Nonosseous Cells Research Articles

Redefining the activity of a bone-specific transcription factor: Novel insights for understanding bone formation

Redefining the activity of a bone-specific transcription factor: Novel insights for understanding bone formation

Functional analysis of the two Runx3 promoters in osseous and non-osseous cells: implications for tissue/differentiation specific transcription of distinct isoforms

Searchable abstracts of presentations at key conferences on calcified tissues ISSN 2052-1219 (online)

The Osteogenic Transcription Factor Runx2 Controls Genes Involved in Sterol/Steroid Metabolism, Including Cyp11a1 in Osteoblasts

Steroid hormones including (1,25)-dihydroxyvitamin D3, estrogens, and glucocorticoids control bone development and homeostasis. We show here that the osteogenic transcription factor Runx2 controls genes involved in sterol/steroid metabolism, including Cyp11a1, Cyp39a1, Cyp51, Lss, and Dhcr7 in murine osteoprogenitor cells. Cyp11a1 (P450scc) encodes an approximately 55-kDa mitochondrial enzyme that catalyzes side-chain cleavage of cholesterol and is rate limiting for steroid hormone biosynthesis. Runx2 is coexpressed with Cyp11a1 in osteoblasts as well as nonosseous cell types (e.g. testis and breast cancer cells), suggesting a broad biological role for Runx2 in sterol/steroid metabolism. Notably, osteoblasts and breast cancer cells express an approximately 32-kDa truncated isoform of Cyp11a1 that is nonmitochondrial and localized in both the cytoplasm and the nucleus. Chromatin immunoprecipitation analyses and gel shift assays show that Runx2 binds to the Cyp11a1 gene promoter in osteoblasts, indicating that Cyp11a1 is a direct target of Runx2. Specific Cyp11a1 knockdown with short hairpin RNA increases cell proliferation, indicating that Cyp11a1 normally suppresses osteoblast proliferation. We conclude that Runx2 regulates enzymes involved in sterol/steroid-related metabolic pathways and that activation of Cyp11a1 by Runx2 may contribute to attenuation of osteoblast growth.

Analysis of the Runx2 promoter in osseous and non-osseous cells and identification of HIF2A as a potent transcription activator

Little is known about the upstream regulator of Runx2, a master regulator of osteoblast differentiation in bone tissues. To elucidate the molecular mechanism of Runx2 gene expression, we analyzed Runx2 promoter activity in osseous (MC3T3-E1, KS483, Kusa) and non-osseous (NIH3T3, C3H10T1/2, mouse embryonic fibroblasts) cells and also identified Runx2 upstream regulator using a Runx2 promoter-derived luciferase reporter system. After cloning 15 serial deletion constructs from − 6832 bp/+ 390 bp to − 37 bp/+ 390 bp of the Runx2-P1 promoter, we performed a transient transfection assay in osseous and non-osseous cells. A reduction in Runx2 promoter activity was observed in two regions; one was between − 3 kb and − 1 kb, and the other was between − 155 bp and − 75 bp. The step-down pattern in promoter activity between − 3 kb and − 1 kb was observed only in osseous cells. Interestingly, the step-down pattern between − 155 bp and − 75 bp was revealed in both cell types. Consistently, β-galactosidase staining in axial skeleton of − 3 kb-Runx2-P1- LacZ transgenic mice was positive, but that of all skeletal tissues of − 1 kb-Runx2-P1- LacZ transgenic mice was negative. To identify upstream regulators of the Runx2-P1 promoter, we screened 100 transcription factors using Runx2-P1-luciferase reporter constructs in NIH3T3 fibroblasts and HeLa cells. Among them, HIF2A was identified as the strongest activator of Runx2-P1 promoter activity. A HIF2A-responsive site on the Runx2 promoter was identified between − 106 bp and − 104 bp by mutation analysis. An electrophoretic mobility shift assay and chromatin immunoprecipitation assay confirmed the binding of HIF2A to the Runx2-P1 promoter in vitro and in vivo, respectively. Knock-down using siRNA against HIF2A confirmed that HIF2A is an important regulator of Runx2 gene expression. Collectively, these results suggest that the region between − 3 kb and − 1 kb is required for the minimal skeletal tissue-specific expression of Runx2, and that the region between − 155 bp and − 75 bp is important for its basal transcription, which may be in part mediated by HIF2A in bone tissues.

