Constitutive transcription of the osteocalcin gene in osteosarcoma cells is reflected by altered protein-DNA interactions at promoter regulatory elements.

Bortell, Rita; Owen, Thomas A.; Shalhoub, Victoria; Heinrichs, Arianne; Aronow, Michael S.; Rochette-Egly, Cicile; Lutz, Yves; Stein, Janet L.; Lian, Jane B.; Stein, Gary S.

doi:10.1073/pnas.90.6.2300

Cited by 46 publications

(32 citation statements)

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“…Osteosarcoma cells such as the ROS 1712.8 line exhibit loss of the developmental sequence of gene expression characteristic of normal diploid osteoblasts [Bortell et al, 1993;Rodan and Noda, 19911. Abrogation of stringent growth control in these rodent osteosarcoma cells is accompanied by and potentially functionally related to concomitant expression of genes involved with both proliferation and post-proliferative phenotypic properties of mature bone cells.…”

Section: Aberrations In Gene Expression a N D Cell Phenotypic Propertmentioning

confidence: 99%

Nuclear architecture supports integration of physiological regulatory signals for transcription of cell growth and tissue‐specific genes during osteoblast differentiation

Stein

Wijnen

Lian

et al. 1994

J of Cellular Biochemistry

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During the past several years it has become increasingly evident that the three-dimensional organization of the nucleus plays a critical role in transcriptional control. The principal theme of this prospect will be the contribution of nuclear structure to the regulation of gene expression as functionally related to development and maintenance of the osteoblast phenotype during establishment of bone tissue-like organization. The contributions of nuclear structure as it regulates and i s regulated by the progressive developmental expression of cell growth and bone cell related genes will be examined. We will consider signalling mechanisms that integrate the complex and interdependent responsiveness to physiological mediators of osteoblast proliferation and differentiation. The focus will be on the involvement of the nuclear matrix, chromatin structure, and nucleosome organization in transcriptional control of cell growth and bone cell related genes. Findings are presented which are consistent with involvement of nuclear structure in gene regulatory mechanisms which support osteoblast differentiation by addressing four principal questions: 1 ) Does the representation of nuclear matrix proteins reflect the developmental stage-specific requirements for modifications in transcription during osteoblast differentiation? 2 ) Are developmental stage-specific transcription factors components of nuclear matrix proteins? 3) Can the nuclear matrix facilitate interrelationships between physiological regulatory signals that control transcription and the integration of activities of multiple promoter regulatory elements? 4) Are alterations in gene expression and cell phenotypic properties in transformed osteoblasts and osteosarcoma cells reflected by modifications in nuclear matrix proteins? There is a striking representation of nuclear matrix proteins unique to cells, tissues as well as developmental stages of differentiation, and tissue organization. Together with selective association of regulatory molecules with the nuclear matrix in a growth and differentiation-specific manner, there is a potential for application of nuclear matrix proteins in tumor diagnosis, assessment of tumor progression, and prognosis of therapies where properties of the transformed state of cells is modified. It i s realistic to consider the utilization of nuclear matrix proteins for targeting regions of cell nuclei and specific genomic domains on the basis of developmental phenotypic properties or tissue pathology. z 1994 WlIey-Llss, Inc

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Section: Aberrations In Gene Expression a N D Cell Phenotypic Propertmentioning

confidence: 99%

Nuclear architecture supports integration of physiological regulatory signals for transcription of cell growth and tissue‐specific genes during osteoblast differentiation

Stein

Wijnen

Lian

et al. 1994

J of Cellular Biochemistry

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“…Under normal growth conditions, these cells express low levels of osteocalcin, but its expression can be enhanced by treating the cells with vitamin D 3 (46). Vitamin D 3 increases the expression of osteocalcin through a vitamin D 3 -responsive element at positions Ϫ465 to Ϫ438 in the rat osteocalcin promoter (46).…”

Section: Identification Of Proteins Bound Differentially To the Osteomentioning

confidence: 99%

Identification of the GATA Factor TRPS1 as a Repressor of the Osteocalcin Promoter

