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PubMed1999

Core-binding factor: a central player in hematopoiesis and leukemia.

Consistent chromosomal rearrangements are found in a large number of hematopoietic tumors. In many cases, these rearrangements disrupt genes whose normal function is required for the proper development of blood cells. Excellent examples are the chromosomal rearrangements t(8;21)(q22;q22), t(12;21)(p13;q22), and inv(16)(p13q22) that disrupt two of the genes encoding a small family of heterodimeric transcription factors, core-binding factors (CBFs). CBFs consist of a DNA-binding CBFalpha subunit and a non-DNA-binding CBFbeta subunit. The t(8;21), associated with de novo acute myeloid leukemias, disrupts the CBFA2 (AML1) gene, which encodes a DNA-binding CBFalpha subunit. The t(12;21), the most common translocation in pediatric acute lymphocytic leukemias, also disrupts CBFA2. The CBFB gene, which encodes the non-DNA-binding subunit of the CBFs, is disrupted by the inv(16) in de novo acute myeloid leukemias. All chromosomal rearrangements involving the CBFA2 and CBFB genes create chimeric proteins, two of which have been unequivocally demonstrated to function as transdominant negative inhibitors of CBF function. Both the Cbfa2 and Cbfb genes are essential for normal hematopoiesis in mice, because homozygous disruption of either gene blocks definitive hematopoiesis. Recent data suggest that Cbfa2 and Cbfb are required for the emergence of definitive hematopoietic stem cells in the embryo from a putative definitive hemangioblast precursor. The transdominant negative inhibitor of CBF created by the inv(16), when present from the beginning of embryogenesis, also blocks the emergence of definitive hematopoietic cells in the embryo. On the other hand, chromosomal translocations involving the CBFA2 and CBFB genes in leukemias block hematopoiesis at later steps. This may reflect a difference in the timing at which translocations are acquired in the leukemias, which presumably is subsequent to emergence of the definitive hematopoietic stem cell. The cumulative data suggest that although the earliest requirement for Cbfa2 and Cbfb is for emergence of definitive hematopoietic stem cells, both genes are also required at later stages in the differentiation of some hematopoietic lineages.

Animals↗

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

The Ig fold of the core binding factor alpha Runt domain is a member of a family of structurally and functionally related Ig-fold DNA-binding domains.

BACKGROUND: CBFA is the DNA-binding subunit of the transcription factor complex called core binding factor, or CBF. Knockout of the Cbfa2 gene in mice leads to embryonic lethality and a profound block in hematopoietic development. Chromosomal disruptions of the human CBFA gene are associated with a large percentage of human leukemias. RESULTS: Utilizing nuclear magnetic resonance spectroscopy we have determined the three-dimensional fold of the CBFA Runt domain in its DNA-bound state, showing that it is an s-type immunoglobulin (Ig) fold. DNA binding by the Runt domain is shown to be mediated by loop regions located at both ends of the Runt domain Ig fold. A putative site for CBFB binding has been identified; the spatial location of this site provides a rationale for the ability of CBFB to modulate the affinity of the Runt domain for DNA. CONCLUSIONS: Structural comparisons demonstrate that the s-type Ig fold found in the Runt domain is conserved in the Ig folds found in the DNA-binding domains of NF-kappaB, NFAT, p53, STAT-1, and the T-domain. Thus, these proteins form a family of structurally and functionally related DNA-binding domains. Unlike the other members of this family, the Runt domain utilizes loops at both ends of the Ig fold for DNA recognition.

Animals↗

PubMed2003

Lymphoid enhancer factor-1 links two hereditary leukemia syndromes through core-binding factor alpha regulation of ELA2.

Two hereditary human leukemia syndromes are severe congenital neutropenia (SCN), caused by mutations in the gene ELA2, encoding the protease neutrophil elastase, and familial platelet disorder with acute myelogenous leukemia (AML), caused by mutations in the gene AML1, encoding the transcription factor core-binding factor alpha (CBFalpha). In mice, CBFalpha regulates the expression of ELA2, suggesting a common link for both diseases. However, gene-targeted mouse models have failed to reproduce either human disease, thus prohibiting further in vivo studies in mice. Here we investigate CBFalpha regulation of the human ELA2 promoter, taking advantage of bone marrow obtained from patients with either illness. In particular, we have identified novel ELA2 promoter substitutions (-199 C to A) within a potential motif for lymphoid enhancer factor-1 (LEF-1), a transcriptional mediator of Wnt/beta-catenin signaling, in SCN patients. The LEF-1 motif lies adjacent to a potential CBFalpha binding site that is in a different position in human compared with mouse ELA2. We find that LEF-1 and CBFalpha co-activate ELA2 expression. In vitro, the high mobility group domain of LEF-1 interacts with the runt DNA binding and proline-, serine-, threonine-rich activation domains of CBFalpha. ELA2 transcript levels are up-regulated in bone marrow of an SCN patient with the -199 C to A substitution. Conversely, a mutation of the CBFalpha activation domain, found in a patient with familial platelet disorder with AML, fails to stimulate the ELA2 promoter in vitro, and bone marrow correspondingly demonstrates reduced ELA2 transcript. Observations in these complementary patients indicate that LEF-1 cooperates with CBFalpha to activate ELA2 in vivo and also suggest the possibility that up-regulating promoter mutations can contribute to SCN. Two hereditary AML predisposition syndromes may therefore intersect via LEF-1, potentially linking them to more generalized cancer mechanisms.

