Villagra Alejandro, Gutiérrez José, Paredes Roberto, Sierra José, Puchi Marcia, Imschenetzky Maria, Wijnen Av Andre van, Lian Jane, Stein Gary, Stein Janet, Montecino Martin
Departamento de Biologia Molecular, Facultad de Ciencias Biologicas, Universidad de Concepcion, Casilla 160-C, Concepcion, Chile.
J Cell Biochem. 2002;85(1):112-22.
Chromatin remodeling of the bone-specific rat osteocalcin (OC) gene accompanies the onset and increase in OC expression during osteoblast differentiation. In osseous cells expressing OC, the promoter region contains two nuclease hypersensitive sites that encompass the elements that regulate basal tissue-specific and vitamin D-enhanced OC transcription. Multiple lines of evidence indicate that DNA methylation is involved in maintaining a stable and condensed chromatin organization that represses eukaryotic transcription. Here we report that DNA methylation at the OC gene locus is associated with the condensed chromatin structure found in cells not expressing OC. In addition, we find that reduced CpG methylation of the OC gene accompanies active transcription in ROS 17/2.8 rat osteosarcoma cells. Interestingly, during differentiation of primary diploid rat osteoblasts in culture, as the OC gene becomes increasingly expressed, CpG methylation of the OC promoter is significantly reduced. Inhibition of OC transcription does not occur by a direct mechanism because in vitro methylated OC promoter DNA is still recognized by the key regulators Runx/Cbfa and the vitamin D receptor complex. Furthermore, CpG methylation affects neither basal nor vitamin D-enhanced OC promoter activity in transient expression experiments. Together, our results indicate that DNA methylation may contribute indirectly to OC transcriptional control in osteoblasts by maintaining a highly condensed and repressed chromatin structure.
在成骨细胞分化过程中，骨特异性大鼠骨钙素（OC）基因的染色质重塑伴随着OC表达的起始和增加。在表达OC的骨细胞中，启动子区域包含两个核酸酶超敏位点，这些位点包含调节基础组织特异性和维生素D增强的OC转录的元件。多条证据表明，DNA甲基化参与维持稳定且紧密的染色质组织，从而抑制真核转录。在此我们报告，OC基因位点的DNA甲基化与在不表达OC的细胞中发现的紧密染色质结构相关。此外，我们发现OC基因的CpG甲基化减少伴随着ROS 17/2.8大鼠骨肉瘤细胞中的活跃转录。有趣的是，在培养的原代二倍体大鼠成骨细胞分化过程中，随着OC基因表达增加，OC启动子的CpG甲基化显著降低。OC转录的抑制并非通过直接机制发生，因为体外甲基化的OC启动子DNA仍能被关键调节因子Runx/Cbfa和维生素D受体复合物识别。此外，在瞬时表达实验中，CpG甲基化既不影响基础OC启动子活性，也不影响维生素D增强的OC启动子活性。总之，我们的结果表明，DNA甲基化可能通过维持高度紧密且受抑制的染色质结构，间接促进成骨细胞中OC的转录调控。
