Methylation status of a single CpG locus 3 bases upstream of TATA-box of receptor activator of nuclear factor-kappaB ligand (RANKL) gene promoter modulates cell- and tissue-specific RANKL expression and osteoclastogenesis.
Receptor activator of nuclear factor-kappaB ligand (RANKL) expression is tissue specific and limited to certain subsets of T-lymphocytes and stromal/osteoblastic cells. Even among osteoblasts, RANKL is expressed on about 20% of osteoblasts of the normal mouse. To clarify the mechanism of population-specific RANKL expression, we analyzed the effect of CpG methylation on its transcription, mRNA and protein expression as well as on osteoclastogenesis. Subpopulations of ST2 cells were used: P9, which expresses RANKL and supports osteoclastogenesis, and P16, which does not. By sodium bisulfite mapping, the rate of CpG methylation of the -65/+350 region, especially of CpG locus no. 1 three bases upstream of the TATA-box, was higher in P16 than in P9 ST2 cells. ChIP and gel shift assay showed that methylated CpG locus no. 1 was a target of MeCP2 binding that, in turn, blocked the binding of the TATA-box binding protein to the TATA-box. In vitro methylation by SssI of the promoter construct reduced its transcriptional activity at the steady state and its response to 1alpha,25(OH)2 vitamin D3. Conversely, treatment with DNA methylase inhibitor, 5-aza-2'-deoxycytidine, significantly restored RANKL expression and osteoclastogenesis in P16 cells. Except for primary cultured osteoblasts, CpG locus no. 1 was frequently methylated in various normal mouse tissues. We propose that the methylation status of the CpG locus three bases upstream of the TATA-box modulates the control of cell- and tissue-specific expression of RANKL gene and osteoclastogenesis. The heterogeneity of stromal/ osteoblastic cells in response to bone-resorbing stimuli may be attributed, in part, to the methylation status of the RANKL gene promoter.
Kitazawa
Riko
R
Division of Molecular Pathology, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan.
Kitazawa
Sohei
S
D000818:Animals; D001374:Azacitidine / Q000031:analogs & derivatives / Q000737:chemistry; D001854:Bone Marrow Cells / Q000378:metabolism; D018899:CpG Islands; D019175:DNA Methylation; D000077209:Decitabine; D005786:Gene Expression Regulation; D051379:Mice; D008954:Models, Biological; D010006:Osteoblasts / Q000378:metabolism; D010010:Osteoclasts / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D053245:RANK Ligand / Q000235:genetics*; D012333:RNA, Messenger / Q000378:metabolism; D018167:Receptors, Calcitriol / Q000378:metabolism; D017154:Stromal Cells / Q000378:metabolism
D053245:RANK Ligand; D012333:RNA, Messenger; D018167:Receptors, Calcitriol; D000077209:Decitabine; D001374:Azacitidine