Animals↗

PubMed2001

Core binding factor and its role in normal hematopoietic development.

The core binding factors are a small family of transcription factors comprising a DNA binding CBFalpha subunit and a non-DNA binding CBFbeta subunit. One gene encoding a CBFalpha subunit, RUNX1 (also known as AML1, CBFA2, and PEBPA2A), and the gene encoding CBFbeta (CBFB) are essential for hematopoiesis and are frequently mutated in human leukemias. Both genes are required for the generation of hematopoietic stem cells (HSCs) during embryonic development. Expression studies in fish and frogs and functional analyses in flies indicate that a role for these genes in hematopoiesis is evolutionarily conserved.

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↗

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↗

PubMed2006

Incidence and prognostic impact of c-Kit, FLT3, and Ras gene mutations in core binding factor acute myeloid leukemia (CBF-AML).

In core binding factors (CBF) acute myeloid leukemia (AML), the disruption of CBFalpha/beta genes impairs normal hematopoietic differentiation and is supposed to cooperate with additional mutations promoting proliferation. The incidence and the prognosis of receptor tyrosine kinase (RTK) c-Kit and FLT3 mutations and Ras mutations were evaluated in 103 pediatric and adult patients with CBF-AML. c-Kit mutations were present in 17% patients. c-Kit exon 8 mutations were more frequent in inv(16) than in t(8;21) subset (20 versus 6%). Only one patient had FLT3-ITD but FLT3-D835 was as frequent as reported in AML population (7%). Ras mutations were significantly more frequent in inv(16) than in t(8;21) subset (36 versus 8%, P=0.001). RTK mutations were associated with a higher white blood cell count (WBC) (36 versus 21 G/L, P=0.05). FLT3 mutations were significantly associated with a shorter EFS and survival (P<0.0001 and P=0.0002) owing to an excess of early events. c-Kit mutations were associated with a shorter EFS and RFS (P=0.002 and P=0.003) in t(8;21) but not inv(16) patients. As previously observed, Ras mutations did not affect prognosis. Screening for RTK mutations may help to identify patients with a more adverse outcome and thus susceptible to benefit from intensified protocols or RTK inhibitors.

Acute Disease↗

PubMed1992

Purification of core-binding factor, a protein that binds the conserved core site in murine leukemia virus enhancers.

The Moloney murine leukemia virus causes thymic leukemias when injected into newborn mice. A major genetic determinant of the thymic disease specificity of the Moloney virus genetically maps to two protein binding sites in the Moloney virus enhancer, the leukemia virus factor b site and the adjacent core site. Point mutations introduced into either of these sites significantly shifts the disease specificity of the Moloney virus from thymic leukemia to erythroleukemia (N. A. Speck, B. Renjifo, E. Golemis, T. Frederickson, J. Hartley, and N. Hopkins, Genes Dev. 4:233-242, 1990). We have purified several polypeptides that bind to the core site in the Moloney virus enhancer. These proteins were purified from calf thymus nuclear extracts by selective pH denaturation, followed by chromatography on heparin-Sepharose, nonspecific double-stranded DNA-cellulose, and core oligonucleotide-coupled affinity columns. We have achieved greater than 13,000-fold purification of the core-binding factors (CBFs), with an overall yield of approximately 19%. Analysis of purified protein fractions by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis reveals more than 10 polypeptides. Each of the polypeptides was recovered from an SDS-polyacrylamide gel, and those in the molecular size range of 19 to 35 kDa were demonstrated to have core-binding activity. The purified CBFs were shown by DNase I footprint analyses to bind the core site in the Moloney virus enhancer specifically, and also to core motifs in the enhancers from a simian immunodeficiency virus, the immunoglobulin mu chain, and T-cell receptor gamma-chain genes.

