Polyomavirus Enhancer Binding Protein Research Articles

Role of runt-related transcription factor 2 in signal network of tumors as an inter-mediator

Runt-related transcription factor 2 (RUNX2) is a member of the polyomavirus enhancer-binding protein 2/core-binding factor superfamily. RUNX2 is known for its contribution to osteoblast phenotype and bone formation. In recent years, increasing attention has been focused on the relationship of Runx2 with tumorigenesis. In different types of tumor cells, RUNX2 cooperates with its co-activators or co-inhibitors, and mediates the responses of cells to various signaling pathways that are hyperactive in tumors. Thus, several downstream target genes of RUNX2 are activated when RUNX2 interacts with its co-factors, leading to a variety of effects on tumor cells (epithelial–mesenchymal transition, metastasis, proliferation, and osteolytic lesion). This review focuses on the involvement of RUNX2 in tumor cells in the crosstalk of diverse signaling pathways and its multiple functions to develop optimal and feasible approaches for clinical treatment based on the functions of RUNX2.

The role of redox-sensitive cysteines in multi sub-unit transcription factors

In response to oxidative stress, cells activate numerous processes and initiate the expression of genes via redox-sensitive transcription factors including the multi sub-unit transcription factors nuclear factor-κB (NFκB), activator protein 1 (AP-1), Hypoxia-inducible factor 1 α (HIF1α), Polyoma virus enhancer-binding protein 2 (PEBP2), cAMP-responsive element-bindingprotein (CREB), Nuclear Factor I/CCAAT Transcription Factor (NFI/CTF), Nuclear transcription factor Y (NF-Y) and E4 Transcription Factor 1 - 60kDa (E4TF1-60). Thioredoxin plays a critical role in this by directly or indirectly (via redox factor 1) reducing redox-sensitive cysteine residues in these transcription factors to modify their tertiary structure, sub-cellular localisation,proteinprotein interactions or DNA-binding affinity. The identity of the redox-sensitive cysteine in E4TF1-60 has not been investigated so far. Determining which cysteines are redox-sensitive within the E4TF1-60 protein sequence together with elucidation of which property is modified will help to better understand the mechanism by which the E4TF1 complex is involved in regulating the cellular oxidative stress response.

A novel in-frame deletion of the RUNX2 gene causes a classic form of cleidocranial dysplasia

The runt-related transcription factor 2 (RUNX2) is a physiological regulatory gene implicated in the development of cleidocranial dysplasia (CCD). Molecular analysis of the RUNX2 gene in a 2-year-old boy with a diagnosis of CCD demonstrated a heterozygous in-frame 9-bp deletion (c.593_601delCCTTGACCA, corresponding to the amino-acid deletion p.ΔTLT198_200). Transcription activity of the ΔTLT198_200 mutant decreased in a similar degree to that of the L199F mutant, which was previously reported in the case with classic CCD. Atomic model assessment demonstrated that the ΔTLT198_200 mutation abolished the heterodimerization of the RUNX2 protein with the partner subunit, polyomavirus enhancer-binding protein 2β (PEBP2β). Destruction of RUNX2/PEBP2β heterodimerization activity appears to impair the function of the RUNX2 protein and cause the disease.

Ectopic bone formation facilitated by human mesenchymal stem cells and osteogenic cytokines via nutrient vessel injection in a nude rat model

In vivo studies using bone marrow-derived mesenchymal stem cells are still uncommon. Applications for bone defect replacement in undesirable clinical circumstances such as large defects, bacterial or other pathogen-contaminated fields, and irradiated surgical wound bed necessitate vascularized bone regeneration. Use of a fascial flap including regenerated bone would be a very powerful tool for treatment. It would be especially beneficial in cases where normal bone regeneration is not expected due to a lack of sufficient blood supply, extensive surgical scarring, or bacterial contamination. In this study, we used nude rats in which the superficial epigastric flap of the experimental group was used to wrap around a mixture of human mesenchymal stem cells, bone morphogenetic protein-2, and basic fibroblast growth factor cytokines in a gelatin carrier. These rats showed significantly higher bone mineral density at 4 weeks compared to the other experimental groups containing phosphate buffered saline, human mesenchymal stem cells alone, or the two cytokines alone (p < 0.01). There were no remarkable histologic differences up to 7 days. At 2 weeks, more progressive vascularity and perivascular tissue deposits were seen in the experimental group. Basophilic mineral structure surrounded the fibroblast-like mesenchymal stem cells at 4 weeks, presumably osteoblastic or osteoclastic cell lining. Bone marker immunohistochemistry against alkaline phosphatase and osteocalcin revealed diffuse and distinct immunoreactivity in osteoblastic cells in the experimental group at 4 weeks. Further transcriptional expression of polyomavirus enhancer binding protein 2alphaA suggested that the human transplanted cells proceeded to osteogenic lineage in 4 weeks. These results may be useful as a new approach for bone regeneration.

