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Results for “Core Binding Factor beta Subunit”

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At least 19 recordsLinked to original sources

PubMed1998

Overexpression, purification, and biophysical characterization of the heterodimerization domain of the core-binding factor beta subunit.

Core-binding factors (CBF) are heteromeric transcription factors essential for several developmental processes, including hematopoiesis. CBFs contain a DNA-binding CBF alpha subunit and a non-DNA binding CBF beta subunit that increases the affinity of CBF alpha for DNA. We have developed a procedure for overexpressing and purifying full-length CBF beta as well as a truncated form containing the N-terminal 141 amino acids using a novel glutaredoxin fusion expression system. Substantial quantities of the CBF beta proteins can be produced in this manner allowing for their biophysical characterization. We show that the full-length and truncated forms of CBF beta bind to a CBF alpha DNA complex with very similar affinities. Sedimentation equilibrium measurements show these proteins to be monomeric. Circular dichroism spectroscopy demonstrates that CBF beta is a mixed alpha/beta protein and NMR spectroscopy shows that the truncated and full-length proteins are structurally similar and suitable for structure determination by NMR spectroscopy.

Circular Dichroism↗

PubMed2003

Mutagenesis of the Runt domain defines two energetic hot spots for heterodimerization with the core binding factor beta subunit.

Core-binding factors (CBFs) are a small family of heterodimeric transcription factors that play critical roles in several developmental pathways and in human disease. Mutations in CBF genes are found in leukemias, bone disorders, and gastric cancers. CBFs consist of a DNA-binding CBF alpha subunit (Runx1, Runx2, or Runx3) and a non-DNA-binding CBF beta subunit. CBF alpha binds DNA in a sequence-specific manner, whereas CBF beta enhances DNA binding by CBF alpha. Both DNA binding and heterodimerization with CBF beta are mediated by a single domain in the CBF alpha subunits known as the "Runt domain." We analyzed the energetic contribution of amino acids in the Runx1 Runt domain to heterodimerization with CBF beta. We identified two energetic "hot spots" that were also found in a similar analysis of CBF beta (Tang, Y.-Y., Shi, J., Zhang, L., Davis, A., Bravo, J., Warren, A. J., Speck, N. A., and Bushweller, J. H. (2000) J. Biol. Chem. 275, 39579-39588). The importance of the hot spot residues for Runx1 function was demonstrated in in vivo transient transfection assays. These data refine the structural analyses and further our understanding of the Runx1-CBF beta interface.

Alanine↗

PubMed2002

The core-binding factor beta subunit is required for bone formation and hematopoietic maturation.

Core-binding factor beta (Cbfbeta) is the common non-DNA-binding subunit of the Cbf family of heterodimeric transcription factors. Mice deficient in Cbfbeta have a severe block in fetal liver hematopoiesis at the stage of hematopoietic stem cell (HSC) emergence. Here we show that by providing Cbfbeta function in endothelial cells and hematopoietic progenitors we can rescue fetal liver hematopoiesis in Cbfbeta-deficient embryos. The rescued mice die at birth, however, with severe defects in skeletal development, though intramembranous ossification occurs to some extent. Fetal liver hematopoiesis is restored at embryonic day (E) 12.5, but by E17.5 significant impairments in lymphopoiesis and myelopoiesis are observed. Thus, we conclude that the Cbfbeta subunit is required for HSC emergence, bone formation and normal differentiation of lymphoid and myeloid lineage cells.

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↗

PubMed1998

The leukemic protein core binding factor beta (CBFbeta)-smooth-muscle myosin heavy chain sequesters CBFalpha2 into cytoskeletal filaments and aggregates.

