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Results for “Core Binding Factor alpha Subunits”

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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↗

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↗

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↗

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↗

PubMed2000

Cloning and embryonic expression patterns of the zebrafish Runt domain genes, runxa and runxb.

We isolated zebrafish homologues of the Runt-related transcription factor gene family (Runx family), runxa and runxb, and analyzed their developmental expression patterns. The deduced amino acid sequence of Runxa was highly homologous to that of AML1 (also called CBFA2, PEBP2alphaB or Runx1), a critical regulator of mammalian hematopoiesis expressed in cells of the hematopoietic lineage as well as other tissues. During zebrafish development, the runxa gene was not expressed in hematopoietic tissues but in the olfactory placodes and cells attached to the otic vesicles. We identified three kinds of runxb transcripts, which encoded two types of proteins with different N-terminal regions. The Runxb proteins were highly similar to AML2 (CBFA3, PEBP2alphaC or Runx3). The expression sites of the shared region of runxb mRNAs during development were the trigeminal ganglions, dorsal neurons of the neural tube and the lateral line primordia. These findings show that expression patterns of the zebrafish Runx genes are distinct from that of the mammalian genes.

Animals↗

PubMedDate not supplied

Intranuclear organization of RUNX transcriptional regulatory machinery in biological control of skeletogenesis and cancer.

RUNX (AML/CBFA/PEBP2) transcription factors serve as paradigms for obligatory relationships between nuclear structure and physiological control of phenotypic gene expression. The RUNX proteins contribute to tissue restricted transcription by sequence-specific binding to promoter elements of target genes and serving as scaffolds for the assembly of coregulatory complexes that mediate biochemical and architectural control of activity. We will present an overview of approaches we are pursuing to address: (1) the involvement of RUNX proteins in governing competency for protein/DNA and protein/protein interactions at promoter regulatory sequences; (2) the recruitment of RUNX factors to subnuclear sites where the machinery for expression or repression of target genes is organized; and (3) the trafficking and integration of regulatory signals that control RUNX-mediated transcription.

Animals↗

PubMed2002

Both the Smad and p38 MAPK pathways play a crucial role in Runx2 expression following induction by transforming growth factor-beta and bone morphogenetic protein.

The Runx family of transcription factors plays pivotal roles during normal development and in neoplasias. In mammals, Runx family genes are composed of Runx1 (Pebp2alphaB/Cbfa2/Aml1), Runx2 (Pebp2alphaA/Cbfa1/Aml3) and Runx3 (Pebp2alphaC/Cbfa3/Aml2). Runx1 and Runx3 are known to be involved in leukemogenesis and gastric carcinogenesis, respectively. Runx2, on the other hand, is a common target of transforming growth factor-beta1 (TGF-beta1) and bone morphogenetic protein-2 (BMP-2) and plays an essential role in osteoblast differentiation. Runx2 is induced by the receptor-activated Smad; Runx2 mediates the blockage of myogenic differentiation and induces osteoblast differentiation in C2C12 pluripotent mesenchymal precursor cells. However, Smad does not directly induce Runx2 expression; an additional step of de novo protein synthesis is required. Here we report that Smad-induced junB functions as an upstream activator of Runx2 expression. Furthermore, not only the Smad pathway but also the mitogen-activated protein kinase (MAPK) cascades are involved in the induction of Runx2 by TGF-beta1 and BMP-2. Our results demonstrate that following TGF-beta and BMP induction, both the Smad and p38 MAPK pathways converge at the Runx2 gene to control mesenchymal precursor cell differentiation.

Animals↗

PubMed2004

Regulation of TGFbeta1-mediated growth inhibition and apoptosis by RUNX2 isoforms in endothelial cells.

Runx transcription factors regulate viral growth, hematopoiesis, bone formation, angiogenesis, and gastric epithelial development through specific DNA-binding motifs on target gene promoters. Vascular endothelial cells (ECs) express RUNX genes that are activated by angiogenic factors. The RUNX2 gene also activates the vascular endothelial growth factor promoter. Alternatively spliced forms of RUNX genes have been described, but their functions in angiogenesis have not been elucidated. In this study, expression of a novel alternatively spliced variant of RUNX2 (RUNX2Delta8), lacking the region encoded by exon 8, was detected in aortic tissue undergoing angiogenesis in vitro and in ECs. Expression of RUNX2 and RUNX2Delta8 increased in vascular sprouts concomitant with expression of cellular proteases and cytokines known to mediate angiogenesis. RUNX2 DNA-binding activity was expressed in proliferating but not quiescent ECs. Ectopic expression of RUNX2 in ECs increased cell sprouting, cell proliferation, DNA synthesis, and phosphorylation of phosphorylated retinoblastoma relative to control transfectants while RUNX2, but not RUNX2Delta8 transfectants, acquired resistance to growth inhibition by transforming growth factor (TGFbeta1). Furthermore, RUNX2Delta8-transfected cells were more sensitive to TGFbeta1-induced apoptosis than RUNX2 transfectants. Consistent with these data, the RUNX2 gene was a strong repressor of the promoter of the cyclin-dependent kinase inhibitor, p21(CIP1), while RUNX2Delta8 was a competitive inhibitor of RUNX2 and exhibited weak repression activity. These results support the hypothesis that ECs regulate growth and apoptosis, in part, by alternative splicing events in the RUNX2 transcription factor to affect the TGFbeta1 signaling pathway. The exon 8 domain of RUNX2 may contribute to the strong repression activity of RUNX2 for some target gene promoters.

