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Results for “Core Binding Factor alpha Subunits”

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PubMed2001

Smad-Runx interactions during chondrocyte maturation.

BACKGROUND: Intracellular signaling triggered by bone morphogenetic proteins (BMPs) results in activated Smad complexes that regulate transcription of BMP-responsive genes. However, the low specificity of Smad binding to regulatory sequences implies that additional tissue-specific transcription factors are also needed. Runx2 (Cbfal) is a transcription factor required for bone formation. We have examined the role of Smads and Runx2 in BMP induction of type X collagen, which is a marker of chondrocyte hypertrophy leading to endochondral bone formation. METHODS: Pre-hypertrophic chondrocytes from the cephalic portion of the chick embryo sternum were placed in culture in the presence or absence of rhBMP-2. Cultures were transiently transfected with DNA containing the BMP-responsive type X collagen promoter upstream of the luciferase gene. The cultures were also transfected with plasmids, causing over-expression of Smads or Runx2, or both. After 24-48 hours, cell extracts were examined for levels of luciferase expression. RESULTS: In the presence of BMP-2, chondrocytes over-expressing BMP-activated Smadl or Smad5 showed significant enhancement of luciferase production compared with that seen with BMP alone. This enhancement was not observed with over-expression of Smad2, a transforming growth factor beta (TGF-beta)-activated Smad. Overexpression of Runx2 in BMP-treated cultures increased transcriptional activity to levels similar to those seen with Smads 1 or 5. When chondrocytes were simultaneously transfected with both Runx2 and Smad 1 or 5, promoter activity was further increased, indicating that BMP-stimulated Smad activity can be augmented by increasing the levels of Runx2. CONCLUSIONS: These results implicate the skeletal tissue transcription factor Runx2 in regulation of chondrocyte hypertrophy and suggest that maximal transcription of the type X collagen gene in pre-hypertrophic chondrocytes involves interaction of BMP-stimulated Smads with Runx2. CLINICAL RELEVANCE: Many skeletal abnormalities are associated with impaired regulation of chondrocyte hypertrophy in growth plates. These studies demonstrate that both BMP-activated Smads and Runx2 levels can modulate chondrocyte transition to hypertrophy.

Animals↗
PubMed2003

RUNX and cancer.

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Animals↗
PubMedDate not supplied

Down-regulation of RUNX1, RUNX3 and CBFbeta in hepatocellular carcinomas in an early stage of hepatocarcinogenesis.

BACKGROUND: Our previous studies suggested that deficient function of RUNX3 protein is causally related to development and progression of human gastric cancer. RUNX3 is mapped to 1p36, which is frequently deleted in hepatocellular carcinomas (HCC), therefore, these tumors were investigated for expression and copy number changes of RUNX3 and other Runt-related genes, RUNX1, RUNX2, and their co-factor CBFP. Similarly nearby uninvolved liver showing cirrhosis or normal histology was investigated in conjunction with various clinicopathological factors. MATERIALS AND METHODS: Copy number change and expression change of RUNX family genes in 35 hepatocellular carcinoma specimens and adjoining liver with cirrhosis (LC) or normal histology were estimated using quantitative reverse transcription polymerase chain reaction (RT-PCR) and in situ hybridization.

Base Sequence↗
PubMed2005

Shared and distinct roles mediated through C-terminal subdomains of acute myeloid leukemia/Runt-related transcription factor molecules in murine development.