Runx2- and Histone Deacetylase 3-mediated Repression Is Relieved in Differentiating Human Osteoblast Cells to Allow High Bone Sialoprotein Expression

Bone sialoprotein (BSP) is a bone matrix glycoprotein whose expression coincides with terminal osteoblastic differentiation and the onset of mineralization. In this study we show that BSP expression is considerably increased in confluent Saos-2 human osteosarcoma cells and in differentiating normal human osteoblasts, concomitantly with the decrease of Runx2, a key transcription factor controlling bone formation. Therefore, we investigated the role of Runx2 in the regulation of BSP expression in Saos-2 cells. Using a mobility shift assay, we demonstrated that Runx2 binds to the BSP promoter only in preconfluent cells. Histone deacetylase 3 (HDAC3) has been recently shown to act as a Runx2 co-repressor. Chromatin immunoprecipitation assays demonstrated that both Runx2 and HDAC3 are detectable at the BSP promoter in preconfluent Saos-2 cells but not when they are confluent and overexpress BSP. Consistently, nuclear Runx2 protein level is down-regulated, whereas Saos-2 cells became increasingly confluent. Finally, the suppression of HDAC3, Runx2, or both by RNA interference induced the expression of BSP at both mRNA and protein levels in Saos-2 cells. Our data demonstrate that Runx2 and HDAC3 repress BSP gene expression and that this repression is suspended upon osteoblastic cell differentiation. Both the nuclear disappearance of Runx2 and the non-recruitment of HDAC3 represent new means to relieve Runx2-mediated suppression of BSP expression, thus allowing the acquisition of a fully differentiated and mineralization-competent phenotype by osteoblast cells.

BMP2 Commitment to the Osteogenic Lineage Involves Activation of Runx2 by DLX3 and a Homeodomain Transcriptional Network

Several homeodomain (HD) proteins are critical for skeletal patterning and respond directly to BMP2 as an early step in bone formation. RUNX2, the earliest transcription factor proven essential for commitment to osteoblastogenesis, is also expressed in response to BMP2. However, there is a gap in our knowledge of the regulatory cascade from BMP2 signaling to the onset of osteogenesis. Here we show that BMP2 induces DLX3, a homeodomain protein that activates Runx2 gene transcription. Small interfering RNA knockdown studies in osteoblasts validate that DLX3 is a potent regulator of Runx2. Furthermore in Runx2 null cells, DLX3 forced expression suffices to induce transcription of Runx2, osteocalcin, and alkaline phosphatase genes, thus defining DLX3 as an osteogenic regulator independent of RUNX2. Our studies further show regulation of the Runx2 gene by several homeodomain proteins: MSX2 and CDP/cut repress whereas DLX3 and DLX5 activate endogenous Runx2 expression and promoter activity in non-osseous cells and osteoblasts. These HD proteins exhibit distinct temporal expression profiles during osteoblast differentiation as well as selective association with Runx2 chromatin that is related to Runx2 transcriptional activity and recruitment of RNA polymerase II. Runx2 promoter mutagenesis shows that multiple HD elements control expression of Runx2 in relation to the stages of osteoblast maturation. Our studies establish mechanisms for commitment to the osteogenic lineage directly through BMP2 induction of HD proteins DLX3 and DLX5 that activate Runx2, thus delineating a transcriptional regulatory pathway mediating osteoblast differentiation. We propose that the three homeodomain proteins MSX2, DLX3, and DLX5 provide a key series of molecular switches that regulate expression of Runx2 throughout bone formation.

Expression of Runx2/Cbfa1/Pebp2αA During Angiogenesis in Postnatal Rodent and Fetal Human Orofacial Tissues

Transient expression of Runx2 is reported in endothelial cells and vascular smooth muscle cells during vessel formation in skin, stroma of forming bones and developing periodontal ligament, developing skeletal muscle cells, and fat tissue. The data suggest that Runx2 is expressed in a multipotential mesenchymal cell population that gives rise to various osseous and nonosseous cell lineages. Runx2/Cbfa1 is a transcription factor essential for cells of osteogenic and dentinogenic lineages. Here we examined expression of Runx2/Cbfa1 (all isotypes) in several nonskeletal cell types present in developing orofacial tissues of neonatal rodents and human fetuses with special emphasis on vessel formation. Sections obtained from heads or jaws of postnatal mice, hamster, and human fetuses were immunostained with monoclonal anti-Pebp2aA antibody. Mouse and human tissues were also examined by in situ hybridization. Sections of Runx2 null mutant mice with a LacZ reporter construct inserted in the Runx2 locus were stained for Runx2 promoter activity with anti-galactosidase. We found transient mRNA and protein expression in endothelial cells and in vascular smooth muscle cells of forming vessels in skin, alveoli of forming bone, and forming periodontal ligament. We also noticed weak and variable expression in some fibroblasts of embryonic skin, early differentiating cross-striated muscle cells, and differentiating fat cells. Runx2 is not an exclusive marker for chondrogenic, osteogenic, and dentinogenic tissues, but is much more widely present in an early multipotential mesenchymal cell population that gives rise to several other lineages.