Identification of TAZ as a Binding Partner of the Polyomavirus T Antigens

A polyomavirus mutant isolated by the tumor host range selection procedure (19) has a three-amino-acid deletion (Delta2-4) in the common N terminus of the T antigens. To search for a cellular protein bound by wild-type but not the mutant T antigen(s), a yeast two-hybrid screen of a mouse embryo cDNA library was carried out with a bait of wild-type small T antigen (sT) fused N terminally to the DNA-binding domain of Gal4. TAZ, a transcriptional coactivator with a WW domain and PDZ-binding motif (17), was identified as a binding partner. TAZ bound in vivo to all three T antigens with different apparent affinities estimated as 1:7:100 (large T antigen [lT]:middle T antigen [mT]:sT). The Delta2-4 mutant T antigens showed no detectable binding. The sT and mT of the host range transformation-defective (hr-t) mutant NG59 with an alteration in the common sT/mT region (179 D-->NI) and a normal N terminus also failed to bind TAZ, while the unaltered lT bound but with reduced affinity compared to that seen in a wild-type virus infection. The WW domain but not the PDZ-binding motif of TAZ was essential for T antigen binding. The Delta2-4 mutant was defective in viral DNA replication. Forced overexpression of TAZ blocked wild-type DNA replication in a manner dependent on the binding site for the polyomavirus enhancer-binding protein 2alpha. Wild-type polyomavirus T antigens effectively block transactivation by TAZ. The functional significance of TAZ interactions with polyomavirus T antigens is discussed.

Oncogenic potential of the RUNX gene family: 'overview'.

Runt-related (RUNX) gene family is composed of three members, RUNX1/AML1, RUNX2 and RUNX3, and encodes the DNA-binding (alpha) subunits of the Runt domain transcription factor polyomavirus enhancer-binding protein 2 (PEBP2)/core-binding factor (CBF), which is a heterodimeric transcription factor. RUNX1 is most frequently involved in human acute leukemia. RUNX2 shows oncogenic potential in mouse experimental system. RUNX3 is a strong candidate as a gastric cancer tumor suppressor. The beta subunit gene of PEBP2/CBF is also frequently involved in chromosome rearrangements associated with human leukemia. In this Overview, I will summarize how this growing field has been formed and what are the challenging new frontiers for better understanding of the oncogenic potential of this gene family.

Alpha-defensin expression during myelopoiesis: identification of cis and trans elements that regulate expression of NP-3 in rat promyelocytes.

Alpha-defensins are antimicrobial peptides that contribute to innate-immune functions of neutrophils and intestinal Paneth cells. Transcription of alpha-defensin genes occurs early in neutrophilic myelopoeisis. To examine the mechanisms that regulate alpha-defensin gene expression, we analyzed transcription of rat neutrophil alpha-defensin NP-3 in D4 cells, a subclone of the promyelocytic cell line IPC-81. Northern blot analysis showed that D4 cells express fivefold higher levels of alpha-defensin mRNA than the parental cell line in a manner relatively independent of passage number. Increased levels of steady-state mRNA in D4 cells correlated with markedly elevated peptide levels detected by immunocytochemical staining. To identify the cis-acting DNA elements involved in tissue-specific expression, D4 cells were transfected with luciferase reporter constructs containing NP-3 gene 5'-flanking sequences. Analyses of transfected D4 cells demonstrated that the proximal 87 base pair (bp) sequence contained cis-acting DNA elements necessary for optimal promoter activity. Mutational analyses within the 87-bp region suggested the involvement of the CAAT box and a putative polyoma enhancer-binding protein 2/core-binding factor (PEBP2/CBF) site in defensin gene transcription. Transient transfection analyses using tandem repeats of oligonucleotides containing these sequences demonstrated that proximity of the CAAT box and PEBP2/CBF site was important for defensin promoter activity. Electrophoretic mobility shift assays indicated that PEBP2/CBF or a PEBP2/CBF-related protein was involved in a specific protein-DNA interaction occurring within a DNA fragment containing the CAAT and PEBP2/CBF sequences. These data identify functional trans- and cis-elements that regulate rat defensin gene expression in high defensin-expressing promyelocytic cells.

