STATE OF METHYLATION OF THE HUMAN OSTEOCALCIN GENE IN BONE-DERIVED AND OTHER TYPES OF CELLS

DNA methylation is a general mechanism of controlling tissue-specific gene expression. Osteocalcin is a bone matrix protein whose expression is limited almost entirely to osteoblasts. We were interested in dete rmining whether the state of methylation of the osteocalcin gene plays a role in its expression by studying human bone-derived (MG-63, U2-Os , SaOs-2) and other types (normal lymphocytes, A-498, Hep G2) of cells . Reverse transcription-polymerase chain reaction (RT-PCR) analysis re vealed that osteocalcin mRNA production is stimulated by 1,25(OH)(2)D- 3 in MG-63 and induced in SaOs-2 but not in U2-Os osteoblast-like oste osarcoma cells. Genomic analysis of the human osteocalcin gene showed that the local surroundings of this single-copy gene are identical in ail cell lines studied. Using an isoschizomeric pair of restriction en zymes and Southern analysis, we found that the osteocalcin gene is ide ntically methylated in all three osteosarcoma cell lines, The same sit es are also methylated in human normal lymphocytes and A-498 kidney ce lls, whereas the degree of methylation is higher in Hep G2 human hepat ocellular carcinoma cells. Furthermore, the osteocalcin gene was ident ically protected against enzymatic digestion at the chromatin lever in normal lymphocytes and in all cell lines studied. Induction of hypome thylation of DNA by 5-azacytidine treatment did not cause an induction of osteocalcin synthesis in these cell lines. On the contrary, it att enuated the induction by 1,25(OH)(2)D-3 in MG-63 cells. In gel mobilit y shift assays, human vitamin D receptor and the AP-1 transcription fa ctor bound to an unmethylated response element oligonucleotide of the osteocalcin gene with greater affinity than to an in vitro methylated response element. These results indicate that the in vivo methylation state of the osteocalcin gene at sites determined in this study does n ot correlate with the inducibility of this gene. Nevertheless, the in vitro results clearly indicated that hypomethylation of critical regio ns of the osteocalcin gene promoter is a potential mechanism influenci ng effective binding of specific nuclear factors and, consequently, ge ne expression. (C) 1997 Wiley-Liss, Inc.