Alternative Splicing↗

PubMed2004

Molecular origins of rapid and continuous morphological evolution.

Mutations in cis-regulatory sequences have been implicated as being the predominant source of variation in morphological evolution. We offer a hypothesis that gene-associated tandem repeat expansions and contractions are a major source of phenotypic variation in evolution. Here, we describe a comparative genomic study of repetitive elements in developmental genes of 92 breeds of dogs. We find evidence for selection for divergence at coding repeat loci in the form of both elevated purity and extensive length polymorphism among different breeds. Variations in the number of repeats in the coding regions of the Alx-4 (aristaless-like 4) and Runx-2 (runt-related transcription factor 2) genes were quantitatively associated with significant differences in limb and skull morphology. We identified similar repeat length variation in the coding repeats of Runx-2, Twist, and Dlx-2 in several other species. The high frequency and incremental effects of repeat length mutations provide molecular explanations for swift, yet topologically conservative morphological evolution.

Alleles↗

PubMed2005

Impaired intranuclear trafficking of Runx2 (AML3/CBFA1) transcription factors in breast cancer cells inhibits osteolysis in vivo.

Runx transcription factors comprise a family of proteins that are essential for organogenesis. A unique nuclear matrix-targeting signal in the C terminus directs these factors to their appropriate subnuclear domains. At these sites, they interact with coregulatory proteins and target genes. We have previously shown that aberrant expression of the Runx2 DNA binding domain in metastatic breast cancer cells can prevent production of osteolytic lesions in bone. Here, we show that proper Runx2 subnuclear targeting is required for osteolysis. We have identified point mutations of the Runx2 nuclear matrix-targeting signal sequence that impair its targeting to nuclear matrix sites. These mutations block the invasive and osteolytic properties of MDA-MB-231 breast cancer cells in vivo. Cell lines expressing this Runx2 mutant protein inhibit the osteogenic properties of bone marrow stromal cells in coculture assays. The mutant breast cancer cells also exhibit reduced invasiveness in vitro and do not express genes involved in invasion and angiogenesis (VEGF and MMP13). Our findings suggest that fidelity of Runx2 intranuclear organization is necessary for expression of target genes that mediate the osteolytic activity of metastatic breast cancer cells.

Active Transport, Cell Nucleus↗

PubMed2001

Subnuclear targeting of Runx/Cbfa/AML factors is essential for tissue-specific differentiation during embryonic development.

Runx (Cbfa/AML) transcription factors are critical for tissue-specific gene expression. A unique targeting signal in the C terminus directs Runx factors to discrete foci within the nucleus. Using Runx2/CBFA1/AML3 and its essential role in osteogenesis as a model, we investigated the fundamental importance of fidelity of subnuclear localization for tissue differentiating activity by deleting the intranuclear targeting signal via homologous recombination. Mice homozygous for the deletion (Runx2 Delta C) do not form bone due to maturational arrest of osteoblasts. Heterozygotes do not develop clavicles, but are otherwise normal. These phenotypes are indistinguishable from those of the homozygous and heterozygous null mutants, indicating that the intranuclear targeting signal is a critical determinant for function. The expressed truncated Runx2 Delta C protein enters the nucleus and retains normal DNA binding activity, but shows complete loss of intranuclear targeting. These results demonstrate that the multifunctional N-terminal region of the Runx2 protein is not sufficient for biological activity. We conclude that subnuclear localization of Runx factors in specific foci together with associated regulatory functions is essential for control of Runx-dependent genes involved in tissue differentiation during embryonic development.

Animals↗

PubMed2000

A mechanism of repression by acute myeloid leukemia-1, the target of multiple chromosomal translocations in acute leukemia.

AML1 is one of the most frequently translocated genes in human leukemia. Here we demonstrate that acute myeloid leukemia-1 (AML-1) (Runx-1) represses transcription from a native promoter, p21(Waf1/Cip1). Unexpectedly, this repression did not require interactions with the Groucho co-repressor. To define the mechanism of repression, we asked whether other co-repressors could interact with AML-1. We demonstrate that AML-1 interacts with the mSin3 co-repressors. Moreover, endogenous AML-1 associated with endogenous mSin3A in mammalian cells. A deletion mutant of AML-1 that did not interact with mSin3A failed to repress transcription. The AML-1/mSin3 association suggests a mechanism of repression for the chromosomal translocation fusion proteins that disrupt AML-1.