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.

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.

HLA-G exhibits low level of polymorphism in indigenous East Africans

Human leukocyte antigen G (HLA-G) is a nonclassical HLA class I antigen that is predominantly expressed on invasive cytotrophoblastic cells, and is postulated to be a mediator of maternal-fetal tolerance. Almost all studies in Caucasian and Asian populations have consistently reported that HLA-G exhibits low levels of allelic polymorphism unlike the classical class I genes. However, the concept that HLA-G is nonpolymorphic has recently been challenged in a single study of African-American subjects. We have examined the DNA sequences of the first seven HLA-G exons by single-strand conformational polymorphism (SSCP) and DNA direct sequencing procedures in 45 healthy individuals from an indigenous African population. Overall, we detected 14 sequence variations: 3 in the signal peptide (exon 1); 2 in the α-1 domain (exon 2); 5 in the α-2 domain (exon 3); 2 in α-3 domain (exon 4); 2 in transmembrane domain (exon 5); and none in the cytoplasmic tail (exons 6 and 7). Of these variants, only three result in amino acid substitutions at the protein level. Of particular interest, we identified a novel nucleotide substitution (C727T), 56 bp before the HLA-G gene transcription start site, located in the putative binding site for polyomavirus enhancer-binding protein 2 (PEBP2) transcriptor factor. These data confirm previous reports describing HLA-G exhibiting limited allelic polymorphism. Further studies are needed to determine the impact of the C727T polymorphism on the level or developmental regulation of HLA-G expression.

Two novel cucurbitacins, neocucurbitacins A and B, from the Brazilian folk medicine "Buchinha" (Luffa operculata) and their effect on PEBP2alphaA and OCIF gene expression in a human osteoblast-like Saos-2 cell line.

Two novel cucurbitacins designated as neocucurbitacins A (1), possessing inhibitory activity of polyoma enhancer binding protein 2alphaA (PEBP2alphaA) and osteoclastogenesis-inhibitory factor (OCIF) gene expression in human osteoblast-like cells, and B (2) were isolated from the fruit of Luffa operculata. Their structures have been determined by extensive spectroscopic investigation.

Mutations of the AML1 gene in myelodysplastic syndrome and their functional implications in leukemogenesis

AbstractThe AML1 gene encodes a DNA-binding protein that contains the runt domain and is the most frequent target of translocations associated with human leukemias. Here, point mutations of the AML1 gene, V105ter (single-letter amino acid code) and R139G, (single-letter amino acid codes) were identified in 2 cases of myelodysplastic syndrome (MDS) by means of the reverse transcriptase–polymerase chain reaction single-strand conformation polymorphism method. Both mutations are present in the region encoding the runt domain of AML1 and cause loss of the DNA-binding ability of the resultant products. Of these mutants, V105ter has also lost the ability to heterodimerize with polyomavirus enhancer binding protein 2/core binding factor β (PEBP2β/CBFβ). On the other hand, the R139G mutant acts as a dominant negative inhibitor by competing with wild-type AML1 for interaction with PEBP2β/CBFβ. This study is the first report that describes mutations of AML1 in patients with MDS and the mechanism whereby the mutant acts as a dominant negative inhibitor of wild-type AML1.

Mutations of the AML1 gene in myelodysplastic syndrome and their functional implications in leukemogenesis

The AML1 gene encodes a DNA-binding protein that contains the runt domain and is the most frequent target of translocations associated with human leukemias. Here, point mutations of the AML1 gene, V105ter (single-letter amino acid code) and R139G, (single-letter amino acid codes) were identified in 2 cases of myelodysplastic syndrome (MDS) by means of the reverse transcriptase-polymerase chain reaction single-strand conformation polymorphism method. Both mutations are present in the region encoding the runt domain of AML1 and cause loss of the DNA-binding ability of the resultant products. Of these mutants, V105ter has also lost the ability to heterodimerize with polyomavirus enhancer binding protein 2/core binding factor beta (PEBP2beta/CBFbeta). On the other hand, the R139G mutant acts as a dominant negative inhibitor by competing with wild-type AML1 for interaction with PEBP2beta/CBFbeta. This study is the first report that describes mutations of AML1 in patients with MDS and the mechanism whereby the mutant acts as a dominant negative inhibitor of wild-type AML1.

