immunogenic in cancer patients and limited expression in normal tissue expression makes SEMG 1 a potential candidate protein for tumor vaccines. In this study, we have set out to determine the molecular mechanisms associated with SEMG 1 expression in MM. Treatment of SEMG 1-positive MM cells with IL-4 and IL-6 resulted in the upregulation of SEMG 1 expression. In SEMG 1-negative MM cells, SEMG 1 expression could only be upregulated by IL-4 and IL-6 after pre-treatment with 5-azacytidine, suggesting that DNA methylation is likely the primary regulatory mechanism for SEMG 1 expression. Treatment of SEMG 1-negative MM cells induced SEMG 1 gene and protein expression. SEMG 1 promoter only has one CpG dinucleotide, located at position -11 of the gene. Bisulfite conversion and nucleotide sequencing was carried out on the genomic DNA from MM cells. MM cells that did not express SEMG 1 were 100% methylated. In contrast, 100% of the sequences obtained from SEMG 1-positive MM cells were unmethylated at the cytosine residue of the CpG dinucleotide. Induction of SEMG 1 expression by 5-azacytidine was associated with a decrease in the % of methylation of this cytosine residue, from 100% to 20%. These results, therefore, further implicate the role of DNA methylation in the primary regulation of SEMG 1 expression. Applying antibodies directed at MeCP2 in chromatin immunoprecipiation, MeCP2 protein binding to the SEMG 1 promoter sequence of MM cell lines and fresh MM cells was correlated to SEMG 1 gene silencing, suggesting the likely role of the MeCP2 protein in SEMG 1 gene repression. Further analysis by promoter truncation studies indicated the dependence of the promoter function on the sequence spanning the two putative GATA-1 binding sites within the gene. Using a reporter gene expression system, both IL-4 and IL-6 were found to upregulate SEMG 1 via their effect on the hypomethylated promoter gene. The effects of IL-4 and IL-6 on the function of the SEMG 1 promoter were dose dependent. In conclusion, the present study demonstrates that SEMG 1 expression in MM cells is regulated through the interaction between primary regulatory effect of promoter methylation, MeCP2 protein binding and the secondary effect of specific cytokines. Our findings provide insight into the molecular mechanisms affecting SEMG 1 expression and suggest the possible use of hypomethylating agents to upregulate SEMG 1 expression in tumor cells. Obviously, it remains to be determined whether or not there is a differential dose response among the different normal tissues in their sensitivity to the antigen-inducing effect of these agents.