Methylation analysis of the promoter F of estrogen receptor α gene: effects on the level of transcription on human osteoblastic cells

In this study, the methylation status of the distal promoter F of estrogen receptor alfa ( ERα) gene in human osteoblastic cells was investigated. The activity of this promoter is responsible for the ERα gene transcription in bone tissue. The methylation status of promoter F was here evaluated, for the first time, by direct sequencing of bisulfite-treated genomic DNA, at 10 CpG specific sites localized in a region of about 800 bp. An heterogeneous methylation pattern was observed. The most notable difference was found at four particular CpGs, distant from the exon F transcription start site, showing a methylation status that correlates with the expression level, being ERα mRNA transcription reduced in a partially methylated cells but preserved in demethylated cells. The other CpG sites, localized around the transcription start site, were always demethylated except for MG-63 cells showing the lowest level of ERα expression. By quantitative RT-PCR analysis we demonstrated that ERα gene expression was higher in primary osteoblasts than in bone-derived cells (MG-63 and SaOS-2) and in all cases the ERα mRNA is represented by the isoform F. The same 10 CpG sites were investigated in non-osseous cell lines and were found fully methylated in ERα-negative breast cancer cells (MDA-MB-231) and completely demethylated in ERα-positive breast cancer cells (MCF7). The overall results suggest that methylation of the CpG sites inside ERα gene promoter F here analyzed may contribute to ERα transcriptional control, directly or indirectly, influencing the tissue specific expression of the gene.

Differential Regulation of the Two Principal Runx2/Cbfa1 N-Terminal Isoforms in Response to Bone Morphogenetic Protein-2 during Development of the Osteoblast Phenotype

Cbfa1/Runx2 is a transcription factor essential for bone formation and osteoblast differentiation. Two major N-terminal isoforms of Cbfa1, designated type I/p56 (PEBP2aA1, starting with the sequence MRIPV) and type II/p57 (til-1, starting with the sequence MASNS), each regulated by distinct promoters, are known. Here, we show that the type I transcript is constitutively expressed in nonosseous mesenchymal tissues and in osteoblast progenitor cells. Cbfa1 type I isoform expression does not change with the differentiation status of the cells. In contrast, the type II transcript is increased during differentiation of primary osteoblasts and is induced in osteoprogenitors and in premyoblast C2C12 cells in response to bone morphogenetic protein-2. The functional equivalence of the two isoforms in activation and repression of bone-specific genes indicates overlapping functional roles. The presence of the ubiquitous type I isoform in nonosseous cells and before bone morphogenetic protein-2 induced expression of the type II isoform suggests a regulatory role for Cbfa1 type I in early stages of mesenchymal cell development, whereas type II is necessary for osteogenesis and maintenance of the osteoblast phenotype. Our data indicate that Cbfa1 function is regulated by transcription, cellular protein levels, and DNA binding activity during osteoblast differentiation. Taken together, our studies suggest that developmental timing and cell type- specific expression of type I and type II Cbfa isoforms, and not necessarily molecular properties or sequences that reside in the N-terminus of Cbfa1, are the principal determinants of the osteogenic activity of Cbfa1.

Differential regulation of the two principal Runx2/Cbfa1 n-terminal isoforms in response to bone morphogenetic protein-2 during development of the osteoblast phenotype.