Polyomavirus Enhancer-binding Protein 2/Core Binding Factor/Acute Myeloid Leukemia Factors Contribute to the Cell Type-specific Activity of the CD11a Integrin Gene Promoter

The CD11a/CD18 leukocyte integrin (LFA-1; also known as alphaL/beta2) mediates leukocyte transendothelial migration during immune and inflammatory responses and participates in lymphoma metastasis. CD11a/CD18 leukocyte-restricted expression is controlled by the CD11a gene promoter, which confers tissue-specific expression to reporter genes in vitro and in vivo. DNase I protection analysis of the CD11a proximal gene promoter revealed DNA-protein interactions centered at position -110 (CD11a-110). Disruption of CD11a-110 reduced CD11a promoter activity in a cell type-specific manner, as it reduced its activity by 70% in Jurkat lymphoid cells, whereas the effect was considerably lower in K562 and HepG2 cells. Electrophoretic mobility shift assays showed evidence of cell type-specific differences in CD11a-110 binding and indicated its specific recognition by members of the polyomavirus enhancer-binding protein 2/core binding factor (CBF)/acute myeloid leukemia (AML) family of transcription factors. AML1B/CBFbeta transactivated the CD11a promoter, with AML1B/CBFbeta-mediated transactivation being completely dependent on the integrity of the CD11a-110 element. Therefore, CBF/AML factors play a role in the cell type-restricted transcription of the CD11a integrin gene through recognition of CD11a-110. The involvement of CBF/AML factors in CD11a expression raises the possibility that CD11a/CD18 expression might be deregulated in acute myeloid and B-lineage acute lymphoblastic leukemias, thus contributing to their altered adhesion and metastatic potential.

A RUNX2/PEBP2alpha A/CBFA1 mutation displaying impaired transactivation and Smad interaction in cleidocranial dysplasia.

Cleidocranial dysplasia (CCD), an autosomal-dominant human bone disease, is thought to be caused by heterozygous mutations in runt-related gene 2 (RUNX2)/polyomavirus enhancer binding protein 2alphaA (PEBP2alphaA)/core-binding factor A1 (CBFA1). To understand the mechanism underlying the pathogenesis of CCD, we studied a novel mutant of RUNX2, CCDalphaA376, originally identified in a CCD patient. The nonsense mutation, which resulted in a truncated RUNX2 protein, severely impaired RUNX2 transactivation activity. We show that signal transducers of transforming growth factor beta superfamily receptors, Smads, interact with RUNX2 in vivo and in vitro and enhance the transactivation ability of this factor. The truncated RUNX2 protein failed to interact with and respond to Smads and was unable to induce the osteoblast-like phenotype in C2C12 myoblasts on stimulation by bone morphogenetic protein. Therefore, the pathogenesis of CCD may be related to the impaired Smad signaling of transforming growth factor beta/bone morphogenetic protein pathways that target the activity of RUNX2 during bone formation.

Identification of the regions involved in DNA binding by the mouse PEBP2α protein

The polyomavirus enhancer binding protein 2α (PEBP2α) is a DNA binding transcriptional regulatory protein that binds conserved sites in the polyomavirus enhancer, mammalian type C retroviral enhancers and T-cell receptor gene enhancers. Binding of PEBP2α and homologous proteins to the consensus DNA sequence TGPyGGTPy is mediated through a protein domain known as the runt domain. Although recent NMR studies of DNA-bound forms of the runt domain have shown an immunoglobulin-like (Ig) fold, the identification of residues of the protein that are involved in DNA binding has been obscured by the low solubility of the runt domain. Constructs of the mouse PEBP2αA1 gene were generated with N- and C-terminal extensions beyond the runt homology region. The construct containing residues Asp90 to Lys225 of the sequence (PEBP2α90–225) yielded soluble protein. The residues that participate in DNA binding were determined by comparing the NMR spectra of free and DNA-bound PEBP2α90–225. Analysis of the changes in the NMR spectra of the two forms of the protein by chemical shift deviation mapping allowed the unambiguous determination of the regions that are responsible for specific DNA recognition by PEBP2α. Five regions in PEBP2α90–225 that are localized at one end of the β-barrel were found to interact with DNA, similar to the DNA binding interactions of other Ig fold proteins.