Base Sequence↗

PubMed2005

Core binding factor in the early avian embryo: cloning of Cbfbeta and combinatorial expression patterns with Runx1.

We have isolated the avian ortholog for CBFbeta, the common non-DNA binding subunit of the core binding factor (CBF) that has important regulatory roles in major developmental pathways. CBFbeta forms heterodimers with the DNA-binding Runx proteins and increases their affinity for DNA and their protein stability. Here, we describe the Cbfbeta expression pattern during the first 4 days of chick embryo development, with a special interest in the developing hematopoietic system. We have compared its expression pattern to that of Runx1, which is crucial for the generation of definitive hematopoietic cells, and to other hematopoietic- or endothelial-specific markers (c-Myb, Pu.1, CD45, c-Ets-1 and VE-Cadherin). Initially, Cbfbeta is widely expressed in the early mesoderm in both the yolk sac and the embryo proper, but later its expression becomes restricted to specific organs or cell types. We have found that Cbfbeta expression overlaps with Runx1 in the hematopoietic system and neural tube. The somitic and mesonephric structures, however, express Cbfbeta in the absence of detectable Runx1. Finally, Cbfbeta and Runx1 display multiple combinatorial patterns in the endoderm and in specific nerves or ganglia. Taken together, we show that Cbfbeta exhibits a dynamic expression pattern that varies according to the organ, cell type or developmental stage. By revealing multiple combinatorial patterns between Cbfbeta and Runx1, these data provide new insights into the role of CBF during early development.

Amino Acid Sequence↗

PubMed2001

Transient suppression of core-binding factor alpha 1 expression by basic fibroblast growth factor in rat osteoblast-like osteosarcoma ROS17/2.8 cells.

Core-binding factor alpha 1 (Cbfa1) is considered a prerequisite transcription factor for osteoblastic differentiation, based on experiments performed in knock-out mice. Cbfa1 binding sequences have been located in the promoter regions of various osteoblast phenotype-related genes, which are also known to be regulated by fibroblast growth factor (FGF). To elucidate the molecular mechanisms of FGF regulation of osteoblasts, we investigated the effects of FGF on Cbfa1 expression in osteoblast-like cells. We found that basic FGF, at 10 ng/ml, suppressed Cbfa1 mRNA expression in ROS17/2.8 cells. To detect the two distinct mRNA species that give rise to Pebp2alphaA/Cbfa1 and Osf2/Til-1/Cbfa1, we used semi-quantitative reverse transcription-polymerase chain reaction analysis using specific sets of primers. This analysis indicated that treatment with FGF transiently decreased the abundance of only the Osf2/Til-1/Cbfa1 isoform and not the Pebp2alphaA/Cbfa1 isoform in ROS17/2.8 cells. The suppression of Osf2/Til-1/Cbfa1 was time dependent and was observed up until 48h, and Cbfa1 mRNA levels then returned closer to control levels. Dose-dependent suppression by FGF was observed only with regard to mRNA levels of Osf2/Til-1/Cbfa1 and not for Pebp2alphaA/Cbfa1 mRNA. These data indicate that FGF suppresses Cbfa1 mRNA expression and that the Osf2/Til-1/Cbfa1 isoform is the specific target of FGF regulation in osteoblastic osteosarcoma ROS17/2.8 cells.

Animals↗

PubMed2000

Parathyroid hormone regulation of the rat collagenase-3 promoter by protein kinase A-dependent transactivation of core binding factor alpha1.

Previously we showed that the activator protein-1 site and the runt domain binding site in the collagenase-3 promoter act cooperatively in response to parathyroid hormone (PTH) in the rat osteoblastic osteosarcoma cell line, UMR 106-01. Our results of the expression pattern of core binding factor alpha1 (Cbfa1), which binds to the runt domain site, indicated that there is no change in the levels of Cbfa1 protein or RNA under either control conditions or after PTH treatment. The importance of posttranslational modification of Cbfa1 in the signaling pathway for PTH-induced collagenase-3 promoter activity was analyzed. PTH stimulation of collagenase-3 promoter activity was completely abrogated by protein kinase A (PKA) inhibition. To determine the role of PKA activity with respect to Cbfa1 activation (in addition to its known activity of phosphorylating cAMP-response element-binding protein to enhance c-fos promoter activity), we utilized the heterologous Gal4 transcription system. PTH stimulated the transactivation of activation domain-3 in Cbfa1 through the PKA site. In vitro phosphorylation studies indicated that the PKA site in the wild type activation domain-3 is a substrate for phosphorylation by PKA. Thus, we demonstrate that PTH induces a PKA-dependent transactivation of Cbfa1, and this transactivation is required for collagenase-3 promoter activity in UMR cells.

