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PubMed1994

PEBP2--a modulator of polyoma DNA replication.

Previously, we have shown that integrated copies of polyoma DNA can be induced to replicate in rat fibroblasts (H3 cells) exposed to a DNA-damaging agent. In the current study, we demonstrate that UV-irradiation of mouse fibroblasts (WOP cells), transiently transfected with polyoma DNA, results in repression of polyoma replication. Cotransfection of oligomers representing wild-type but not mutated forms of the PEBP2 target sequence restored levels of viral replication indicating a role of PEBP2 binding proteins in mediating this effect. DNA-binding assays revealed that a different subset of complexes was formed with the PEBP2 target sequence when nuclear proteins from sham and UV-irradiated WOP and H3 cells were compared, suggesting that the activities of PEBP2 binding proteins are differentially regulated upon UV-irradiation in these two cell types. The ability of PEBP2 to modulate polyoma replication following UV-irradiation in WOP cells suggests a potential role of PEBP2 proteins in the cellular response to DNA damage.

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↗
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↗
PubMed2004

Large fontanelles are a shared feature of haploinsufficiency of RUNX2 and its co-activator CBFB.

CBFB at 16q22 heterodimerizes with either RUNX2 (also known as CBFA1) or RUNX1 (CBFA2) to activate the transcription of downstream molecules. RUNX2 regulates osteoblast differentiation and chondrocyte maturation and its haploinsufficiency leads to cleidocranial dysplasia, characterized large fontanelles, hypoplasia or aplasia of the clavicles, hypoplasia of the distal phalanges, and a wide pubic symphysis. Complete loss of Runx1 or Cbfb in mice is lethal because of the absence of fetal liver hematopoiesis. Fetal rescue in Cbfb(-/-) mice by providing the Cbfb functions in the hematopoietic progenitors leads to wide fontanelle and delayed chondrocyte maturation, presumably resulting from the incomplete function of the transcriptional pathway mediated by the Cbfb-Runx2 heterodimer. The present report describes a patient with a small deletion of chromosome 16q22.1 encompassing CBFB. Skeletal abnormalities included a widely open fontanelle, multiple wormian bones along the sagittal suture, hypoplasia of the distal phalanges, and mildly shortened clavicles. G-banding analysis revealed a shortening of the 16q22.1 band. A fluorescence in situ hybridization analysis, using the BAC probe spanning the CBFB locus at 16q22.1, revealed that the CBFB probe hybridized to only one of the two homologous chromosome 16 regions. Array-comparative genomic hybridization analysis revealed that the deletion spans 1.2 megabases. In reviewing eight previously reported cases of 16q interstitial deletions involving band q22, large cranial sutures were noted in all but one case. Considering the phenotypic similarity of the 16q22 deletion case and Cbfb(-/-) mice rescued for hematopoiesis and the consistency of the phenotype among 16q22 deletion cases, we suggest that the common phenotypic feature of the 16q22 deletion, large fontanelles, can be attributed to a haploinsufficiency of CBFB.

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↗
PubMed1998

Transcription factors and osteoblasts.

Transcription factors play a key role in determination of the fate of the cells in osteoblastic and chondrocytic lineage. A runt family member, Cbfa, is indispensable for osteoblastic differentiation. Sox 9 and scleraxis are involved in the phenotypic expression in chondrocytes and the cells of early stage connective tissues. These transcription factors will give us a clue to unravel interaction of these known and yet unknown transcription factors to fully understand the mechanisms of skeletal cells ' differentiation and regulation of their functions.

Animals↗
PubMed2002

Core binding factor genes and human leukemia.