AML1/Runx1 is a frequent target of human leukemia-associated gene aberration and encodes a transcription factor with nonredundant biologic functions in initial development of definitive hematopoiesis, T-cell development, and steady-state platelet production. AML1/Runx1 and 2 closely related family genes, AML2/Runx3 and AML3/Runx2/Cbfa1, present in mammals, comprise the Runt-domain transcription factor family. Although they have similar structural and biochemical properties, gene-targeting experiments have identified distinct biologic roles. To directly determine the presence of functional overlap among runt-related transcription factor (Runx) family molecules, we replaced the C-terminal portion of acute myeloid leukemia 1 (AML1) with that derived from its family members, which are variable in contrast to conserved Runt domain, using the gene knock-in method. We found that C-terminal portions of either AML2 or AML3 could functionally replace that of AML1 for myeloid development in culture and within the entire mouse. However, while AML2 substituted for AML1 could effectively rescue lymphoid lineages, AML3 could not, resulting in a smaller thymus and lymphoid deficiency in peripheral blood. Substitution by the C-terminal portion of AML3 also led to high infantile mortality and growth retardation, suggesting that AML1 has as yet unidentified effects on these phenotypes. Thus, the C-terminal portions of Runx family members have both similar and distinct biologic functions.

Animals↗
PubMed1995

Overexpression of core-binding factor alpha (CBF alpha) reverses cellular transformation by the CBF beta-smooth muscle myosin heavy chain chimeric oncoprotein.

A fusion between the transcription factor core-binding factor beta (CBF beta; also known as PEBP2 beta) and the tail region of smooth muscle myosin heavy chain (SMMHC) is generated by an inversion of chromosome 16 [inv(16) (p13q22)] associated with the M4Eo subtype of acute myeloid leukemia. We have previously shown that this CBF beta-SMMHC chimeric protein can transform NIH 3T3 cells and that this process requires regions of the chimeric protein necessary for association with the CBF alpha subunit. In this study, we show that NIH 3T3 cells overexpressing murine Cbf alpha 2 (also known as Aml1) cannot be transformed by CBF beta-SMMHC and that overexpression of Cbf alpha 2 in cells previously transformed by CBF beta-SMMHC reverts the cells to a less transformed phenotype. Cbf alpha 2 overexpression does not cause any gross morphological changes to NIH 3T3 cells but does result in increased CBF activity, as indicated by electrophoretic mobility shift assays and transactivation of reporter constructs. Cells transformed by CBF beta-SMMHC lack normal CBF-DNA complexes and have decreased levels of transactivation. Reversion of CBF beta-SMMHC transformation by Cbf alpha 2 is associated with a restoration of normal CBF-DNA complexes and transactivation activity. A Cbf alpha 2 mutant lacking transactivation properties does not transform cells when overexpressed, nor does it protect cells from CBF beta-SMMHC transformation. These results suggest that CBF beta-SMMHC interferes with the normal function of CBF and that this interference is necessary but not sufficient for cellular transformation.

3T3 Cells↗
PubMed1994

Identification of a region which directs the monocytic activity of the colony-stimulating factor 1 (macrophage colony-stimulating factor) receptor promoter and binds PEBP2/CBF (AML1).

The receptor for the macrophage colony-stimulating factor (or colony-stimulating factor 1 [CSF-1]) is expressed from different promoters in monocytic cells and placental trophoblasts. We have demonstrated that the monocyte-specific expression of the CSF-1 receptor is regulated at the level of transcription by a tissue-specific promoter whose activity is stimulated by the monocyte/B-cell-specific transcription factor PU.1 (D.-E. Zhang, C.J. Hetherington, H.-M. Chen, and D.G. Tenen, Mol. Cell. Biol. 14:373-381, 1994). Here we report that the tissue specificity of this promoter is also mediated by sequences in a region II (bp -88 to -59), which lies 10 bp upstream from the PU.1-binding site. When analyzed by DNase footprinting, region II was protected preferentially in monocytic cells. Electrophoretic mobility shift assays confirmed that region II interacts specifically with nuclear proteins from monocytic cells. Two gel shift complexes (Mono A and Mono B) were formed with separate sequence elements within this region. Competition and supershift experiments indicate that Mono B contains a member of the polyomavirus enhancer-binding protein 2/core-binding factor (PEBP2/CBF) family, which includes the AML1 gene product, while Mono A is a distinct complex preferentially expressed in monocytic cells. Promoter constructs with mutations in these sequence elements were no longer expressed specifically in monocytes. Furthermore, multimerized region II sequence elements enhanced the activity of a heterologous thymidine kinase promoter in monocytic cells but not other cell types tested. These results indicate that the monocyte/B-cell-specific transcription factor PU.1 and the Mono A and Mono B protein complexes act in concert to regulate monocyte-specific transcription of the CSF-1 receptor.

