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Results for “Core Binding Factor alpha Subunits”

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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↗

PubMed2006

JunB is required for endothelial cell morphogenesis by regulating core-binding factor beta.

The molecular mechanism triggering the organization of endothelial cells (ECs) in multicellular tubules is mechanistically still poorly understood. We demonstrate that cell-autonomous endothelial functions of the AP-1 subunit JunB are required for proper endothelial morphogenesis both in vivo in mouse embryos with endothelial-specific ablation of JunB and in in vitro angiogenesis models. By cDNA microarray analysis, we identified core-binding factor beta (CBFbeta), which together with the Runx proteins forms the heterodimeric core-binding transcription complex CBF, as a novel JunB target gene. In line with our findings, expression of the CBF target MMP-13 was impaired in JunB-deficient ECs. Reintroduction of CBFbeta into JunB-deficient ECs rescued the tube formation defect and MMP-13 expression, indicating an important role for CBFbeta in EC morphogenesis.

Animals↗

PubMed2006

T-lymphoid, megakaryocyte, and granulocyte development are sensitive to decreases in CBFbeta dosage.

The family of core-binding factors includes the DNA-binding subunits Runx1-3 and their common non-DNA-binding partner CBFbeta. We examined the collective role of core-binding factors in hematopoiesis with a hypomorphic Cbfb allelic series. Reducing CBFbeta levels by 3- or 6-fold caused abnormalities in bone development, megakaryocytes, granulocytes, and T cells. T-cell development was very sensitive to an incremental reduction of CBFbeta levels: mature thymocytes were decreased in number upon a 3-fold reduction in CBFbeta levels, and were virtually absent when CBFbeta levels were 6-fold lower. Partially penetrant consecutive differentiation blocks were found among early T-lineage progenitors within the CD4- CD8- double-negative 1 and downstream double-negative 2 thymocyte subsets. Our data define a critical CBFbeta threshold for normal T-cell development, and situate an essential role for core-binding factors during the earliest stages of T-cell development.

Alleles↗

PubMed2006

A mutation in the S-switch region of the Runt domain alters the dynamics of an allosteric network responsible for CBFbeta regulation.

The Runt domain is the DNA binding domain of the core binding factor (CBF) Runx subunits. The CBFs are transcription factors that play critical roles in hematopoiesis, bone, and neuron development in mammals. A common non-DNA binding CBFbeta subunit heterodimerizes with the Runt domain of the Runx proteins and allosterically regulates its affinity for DNA. Previous NMR dynamics studies suggested a model whereby CBFbeta allosterically regulates DNA binding by quenching conformational exchange in the Runt domain, particularly in the S-switch region and the betaE'-F loop. We sought to test this model, and to this end introduced all possible single amino acid substitutions into the S-switch region and the betaE'-F loop, and screened for mutations that enhanced DNA-binding. We demonstrate that one Runt domain mutant, R164N, binds both DNA and CBFbeta with higher affinity, but it is less sensitive to allosteric regulation by CBFbeta. Analysis of NMR relaxation data shows that the chemical exchange exhibited by the wild-type Runt domain is largely quenched by the R164N substitution. These data support a model in which the dynamic behavior of a network of residues connecting the CBFbeta and DNA binding sites on the Runt domain plays a critical role in the mechanism of allosteric regulation. This study provides an important functional link between dynamic behavior and protein allosteric function, consistent with results on other allosterically regulated proteins.

Allosteric Regulation↗

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↗

PubMed2004

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

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

Alternative Splicing↗

PubMed2002

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↗

PubMed2005

Cooperative binding of DNA and CBFbeta to the Runt domain of the CBFalpha studied via MD simulations.

The Runt domain (RD) is the DNA-binding region of the Runx genes. A related protein, known as core binding factor beta (CBFbeta) also binds to the RD to enhance RD-DNA interaction by 6- to 10-fold. Here, we report results from molecular dynamics (MD) simulations of RD alone, as a dimer in complexes with DNA and CBFbeta and in a ternary complex with DNA and CBFbeta. Consistent with the experimental findings, in the presence of CBFbeta the estimated free energy of binding of RD to the DNA is more favorable, which is shown to be due to more favorable intermolecular interactions and desolvation contributions. Also contributing to the enhanced binding are favorable intramolecular interactions between the 'wing' residues (RD residues 139-145) and the 'wing1' residues (RD residues 104-116). The simulation studies also indicate that the RD-CBFbeta binding is more favorable in the presence of DNA due to a more favorable RD-CBFbeta interaction energy. In addition, it is predicted that long-range interactions involving ionic residues contribute to binding cooperativity. Results from the MD calculations are used to interpret a variety of experimental mutagenesis data. A novel role for RD Glu116 to the RD-CBFbeta interaction is predicted.

Amino Acids↗

PubMed2001

Function of the inv(16) fusion gene CBFB-MYH11.

