Bisulfite sequencing analysis of mouse osteocalcin and osteopontin promoter regions

Early studies proposed that DNA methylation might regulate gene expression during stem cell differentiation. However, little is known about DNA methylation during osteogenic differentiation of mesenchymal stem cells. Objectives: To analyze the methylation status of osteocalcin and osteopontin promoter regions in genomes of bone marrow-derived stromal cells (BMSCs) before and after osteogenic differentiation. Methods: Clonal cultures of C57BL/6J mouse BMSCs were established. Genomic DNA and total RNA were extracted from uninduced or osteogenically induced BMSCs, gingival fibroblasts, brain, calvaria, and C2C12 myoblasts. DNA was treated with bisulfite conversion and amplified by PCR using primer sets for osteocalcin and osteopontin promoter regions (amplified regions contained 6 and 4 CpG sites, respectively). Subsequently, PCR products were sequenced and the methylation status of the CpG sites was analyzed. Relative expression levels were determined by RT-PCR. Results: The overall methylation pattern in the BMSC osteocalcin promoter region did not significantly change after osteogenic differentiation. However, the fourth CpG site was methylated less frequently (<15%) in osteogenic BMSCs, C2C12, and calvaria, all of which highly expressed osteocalcin mRNA. In contrast, the frequency of methylation at this site was high (>60%) in fibroblasts and brain, which did not express osteocalcin transcripts. In the BMSC osteopontin promoter region, the frequency of methylation at the fourth CpG site was specifically decreased after osteogenic differentiation. In addition, this CpG site was hypomethylated (<30%) in osteogenic BMSCs, fibroblasts, C2C12, and calvaria, all of which highly expressed osteopontin mRNA, and hypermethylated (>60%) in uninduced BMSCs and brain, which expressed osteopontin at lower levels. Conclusion: These results suggest that the methylation status of specific CpG loci in mouse osteocalcin and osteopontin promoter regions vary depending on cell type and differentiation stage, and may thus be involved in the regulation of gene expression.