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Results for “Core Binding Factor Alpha 1 Subunit”

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PubMed2004

Establishment and characterization of a stable cell line to evaluate cellular Runx2 activity.

Runx2 is an essential transcription factor for osteoblast differentiation from early commitment step to final differentiation. Based on its crucial role in osteoblast differentiation, the transcriptional activity of Runx2 protein implies more valuable information for osteoblast differentiation than any other parameters, such as Runx2 mRNA or protein level. Thus, a sensitive, specific, and consistent method to determine the Runx2 transcriptional activity has long been expected. Here we suggest a stable cell line that carries 6xOSE2-Luciferase reporter vector would be a good evaluation system to determine biological Runx2 transcriptional activity. The proliferation rate, cell shape, and the myogenic differentiation potential of the cloned cell line were similar to those of parental premyoblastic C2C12 cells. The cells specifically responded to Runx2 modulating agent such as FGF2. The stable cell line responded 5-6 folds more sensitively than the transiently transfected cells with Runx2. Though overexpression of any Runx gene stimulated the luciferase activity, Runx2 enhanced the reporter activity the highest. Collectively, the 6xOSE2-luc stable cells would be a good biological evaluation system to assess the activity of extracellular Runx2 modulating stimulations as well as the signal transduction pathways involved in the stimulations.

Animals↗

PubMed2005

Control of RUNX2 isoform expression: the role of promoters and enhancers.

The three mammalian RUNX genes constitute the family of runt domain transcription factors that are involved in the regulation of a number of developmental processes such as haematopoiesis, osteogenesis and neuronal differentiation. All three genes show a complex temporo-spatial pattern of expression. Since the three proteins are probably mutually interchangeable with regard to function, most of the specificity of each family member seems to be based on a tightly controlled regulation of expression. While RUNX gene expression is driven by two promoters for each gene, the promoter sequence alone does not seem to suffice for a proper expressional control. This review focuses on the available evidence for the existence of such control mechanisms and studies aiming at discovering cis-acting regulatory sequences of the RUNX2 gene.

Animals↗

PubMed2006

Overexpression of Runx2 directed by the matrix metalloproteinase-13 promoter containing the AP-1 and Runx/RD/Cbfa sites alters bone remodeling in vivo.

The activator protein-1 (AP-1) and runt domain binding (Runx/RD/Cbfa) sites and their respective binding proteins, c-Fos/c-Jun and Runx2 (Cbfa1), regulate the rat matrix metalloproteinase-13 (MMP-13) promoter in both parathyroid hormone (PTH)-treated and differentiating osteoblastic cells in culture. To determine the importance of these regulatory sites in the expression of MMP-13 in vivo, transgenic mice containing either wild-type (-456 or -148) or AP-1 and Runx/RD/Cbfa sites mutated (-148A3R3) MMP-13 promoters fused with the E. coli lacZ reporter were generated. The wild-type transgenic lines expressed higher levels of bacterial beta-galactosidase in bone, teeth, and skin compared to the mutant and non-transgenic lines. Next, we investigated if overexpression of Runx2 directed by the MMP-13 promoter regulated expression of bone specific genes in vivo, and whether this causes morphological changes in these animals. Real time RT-PCR experiments identified increased mRNA expression of bone forming genes and decreased MMP-13 in the tibiae of transgenic mice (14 days and 6 weeks old). Histomorphometric analyses of the proximal tibiae showed increased bone mineralization surface, mineral apposition rate, and bone formation rate in the transgenic mice which appears to be due to decreased osteoclast number. Since MMP-13 is likely to play a role in recruiting osteoclasts to the bone surface, decreased expression of MMP-13 may cause reduced osteoclast-mediated bone resorption, resulting in greater bone formation in transgenic mice. In summary, we show here that the 148 bp upstream of the MMP-13 transcriptional start site is sufficient and necessary for gene expression in bone, teeth, and skin in vivo and the AP-1 and Runx/RD/Cbfa sites are likely to regulate this. Overexpression of Runx2 by these regulatory elements appears to alter the balance between the bone formation-bone resorption processes in vivo.

Animals↗

PubMed2005

Bone morphogenetic protein-2 suppresses collagenase-3 promoter activity in osteoblasts through a runt domain factor 2 binding site.