Cbfa1/Runx2 is a transcription factor essential for bone formation and osteoblast differentiation. Two major N-terminal isoforms of Cbfa1, designated type I/p56 (PEBP2aA1, starting with the sequence MRIPV) and type II/p57 (til-1, starting with the sequence MASNS), each regulated by distinct promoters, are known. Here, we show that the type I transcript is constitutively expressed in nonosseous mesenchymal tissues and in osteoblast progenitor cells. Cbfa1 type I isoform expression does not change with the differentiation status of the cells. In contrast, the type II transcript is increased during differentiation of primary osteoblasts and is induced in osteoprogenitors and in premyoblast C2C12 cells in response to bone morphogenetic protein-2. The functional equivalence of the two isoforms in activation and repression of bone-specific genes indicates overlapping functional roles. The presence of the ubiquitous type I isoform in nonosseous cells and before bone morphogenetic protein-2 induced expression of the type II isoform suggests a regulatory role for Cbfa1 type I in early stages of mesenchymal cell development, whereas type II is necessary for osteogenesis and maintenance of the osteoblast phenotype. Our data indicate that Cbfa1 function is regulated by transcription, cellular protein levels, and DNA binding activity during osteoblast differentiation. Taken together, our studies suggest that developmental timing and cell type- specific expression of type I and type II Cbfa isoforms, and not necessarily molecular properties or sequences that reside in the N-terminus of Cbfa1, are the principal determinants of the osteogenic activity of Cbfa1.

Runt homology domain transcription factors (Runx, Cbfa, and AML) mediate repression of the bone sialoprotein promoter: evidence for promoter context-dependent activity of Cbfa proteins.

Expression of the bone sialoprotein (BSP) gene, a marker of bone formation, is largely restricted to cells in mineralized tissues. Recent studies have shown that the Cbfa1 (also known as Runx2, AML-3, and PEBP2alphaA) transcription factor supports commitment and differentiation of progenitor cells to hypertrophic chondrocytes and osteoblasts. This study addresses the functional involvement of Cbfa sites in expression of the Gallus BSP gene. Gel mobility shift analyses with nuclear extracts from ROS 17/2.8 osteoblastic cells revealed that multiple Cbfa consensus sequences are functional Cbfa DNA binding sites. Responsiveness of the 1.2-kb Gallus BSP promoter to Cbfa factors Cbfa1, Cbfa2, and Cbfa3 was assayed in osseous and nonosseous cells. Each of the Cbfa factors mediated repression of the wild-type BSP promoter, in contrast to their well known activation of various hematopoietic and skeletal phenotypic genes. Suppression of BSP by Cbfa factors was not observed in BSP promoters in which Cbfa sites were deleted or mutated. Expression of the endogenous BSP gene in Gallus osteoblasts was similarly downregulated by forced expression of Cbfa factors. Our data indicate that Cbfa repression of the BSP promoter does not involve the transducin-like enhancer (TLE) proteins. Neither coexpression of TLE1 or TLE2 nor the absence of the TLE interaction motif of Cbfa1 (amino acids 501 to 513) influenced repressor activity. However, removal of the C terminus of Cbfa1 (amino acids 362 to 513) relieved suppression of the BSP promoter. Our results, together with the evolutionary conservation of the seven Cbfa sites in the Gallus and human BSP promoters, suggest that suppressor activity by Cbfa is of significant physiologic consequence and may contribute to spatiotemporal expression of BSP during bone development.

Transient upregulation of CBFA1 in response to bone morphogenetic protein‐2 and transforming growth factor β1 in C2C12 myogenic cells coincides with suppression of the myogenic phenotype but is not sufficient for osteoblast differentiation

The bone morphogenetic protein (BMP)-2 is a potent osteoinductive signal, inducing bone formation in vivo and osteoblast differentiation from non-osseous cells in vitro. The runt domain-related protein Cbfa1/PEBP2αA/AML-3 is a critical component of bone formation in vivo and transcriptional regulator of osteoblast differentiation. To investigate the relationship between the extracellular BMP-2 signal, Cbfa1, and osteogenesis, we examined expression of Cbfa1 and osteoblastic genes during the BMP-2 induced osteogenic transdifferentiation of the myoblastic cell line C2C12. BMP-2 treatment completely blocked myotube formation and transiently induced expression of Cbfa1 and the bone-related homeodomain protein Msx-2 concomitant with loss of the myoblast phenotype. While induction of collagen type I and alkaline phosphatase (AP) expression coincided with Cbfa1 expression, Cbfa1 mRNA was strikingly downregulated at the onset of expression of osteopontin (OPN) and osteocalcin (OCN) genes, reflecting the mature osteoblast phenotype. TGF-β1 treatment effectively suppressed myogenesis and induced Cbfa1 expression but was insufficient to support osteoblast differentiation reflected by the absence of ALP, OPN, and OCN. We addressed whether induction of Cbfa1 in response to BMP-2 results in the transcriptional activation of the OC promoter which contains three enhancer Cbfa1 elements. Transfection studies show BMP-2 suppresses OC promoter activity in C2C12, but not in osteoblastic ROS 17/2.8 cells. Maximal suppression of OC promoter activity in response to BMP-2 requires sequences in the proximal promoter (up to nt -365) and may occur independent of the three Cbfa sites. Taken together, our results demonstrate a dissociation of Cbfa1 expression from development of the osteoblast phenotype. Our findings suggest that Cbfa1 may function transiently to divert a committed myoblast to a potentially osteogenic cell. However, other factors induced by BMP-2 appear to be necessary for complete expression of the osteoblast phenotype. J. Cell. Biochem. 73:114–125, 1999. © 1999 Wiley-Liss, Inc.