Animals↗

PubMed2006

[Apoptosis of mesenchymal cell line MBA-1 induced by core binding factor alpha 1].

OBJECTIVE: To investigate the effect of two core binding factors alpha 1 (Cbfa1) isfroms (Cbfa1/P56 and Cbfa1/P57) on the apoptosis of mesenchymal cell line MBA-1. METHODS: The two Cbfal isfroms were transiently transfected into MBA-1 cells, then the changes of apoptosis rate were observed by flow cytometer. The protein expressions of Cbfa1, Bax, Bcl-2, caspase-3, and caspase-9, cytochrome-C and TNF-alpha were determined by Western immunoblot. RESULTS: After the transient transfection with the two isforms of Cbfa1, MBA-1, the cells apoptotic rates increased, and the ratio of Bax/Bcl-2, the expressions of cytochrome-C, caspase-3, caspase-9, and TNF-alpha were significantly increased. CONCLUSION: Cbfa1 can promote the apoptosis in mesenchymal cell line MBA-1. Bax/Bcl-2, cytochrome-C, caspase-9, caspase-3, and TNF-alpha are also involved in the apoptosis pathway.

Animals↗

PubMed2006

Molecular analysis of PDGFRalpha/beta genes in core binding factor leukemia with eosinophilia.

Eosinophilia sometimes occurs in acute myeloid leukemia (AML), especially in core binding factor (CBF) leukemia. However, the pathogenesis of the differentiation from leukemic progenitors to eosinophils is not well understood in this type of leukemia. Recent reports showed that a novel fusion tyrosine kinase, Fip1-like1 (FIP1L1) platelet-derived growth factor receptor alpha (PDGFRalpha), is found in idiopathic hypereosinophilic syndrome. The involvement of another chimeric gene, PDGFRbeta, was also reported in myeloproliferative disorder with eosinophilia. These chimeric genes cause constitutive activation of PDGFR tyrosine kinases. On the other hand, a two-hit model for the pathogenesis of AML, which seems to be caused by inactivating mutations in transcription factors and genetic lesions in tyrosine kinase resulting in constitutive activation, has been proposed. On the basis of these findings, we screened for the expression of the FIP1L1-PDGFRalpha fusion gene and for mutations in the juxtamembrane and tyrosine kinase domains of PDGFRalpha/beta genes in 22 cases of CBF leukemia with eosinophilia. Among these cases, no FIP1L1-PDGFRalpha fusion gene was found. Although cDNA sequencing also detected three types of single-nucleotide alterations at kinase domains in PDGFRalpha/beta genes, all of them were silent changes and polymorphisms. Therefore, PDGFRalpha/beta genes do not appear to play a significant pathogenetic role in eosinophilia or leukemogenesis of CBF leukemia.

Adult↗

PubMed2003

Expression of core binding factor alpha1 up-regulated by IGF-I, GM-CSF, and EGF through MAPK pathway in MC3T3-E1 and C2C12 cells.

AIM: To study the regulating function and mechanism of insulin-like growth factor-I (IGF-I), granulocyte-macrophage colony-stimulating factor (GM-CSF), and epidermal growth factor (EGF) on murine core binding factor alpha1 (Cbfalpha1) gene expression. METHODS: Luciferase reporter gene method and RT-PCR technique were used to examine the effects of these growth factors on the promoter activity and mRNA expression of Cbfalpha1 gene in MC3T3-E1 and C2C12 cells. RESULTS: IGF-I (from 1 nmol/L to 1 micromol/L), GM-CSF (100 nmol/L), and EGF (1 micromol/L) increased the luciferase expression in MC3T3-E1 cells (P<0.05). And mitogen-activated protein kinase (MAPK) inhibitor, PD 98059 (10 micromol/L), completely blocked IGF-1, GM-CSF, and EGF-induced expression of Cbfa1 promoter activity (P<0.01). In C2C12 cells, IGF-I (from 1 nmol/L to 10 micromol/L), GM-CSF (100 nmol/L and 1 micromol/L), and EGF (100 nmol/L) enhanced the expression of luciferase reporter plasmid driven by mCbfalpha1 promoter (P<0.05). Addition of PD 98059 also blocked the stimulatory effects of these growth factors on Cbfalpha1 promoter activity (P<0.01). Moreover, Cbfalpha1 mRNA expression was significantly increased after treatment with IGF-I (1 nmol/L, 100 nmol/L), GM-CSF (100 nmol/L, 1 micromol/L), and EGF (1 micromol/L, 100 nmol/L) in MC3T3-E1 and C2C12 cells, respectively (P<0.05). These stimulatory effects of IGF-I, GM-CSF, and EGF on Cbfalpha1 mRNA expression were abolished by PD 98059. CONCLUSION: IGF-I, GM-CSF, and EGF could increase the promoter activity and the mRNA expression of murine Cbfalpha1 gene in MC3T3-E1 and C2C12 cells. These stimulatory effects might be mediated by activating the intracellular MAPK-dependent signaling pathway.