Piscopo

Johansen

Derynck

2009

Journal of Biological Chemistry

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A proteomic analysis of proteins bound to the osteocalcin OSE2 sequence of the mouse osteocalcin promoter identified TRPS1 as a regulator of osteocalcin transcription. Mutations in the TRPS1 gene are responsible for human tricho-rhino-phalangeal syndrome, which is characterized by skeletal and craniofacial abnormalities. TRPS1 has been shown to bind regulatory promoter sequences containing GATA consensus binding sites and to repress transcription of genes involved in chondrocyte differentiation. Here we show that TRPS1 can directly bind the osteocalcin promoter in the presence or absence of Runx2. TRPS1 binds through a GATA binding sequence in the proximal promoter of the osteocalcin gene. The GATA binding site is conserved in mice, humans, and rats, although its location and orientation are not. Mutation of the mouse or human GATA binding sequence abrogates binding of TRPS1 to the osteocalcin promoter. We show that TRPS1 is expressed in osteosarcoma cells and upon induction of osteoblast differentiation in primary mouse bone marrow stromal cells and that TRPS1 regulates the expression of osteocalcin in both cell types. The expression of TRPS1 modulates mineralized bone matrix formation in differentiating osteoblast cells. These data suggest a role for TRPS1 in osteoblast differentiation, in addition to its previously described role in chondrogenesis.Runx2, previously described as CBFA1, OSF2, AML3, or PEPB2␣A, is a runt homology domain transcription factor and plays a key role in driving osteoblast differentiation. Runx2 expression, which occurs in differentiating and mature osteoblasts, is essential for bone formation, as illustrated by the phenotype of Runx2-deficient mice, which lack osteoblasts and have skeletons that are primarily composed of immature chondrocytes (1-3). Haploinsufficiency for RUNX2 in humans results in cleidocranial dysplasia, a syndrome that is characterized by delayed endochondral and intramembranous ossification (4). Runx2 transactivates genes involved in the deposition of bone matrix, e.g. those encoding osteocalcin, collagen IA1, osteopontin, and matrix metalloproteinase 13 (MMP13), and regulation of osteoclastogenesis, e.g. the genes encoding RankL and osteoprotegerin (reviewed in Ref. 5). Although Runx2 is expressed in cells of the osteoblast lineage throughout skeletal development, its levels increase only 1.5-2-fold as preosteoblasts differentiate into mature osteoblasts. Furthermore, the amount of Runx2 bound to target promoters, as measured by chromatin immunoprecipitation, is relatively unchanged throughout differentiation (6), yet the targets it regulates have distinct patterns of expression. These observations suggest that the regulation of Runx2 activity, either by post-translational modification or by the binding of co-regulators, plays a substantial role in defining the Runx2-mediated activation of target genes. Various proteins have been shown to interact with Runx2 and modulate its activity, including Smad3, CREB 2 -binding protein (CBP) HES1, Groucho/TLE, and histone d...

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“…The VDRE is a direct repeat of two steroid half elements that mediate selective recognition and protein binding (17)(18)(19)(20)(21). While homodimers of overexpressed VDR are capable of binding the osteocalcin VDRE in vitro, heteromerization of the VDR with additional nuclear factors greatly enhances in vitro protein-VDRE interactions (10,(22)(23)(24). To date, the base-pair contacts that form upon binding of VDR-protein complexes to the VDRE have been analyzed only in vitro.…”

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confidence: 99%

“…This complex transcriptional mechanism involves a specific ligand-receptor interaction, enhanced receptor gene expression, critical receptor phosphorylation sites, formation of multiple receptor-protein complexes, and protein-DNA interactions, as well as overlapping DNA elements at the vitamin D responsive element (VDRE) locus (9)(10)(11)(12)(13)(14)(15). These components of transcriptional control contribute to developmental and physiologic responsiveness of the osteocalcin gene to 1,25(OH)2D3 (16).…”

mentioning

confidence: 99%

“…with a single dose of the active metabolite 1,25(OH)2D3 results in a ligand-dependent increase in transcription of the target gene osteocalcin (9)(10)(11). This complex transcriptional mechanism involves a specific ligand-receptor interaction, enhanced receptor gene expression, critical receptor phosphorylation sites, formation of multiple receptor-protein complexes, and protein-DNA interactions, as well as overlapping DNA elements at the vitamin D responsive element (VDRE) locus (9)(10)(11)(12)(13)(14)(15).…”

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In vivo occupancy of the vitamin D responsive element in the osteocalcin gene supports vitamin D-dependent transcriptional upregulation in intact cells.