Inv(16)(p13q22) is associated with acute myeloid leukemia subtype M4Eo, which is characterized by the presence of myelomonocytic blasts and atypical eosinophils. This chromosomal rearrangement results in the fusion of CBFB and MYH11 genes. Mouse models indicate that the fusion gene, Cbfb-MYH11, inhibits differentiation of hematopoietic cells. Although expression of Cbfb-MYH11 is not sufficient for leukemogenesis, a combination of Cbfb-MYH11 and additional mutations can lead specifically to the development of myeloid leukemia. Normally, CBFbeta interacts with CBFalpha to form a transcriptionally active nuclear complex. In vitro studies indicate that expression of CBFB-MYH11 leads to sequestration of CBFalpha2 in the cytoplasm. It also has been shown to inhibit CBF-mediated transactivation, slow cell cycle progression, delay the apoptotic response to DNA damaging agents, and protect CBFalpha2 from degradation. The importance of these functions in vivo remains to be determined.

Animals↗

PubMed2006

CBFbeta is a facultative Runx partner in the sea urchin embryo.

BACKGROUND: Runx proteins are developmentally important metazoan transcription factors that form a heterodimeric complex with the non-homologous protein Core Binding Factor beta (CBFbeta). CBFbeta allosterically enhances Runx DNA binding but does not bind DNA itself. We report the initial characterization of SpCBFbeta, the heterodimeric partner of SpRunt-1 from the sea urchin Stronylocentrotus purpuratus. RESULTS: SpCBFbeta is remarkably similar to its mammalian homologues, and like them it enhances the DNA binding of the Runt domain. SpCBFbeta is entirely of zygotic provenance and its expression is similar that of SpRunt-1, accumulating globally at late blastula stage then later localizing to endoderm and oral ectoderm. Unlike SpRunt-1, however, SpCBFbeta is enriched in the endodermal mid- and hindgut of the pluteus larva, and is not highly expressed in the foregut and ciliated band. We showed previously that morpholino antisense-mediated knockdown of SpRunt-1 leads to differentiation defects, as well as to extensive post-blastula stage apoptosis caused by under-expression of the Runx target gene SpPKC1. In contrast, we show here that knockdown of SpCBFbeta does not negatively impact cell survival or SpPKC1 expression, although it does lead to differentiation defects similar to those associated with SpRunt-1 deficiency. Moreover, SpRunt-1 containing a single amino acid substitution that abolishes its ability to interact with SpCBFbeta retains the ability to rescue cell survival in SpRunt-1 morphant embryos. Chromatin immunoprecipitation shows that while the CyIIIa promoter engages both proteins, the SpPKC1 promoter only engages SpRunt-1. CONCLUSION: SpCBFbeta is a facultative Runx partner that appears to be required specifically for cell differentiation.

Amino Acid Substitution↗

PubMed2003

Diabetes interferes with the bone formation by affecting the expression of transcription factors that regulate osteoblast differentiation.

Type 1 diabetes in humans has as one of its complications inadequate bone formation, resulting in osteopenia and delayed fracture healing. To investigate the mechanisms by which diabetes affects bone formation, experiments were performed in a marrow ablation model. Mice were made diabetic by multiple low-dose streptozotocin treatment, and controls were treated with vehicle alone. Killing occurred 0, 2, 4, 6, 10, and 16 d following marrow ablation. Histologic analysis demonstrated that the amount of immature mesenchymal tissue was equivalent in both the experimental and control groups on d 4. On d 6 a burst of bone formation occurred in the control group that was significantly reduced in the diabetic group. This deficit was evident at the molecular level as shown by diminished expression of osteocalcin, collagen types I. When transcription factors were examined, core-binding factor alpha1 (Cbfa1)/runt domain factor-2 (Runx-2) and human homolog of the drosophila distal-less gene (Dlx5) expression were substantially reduced in the diabetic, compared with control, groups on d 4 and 6. C-fos but not c-jun expression was also suppressed in the diabetic group but not closely linked to bone formation. Insulin treatment substantially reversed the effect of diabetes on the expression of bone matrix osteocalcin and collagen type I and transcription factors Cbfa1/Runx2 and Dlx5. These results indicate that diabetic animals produce sufficient amounts of immature mesenchymal tissue but fail to adequately express genes that regulate osteoblast differentiation, Cbfa1/Runx-2 and Dlx5, which in turn, leads to decreased bone formation.

Animals↗

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↗

PubMed2004

Core-binding factors in hematopoiesis and immune function.

Core binding factors are heterodimeric transcription factors containing a DNA binding Runx1, Runx2, or Runx3 subunit, along with a non DNA binding CBF beta subunit. All four subunits are required at one or more stages of hematopoiesis. This review describes the role of Runx1 and CBF beta in the initiation of hematopoiesis in the embryo, and in the emergence of hematopoietic stem cells. We also discuss the later stages of hematopoiesis for which members of the core binding factor family are required, as well as the recently described roles for these proteins in autoimmunity.

Animals↗