Animals↗

PubMed2002

Core-binding factor alpha 1 (Cbfa1) induces osteoblastic differentiation of C2C12 cells without interactions with Smad1 and Smad5.

Core-binding factor alpha(1) (Cbfa1) is an essential transcription factor for osteoblastic differentiation and osteogenesis. Bone morphogenetic protein (BMP) is also a powerful inducer of differentiation of pluripotent mesenchymal cells to osteoblast lineage and bone formation. Recent studies suggest that Cbfa1 plays a critical role during BMP-induced osteoblastic differentiation through association with cytoplasmic BMP signaling molecules, Smads. However, other studies have suggested that Cbfa1 may exhibit its osteogenic function without interaction with Smads. Therefore, it remains unclear whether association with Smad is essential for Cbfa1 function. In this study we examine the effects of Cbfa1 on osteoblastic differentiation in the presence or absence of interactions with Smad1 or Smad5 using C2C12 undifferentiated mesenchymal cells. Cbfa1 expression was induced upon stimulation with BMP-2 in C2C12 cells. Introduction of Cbfa1 into C2C12 cells induced osteoblastic differentiation and promoted transactivation of osteocalcin gene promoter without forming the complex with Smad1 or Smad5. Furthermore, in C2C12 cells in which the association of Cbfa1 with Smad1/Smad5 was prevented by the overexpression of the natural antagonist, Smad6, Cbfa1 still induced osteoblastic differentiation and transactivated osteocalcin gene promoter, regardless of BMP-2 stimulation. These results suggest that the interactions with Smad1 or Smad5 are not essential for Cbfa1 to demonstrate its osteogenic actions. However, interactions with Smad1/Smad5 enhance these osteogenic actions of Cbfa1. Of note, BMP-2-induced or Smad-induced osteoblastic differentiation was inhibited by dominant-negative Cbfa1, suggesting that the function of Cbfa1 is critical for BMP-2-induced osteoblastic differentiation. Our results suggest that Cbfa1 is essential and also sufficient to induce osteoblastic differentiation in undifferentiated mesenchymal cells, and establishment of an association with Smad1/Smad5 enhances the osteogenic actions of Cbfa1. On the other hand, Cbfa1 expression requires the activation of Smad1/Smad5 by BMP-2.

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↗

PubMed2000

Auto-inhibition and partner proteins, core-binding factor beta (CBFbeta) and Ets-1, modulate DNA binding by CBFalpha2 (AML1).

Core-binding factor alpha2 (CBFalpha2; otherwise known as AML1 or PEBP2alphaB) is a DNA-binding subunit in the family of core-binding factors (CBFs), heterodimeric transcription factors that play pivotal roles in multiple developmental processes in mammals, including hematopoiesis and bone development. The Runt domain in CBFalpha2 (amino acids 51 to 178) mediates DNA binding and heterodimerization with the non-DNA-binding CBFbeta subunit. Both the CBFbeta subunit and the DNA-binding protein Ets-1 stimulate DNA binding by the CBFalpha2 protein. Here we quantify and compare the extent of cooperativity between CBFalpha2, CBFbeta, and Ets-1. We also identify auto-inhibitory sequences within CBFalpha2 and sequences that modulate its interactions with CBFbeta and Ets-1. We show that sequences in the CBFalpha2 Runt domain and sequences C terminal to amino acid 214 inhibit DNA binding. Sequences C terminal to amino acid 214 also inhibit heterodimerization with the non-DNA-binding CBFbeta subunit, particularly heterodimerization off DNA. CBFbeta rescinds the intramolecular inhibition of CBFalpha2, stimulating DNA binding approximately 40-fold. In comparison, Ets-1 stimulates CBFalpha2 DNA binding 7- to 10-fold. Although the Runt domain alone is sufficient for heterodimerization with CBFbeta, sequences N terminal to amino acid 41 and between amino acids 190 and 214 are required for cooperative DNA binding with Ets-1. Cooperative DNA binding with Ets-1 is less pronounced with the CBFalpha2-CBFbeta heterodimer than with CBFalpha2 alone. These analyses demonstrate that CBFalpha2 is subject to both negative regulation by intramolecular interactions, and positive regulation by two alternative partnerships.

Animals↗