Acute Disease↗

PubMed2000

Runt domain factor (Runx)-dependent effects on CCAAT/ enhancer-binding protein delta expression and activity in osteoblasts.

Transcription factor CCAAT/enhancer-binding protein delta (C/EBPdelta) is normally associated with acute-phase gene expression. However, it is expressed constitutively in primary osteoblast cultures where it increases insulin-like growth factor I synthesis in a cAMP-dependent way. Here we show that the 3' proximal region of the C/EBPdelta gene promoter contains a binding sequence for Runt domain factor Runx2, which is essential for osteogenesis. This region of the C/EBPdelta promoter directed high reporter gene expression in osteoblasts, and specifically bound Runx2 in osteoblast-derived nuclear extract. C/EBPdelta gene promoter activity was reduced by mutating the Runx binding sequence or by co-transfecting with Runx2 antisense expression plasmid, and was enhanced by overexpression of Runx-2. Exposure to prostaglandin E(2) increased Runx-dependent gene transactivation independently of Runx2 binding to DNA. Runx2 bound directly to the carboxyl-terminal region of C/EBPdelta itself, and its ability to drive C/EBPdelta expression was suppressed when C/EBPdelta or its carboxyl-terminal fragment was increased by overexpression. Consistent effects also occurred on C/EBPdelta-dependent increases in gene expression driven by synthetic or insulin-like growth factor I gene promoter fragments. These interactions between Runx2 and C/EBPdelta, and their activation by prostaglandin E(2), provide new evidence for their importance during skeletal remodeling, inflammatory bone disease, or fracture repair.

Animals↗

PubMed2001

Association with the nuclear matrix and interaction with Groucho and RUNX proteins regulate the transcription repression activity of the basic helix loop helix factor Hes1.

Hairy/Enhancer of split 1 (Hes1) is a mammalian transcriptional repressor that plays crucial roles in the regulation of several developmental processes, including neuronal differentiation. The aim of this study was to elucidate the molecular mechanisms that regulate the transcription repression activity of Hes1. It is shown here that Hes1 associates with the nuclear matrix, the ribonucleoprotein network of the nucleus that plays important roles in transcriptional regulation. Nuclear matrix binding is mediated by the same Hes1 C-terminal domain that is also required for transcriptional repression. This domain contains the WRPW motif that acts as a binding site for the transcriptional corepressor Groucho, which also localizes to the nuclear matrix. Both the nuclear matrix association and transcription repression activity of Hes1 are inhibited by deletion of the WRPW motif, indicating that Groucho acts as a transcriptional corepressor for Hes1. This corepressor role is not modulated by the Groucho-related gene product Grg5. In contrast, the Runt-related protein RUNX2, which localizes to the nuclear matrix and interacts with Groucho and Hes1, can inhibit both the Groucho.Hes1 interaction and the transcription repression ability of Hes1. Together, these observations suggest that transcriptional repression by Hes1 requires interactions with Groucho at the nuclear matrix and that RUNX proteins act as negative regulators of the repressive activity of Groucho.Hes1 complexes.

Amino Acid Sequence↗

PubMed2003

Runx2 integrates estrogen activity in osteoblasts.

Steroids significantly effect skeletal integrity. For example, bone mass decreases with glucocorticoid excess or with estrogen depletion after menopause. Glucocorticoid suppresses gene expression by an essential skeletal tissue transcription factor, Runx2, in rat osteoblasts. We now report that estrogen enhances Runx2 activity in dose- and estrogen receptor-dependent ways independently of changes in Runx2 levels or its DNA binding potential. Estrogen receptor and Runx2 can be collected by co-immunoprecipitation. By two-hybrid gene expression analysis, high affinity complex formation involves portions of Runx2 outside of its own DNA binding domain and the DNA binding domain of the estrogen receptor. Consistent with this interaction, the stimulatory effect of estrogen on Runx2 activity is lost when the DNA binding domain of the estrogen receptor is eliminated. Unlike the stimulatory effect of estrogen and the inhibitory effect of glucocorticoid, androgen fails to increase Runx2 activity, whereas Runx2 potently suppresses gene expression induced by all three steroids. Finally, estrogen increases gene transcription by the transforming growth factor-beta type I receptor gene promoter, which contains several Runx binding sequences, and enhances Smad dependent gene expression by transforming growth factor-beta in osteoblasts. These results reveal that Runx2 can integrate complex effects on gene transcription in hormone-, growth factor-, and tissue-restricted ways.