The fusion gene CBFB-MYH11 is generated by the chromosome 16 inversion associated with acute myeloid leukemias. This gene encodes a chimeric protein involving the core binding factor beta (CBFbeta) and the smooth-muscle myosin heavy chain (SMMHC). Mouse model studies suggest that this chimeric protein CBFbeta-SMMHC dominantly suppresses the function of CBF, a heterodimeric transcription factor composed of DNA binding subunits (CBFalpha1 to 3) and a non-DNA binding subunit (CBFbeta). This dominant suppression results in the blockage of hematopoiesis in mice and presumably contributes to leukemogenesis. We used transient-transfection assays, in combination with immunofluorescence and green fluorescent protein-tagged proteins, to monitor subcellular localization of CBFbeta-SMMHC, CBFbeta, and CBFalpha2 (also known as AML1 or PEBP2alphaB). When expressed individually, CBFalpha2 was located in the nuclei of transfected cells, whereas CBFbeta was distributed throughout the cell. On the other hand, CBFbeta-SMMHC formed filament-like structures that colocalized with actin filaments. Upon cotransfection, CBFalpha2 was able to drive localization of CBFbeta into the nucleus in a dose-dependent manner. In contrast, CBFalpha2 colocalized with CBFbeta-SMMHC along the filaments instead of localizing to the nucleus. Deletion of the CBFalpha-interacting domain within CBFbeta-SMMHC abolished this CBFalpha2 sequestration, whereas truncation of the C-terminal-end SMMHC domain led to nuclear localization of CBFbeta-SMMHC when coexpressed with CBFalpha2. CBFalpha2 sequestration by CBFbeta-SMMHC was further confirmed in vivo in a knock-in mouse model. These observations suggest that CBFbeta-SMMHC plays a dominant negative role by sequestering CBFalpha2 into cytoskeletal filaments and aggregates, thereby disrupting CBFalpha2-mediated regulation of gene expression.

3T3 Cells↗

PubMed1993

Fusion between transcription factor CBF beta/PEBP2 beta and a myosin heavy chain in acute myeloid leukemia.

The pericentric inversion of chromosome 16 [inv(16)(p13q22)] is a characteristic karyotypic abnormality associated with acute myeloid leukemia, most commonly of the M4Eo subtype. The 16p and 16q breakpoints were pinpointed by yeast artificial chromosome and cosmid cloning, and the two genes involved in this inversion were identified. On 16q the inversion occurred near the end of the coding region for CBF beta, also known as PEBP2 beta, a subunit of a heterodimeric transcription factor regulating genes expressed in T cells; on 16p a smooth muscle myosin heavy chain (SMMHC) gene (MYH11) was interrupted. In six of six inv(16) patient samples tested, an in-frame fusion messenger RNA was demonstrated that connected the first 165 amino acids of CBF beta with the tail region of SMMHC. The repeated coiled coil of SMMHC may result in dimerization of the CBF beta fusion protein, which in turn would lead to alterations in transcriptional regulation and contribute to leukemic transformation.

Amino Acid Sequence↗

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↗

PubMed2002

Core-binding factor beta interacts with Runx2 and is required for skeletal development.

Core-binding factor beta (CBFbeta, also called polyomavirus enhancer binding protein 2beta (PEBP2B)) is associated with an inversion of chromosome 16 and is associated with acute myeloid leukemia in humans. CBFbeta forms a heterodimer with RUNX1 (runt-related transcription factor 1), which has a DNA binding domain homologous to the pair-rule protein runt in Drosophila melanogaster. Both RUNX1 and CBFbeta are essential for hematopoiesis. Haploinsufficiency of another runt-related protein, RUNX2 (also called CBFA1), causes cleidocranial dysplasia in humans and is essential in skeletal development by regulating osteoblast differentiation and chondrocyte maturation. Mice deficient in Cbfb (Cbfb(-/-)) die at midgestation, so the function of Cbfbeta in skeletal development has yet to be ascertained. To investigate this issue, we rescued hematopoiesis of Cbfb(-/-) mice by introducing Cbfb using the Gata1 promoter. The rescued Cbfb(-/-) mice recapitulated fetal liver hematopoiesis in erythroid and megakaryocytic lineages and survived until birth, but showed severely delayed bone formation. Although mesenchymal cells differentiated into immature osteoblasts, intramembranous bones were poorly formed. The maturation of chondrocytes into hypertrophic cells was markedly delayed, and no endochondral bones were formed. Electrophoretic mobility shift assays and reporter assays showed that Cbfbeta was necessary for the efficient DNA binding of Runx2 and for Runx2-dependent transcriptional activation. These findings indicate that Cbfbeta is required for the function of Runx2 in skeletal development.