Transient upregulation of CBFA1 in response to bone morphogenetic protein-2 and transforming growth factor ?1 in C2C12 myogenic cells coincides with suppression of the myogenic phenotype but is not sufficient for osteoblast differentiation

The bone morphogenetic protein (BMP)-2 is a potent osteoinductive signal, inducing bone formation in vivo and osteoblast differentiation from non-osseous cells in vitro. The runt domain-related protein Cbfa1/PEBP2alphaA/AML-3 is a critical component of bone formation in vivo and transcriptional regulator of osteoblast differentiation. To investigate the relationship between the extracellular BMP-2 signal, Cbfa1, and osteogenesis, we examined expression of Cbfa1 and osteoblastic genes during the BMP-2 induced osteogenic transdifferentiation of the myoblastic cell line C2C12. BMP-2 treatment completely blocked myotube formation and transiently induced expression of Cbfa1 and the bone-related homeodomain protein Msx-2 concomitant with loss of the myoblast phenotype. While induction of collagen type I and alkaline phosphatase (AP) expression coincided with Cbfa1 expression, Cbfa1 mRNA was strikingly downregulated at the onset of expression of osteopontin (OPN) and osteocalcin (OCN) genes, reflecting the mature osteoblast phenotype. TGF-beta1 treatment effectively suppressed myogenesis and induced Cbfa1 expression but was insufficient to support osteoblast differentiation reflected by the absence of ALP, OPN, and OCN. We addressed whether induction of Cbfa1 in response to BMP-2 results in the transcriptional activation of the OC promoter which contains three enhancer Cbfa1 elements. Transfection studies show BMP-2 suppresses OC promoter activity in C2C12, but not in osteoblastic ROS 17/2.8 cells. Maximal suppression of OC promoter activity in response to BMP-2 requires sequences in the proximal promoter (up to nt -365) and may occur independent of the three Cbfa sites. Taken together, our results demonstrate a dissociation of Cbfa1 expression from development of the osteoblast phenotype. Our findings suggest that Cbfal may function transiently to divert a committed myoblast to a potentially osteogenic cell. However, other factors induced by BMP-2 appear to be necessary for complete expression of the osteoblast phenotype.

An AML-1 consensus sequence binds an osteoblast-specific complex and transcriptionally activates the osteocalcin gene.

Tissue and cell-type specific expression of the rat osteocalcin (rOC) gene involves the interplay of multiple transcriptional regulatory factors. In this report we demonstrate that AML-1 (acute myeloid leukemia-1), a DNA-binding protein whose genes are disrupted by chromosomal translocations in several human leukemias, interacts with a sequence essential for enhancing tissue-restricted expression of the rOC gene. Deletion analysis of rOC promoter-chloramphenicol acetyltransferase constructs demonstrates that an AML-1-binding sequence within the proximal promoter (-138 to -130 nt) contributes to 75% of the level of osteocalcin gene expression. The activation potential of the AML-1-binding sequence has been established by overexpressing AML-1 in osteoblastic as well as in nonosseous cell lines. Overexpression not only enhances rOC promoter activity in osteoblasts but also mediates OC promoter activity in a nonosseous human fibroblastic cell line. A probe containing this site forms a sequence specific protein-DNA complex with nuclear extracts from osteoblastic cells but not from nonosseous cells. Antisera supershift experiments indicate the presence of AML-1 and its partner protein core-binding factor beta in this osteoblast-restricted complex. Mutations of the critical AML-1-binding nucleotides abrogate formation of the complex and strongly diminish promoter activity. These results indicate that an AML-1 related protein is functional in cells of the osteoblastic lineage and that the AML-1-binding site is a regulatory element important for osteoblast-specific transcriptional activation of the rOC gene.