Transforming growth factor-beta (TGFbeta) superfamily of growth factors, which include bone morphogenetic proteins (BMPs), have multiple effects in osteoblasts. In this study, we examined the regulation of collagenase-3 promoter activity by BMP-2 in osteoblast-enriched (Ob) cells from fetal rat calvariae. BMP-2 suppressed the activity of a -2 kb collagenase-3 promoter/luciferase recombinant in a time- and dose-dependent manner. The BMP-2 effect on the collagenase-3 promoter was further tested in several collagenase-3 promoter deletion constructs and it was narrowed down to a -148 to -94 nucleotide segment of the promoter containing a runt domain factor 2 (Runx2) site at nucleotide -132 to -126. The effect of BMP-2 was obliterated in a collagenase-3 promoter/luciferase construct containing a mutated Runx2 (mRunx2) sequence indicating that the Runx2 site mediates the BMP-2 response. Electrophoretic mobility shift assays, using nuclear extracts from control and BMP-2-treated Ob cells, indicated that the Runx2 protein is a component of the specific DNA-protein complex formed on the Runx2 site and that the BMP-2 effect may be associated with minor protein modifications rather than major changes in the composition of specific proteins interacting with the Runx2 site. We confirmed that other members of the TGFbeta family can down-regulate the collagenase-3 promoter by showing that TGFbeta1 also suppresses the promoter activity in a time- and dose-dependent manner. In conclusion, this study demonstrates that BMP-2 and TGFbeta1 suppress collagenase-3 promoter activity in osteoblasts and establishes a link between BMP-2 action and collagenase-3 expression via Runx2, a major regulator of osteoblast formation and function.

Animals↗

PubMed2005

Overlapping expression of Runx1(Cbfa2) and Runx2(Cbfa1) transcription factors supports cooperative induction of skeletal development.

Identifying the genetic pathways that regulate skeletal development is necessary to correct a variety of cartilage and bone abnormalities. The Runx family of transcription factors play a fundamental role in organ development and cell differentiation. Initial studies have shown that both Runx1 and Runx2 are expressed in pre-chondrogenic mesenchyme of the developing embryo at E12.5. Abrogation of the Runx2 gene completely inhibits bone formation yet the cartilage anlagen in these mice is fully formed. In the present study, we hypothesized that Runx1 may compensate for the lack of Runx2 in vivo to induce the early stages of skeletal formation and development. Histologic beta-gal stained sections using the Runx1(+/-)-Lac-Z mice demonstrate Runx1 promoter activity in pre-chondrocytic cell populations. In situ hybridization using Runx1 and Runx2 specific probes indicate that both factors are expressed in mesenchymal stem cell progenitors during early embryonic development. During later stages of mouse skeletal formation, Runx1 is excluded from the hypertrophic cartilage while Runx2 is present in these matured chondrocyte populations. Quantification of Runx expression by real time RT-PCR and Western blot analyses reveals that Runx1 and Runx2 are differentially modulated during embryogenesis suggesting a temporal role for each of these transcriptional regulators during skeletal formation. We provide evidence that haploinsufficiency results in normal appearing embryo skeletons of heterozygote Runx2 and Runx1 mutant mouse models; however, a delay in bone formation was identified in the calvarium. In summary, our results support a function for Runx1 and Runx2 during skeletal development with a possible role for Runx1 in mediating early events of endochondral and intramembranous bone formation, while Runx2 is a potent inducer of late stages of chondrocyte and osteoblast differentiation.

Animals↗

PubMed2002

Role of Runx genes in chondrocyte differentiation.