Animals↗
PubMed2004

Oncogenic potential of the RUNX gene family: 'overview'.

Runt-related (RUNX) gene family is composed of three members, RUNX1/AML1, RUNX2 and RUNX3, and encodes the DNA-binding (alpha) subunits of the Runt domain transcription factor polyomavirus enhancer-binding protein 2 (PEBP2)/core-binding factor (CBF), which is a heterodimeric transcription factor. RUNX1 is most frequently involved in human acute leukemia. RUNX2 shows oncogenic potential in mouse experimental system. RUNX3 is a strong candidate as a gastric cancer tumor suppressor. The beta subunit gene of PEBP2/CBF is also frequently involved in chromosome rearrangements associated with human leukemia. In this Overview, I will summarize how this growing field has been formed and what are the challenging new frontiers for better understanding of the oncogenic potential of this gene family.

Animals↗
PubMed2003

Energetic contribution of residues in the Runx1 Runt domain to DNA binding.

Core-binding factors (CBFs) are a small family of heterodimeric transcription factors that play critical roles in hematopoiesis and in the development of bone, stomach epithelium, and proprioceptive neurons. Mutations in CBF genes are found in leukemias, bone disorders, and gastric cancer. CBFs consist of a DNA-binding CBF alpha subunit and a non-DNA-binding CBF beta subunit. DNA binding and heterodimerization with CBF beta are mediated by the Runt domain in CBF alpha. Here we report an alanine-scanning mutagenesis study of the Runt domain that targeted amino acids identified by structural studies to reside at the DNA or CBF beta interface, as well as amino acids mutated in human disease. We determined the energy contributed by each of the DNA-contacting residues in the Runt domain to DNA binding both in the absence and presence of CBF beta. We propose mechanisms by which mutations in the Runt domain found in hematopoietic and bone disorders affect its affinity for DNA.

Alanine↗
PubMed1993

PEBP2/PEA2 represents a family of transcription factors homologous to the products of the Drosophila runt gene and the human AML1 gene.

cDNAs representing the alpha subunit of polyomavirus enhancer binding protein 2 (PEBP2; also called PEA2) were isolated. The products of the cDNAs are highly homologous to that of Drosophila segmentation gene runt (run) for an N-proximal 128-amino acid region showing 66% identity. The run homology region encompasses the domain capable of binding to a specific nucleotide sequence motif and of dimerizing with the companion beta subunit. The human AML1 gene related to t(8;21) acute myeloid leukemia also had a run homology region. Together with the beta subunit, which increases the affinity of the alpha subunit to DNA without binding to DNA by itself, PEBP2 represents a newly discovered family of transcription factor. The major species of PEBP2 alpha mRNA was expressed in T-cell lines but not in B-cell lines tested. Evidence indicated that PEBP2 functions as a transcriptional activator and is involved in regulation of T-cell-specific gene expression.

Amino Acid Sequence↗
PubMed2000

Energetic and functional contribution of residues in the core binding factor beta (CBFbeta ) subunit to heterodimerization with CBFalpha.

Core-binding factors (CBFs) are a small family of heterodimeric transcription factors that play critical roles in several developmental pathways, including hematopoiesis and bone development. Mutations in CBF genes are found in leukemias and bone disorders. CBFs consist of a DNA-binding CBFalpha subunit (Runx1, Runx2, or Runx3) and a non-DNA-binding CBFbeta subunit. CBFalpha binds DNA in a sequence-specific manner, whereas CBFbeta enhances DNA binding by CBFalpha. Recent structural analyses of the DNA-binding Runt domain of CBFalpha and the CBFbeta subunit identified the heterodimerization surfaces on each subunit. Here we identify amino acids in CBFbeta that mediate binding to CBFalpha. We determine the energy contributed by each of these amino acids to heterodimerization and the importance of these residues for in vivo function. These data refine the structural analyses and further support the hypothesis that CBFbeta enhances DNA binding by inducing a conformational change in the Runt domain.

