S. Ryhanen et al., STATE OF METHYLATION OF THE HUMAN OSTEOCALCIN GENE IN BONE-DERIVED AND OTHER TYPES OF CELLS, Journal of cellular biochemistry, 66(3), 1997, pp. 404-412
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.