Runx2/Cbfa1 plays a central role in skeletal development as demonstrated by the absence of osteoblasts/bone in mice with inactivated Runx2/Cbfa1 alleles. To further investigate the role of Runx2 in cartilage differentiation and to assess the potential of Runx2 to induce bone formation, we cloned chicken Runx2 and overexpressed it in chick embryos using a retroviral system. Infected chick wings showed multiple phenotypes consisting of (1) joint fusions, (2) expansion of carpal elements, and (3) shortening of skeletal elements. In contrast, bone formation was not affected. To investigate the function of Runx2/Cbfa1 during cartilage development, we have generated transgenic mice that express a dominant negative form of Runx2 in cartilage. The selective inactivation of Runx2 in chondrocytes results in a severe shortening of the limbs due to a disturbance in chondrocyte differentiation, vascular invasion, osteoclast differentiation, and periosteal bone formation. Analysis of the growth plates in transgenic mice and in chick limbs shows that Runx2 is a positive regulator of chondrocyte differentiation and vascular invasion. The results further indicate that Runx2 promotes chondrogenesis either by maintaining or by initiating early chondrocyte differentiation. Furthermore, Runx2 is essential but not sufficient to induce osteoblast differentiation. To analyze the role of runx genes in skeletal development, we performed in situ hybridization with Runx2- and Runx3-specific probes. Both genes were coexpressed in cartilaginous condensations, indicating a cooperative role in the regulation of early chondrocyte differentiation and thus explaining the expansion/maintenance of cartilage in the carpus and joints of infected chick limbs.

Animals↗

PubMed2003

Requisite roles of Runx2 and Cbfb in skeletal development.

Each Runx (runt-related gene) protein exerts a fundamental role in different cell lineages. Runx2 is essential for osteoblast differentiation and plays an important role in chondrocyte maturation. Runx2 determines the lineage of osteoblastic cells from multipotent mesenchymal cells, enhances osteoblast differentiation at an early stage, and inhibits osteoblast differentiation at a late stage. In addition, Runx2 is involved in the production of bone matrix proteins. Further, Runx2 is a positive regulator of chondrocyte maturation and is involved in vascular invasion into the cartilage. Core binding factor beta (Cbfb) is a cotranscription factor which forms a heterodimer with Runx proteins. Cbfb is required for the functions of Runx1 and Runx2. Thus, Runx2/Cbfb heterodimers play essential roles in skeletal development.

Animals↗

PubMed1996

Comparison of the human genomic structure of the Runt domain-encoding PEBP2/CBFalpha gene family.

We cloned the human cDNA corresponding to the cDNA (PEBP2alphaB-451) encoding the mouse polyomavirus enhancer-binding protein 2alphaB-451, representing a major splice variant from acute myeloid leukemia gene 1 (AML1). Genomic DNA clones of AML1 were also isolated and the exon/intron structure was determined. Furthermore, we determined and compared the genomic structures of three mammalian Runt domain-containing genes, PEBP2alphaA,AML/PEBE2alphaB and PEBP2alphaC.

Acute Disease↗

PubMed2004

Runx-2 is not essential for the vitamin D-regulated expression of RANKL and osteoprotegerin in osteoblastic cells.

The differentiation and activity of osteoclasts are positively and negatively controlled by receptor activator of nuclear factor-kappaB ligand (RANKL), which is expressed on the surface of osteoblasts and stromal cells, and its decoy receptor osteoprotegerin (OPG), which is secreted by osteoblasts and stromal cells, respectively. The expression of the genes for RANKL and OPG is regulated by 1alpha,25-dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)]. Runt-related gene-2 (Runx-2) is essential for osteoblast differentiation and there are several reports that Runx-2 is involved in osteoclast formation. Therefore, to clarify the role of Runx-2 in osteoclastogenesis, we designed a series of experiments using C2 cells and C6 cells, which are derived from calvariae of runx2-deficient mice. Treatment of C2 cells and C6 cells with 1alpha,25(OH)(2)D(3) for 2-4 days increased and decreased the levels of expression of the mRNAs for RANKL and OPG, respectively, and the effects were dose-dependent. However, by day 8, the level of RANKL mRNA had fallen and that of OPG mRNA had risen. Furthermore, C6 cells induced the differentiation of mouse spleen cells into tartrate-resistant acid phosphatase-positive (TRAP-positive) multinucleated cells (osteoclast-like cells) in the presence of 10(-7)M 1alpha,25(OH)(2)D(3). Such formation of osteoclast-like cells was inhibited by exogenous OPG in a dose-dependent manner. Thus, our findings indicate that Runx-2 is not essential for the expression of RANKL and OPG, and the formation of osteoclast-like cells.

3T3 Cells↗

PubMed2004

Conservation and expression of an alternative 3' exon of Runx2 encoding a novel proline-rich C-terminal domain.

