Methylation status of CpG islands in the promoter regions of signature genes during chondrogenesis of human synovium-derived mesenchymal stem cells.
OBJECTIVE
Human synovium-derived mesenchymal stem cells (MSCs) can efficiently differentiate into mature chondrocytes. It has been suggested that DNA methylation is one mechanism that regulates human chondrogenesis; however, the methylation status of genes related to chondrogenic differentiation is not known. The purpose of this study was to investigate the CpG methylation status in human synovium-derived MSCs during experimental chondrogenesis, with a view toward potential therapeutic use in osteoarthritis.


METHODS
Human synovium-derived MSCs were subjected to chondrogenic pellet culture for 3 weeks. The methylation status of 12 regions in the promoters of 10 candidate genes (SOX9, RUNX2, CHM1, FGFR3, CHAD, MATN4, SOX4, GREM1, GPR39, and SDF1) was analyzed by bisulfite sequencing before and after differentiation. The expression levels of these genes were analyzed by real-time reverse transcription-polymerase chain reaction. Methylation status was also examined in human articular cartilage.


RESULTS
Bisulfite sequencing analysis indicated that 10 of the 11 CpG-rich regions analyzed were hypomethylated in human progenitor cells before and after 3 weeks of pellet culture, regardless of the expression levels of the genes. The methylation status was consistently low in SOX9, RUNX2, CHM1, CHAD, and FGFR3 following an increase in expression upon differentiation and was low in GREM1 and GPR39 following a decrease in expression upon chondrogenesis. One exceptional instance of a differentially methylated CpG-rich region was in a 1-kb upstream sequence of SDF1, the expression of which decreased upon differentiation. Paradoxically, the hypermethylation status of this region was reduced after 3 weeks of pellet culture.


CONCLUSIONS
The DNA methylation levels of CpG-rich promoters of genes related to chondrocyte phenotypes are largely kept low during chondrogenesis in human synovium-derived MSCs.
Ezura
Yoichi
Y
Department of Molecular Pharmacology, Tokyo Medical and Dental University, Tokyo, Japan. ezura.mph@mri.tmd.ac.jp
Sekiya
Ichiro
I
Koga
Hideyuki
H
Muneta
Takeshi
T
Noda
Masaki
M
D020219:Chondrogenesis / Q000502:physiology*; D050659:Core Binding Factor Alpha 1 Subunit / Q000235:genetics; D018899:CpG Islands / Q000502:physiology*; D019175:DNA Methylation; D016326:Extracellular Matrix Proteins / Q000235:genetics; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000235:genetics; D064235:Matrilin Proteins; D008565:Membrane Proteins / Q000235:genetics; D059630:Mesenchymal Stem Cells / Q000502:physiology*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D051498:Receptor, Fibroblast Growth Factor, Type 3 / Q000235:genetics; D043562:Receptors, G-Protein-Coupled / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D055758:SOX9 Transcription Factor / Q000235:genetics; D055751:SOXC Transcription Factors / Q000235:genetics; D013583:Synovial Membrane / Q000166:cytology* / Q000378:metabolism
D050659:Core Binding Factor Alpha 1 Subunit; D016326:Extracellular Matrix Proteins; C495797:GPR39 protein, human; C486399:GREM1 protein, human; D036341:Intercellular Signaling Peptides and Proteins; C577548:MATN4 protein, human; D064235:Matrilin Proteins; D008565:Membrane Proteins; C493709:RUNX2 protein, human; D043562:Receptors, G-Protein-Coupled; C083431:SOX4 protein, human; D055758:SOX9 Transcription Factor; C527932:SOX9 protein, human; D055751:SOXC Transcription Factors; C422444:TNMD protein, human; C496365:FGFR3 protein, human; D051498:Receptor, Fibroblast Growth Factor, Type 3