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At least 19 recordsLinked to original sources
PubMed2000

Biophysical characterization of interactions between the core binding factor alpha and beta subunits and DNA.

Core binding factors (CBFs) play key roles in several developmental pathways and in human disease. CBFs consist of a DNA binding CBFalpha subunit and a non-DNA binding CBFbeta subunit that increases the affinity of CBFalpha for DNA. We performed sedimentation equilibrium analyses to unequivocally establish the stoichiometry of the CBFalpha:beta:DNA complex. Dissociation constants for all four equilibria involving the CBFalpha Runt domain, CBFbeta, and DNA were defined. Conformational changes associated with interactions between CBFalpha, CBFbeta, and DNA were monitored by nuclear magnetic resonance and circular dichroism spectroscopy. The data suggest that CBFbeta 'locks in' a high affinity DNA binding conformation of the CBFalpha Runt domain.

Animals↗
PubMed1999

Solution structure of core binding factor beta and map of the CBF alpha binding site.

The core binding factor beta subunit (CBF beta) is the non-DNA binding subunit of the core-binding factors, transcription factors essential for multiple developmental processes including hematopoiesis and bone development. Chromosomal translocations involving the human CBFB gene are associated with a large percentage of human leukemias. The N-terminal 141 amino acids of CBF beta contains the heterodimerization domain for the DNA-binding CBF alpha subunits, and is sufficient for CBF beta function in vivo. Here we present the high-resolution solution structure of the CBF beta heterodimerization domain. It is a novel alpha/beta structure consisting of two three-stranded beta-sheets packed on one another in a sandwich arrangement, with four peripheral alpha-helices. The CBF alpha binding site on CBF beta has been mapped by chemical shift perturbation analysis.

Binding Sites↗
PubMed1995

A new transcription factor family associated with human leukemias.

The proteins known as the polyoma enhancer binding protein 2 or core-binding factor (PEBP2/CBF) are heterodimeric transcription factors that contain a DNA-binding subunit (CBF alpha), and a subunit that does not bind DNA independently (CBF beta). PEBP2 and CBF were identified independently as proteins that regulate transcription of viral and cellular enhancers, and also as proteins whose genes are disrupted by chromosomal abnormalities in human leukemias. This review traces the history of the discovery of the PEBP2 and CBF protein family and describes the biochemical properties of the normal and oncogenic forms of these proteins.

Amino Acid Sequence↗
PubMed1994

AML1 is expressed in skeletal muscle and is regulated by innervation.

Although most skeletal muscle genes are expressed at similar levels in electrically active, innervated muscle and in electrically inactive, denervated muscle, a small number of genes, including those encoding the acetylcholine receptor, N-CAM, and myogenin, are expressed at significantly higher levels in denervated than in innervated muscle. The mechanisms that mediate electrical activity-dependent gene regulation are not understood, but these mechanisms are likely to be responsible, at least in part, for the changes in muscle structure and function that accompany a decrease in myofiber electrical activity. To understand how muscle activity regulates muscle structure and function, we used a subtractive-hybridization and cloning strategy to identify and isolate genes that are expressed preferentially in innervated or denervated muscle. One of the genes which we found to be regulated by electrical activity is the recently discovered acute myeloid leukemia 1 (AML1) gene. Disruption and translocation of the human AML1 gene are responsible for a form of acute myeloid leukemia. AML1 is a DNA-binding protein, but its normal function is not known and its expression and regulation in skeletal muscle were not previously appreciated. Because of its potential role as a transcriptional mediator of electrical activity, we characterized expression of the AML1 gene in innervated, denervated, and developing skeletal muscle. We show that AML1 is expressed at low levels in innervated skeletal muscle and at 50- to 100-fold-higher levels in denervated muscle. Four AML1 transcripts are expressed in denervated muscle, and the abundance of each transcript increases after denervation. We transfected C2 muscle cells with an expression vector encoding AML1, tagged with an epitope from hemagglutinin, and we show that AML1 is a nuclear protein in muscle. AML1 dimerizes with core-binding factor beta (CBF beta), and we show that CGF beta is expressed at high levels in both innervated and denervated skeletal muscle. PEBP2 alpha, which is structurally related to AML1 and which also dimerizes with CBF beta, is expressed at low levels in skeletal muscle and is up-regulated only weakly by denervation. These results are consistent with the idea that AML1 may have a role in regulating gene expression in skeletal muscle.