BACKGROUND: The core binding factor (CBF) transcription complex, consisting of the interacting proteins RUNX1 and CBFb, is essential for normal hematopoiesis. Recent studies have shown that mutations and gene rearrangements involving this complex are frequently implicated in leukemogenesis. Understanding the molecular events leading to the disruption of CBF has provided important insights into our understanding of the normal regulatory pathways that control hematopoiesis and has begun to reveal how alterations in these pathways induce leukemia. INFORMATION SOURCES: Both authors are involved in the identification and characterization of chromosomal abnormalities associated with hematologic malignancy. This has led to contributions to multicenter clinical and laboratory investigations as well as publications in peer-reviewed journals. All of the references cited in this review are published in journals covered by Medline. State of the Art. The core binding factor (CBF) is a heterodimeric transcription factor composed of the RUNX1 and CBFb subunits. RUNX1 is the DNA binding element of the complex and its affinity is greatly increased in the presence of CBFb. Knock-out studies in mice have demonstrated that both RUNX1 and CBFb are necessary for definitive hematopoiesis. Furthermore, reciprocal chromosomal translocations involving both partners have been directly implicated in leukemogenesis. Evidence is now emerging that at least some of the resulting fusion proteins, namely ETV6-RUNX1, RUNX1-MTG8 and CBFb-MYH11 dominantly inhibit the function of native CBF by recruiting transcriptional co-repressor complexes. However, knock-in studies have shown that whilst expression of these fusion genes may disrupt normal hematopoiesis, this, by itself, is not sufficient for the subsequent development of leukemia. Mutations of RUNX1 have been identified in familial platelet disorder (FDP), in which there is a congenital predisposition to the development of AML and heterozygous point mutations have been identified in the RUNX1 gene in some leukemias. Moreover, a small number of cases have been reported in which amplification of RUNX1 has been detected in childhood ALL suggesting mechanisms other than loss of function, such as gene dosage may also play a role. CONCLUSIONS: Understanding the role CBF plays in normal hematopoiesis and hematologic malignancies has provided critical reagents for the accurate identification of the broad group of leukemias harboring alterations of CBF. The application of these molecular approaches has already shown an impact on the clinical management of these patients and as more information becomes available, the ability to tailor therapy to improve each patient's chance of a cure becomes feasible.

Animals↗
PubMed1996

Biochemical and biophysical properties of the core-binding factor alpha2 (AML1) DNA-binding domain.

The Runt domain is the DNA-binding domain defining a small family of transcription factors that are involved in important developmental processes. Developmental pathways controlled by Runt domain proteins include sex determination, neurogenesis, segmentation, and eye development in Drosophila and hematopoiesis in mammals. In addition to binding DNA, the Runt domain also mediates heterodimerization with another subunit called the core-binding factor beta (CBFbeta) subunit. In this study we overexpress the Runt domain from the mouse CBFalpha2 (AML1) protein in Escherichia coli, and purify it from the insoluble fraction. We determine the equilibrium constants for Runt domain binding to two different DNA sequences by surface plasmon resonance technology. Circular dichroism spectroscopy demonstrates that the Runt domain is a folded beta-domain with essentially no alpha-helical content. The single tryptophan residue in the CBFalpha2 Runt domain at amino acid 79 is shown by tryptophan fluorescence spectroscopy to reside in a polar environment. Finally, we demonstrate that ATP can be UV cross-linked to the Runt domain and that ATP binding is sensitive to an amino acid substitution in the putative Kinase-1a motif (P-loop).

Adenosine Triphosphate↗
PubMed1995

Structure of the leukemia-associated human CBFB gene.

We have determined the structure of the human CBFB gene, which encodes the beta subunit of the heterodimeric transcription factor core binding factor (CBF). This gene becomes fused to the MYH11 gene encoding smooth muscle myosin heavy chain by an inversion of chromosome 16 that occurs in the M4Eo subtype of acute myeloid leukemia. The CBFB gene contains 6 exons and spans 50 kb. The gene is highly conserved in animal species as distant as Drosophila, and the exon boundaries are in locations identical to those of the murine Cbfb homologue. The CBFB promoter region has typical features of a housekeeping gene, including high G+C content, high frequency of CpG dinucleotides, and lack of canonical TATA and CCAAT boxes. This gene has a single transcriptional start site, 345 nucleotides upstream of the beginning of the coding region. The human and mouse CBFB promoters show conservation of several transcriptional regulatory sequence motifs, including binding sites for Sp1, Ets family members, and Myc, but do not contain any CBF binding sites. The 5' end of the human CBFB gene also contains a highly polymorphic, transcribed CGG repeat that is not present in the murine homologue.

Acute Disease↗
PubMed2025

CDK12 inhibition reveals melanoma dependence on the RUNX1/CBFβ complex for genomic stability.

