Global Methylation Screening in Mesenchymal Stem Cell Genomes during Osteogenesis

Understanding the role of epigenetic changes such as DNA methylation has become critical for understanding many biological processes including adult stem cell differentiation. Restriction landmark genomic scanning (RLGS) uses a two-dimensional display of end-labeled DNA restriction fragments to detect genomic DNA methylation. Virtual (Vi)-RLGS computational software can be used to generate a similar landmark map based on genomic sequence information. Objectives: To identify differentially methylated regions between uninduced- and osteogenically-induced mouse bone marrow-derived stromal stem cell (BMSC) genomes using real- and Vi-RLGS methods. Methods: Clonal cultures of C57BL/6J mouse BMSCs were established by frequent subculture immortalization and limiting dilution. RLGS was performed for genomic DNA extracted from uninduced- or osteogenically-induced BMSCs by using a combination of NotI, PvuII, and PstI restriction enzymes. Vi-RLGS was performed using RLGSSim software (RIKEN, Japan) with the same combination of restriction enzymes used for real-RLGS. Correspondence between real and virtual RLGS spots for differentially methylated loci was confirmed by bisulfite sequence analysis with primers derived from sequences predicted by Vi-RLGS. NCBI genome database analysis was used to identify the specific genomic region and gene associated with the differentially methylated region. Results: The real RLGS method found that 3.8% of the spots were differentially expressed between uninduced- and osteogenically-induced BMSCs. Vi-RLGS identified a unique RLGS locus/spot that was present (unmethylated) in uninduced-BMSCs but absent (methylated) in osteogenically-induced BMSCs. NCBI database analysis showed that the RLGS locus/spot was located within an intron of the palladin gene on chromosome 8. Conclusion: These results demonstrate that Vi-RLGS in conjunction with real RLGS successfully associated the palladin gene with differential methylation during osteogenic differentiation of BMSCs.