Animals↗
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↗
PubMed1994

PEBP2/CBF, the murine homolog of the human myeloid AML1 and PEBP2 beta/CBF beta proto-oncoproteins, regulates the murine myeloperoxidase and neutrophil elastase genes in immature myeloid cells.

The myeloperoxidase (MPO) and neutrophil elastase genes are expressed specifically in immature myeloid cells. The integrity of a polyomavirus enhancer core sequence, 5'-AACCACA-3', is critical to the activity of the murine MPO proximal enhancer. This element binds two species, myeloid nuclear factors 1 alpha and 1 beta (MyNF1 alpha and -beta), present in 32D cl3 myeloid cell nuclear extracts. The levels of the MyNF1s increase during early 32D cl3 cell granulocytic differentiation. Both MyNF1 alpha and -beta supershift with an antiserum raised by using a peptide derived from the N terminus of polyomavirus enhancer-binding protein 2/core-binding factor (PEBP2/CBF) alpha subunit. The specific peptide inhibits these supershifts. In vitro-translated PEBP2/CBF DNA-binding domain binds the murine MPO PEBP2/CBF site. An alternate PEBP2/CBF consensus site, 5'-GACCGCA-3', but not a simian virus 40 enhancer core sequence, 5'-TTCCACA-3', binds the MyNF1s in vitro and activates a minimal murine MPO-thymidine kinase promoter in vivo. The murine neutrophil elastase gene 100-bp 5'-flanking sequences contain several functional elements, including potential binding sites for PU.1, C/EBP, c-Myb, and PEBP2/CBF. The functional element 5'-GGCCACA-3' located at positions -66 to 72 differs from the PEBP2/CBF consensus (5'-PuACCPuCA-3') only by an A-to-G transition at position 2. This DNA element binds MyNF1 alpha and -beta weakly. The N terminis of two PEBP2/CBF alpha subunit family members, PEBP2 alpha A and PEBP2 alpha B (murine AML1), are nearly identical, and 32D c13 cl3 cells contain both corresponding mRNAs. Since t(8;21), t(3;21), and inv(16), associated with myeloid leukemias, disrupt subunits of PEBP2/CBF, we speculate that the resulting oncoproteins, AML1-ETO, AML1-EAP, AML1-Evi1, and CBF beta-MYH11, inhibit early myeloid differentiation.

Animals↗
PubMed2000

Indispensable role of the transcription factor PEBP2/CBF in angiogenic activity of a murine endothelial cell MSS31.

Mice lacking the AML1/PEBP2alphaB/CBFa2 gene or PEBP2beta/CBFb gene exhibit a defect in definitive hematopoiesis and die in utero because of hemorrhage in the central nervous system. Hematopoiesis in the embryo is considered to be tightly associated with vascular development. Here we examined whether PEBP2/CBF plays any role in angiogenesis besides that in definitive hematopoiesis. We found that AML1/PEBP2alphaB/CBFa2, PEBP2alphaA/CBFa1, and PEBP2beta/CBFb were expressed in a murine endothelial cell line MSS31. The expression of these molecules as well as the DNA binding activity of PEBP2/CBF were augmented by angiogenic growth factors such as bFGF and VEGF. Moreover, the expression of PEBP2 alpha/CBFa protein in endothelial cells was confirmed at the site of angiogenesis in vivo. To further clarify the role of PEBP2/CBF in angiogenesis, we established permanent transfectants of PEBP2 beta-MYH11 gene, one that interacts with the runt domain of the alpha subunit and deregulates PEBP2/CBF in a dominant interfering manner. Proliferation, migration, and tube formation of the PEBP2 beta-MYH11 transfectants were significantly reduced in comparison with those activities of the mock transfectants. These results suggest that transcription factor PEBP2/CBF plays an important role in angiogenesis.

Animals↗
PubMed2000

Interaction of CBF alpha/AML/PEBP2 alpha transcription factors with nucleosomes containing promoter sequences requires flexibility in the translational positioning of the histone octamer and exposure of the CBF alpha site.