Cutaneous melanoma is the deadliest form of skin cancer, frequently driven by hyperactivation of the RAS/mitogen-activated protein kinase (MAPK) pathway. Cyclin-dependent kinase 12 (CDK12), a downstream effector of MAPK signaling, has emerged as a therapeutic target due to its essential role in transcriptional regulation and DNA damage repair. To identify vulnerabilities associated with CDK12 inhibition, we performed a genome-wide CRISPR-Cas9 screen and identified the Runt-related transcription factor RUNX1 and its cofactor CBFβ as synthetic lethal partners of CDK12. RUNX1 inhibition enhanced melanoma sensitivity to CDK12 inhibitors in a p53-independent manner, resulting in DNA damage accumulation and impaired repair capacity. Combined inhibition of CDK12 and RUNX1 suppressed melanoma growth in vivo. These findings identify RUNX1/CBFβ as a compensatory mechanism in CDK12-inhibited melanoma and define a synthetic lethal interaction with translational potential for combinatorial therapy.

Core Binding Factor Alpha 2 Subunit↗
PubMed1995

The leukemic core binding factor beta-smooth muscle myosin heavy chain (CBF beta-SMMHC) chimeric protein requires both CBF beta and myosin heavy chain domains for transformation of NIH 3T3 cells.

An inversion of chromosome 16 associated with the M4Eo subtype of acute myeloid leukemia produces a chimeric protein fusing the beta subunit of the transcription factor core binding factor (CBF beta) to the tail region of smooth muscle myosin heavy chain (SMMHC). We investigated the oncogenic properties of this CBF beta-SMMHC chimeric protein using a 3T3 transformation assay. NIH 3T3 cells expressing CBF beta-SMMHC acquired a transformed phenotype, as indicated by their ability to form foci, grow in soft agarose, and form tumors in nude mice. Cells expressing normal CBF beta or the SMMHC tail domain did not become transformed. Electrophoretic mobility-shift assays showed that extracts from cells transformed by CBF beta-SMMHC no longer formed the normal CBF/DNA complex but instead formed a much larger complex that did not migrate into the gel. Analysis of CBF beta-SMMHC deletion mutants demonstrated that the chimeric protein was transforming only if two domains were both present: (i) CBF beta sequences necessary for association with the CBF alpha subunit, and (ii) SMMHC sequences important for the formation of multimeric filaments. These results are direct evidence that CBF beta-SMMHC can function as an oncoprotein.

3T3 Cells↗
PubMed1999

Regulation mechanisms for the heterodimeric transcription factor, PEBP2/CBF.

Members of the new PEBP2 (Polyomavirus Enhancer Binding Protein 2) family of heterodimeric transcriptional regulatory protein are composed of two subunits, alpha and beta. One of the genes encoding the alpha subunit, AML1/PEBP2 alpha B, was identified at the breakpoints of various chromosome translocations, including t(8;21) and t(12;21) associated with acute myeloid leukemia and acute lymphoblastic leukemia, respectively. The gene encoding the beta subunit (PEBP2 beta/CBFB) was also shown to be the target of the inversion of chromosome 16, another chromosomal anomaly associated with acute myeloid leukemia. Targeted disruption of either the Aml1/Pebp2 alpha B or Pebp2 beta/Cbfb gene resulted in strikingly similar phenotypes such as lack of definitive hematopoiesis of the fetal liver and accompanying hemorrhage of the central nervous system. These observations suggest that both alpha and beta subunits of PEBP2 are indispensable for its in vivo function. However, the heterodimerization of the alpha and beta subunit does not seem to occur readily suggesting that their capacity to associate might be an important rate limiting step in PEBP2 site-dependent transcription regulation. In this review, we concentrate on the possible regulatory mechanisms of PEBP2 activity in relation to leukemogenesis.

Animals↗
PubMed1997

The protooncogene product, PEBP2beta/CBFbeta, is mainly located in the cytoplasm and has an affinity with cytoskeletal structures.

The Pebpb2/Cbfb gene encodes the non-DNA binding beta subunit of the heterodimeric transcription factor, PEBP2/CBF, and has been implicated in a subtype of human acute myeloid leukemia, as well as being indispensable for the development of definitive hematopoiesis in the murine fetal liver. By examining a subcellular localization of the PEBP2beta/CBFbeta protein in tissue culture cells, we could reveal an additional aspect of the protein other than to be a subunit of a transcription factor. Immunoblot and immunocytochemical staining showed that PEBP2beta/CBFbeta was mostly present in the cytoplasm. This PEBP2beta/CBFbeta was free from its DNA-binding partner, the alpha subunit of PEBP2/CBF, as judged by the electrophoretic mobility shift assays. Furthermore, a significant amount of PEBP2beta/CBFbeta was retained in the cytoskeleton preparation after detergent extraction of the cells and was found by double immunofluorescence to colocalize with the F-actin on stress fibers and the vinculin in membrane processes. Thus, the present study extends PEBP2beta/CBFbeta to be a cytoskeleton-affinitive as well as nuclear protein. The implications of these results are discussed.