The Runx2 (Cbfa1, Aml3, PEBP2alphaA) gene plays an essential role in bone development and is one of a three-member family of closely related genes that encode the alpha-chain DNA binding components of the heterodimeric core binding factor complex. While all three mammalian Runx genes share a complex dual promoter structure (P1, P2) and display alternative splicing, a distinctive feature of Runx2 is the potential to encode larger isoforms in which the C-terminal domain encoded by the standard 3' terminal exon (exon 6) is replaced by an extended 200-201 amino acid C-terminal sequence including an extensive proline-rich domain and a C-terminal amphipathic helix. We report that the novel exon that gives rise to these variants (exon 6.1) is located over 100 kb downstream of exon 6 in the mouse, rat and human genomes. Exon 6.1 spans a CpG-rich island, and human/rodent conservation is evident through the coding sequence and the 3' untranslated region (UTR). Reverse transcriptase polymerase chain reaction (RT-PCR) and blot hybridisation analyses reveal that exon 6.1 is utilised at low levels in all mouse tissues and cell lines that express Runx2, regardless of which promoter is active, giving Runx2 the potential to encode more than 12 distinct isoforms. RT-PCR analysis of human RUNX2 exon 6.1 expression shows that utilisation of this exon is also conserved. In vitro transcription/translation of cDNAs encoding several exon 6.1 isoforms reveals that the novel Runx proteins are able to bind specifically to canonical Runx DNA target sequences. Antibodies raised to the unique C-terminal domain were shown to be reactive by immunoprecipitation and immunoblot assay, and were used in confocal immunofluorescence microscopy to reveal low level cytoplasmic staining in osteosarcoma and lymphoma cells that express high levels of Runx2 mRNA. However, reactive protein could not be detected in immunoblots of extracts from either cell type, suggesting that these proteins are unstable in lymphoid and osteosarcoma cells. In conclusion, the conservation and widespread utilisation of Runx2 exon 6.1 suggest that its encoded isoforms play an as yet undetermined role in mammalian development.

Alternative Splicing↗

PubMed2002

The RUNX1 Runt domain at 1.25A resolution: a structural switch and specifically bound chloride ions modulate DNA binding.

The evolutionarily conserved Runt homology domain is characteristic of the RUNX family of heterodimeric eukaryotic transcription factors, including RUNX1, RUNX2 and RUNX3. The genes for RUNX1, also termed acute myeloid leukemia protein 1, AML1, and its dimerization partner core-binding factor beta, CBFbeta, are essential for hematopoietic development and are together the most common targets for gene rearrangements in acute human leukemias. Here, we describe the crystal structure of the uncomplexed RUNX1 Runt domain at 1.25A resolution and compare its conformation to previously published structures in complex with DNA, CBFbeta or both. We find that complex formation induces significant structural rearrangements in this immunoglobulin (Ig)-like DNA-binding domain. Most pronounced is the movement of loop L11, which changes from a closed conformation in the free Runt structure to an open conformation in the CBFbeta-bound and DNA-bound forms. This transition, which we refer to as the S-switch, and accompanying structural movements that affect other parts of the Runt domain are crucial for sustained DNA binding. The closed to open transition can be induced by CBFbeta alone; suggesting that one role of CBFbeta is to trigger the S-switch and to stabilize the Runt domain in a conformation enhanced for DNA binding.A feature of the Runt domain hitherto unobserved in any Ig-like DNA-binding domain is the presence of two specifically bound chloride ions. One chloride ion is coordinated by amino acid residues that make direct DNA contact. In a series of electrophoretic mobility-shift analyses, we demonstrate a chloride ion concentration-dependent stimulation of the DNA-binding activity of Runt in the physiological range. A comparable DNA-binding stimulation was observed for negatively charged amino acid residues. This suggests a regulatory mechanism of RUNX proteins through acidic amino acid residues provided by activation domains during cooperative interaction with other transcription factors.

Acute Disease↗

PubMed2001

Identification and comparative analysis of a second runx3 promoter.