Chromatin remodeling at eukaryotic gene promoter sequences accompanies transcriptional activation. Both molecular events rely on specific protein-DNA interactions that occur within these promoter sequences. Binding of CBFalpha/AML/PEBP2alpha (core binding factor alpha/acute myelogenous leukemia/polyoma enhancer binding protein 2alpha) proteins is a key event in both tissue-specific and developmentally regulated osteocalcin (OC) promoter activity. To address linkage between chromatin organization and transcription factor binding, we reconstituted segments of the rat OC gene proximal promoter into mononucleosomes and studied binding of CBFalpha proteins. We analyzed binding of bacterially produced Cbfalpha2Alpha and Cbfalpha2B, two splice variants of the human CBFalpha2 gene, and determined the effect of heterodimerization with the Cbfbeta subunit on binding activity. Our results indicate that binding of the truncated Cbfalpha2A protein to naked DNA is independent of Cbfbeta whereas Cbfalpha2A binding to nucleosomal DNA was enhanced by Cbfbeta. In contrast, the Cbfalpha2B interaction with either naked or nucleosomal DNA was strongly dependent on heterodimerization with the Cbfbeta subunit. Additionally, our results demonstrate that both Cbfalpha2A alone and Cbfalpha2B complexed with Cbfbeta can interact with nucleosomal DNA only if there is a degree of flexibility in the positioning of the histone octamer on the DNA fragment and exposure of the CBFalpha site. This situation was achieved with a DNA segment of 182 bp from the rat OC promoter that preferentially positions mononucleosomes upstream of the CBFalpha binding site and leaves this element partially exposed. Taken together, these results suggest that nucleosomal translational positioning is a major determinant of the binding of CBFalpha factors to nucleosomal DNA.

Animals↗
PubMed1995

Cloning, mapping and expression of PEBP2 alpha C, a third gene encoding the mammalian Runt domain.

PEBP2/CBF is a heterodimeric transcription factor composed of alpha and beta subunits. Previously, we reported two distinct mouse genes, PEBP2 alpha A and PEBP2 alpha B, which encode the alpha subunit. PEBP2 alpha B is the homologue of human AML1, encoding the acute myeloid leukemia 1 protein. AML1 and human PEBP2/CBF beta were detected independently at the breakpoints of two characteristic chromosome translocations observed frequently in two subtypes of acute myeloid leukemia. The PEBP2 alpha proteins contain a 128-amino-acid (aa) region highly homologous to the Drosophila melanogaster segmentation gene runt. The evolutionarily conserved region, named the Runt domain, harbors DNA-binding and heterodimerizing activities. In this study, we identified the third Runt-domain-encoding gene, PEBP2 alpha C, which maps to 1p36.11-p36.13 in the human chromosome and encodes a 415-aa protein. PEBP2 alpha C forms a heterodimer with PEBP2 beta, binds to the PEBP2 site and transactivates transcription, similar to PEBP2 alpha A and PEBP2 alpha B.

Amino Acid Sequence↗
PubMed2000

CBF--a biophysical perspective.

Core binding factor (CBF) is a heterodimeric transcription factor consisting of a DNA-binding subunit (Runx, also referred to as CBFA, AML 1, PEBP2alpha) and a non-DNA-binding subunit (CBFB). Biophysical characterization of the two proteins (and their interactions is providing a detailed understanding of this important transcription factor at the molecular level. Measurements of the relevant binding constants are helping to elucidate the mechanism of leukemogenesis associated with altered forms of these proteins. Determination of the 3D structures of CBFB and the DNA- and CBFB-binding domain of Runx, referred to as the Runt domain, are providing a structural basis for the functioning of the two proteins of CBF.

Animals↗
PubMed1994

PEBP2 alpha B/mouse AML1 consists of multiple isoforms that possess differential transactivation potentials.