3T3 Cells↗
PubMed2005

Biological implications of filamin A-bound PEBP2beta/CBFbeta retention in the cytoplasm.

Multiple mechanisms regulate dynamic cytoplasmic-to-nuclear transport of transcription factors. However, little is known about the involvement of cytoskeletal proteins in this process. The heterodimeric transcription factor PEBP2/CBF is composed of a DNA-binding subunit, Runx1, and a non-DNA-binding subunit, PEBP2beta/CBFbeta. The Runx1 protein possesses nuclear localization signals and is found exclusively in the nucleus, whereas PEBP2beta is located in the cytoplasm in most cells and tissues examined thus far. We investigated the mechanism by which PEBP2beta localizes to the cytoplasm and found that it associates with filamin A, an actin-binding cytoskeletal protein. Filamin A retains PEBP2beta in the cytoplasm, thereby hindering its engagement as a Runx1 partner. When filamin A is absent, PEBP2beta moves into the nucleus and enhances Runx1-dependent transcription. These observations highlight the significance of the subcellular localization of PEBP2beta in regulating its activity as a component of the PEBP2/CBF transcription factor. In humans, PEBP2beta is frequently targeted in the leukemia-associated chromosomal abnormality, inversion 16 (inv 16). Thus, identifying the factors that mediate the subcellular localization of the PEBP2beta-derived chimeric transcription factor produced by inv 16 is an important issue that will need to be resolved in order to understand the mechanism(s) involved in inv 16-induced leukemogenesis.

Animals↗
PubMed2001

Cloning and expression pattern of a novel PEBP2 beta-binding protein (charged amino acid rich leucine zipper-1[Crl-1]) in the mouse.

PEBP2 beta/Cbf beta is the beta subunit of PEBP2/Cbf, which has been demonstrated to have important biological activities in hematopoiesis and osteogenesis. However, PEBP2 beta is ubiquitously expressed, suggesting that PEBP2 has other additionally important physiological activities. In an effort to elucidate other possible functions for PEBP2, we have isolated a novel gene that encodes a PEBP2 beta-interacting protein from a mouse cDNA library. We have called this gene Crl-1 for charged amino acid rich leucine zipper-1 (Crl-1) because it is rich in charged amino acids and contains a putative leucine zipper region. Expression studies in a 17.5 days post-coitum mouse embryo demonstrated Crl-1 expression mainly in the olfactory bulb and cerebral cortex. Post-natally, Crl-1 expression was additionally observed in the cerebellar cortex with strong expression in the hippocampus. These findings show that this novel PEBP2 beta-interacting protein is expressed mainly in subsets of neuronal cells, suggesting that Crl-1 plays some role in the developing mouse brain.

Amino Acid Sequence↗
PubMed1998

The chimeric protein, PEBP2 beta/CBF beta-SMMHC, disorganizes cytoplasmic stress fibers and inhibits transcriptional activation.

The chromosomal inversion 16(p13;q22) associated with human acute myeloid leukemia generates the chimeric PEBP2 beta/CBF beta-SMMHC gene. The PEBP2 beta/CBF beta portion of the chimeric polypeptide harbors most of the amino acid sequence of the PEBP2 beta/CBF beta protein, the non-DNA binding subunit of the heterodimeric transcription factor, PEBP2/CBF, whereas the SMMHC portion of the chimera consists of the rod domain of the smooth muscle myosin heavy chain molecule. In this study we examined the subcellular localization of the chimeric protein and its effect both on stress fibers and transcriptional activation by transfecting cDNA into tissue culture cells. The localization of the chimera was investigated by immunocytochemical staining of cells and was found to be both cytoplasmic and nuclear. One aspect of the effect of expression of the chimera was a drastic alteration of cell morphology. The cells appeared elongated and possessed long cytoplasmic processes. Double fluorescent labeling revealed disorganization of the stress fibers and an altered F-actin staining pattern in the transfected cells. Studies using a deletion mutant showed that both the PEBP2 beta/CBF beta and SMMHC domains are necessary for the induction of the morphological alteration. A significant proportion of the chimeric protein was retained in the cytoskeleton after detergent extraction of the cells and could be recuperated as a membrane fraction, suggesting that this is one of the probable sites of action of the PEBP2 beta/CBF beta-SMMHC protein. Another effect of the chimeric protein was inhibition of transcriptional activation dependent on the PEBP2/CBF binding DNA sequence. However, deregulation of PEBP2/CBF site dependent transcription by itself was not sufficient to induce cell morphological changes. Taken together, these results indicate that the PEBP2 beta/CBF beta-SMMHC chimeric protein acts at two levels, at the level of stress fiber organization and at the level of transcriptional activation. We suggest that the action of PEBP2 beta/CBF beta-SMMHC depends to a great extent on whether it is located in the cytoplasm or in the nucleus.