Breen

Wijnen

Lian

et al. 1994

Proc. Natl. Acad. Sci. U.S.A.

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The steroid hormone vitamin D is a principal mediator of skeletal homeostasis. 1,25-Dihydroxyvitamin D3 treatment of ROS 17/2.8 osteoblast-like cells results in a ligand-dependent increase in transcription of the bone-specific osteocalcin gene. This transcriptional upregulation requires the positive cis-acting vitamin D responsive element (VDRE). We have used the ligation-mediated polymerase chain reaction to demonstrate that protein occupancy of the VDRE within the intact cell correlates with increased synthesis of osteocalcin transcripts. These protein-DNA contacts were not present in the absence of vitamin D or in osteosarcoma cells (ROS 24 Rodan (Merck Sharp & Dohme). Cells were maintained in F12 medium containing calcium and charcoal stripped 5% fetal calf serum for 5 days after plating before hormone treatment or vehicle control. 1,25(OH)2D3 was provided by Milan Uskokovic (Hoffman-La Roche).In Vivo Footprint Analysis. In vivo footprinting was performed by exposing cells to 0.1% dimethy sulfate (DMS) (Aldrich) in culture medium for 5 min at room temperature. Methylated genomic DNA was isolated and cleaved with piperidine (Sigma) (26), and the level of methylation protection of G residues was analyzed by the ligation-mediated single-sided polymerase chain reaction (LMPCR) footprinting technique (27). LMPCR was performed with Vent polymerase (New England Biolabs). First-strand synthesis was accomplished with primer 1 (5'-GACAAACTGGCTCCAAC-TCGCATAGC-3'; nt -358 to -383) in first-strand buffer [40 mM NaCl/10 mM Tris HCl, pH 8.9/4 mM MgSO4/0.01% nonacetylated bovine serum albumin (BSA) (New England Biolabs)/0.1% Triton X-100]. The DNA was ligated with the unidirectional linker primer (27). The DNA was amplified in amplification buffer (10 mM NaCl/20 mM Tris HCl, pH 8.9/2 mM MgSO4/0.01% nonacetylated BSA/0.1% Triton X-100) using primer 2 (5'-GCATAGCCTGTGATTTTCAGTGTCT-GCCGT-3'; nt -377 to -406) and the linker oligonucleotide. Eighteen cycles of PCR were performed, each involving denaturation at 96°C, annealing at 56°C, and elongation at 76°C as described (27). Labeling was performed with 32p-end-labeled primer 3 (5'-AGCCTGTGATTTTCAGTGTCTAbbreviations: 1,25(OH)2D3, 1,25-dihydroxyvitamin D3; VDR, vitamin D receptor; VDRE, vitamin D responsive element; DMS, dimethyl sulfate; LMPCR, ligation-mediated single-sided PCR.

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Constitutive transcription of the osteocalcin gene in osteosarcoma cells is reflected by altered protein-DNA interactions at promoter regulatory elements.

Cited by 46 publications

References 46 publications

Nuclear architecture supports integration of physiological regulatory signals for transcription of cell growth and tissue‐specific genes during osteoblast differentiation

Nuclear architecture supports integration of physiological regulatory signals for transcription of cell growth and tissue‐specific genes during osteoblast differentiation

Identification of the GATA Factor TRPS1 as a Repressor of the Osteocalcin Promoter

In vivo occupancy of the vitamin D responsive element in the osteocalcin gene supports vitamin D-dependent transcriptional upregulation in intact cells.

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