The runx gene family encodes transcriptional regulators that are essential in several processes. Alternative transcription start sites (3' or P1 and 5' or P2) have been described both at runx1 and runx2. Functional domains have been mapped to P2 N-termini. Here, starting from an analysis of cDNA clones, we identify a P2 promoter at mouse runx3. In functional assays in COS-7 cells, this promoter is as active as runx1 P2. By nuclease protection, primer extension and deletion analysis we map a major start site and define a minimal promoter element. The activity of both promoters at all three mouse loci was systematically compared in a comprehensive panel of fresh tissues. The conservation of a two-promoter structure and their distinctive pattern of activity support the hypothesis that alternative 5' isoforms subserve unique functions.

Animals↗

PubMed2002

Genomic characterization of the RUNX2 gene of Fugu rubripes.

A 105 kb Fugu rubripes genomic region containing the RUNX2 ortholog (frunx2) was sequenced and analysed. Spanning 32 kb, frunx2 is seven times smaller than its human orthologue (223 kb). By comparison of Fugu and human genomic environment a stretch of conserved synteny, comprising the neighbouring genes on both sides, was identified. Except one exon that is alternatively spliced in human RUNX2, all other seven exons could be identified in frunx2. The predicted protein sequence of frunx2 shows a high degree of sequence conservation compared with RUNX2 (83% identity). Like all human paralogues, frunx2 possesses two promoter regions separated by a large intron. Both promoter regions are conserved between the two species and contain several RUNX binding sites pointing to a self-regulatory function. Three further conserved non-coding regions were identified possibly functioning as enhancer elements for tissue-specific expression of RUNX2.

Amino Acid Sequence↗

PubMed2002

Expression of Runx1, -2 and -3 during tooth, palate and craniofacial bone development.

We describe the expression of three Runt-related RUNX genes (previously termed AML, Cbfa, or Pebp2alpha) Runx1 and Runx3 during the development of teeth and other craniofacial tissues and compare them to Runx2 expression reported earlier. All three genes were expressed in mesenchymal condensates. Runx1 was expressed in several cartilage primordia earlier than Runx3, and Runx2 was intense in all mesenchymal condensations of bones and teeth. Only Runx1 was expressed in epithelia, and in tooth germs transcripts were detected in outer dental epithelium. Runx1 was also intensely expressed in the midline epithelium of palatal shelves. In early tooth morphogenesis Runx3 was coexpressed with Runx2 in a thin layer of mesenchymal cells underlying dental epithelium. Unlike Runx2, Runx3 was expressed in odontoblasts. However, Runx3 mutant mice did not show obvious tooth phenotype or deviations of Runx1 and Runx2 expression patterns in the tooth.

Animals↗

PubMed2003

RUNX transcription factors as key targets of TGF-beta superfamily signaling.

The Runt domain transcription factors, RUNX1, RUNX2 and RUNX3, are integral components of signaling cascades mediated by both TGF-beta and bone morphogenetic proteins (BMPs) in several important biological systems. RUNX2 functions synergistically with Smad1 and Smad5 to regulate bone-specific genes when BMP induces osteogenesis. RUNX3, which has been mapped to locus 1p36, is a major tumor suppressor of gastric cancer and appears to be an important component of the TGF-beta-induced tumor suppressor pathway. A possible relationship between the TGF-beta-induced tumor-suppressor pathway and a postulated tumor suppressor gene on 1p36 must be examined.

Animals↗

PubMed2003

Runx transcription factors and the developmental balance between cell proliferation and differentiation.

The runt box (Runx) is a highly conserved DNA binding and protein-protein interaction domain that defines a family of heterodimeric transcription factors with essential roles in metazoan development. The first member of this family to be identified was the Drosophila regulatory gene runt, which was named by virtue of its function in establishing segmentation patterns during embryogenesis, and subsequently discovered to have additional functions in sex determination and neurogenesis. A second Drosophila Runx gene, lozenge, is required for cell patterning in the eye and for hematopoiesis. The genome project has revealed the existence of two additional Drosophila Runx genes, which to date have not been functionally characterized. Other invertebrate species with well-characterized Runx transcription factors include the nematode Caenorhabditis elegans and the sea urchin Strongylocentrotus purpuratus, each of which apparently contains only a single Runx gene. There are three Runx genes in mammals; Runx1 is required for definitive hematopoiesis and is a frequently mutated gene in human leukemia, Runx2 is required for osteogenesis and is associated with cleidocranial dysplasia, and Runx3 controls neurogenesis in the dorsal root ganglia and cell proliferation in the gastric epithelium, and is frequently deleted or silenced in human gastric cancer. Studies using mammalian systems and sea urchins indicate that Runx proteins have essential functions in both cell proliferation and differentiation, and in mammals they are both proto-oncogenes and tumor suppressors. Thus, a central question concerning the cell biology of Runx proteins is how are the opposing functions of this class of transcription factors regulated during development? Here I review current knowledge of Runx protein structure, function and regulation, and outline directions for future research aimed at understanding how Runx protein function is modulated during the transition from cell proliferation to differentiation in animal development.