Acute Disease↗
PubMed1997

Differentiation dependent expression and distinct subcellular localization of the protooncogene product, PEBP2beta/CBFbeta, in muscle development.

The Pebpb2/Cbfb gene encodes the non-DNA binding subunit of the heterodimeric transcription factor, PEBP2/CBF. To examine the expression of the PEBP2beta/CBFbeta protein in vivo, we carried out immunohistochemistry using the tissues from adult mice as well as embryos. Although PEBP2beta/CBFbeta was detected in various tissues to various degrees, interesting features of expression were observed in the skeletal myogenic cells. Here PEBP2beta/CBFbeta was found mainly to occur as cytoplasmic staining and the intensity of this staining increased depending on the differentiation stage of the cells. In the undifferentiated myoblasts PEBP2beta/CBFbeta was undetectable, whereas moderate levels of PEBP2beta/ CBFbeta were detected in the elongated and aligned myocytes. PEBP2beta/CBFbeta appeared to accumulate further when the cells fused to each other to become multinucleated myotubes. Once the muscle fibers were established, PEBP2beta/CBFbeta was relocated onto or around the Z-lines. PEBP2beta/CBFbeta was also detected in the cytoplasm of cardiac myocytes and in the smooth muscle cells of the digestive tract. In all the above, the skeletal myotubes were the only case that showed both nuclear and cytoplasmic staining of PEBP2beta/CBFbeta. Thus, we could show differentiation dependent pattern of PEBP2beta/CBFbeta expression in muscle development and establish PEBP2beta/CBFbeta to be a cytoplasmic as well as nuclear protein in vivo.

Actinin↗
PubMed2001

PEBP2 and c-myb sites crucial for lambda5 core enhancer activity in pre-B cells.

The lambda5 gene is expressed exclusively in precursor (pre-) B cells where its gene product, as part of the pre-B cell receptor, is crucial for the proliferation of these cells. Several DNA regions regulate the activity and expression pattern of the lambda5 gene. Amongst these is an enhancer, B(lambda5), located 5' of the gene. Here we analyze the lambda5 enhancer core, b(lambda5), which in earlier experiments was demonstrated to retain 50% of the enhancer activity, and show that this activity is restricted to pre-B cells. We identify a DNA element within b(lambda5), PEBP2(lambda5), which is essential for enhancer activity: mutation within this site dramatically reduces core enhancer activity in pre-B cells. The PEBP2(lambda5) site binds bacterially produced polyoma enhancer binding proteins (PEBP) (Runx/AML/CBFA). Furthermore, PEBP2 proteins present in nuclear extracts from murine pre-B cells bind to the PEBP2(lambda5) element. PEBP2 proteins in mature B cells also bind to the PEBP2(lambda5 )element, implying that if PEBP2 proteins are responsible for the stage-specific expression, they have to be non-activating or inhibiting in mature B cells. We also demonstrate that a described partner of PEBP2, c-myb, binds to a sequence termed myb(lambda5) located just upstream of the PEBP2(lambda5) site in the core enhancer. The myb(lambda5) element is also crucial for enhancer activity, since mutating the myb site reduces core enhancer activity to the same extent as mutating the PEBP2 site. Earlier reports have shown that c-myb is expressed at high levels in pre-B cell lines whereas its expression is down-regulated in more mature B cell lines. Thus, c-myb may be involved in determining the stage-specific expression of the lambda5 gene.

Animals↗