Bone tissue-specific transcription of the osteocalcin gene: role of an activator osteoblast-specific complex and suppressor hox proteins that bind the OC box.

Bone-specific expression of the osteocalcin gene is transcriptionally controlled. Deletion analysis of osteocalcin promoter sequences by transient transfection of osseous (ROS 17/2.8) and nonosseous (R2 fibroblast) cells revealed that the most proximal 108 nucleotides are sufficient to confer tissue-specific expression. By gel mobility shift assays with wild-type and mutated oligonucleotides and nuclear extracts from several different cell lines we identified a novel transcription factor complex which exhibits sequence-specific interactions with the primary transcriptional element, the OC box (nt -99 to -76). This OC box binding protein (OCBP) is present only in osteoblast-like cells. Methylation interference demonstrated association of the factor with OC box sequences overlapping the Msx homeodomain consensus binding site. By assaying several mutations of the OC box, both in gel shift and transient transfection studies using ROS 17/2.8, we show the following. First, binding of OCBP correlates with osteocalcin promoter activity in ROS 17/2.8 cells. Increased binding leads to a 2-3-fold increase in transcription, while decreased binding results in transcription 30-40% of control. Second, homeodomain protein binding suppresses transcription. However, Msx expression is critical for full development of the bone phenotype as determined by antisense studies. Last, we show that one of the mutations of the OC box permits expression of osteocalcin in non-osseous cell lines. In summary, we demonstrate association of at least two classes of tissue-restricted transcription factors with the OC box element, the OCBP and Msx proteins, supporting the concept that these sequences contribute to defining tissue specificity.

The osteocalcin gene promoter provides a molecular blueprint for regulatory mechanisms controlling bone tissue formation: role of transcription factors involved in development.

Characterization of regulatory sequences and their cognate binding factors in the bone-specific osteocalcin (OC) gene promoter has provided insight into mechanisms that control expression of the gene under diverse biological conditions. We present evidence for AP-1 motifs and two multipartite conserved regulatory sequences, the OC Box I (nt-99 to-76) and a site designated OC Box II (nt-136 to-130) in contributing to developmental and tissue-specific expression of osteocalcin. OC Box I is characterized by a homeodomain binding site and OC Box II is a recognition sequence for AML-1 (also called PEBP2 alpha), a runt homology-related DNA binding protein. Functional activity of the elements was established in osseous and non-osseous cell lines and is in part related to the binding of osteoblast-specific complexes which enhance OC transcription. The contribution of several elements and binding of multiple classes of transcription factors to independent elements in both these domains serve to illustrate the complexity of control required for tissue-specific OC expression.

DNase I hypersensitive sites in promoter elements associated with basal and vitamin D dependent transcription of the bone-specific osteocalcin gene.

Nuclease hypersensitive sites were mapped in the proximal promoter of the osteocalcin gene, which is expressed only in bone cells exhibiting the mature osteoblast phenotype. Nuclei from proliferating and confluent rat osteosarcoma (ROS) 17/2.8 cells were subjected to DNase I digestion, and hypersensitivity was assayed by the indirect end-labeling method, using osteocalcin gene probes. Hypersensitive sites were detected in two promoter domains: -590 to -390, which spans the vitamin D responsive element, and -170 to -70, which spans the TATA box and the CCAAT-containing OC box domain. Together, these elements regulate basal and vitamin D enhanced osteocalcin gene transcription. We observed a parallel relationship between the intensity of bands representing the hypersensitive sites and the extent to which the osteocalcin gene is transcribed. Both in confluent cultures and in response to vitamin D, when osteocalcin transcription was upregulated, the hypersensitive bands were significantly intensified. Additionally, the bands were decreased under conditions that downregulate osteocalcin gene transcription. A functional relationship between the presence of hypersensitive sites and osteocalcin gene transcription is further supported by the absence of hypersensitivity in nonosseous cells that do not express osteocalcin, although these proliferating cells exhibited hypersensitivity in a cell cycle regulated histone gene promoter. Our results suggest the involvement of chromatin structure in transcriptional responsiveness of the osteocalcin gene to physiologic modulation.