Amino Acid Sequence↗

PubMedDate not supplied

The Runx genes as dominant oncogenes.

We have shown previously that Runx2 is a frequent target (approximately equal to 30%) for proviral insertion in murine leukemia virus (MLV) induced T cell tumors in CD2-MYC transgenic mice. Further investigation of a large panel of these tumors revealed that a small number also contain insertions at either Runx3 or Runx1. None of the tumors contained insertions at more than one family member, but in each case proviral insertion was associated with a high level of expression from the upstream (P1) promoter of the respective target gene. Moreover, we confirmed that transcriptional activation of Runx1 does not affect the integrity of the coding sequence, as previously observed for Runx2. These observations suggest that the three Runx genes act as functionally redundant oncogenes in T-cell lymphoma development. To explore the oncogenic potential of Runx2 further we created transgenic mice that over-express this gene in the T cell compartment. These CD2-Runx2 animals show a preneoplastic enlargement of the CD8 immature single positive (ISP) thymocyte pool and develop lymphomas at a low incidence. Although the CD8 ISP population is greatly increased, unlike their wild type counterparts these cells are largely non-cycling. Co-expression of c-MYC in this lineage accentuates the CD8 ISP skew and induces rapid tumor development, confirming the potent synergy that exists between these two oncogenes. Experiments designed to understand the nature of the observed synergy are ongoing and are based on the hypothesis that Runx2 may exert a survival effect in c-MYC expressing tumors in vivo while c-MYC may rescue cells from the antiproliferative effects of Runx2. The oncogenic potential of Runx1 is also being assessed using primary murine embryonic fibroblasts (MEFs). These studies have revealed that while Runx1 exerts a growth suppressive effect in wild type cells a growth promoting effect is seen in the absence of p53, suggesting that the Runx genes may harbor latent oncogene-like properties.

Animals↗

PubMed2001

Structure and backbone dynamics of Apo-CBFbeta in solution.

Runx proteins constitute a family of mammalian transcription factors that interact with DNA through their evolutionarily conserved Runt domain. CBFbeta, alternatively denoted PEBP2beta, is the non-DNA-binding heterodimer partner and acts to enhance the DNA binding affinity of Runx proteins. Runx proteins and CBFbeta are associated with a variety of biological functions and human diseases; they are, for example, together the most frequent targets for chromosomal rearrangements in acute human leukemias. We have determined the solution structure and characterized the backbone dynamics of C-terminally truncated fragments containing residues 1-141 of CBFbeta. The present apo-CBFbeta structure is very similar to that seen in a Runt-CBFbeta complex. An evaluation of backbone (15)N NMR relaxation parameters shows that CBFbeta is a rigid molecule with high order parameters throughout the backbone; the only regions displaying significant dynamics are a long loop and the C-terminal alpha-helix. A few residues display relaxation behavior indicating conformational exchange on microsecond to millisecond time scales, but only one of these is located at the Runt binding surface. Our structure and dynamics analysis of CBFbeta therefore suggests that the protein binds to Runt without large conformational changes or induced folding ("lock-and-key" interaction). The apo-CBFbeta structure presented here exhibits several significant differences with two other published NMR ensembles of very similar protein fragments. The differences are located in four regions outside of the central beta-barrel, whereas the beta-barrel itself is almost identical in the three NMR structures. The comparison illustrates that independently determined NMR structures may display rather large differences in backbone conformation in regions that appear to be well-defined in each of the calculated NMR ensembles.

Animals↗