Biological characteristics of the leukemia-associated transcriptional factor AML1 disclosed by hematopoietic rescue of AML1-deficient embryonic stem cells by using a knock-in strategy.

AML1 is one of the most frequently mutated genes associated with human acute leukemia and encodes the DNA-binding subunit of the heterodimering transcriptional factor complex, core-binding factor (CBF) (or polyoma enhancer binding protein 2 [PEBP2]). A null mutation in either AML1 or its dimerizing partner, CBFbeta, results in embryonic lethality secondary to a complete block in fetal liver hematopoiesis, indicating an essential role of this transcription complex in the development of definitive hematopoiesis. The hematopoietic phenotype that results from the loss of AML1 can be replicated in vitro with a two-step culture system of murine embryonic stem (ES) cells. Using this experimental system, we now demonstrate that this hematopoietic defect can be rescued by expressing the PEBP2alphaB1 (AML1b) isoform under the endogenous AML1-regulatory sequences through a knock-in (targeted insertion) approach. Moreover, we demonstrate that the rescued AML1(-/-) ES cell clones contribute to lymphohematopoiesis within the context of chimeric animals. Rescue requires the transcription activation domain of AML1 but does not require the C-terminal VWRPY motif, which is conserved in all AML1 family members and has been shown to interact with the transcriptional corepressor, Groucho/transducin-like Enhancer of split. Taken together, these data provide compelling evidence that the phenotype seen in AML1-deficient mice is due solely to the loss of transcriptionally active AML1.

Crystal Structure of the Nuclear Matrix Targeting Signal of the Transcription Factor Acute Myelogenous Leukemia-1/Polyoma Enhancer-binding Protein 2αB/Core Binding Factor α2

Transcription factors of the acute myelogenous leukemia (AML)/polyoma enhancer-binding protein (PEBP2alpha)/core-binding factor alpha (CBFA) class are key transactivators of tissue-specific genes of the hematopoietic and bone lineages. AML-1/PEBP2alphaB/CBFA2 proteins participating in transcription are associated with the nuclear matrix. This association is solely dependent on a highly conserved C-terminal protein segment, designated the nuclear matrix targeting signal (NMTS). The NMTS of AML-1 is physically distinct from the nuclear localization signal, operates autonomously, and supports transactivation. Our data indicate that the related AML-3 and AML-2 proteins are also targeted to the nuclear matrix in situ by analogous C-terminal domains. Here we report the first crystal structure of an NMTS in an AML-1 segment fused to glutathione S-transferase. The model of the NMTS consists of two loops connected by a flexible U-shaped peptide chain.

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.

Interaction and Functional Cooperation of PEBP2/CBF with Smads: SYNERGISTIC INDUCTION OF THE IMMUNOGLOBULIN GERMLINE Cα PROMOTER

Smads are signal transducers for members of the transforming growth factor-beta (TGF-beta) superfamily. Upon ligand stimulation, receptor-regulated Smads (R-Smads) are phosphorylated by serine/threonine kinase receptors, form complexes with common-partner Smad, and translocate into the nucleus, where they regulate the transcription of target genes together with other transcription factors. Polyomavirus enhancer binding protein 2/core binding factor (PEBP2/CBF) is a transcription factor complex composed of alpha and beta subunits. The alpha subunits of PEBP2/CBF, which contain the highly conserved Runt domain, play essential roles in hematopoiesis and osteogenesis. Here we show that three mammalian alpha subunits of PEBP2/CBF form complexes with R-Smads that act in TGF-beta/activin pathways as well as those acting in bone morphogenetic protein (BMP) pathways. Among them, PEBP2alphaC/CBFA3/AML2 forms a complex with Smad3 and stimulates transcription of the germline Ig Calpha promoter in a cooperative manner, for which binding of both factors to their specific binding sites is essential. PEBP2 may thus be a nuclear target of TGF-beta/BMP signaling.