Animals↗

PubMed2002

Cbfbeta interacts with Runx2 and has a critical role in bone development.

Runx2 (runt-related transcription factor 2, also known as Cbfa1, Osf2 and AML3) is essential for bone development in mice, and mutations in RUNX2 are found in 65-80% of individuals with cleidocranial dysplasia. Although all Runx family members can interact with Cbfbeta (core-binding factor b, encoded by Cbfb), a role for Cbfbeta in bone development has not been demonstrated owing to lethality in Cbfb(-/-) mouse embryos at 12.5 days post coitum (d.p.c.) from hemorrhages and lack of definitive hematopoiesis. Using a 'knock-in' strategy, we generated mouse embryonic stem (ES) cells that express Cbfb fused in-frame to a cDNA encoding green fluorescent protein (GFP). Cbfb(+/GFP) mice had normal life spans and appeared normal, but Cbfb(GFP/GFP) pups died within the first day after birth. The Cbfb(GFP/GFP) mice exhibited a delay in endochondral and intramembranous ossification as well as in chondrocyte differentiation, similar to but less severe than delays observed in Runx2(-/-) mice. We demonstrate that Cbfbeta is expressed in developing bone and forms a functional interaction with Runx2, and that Cbfb(GFP) is a hypomorphic allele. The fusion allele maintains sufficient function in hematopoietic cells to bypass the early embryonic lethality, and identifies a new role for Cbfb in bone development. Our findings raise the possibility that mutations in CBFB may be responsible for some cases of cleidocranial dysplasia that are not linked to mutations in RUNX2.

Alleles↗

PubMed2002

Distinct roles for c-Myb and core binding factor/polyoma enhancer-binding protein 2 in the assembly and function of a multiprotein complex on the TCR delta enhancer in vivo.

Enhancers and promoters within TCR loci functionally collaborate to modify chromatin structure and to confer accessibility to the transcription and V(D)J recombination machineries during T cell development in the thymus. Two enhancers at the TCRalphadelta locus, the TCR alpha enhancer and the TCR delta enhancer (Edelta), are responsible for orchestrating the distinct developmental programs for V(D)J recombination and transcription of the TCR alpha and delta genes, respectively. Edelta function depends critically on transcription factors core binding factor (CBF)/polyoma enhancer-binding protein 2 (PEBP2) and c-Myb as measured by transcriptional activation of transiently transfected substrates in Jurkat cells, and by activation of V(D)J recombination within chromatin-integrated substrates in transgenic mice. To understand the molecular mechanisms for synergy between these transcription factors in the context of chromatin, we used in vivo footprinting to study the requirements for protein binding to Edelta within wild-type and mutant versions of a human TCR delta minilocus in stably transfected Jurkat cells. Our data indicate that CBF/PEBP2 plays primarily a structural role as it induces a conformational change in the enhanceosome that is associated with augmented binding of c-Myb. In contrast, c-Myb has no apparent affect on CBF/PEBP2 binding, but is critical for transcriptional activation. Thus, our data reveal distinct functions for c-Myb and CBF/PEBP2 in the assembly and function of an Edelta enhanceosome in the context of chromatin in vivo.

Base Sequence↗

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↗

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↗

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

Transcriptional activity of core binding factor-alpha (AML1) and beta subunits on murine leukemia virus enhancer cores.