Animals↗

PubMed2003

The osteoblast transcription factor Runx2 is expressed in mammary epithelial cells and mediates osteopontin expression.

Targeted deletion of the Runx2 gene in mice has demonstrated that Runx2 is a master regulator of osteoblast differentiation. Runx2 has therefore largely been regarded as a bone-specific transcription factor. Runx2-/- mice die shortly after birth and therefore the role of Runx2 in later developing tissues remains unclear. Here we show that the Runx2 protein is expressed in several mammary epithelial cell lines and in primary mammary epithelial cells. In addition, we have also found that it has a functionally important role in gene regulation. Osteopontin (OPN) is expressed in mammary epithelial cells during pregnancy and lactation and has been shown to have a role in mammary gland differentiation. Here we show that a Runx2 site in the OPN promoter is required for activation of the promoter in mammary epithelial cells. Moreover, dominant-negative Runx proteins can inhibit both activation of an OPN promoter reporter in transient transfections and expression of the endogenous OPN gene in mammary epithelial cells. Our data suggest, for the first time, that the osteoblast transcription factor Runx2 has a role in the normal regulation of gene expression in mammary epithelial cells.

Animals↗

PubMed2004

The vitamin D response element in the distal osteocalcin promoter contributes to chromatin organization of the proximal regulatory domain.

Vitamin D receptor (VDR) and Runx2 are key regulators of tissue-specific gene transcription. Using the bone-related osteocalcin (OC) gene, we have previously shown that Runx2 is required for the extensive chromatin remodeling that accompanies gene activation. Here, we have addressed the direct contribution of the VDR to chromatin remodeling events necessary for regulation of OC transcription using mutational analysis. Our studies demonstrate that both the distal and proximal DNase I-hypersensitive sites characteristic of the transcriptionally active OC promoter are not enhanced in the absence of a functional vitamin D response element (VDRE). Furthermore, restriction enzyme accessibility studies reveal that nucleosomal reorganization of the proximal promoter occurs in response to vitamin D and this reorganization is abrogated by mutation of the VDRE. These findings indicate that binding of liganded VDR in the distal promoter directly impacts the chromatin structure of the proximal promoter. We find that, in the absence of functional Runx sites, the VDR cannot be recruited to the OC promoter and, therefore, the VDRE is not competent to mediate vitamin D responsiveness. On the other hand, chromatin immunoprecipitation assays show that Runx2 association with the OC promoter is not significantly impaired when the VDRE is mutated. Chromatin immunoprecipitation assays also demonstrate that basal levels of histone acetylation occur in the absence of Runx2 binding but that the VDRE and vitamin D are required for enhanced acetylation of histones H3 and H4 downstream of the VDRE. Together our results support a stepwise model for chromatin remodeling of the OC promoter and show that binding of the liganded VDR.retinoid X receptor directly impacts both the distal and proximal regulatory domains.

Animals↗

PubMed2002

Spatial expression of Cbfa1/Runx2 isoforms in teeth and characterization of binding sites in the DSPP gene.

Cbfa1/Runx2 is an essential transcription factor for osteoblast and odontoblast differentiation. Heterogeneous mutations of Cbfa1 gene result in cleidocranial dysplasia, an autosomal dominant syndrome, characterized by abnormal skeletal genesis and dental disorders. Recently three Cbfa1/Runx isoforms (Pebp2 alpha A/type I, til-1/type II, and Osf2/type III) have been identified that differ in their amino-terminal sequences. The precise roles of Cbfa1/Runx2 isoforms in odontoblast development are not known. The purpose of this study was to determine and compare expression patterns of the three Cbfa1/Runx2 isoforms in newborn tooth organs. Toward this aim, we developed three probes: type I and type II, which specifically hybridize with Pebp2 alpha A and til-1, respectively, and type II/III, which hybridizes with osf2 and partially with til-1. In addition, Cbfa1/Runx2 binding sites were identified in the regulatory elements of mouse dentin sialophosphoprotein (mDSPP) gene, which encodes a matrix protein expressed during odontogenesis. In situ hybridization performed with the specific Cbfa1/Runx2 isoform probes demonstrated that all isoforms are expressed in teeth and bone. The type I isoform was expressed at higher levels than isoforms type II and type II/III in developing newborn mouse incisors. Genomic mDSPP clones were isolated and characterized containing approximately 2.6 kb of the promoter region. Computer analysis of the promoter segment and intron 1 revealed a number of potential transcriptional factor binding sites including five Cbfa1/Runx2 binding sites, three in the promoter region and two within intron 1. DNA-protein assay and antibody supershift experiments showed that these binding sites interact with nuclear extracts isolated from the mouse odontoblast cell line MO6-G3. Further characterization of the functional role of Cbfa1/Runx2 in the regulation of the mDSPP gene expression is being investigated.

Animals↗