Amino Acids↗
PubMed1995

Expression of the Runt domain-encoding PEBP2 alpha genes in T cells during thymic development.

The PEBP2 alpha A and PEBP2 alpha B genes encode the DNA-binding subunit of a murine transcription factor, PEBP2, which is implicated as a T-cell-specific transcriptional regulator. These two related genes share the evolutionarily conserved region encoding the Runt domain. PEBP2 alpha B is the murine counterpart of human AML1, which is located at the breakpoints of the 8;21 and 3;21 chromosome translocations associated with acute myeloid leukemia. Northern (RNA) blots of various adult mouse tissues revealed that the levels of expression of both genes were most prominent in the thymus. Furthermore, transcripts of PEBP2 alpha A and mouse AML1/PEBP2 alpha B were detected in T lymphocytes in the thymuses from day 16 embryos and newborns, as well as 4-week-old adult mice, by in situ hybridization. The expression of the genes persisted in peripheral lymph nodes of adult mice. The transcripts were detected in all the CD4- CD8-, CD4+ CD8+, CD4+ CD8-, and CD4- CD8+ cell populations. The results indicated that both genes are expressed in T cells throughout their development, supporting the notion that PEBP2 is a T-cell-specific transcription factor. Transcripts of mouse AML1/PEBP2 alpha B were also detected in day 12 fetal hematopoietic liver and in the bone marrow cells of newborn mice. The implication of mouse AML1/PEBP2 alpha B expression in hematopoietic cells other than those of T-cell lineage is discussed in relation to myeloid leukemogenesis.

Acute Disease↗
PubMed2001

Structural analyses of DNA recognition by the AML1/Runx-1 Runt domain and its allosteric control by CBFbeta.

The core binding factor (CBF) heterodimeric transcription factors comprised of AML/CBFA/PEBP2alpha/Runx and CBFbeta/PEBP2beta subunits are essential for differentiation of hematopoietic and bone cells, and their mutation is intimately related to the development of acute leukemias and cleidocranial dysplasia. Here, we present the crystal structures of the AML1/Runx-1/CBFalpha(Runt domain)-CBFbeta(core domain)-C/EBPbeta(bZip)-DNA, AML1/Runx-1/CBFalpha(Runt domain)-C/EBPbeta(bZip)-DNA, and AML1/Runx-1/CBFalpha(Runt domain)-DNA complexes. The hydrogen bonding network formed among CBFalpha(Runt domain) and CBFbeta, and CBFalpha(Runt domain) and DNA revealed the allosteric regulation mechanism of CBFalpha(Runt domain)-DNA binding by CBFbeta. The point mutations of CBFalpha related to the aforementioned diseases were also mapped and their effect on DNA binding is discussed.

Allosteric Regulation↗
PubMed2002

Mutations in the RUNX2 gene in patients with cleidocranial dysplasia.

Cleidocranial dysplasia (CCD) is a autosomal dominant disorder characterized by skeletal anomalies such as patent fontanels, late closure of cranial sutures with Wormian bones, late erupting secondary dentition, rudimentary clavicles, and short stature. The locus for this disease was mapped to chromosome 6p21. RUNX2 is a member of the runt family of transcription factors and its expression is restricted to developing osteoblasts and a subset of chondrocytes. Mutations in the RUNX2 gene have been shown to cause CCD. Chromosomal translocations, deletions, insertions, nonsense and splice-site mutations, as well as missense mutations of the RUNX2 gene have been described in CCD patients. Although there is a wide spectrum in phenotypic variability ranging from primary dental anomalies to all CCD features plus osteoporosis, no clear phenotype-genotype correlation has been established. However analysis of the three-dimensional structure of the DNA binding runt domain of the RUNX proteins and its interaction with DNA, as well as the cofactor CBFB, start to provide an insight into how missense mutations affect RUNX2 function.

Amino Acid Sequence↗
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↗