Core binding factor (CBF), also known as polyomavirus enhancer-binding protein 2 and SL3 enhancer factor 1, is a mammalian transcription factor that binds to an element termed the core within the enhancers of the murine leukemia virus family of retroviruses. The core elements of the SL3 virus are important genetic determinants of the ability of this virus to induce T-cell lymphomas and the transcriptional activity of the viral long terminal repeat in T lymphocytes. CBF consists of two subunits, a DNA binding subunit, CBF alpha, and a second subunit, CBF beta, that stimulates the DNA binding activity of CBF alpha. One of the genes that encodes a CBF alpha subunit is AML1, also called Cbf alpha 2. This locus is rearranged by chromosomal translocations in human myeloproliferative disorders and leukemias. An exogenously expressed Cbf alpha 2-encoded subunit (CBF alpha 2-451) stimulated transcription from the SL3 enhancer in P19 and HeLa cells. Activity was mediated through the core elements. Three different isoforms of CBF beta were also tested for transcriptional activity on the SL3 enhancer. The longest form, CBF beta-187, increased the transcriptional stimulation by CBF alpha 2-451 twofold in HeLa cells, although it had no effect in P19 cells. Transcriptional activation by CBF beta required binding to the CBF alpha subunit, as a form of CBF beta that lacked binding ability, CBF beta-148, failed to increase activity. These results indicated that at least in certain cell types, the maximum activity of CBF required both subunits. They also provided support for the hypothesis that CBF is a factor in T lymphocytes that is responsible for recognition of the SL3 cores. We also examined whether CBF could distinguish a 1-bp difference between the enhancer core of SL3 and the core of the nonleukemogenic virus, Akv. This difference strongly affects transcription in T cells and leukemogenicity of SL3. However, no combination of CBF alpha and CBF beta subunits that we tested was able to distinguish the 1-bp difference in transcription assays. Thus, a complete understanding of how T cells recognize the SL3 core remains to be elucidated.

Animals↗

PubMed2001

Core-binding factor beta (CBFbeta), but not CBFbeta-smooth muscle myosin heavy chain, rescues definitive hematopoiesis in CBFbeta-deficient embryonic stem cells.

Core-binding factor beta (CBFbeta) is the non-DNA-binding subunit of the heterodimeric CBFs. Genes encoding CBFbeta (CBFB), and one of the DNA-binding CBFalpha subunits, Runx1 (also known as CBFalpha2, AML1, and PEBP2alphaB), are required for normal hematopoiesis and are also frequent targets of chromosomal translocations in acute leukemias in humans. Homozygous disruption of either the Runx1 or Cbfb gene in mice results in embryonic lethality at midgestation due to hemorrhaging in the central nervous system, and severely impairs fetal liver hematopoiesis. Results of this study show that Cbfb-deficient mouse embryonic stem (ES) cells can differentiate into primitive erythroid colonies in vitro, but are impaired in their ability to produce definitive erythroid and myeloid colonies, mimicking the in vivo defect. Definitive hematopoiesis is restored by ectopic expression of full-length Cbfb transgenes, as well as by a transgene encoding only the heterodimerization domain of CBFbeta. In contrast, the CBFbeta-smooth muscle myosin heavy chain (SMMHC) fusion protein generated by the inv(16) associated with acute myeloid leukemias (M4Eo) cannot rescue definitive hematopoiesis by Cbfb-deficient ES cells. Sequences responsible for the inability of CBFbeta-SMMHC to rescue definitive hematopoiesis reside in the SMMHC portion of the fusion protein. Results also show that the CBFbeta-SMMHC fusion protein transdominantly inhibits definitive hematopoiesis, but not to the same extent as homozygous loss of Runx1 or Cbfb. CBFbeta-SMMHC preferentially inhibits the differentiation of myeloid lineage cells, while increasing the number of blastlike cells in culture.

Animals↗

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↗

PubMed2006

[Mechanism of transcriptional regulation by Runx2 in osteoblasts].

Runt-related transcription factor 2 (Runx2) is an essential transcription factor for osteoblast differentiation, and the expression is regulated by many factors including bone morphogenetic protein (BMP). Core binding factorbeta (Cbf beta) is required for DNA binding of Runx2 and Runx2-dependent transcription, and Cbf beta regulates Runx2 function isoform-dependently. Further, ability of Runx2 for transcriptional activation is regulated by many molecules. Phosphoinositide 3-kinase (PI3K)-Akt signaling enhances DNA binding of Runx2 and Runx2-dependent transcription, and Runx2 upregulates PI3K subunits (p85 and p110 beta) and Akt. Therefore, Runx2 and PI3K-Akt signaling are mutually dependent each other in the regulation of osteoblast and chondrocyte differentiation and their migration.

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↗