A murine transcription factor, PEBP2, is composed of two subunits, alpha and beta. There are two genes in the mouse genome, PEBP2 alpha A and PEBP2 alpha B, which encode the alpha subunit. Two types of the alpha B cDNA clones, alpha B1 and alpha B2, were isolated from mouse fibroblasts and characterized. They were found to represent 3.8- and 7.9-kb transcripts, respectively. The 3.8-kb RNA encodes the previously described alpha B protein referred to as alpha B1, while the 7.9-kb RNA encodes a 387-amino-acid protein, termed alpha B2, which is identical to alpha B1 except that it has an internal deletion of 64 amino acid residues. Both alpha B1 and alpha B2 associate with PEBP2 beta and form a heterodimer. The alpha B2/beta complex binds to the PEBP2 binding site two- to threefold more strongly than the alpha B1/beta complex does. alpha B1 stimulates transcription through the PEBP2 site about 40-fold, while alpha B2 is only about 25 to 45% as active as alpha B1. Transactivation domain is located downstream of the 128-amino-acid runt homology region, referred to as the Runt domain. Mouse chromosome mapping studies revealed that alpha A, alpha B, and beta genes are mapped to chromosomes 17, 16, and 8, respectively. The last two genes are syntenic with the human AML1 on chromosome 21q22 and PEBP2 beta/CBF beta on 16q22 detected at the breakpoints of characteristic chromosome translocations of the two different subtypes of acute myeloid leukemia. These results suggest that previously described chimeric gene products, AML1/MTG8(ETO) and AML1-EAP generated by t(8;21) and t(3;21), respectively, lack the transactivation domain of AML1.

Amino Acid Sequence↗
PubMed2000

Zebrafish homolog of the leukemia gene CBFB: its expression during embryogenesis and its relationship to scl and gata-1 in hematopoiesis.

Mammalian CBFB encodes a transcription factor (CBF beta) that in combination with CBF alpha 2 binds to specific DNA sequences and regulates expression of a number of hematopoietic genes. CBFB is associated with human leukemias through a chromosome 16 inversion and is essential for definitive hematopoiesis during mouse embryo development. We have isolated a zebrafish cbfb complementary DNA (cDNA) clone from a zebrafish kidney cDNA library. This cbfb is highly homologous to human and mouse CBFB/Cbfb genes at both the DNA and protein level. In biochemical analyses, cbfbeta binds to human CBF alpha 2 and enhances its DNA binding. During zebrafish development, cbfb is expressed in the lateral plate mesoderm at tail bud stage and in the intermediate cell mass (ICM, the location of embryonic hematopoiesis) between the 21- to 26-somite stages. The cbfb is also expressed in Rohon-Beard cells, cranial nerve ganglia, hindbrain, retina, branchial arches, jaw, and fin buds. Expression of cbfb is decreased or absent in the ICM and Rohon-Beard cells in some hematopoietic mutants and is unaffected in others. We have also analyzed the expression of scl and gata-1 in the same hematopoietic mutants to ascertain the relative order of these transcription factors to cbfb in zebrafish hematopoiesis. Our results indicate that cbfb is expressed in early hematopoietic progenitors and that its expression pattern in the hematopoietic mutants is similar to that of scl. (Blood. 2000;96:4178-4184)

Amino Acid Sequence↗
PubMed1995

Positive and negative regulation of granulocyte-macrophage colony-stimulating factor promoter activity by AML1-related transcription factor, PEBP2.

The granulocyte-macrophage colony-stimulating factor (GM-CSF) gene promoter contains a consensus sequence for the polyomavirus enhancer binding-protein 2 (PEBP2) transcription factor, which consists of alpha and beta subunits. There are at least two genes, alpha A and alpha B, encoding the alpha subunit. alpha B is the mouse homologue of human AML1 gene detected at the breakpoints of t(8;21) and t(3;21) myeloid leukemias. We examined alpha A1 (an alpha A-gene product) and alpha B1 and alpha B2 (two alpha B-encoded isomers) for their effects on the GM-CSF promoter. PEBP2 alpha A1, alpha B1, and alpha B2 proteins bound the PEBP2 site within the mouse GM-CSF promoter. PEBP2 alpha A1 and alpha B1 enhanced the expression of the GM-CSF promoter-driven reporter plasmid in unstimulated and 12-O-tetradecanoylphorbol 13-acetate/phytohemagglutinin-stimulated human Jurkat T cells. In contrast, the promoter activity was suppressed by alpha B2. Coexpression of alpha B1 and alpha B2 showed that the promoter activity could be determined by the alpha B1/alpha B2 ratio. Jurkat cell extract contained PEBP2 site-binding protein(s) that cross-reacted with antimouse alpha A1 antibodies. Northern blot analysis indicated the expression of human PEBP2 alpha A, alpha B (AML1), and beta genes in Jurkat cells. Although further studies are required to determine the precise role of PEBP2 in the GM-CSF promoter activity, the present findings suggested the importance of the relative ratio of different PEBP2 isoforms in regulating the levels of the promoter activity.

Animals↗
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↗
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↗
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↗