Katsuya Shigesada

A WW domain‐containing Yes‐associated protein (YAP) is a novel transcriptional co‐activator

A protein module called the WW domain recognizes and binds to a short oligopeptide called the PY motif, PPxY, to mediate protein–protein interactions. The PY motif is present in the transcription activation domains of a wide range of transcription factors including c‐Jun, AP‐2, NF‐E2, C/EBPα and PEBP2/CBF, suggesting that it plays an important role in transcriptional activation. We show here that mutation of the PY motif in the subregion of the activation domain of the DNA‐binding subunit of PEBP2, PEBP2α, abolishes its transactivation function. Using yeast two‐hybrid screening, we demonstrate that Yes‐associated protein (YAP) binds to the PY motif of PEBP2α through its WW domain. The C‐terminal region of YAP fused to the DNA‐binding domain of GAL4 showed transactivation as strong as that of GAL4–VP16. Exogenously expressed YAP conferred transcription‐stimulating activity on the PY motif fused to the GAL4 DNA‐binding domain as well as to native PEBP2α. The osteocalcin promoter was stimulated by exogenous PEBP2αA and a dominant negative form of YAP strongly inhibited this activity, suggesting YAP involvement in this promoter activity in vivo. These results indicate that the PY motif is a novel transcription activation domain that functions by recruiting YAP as a strong transcription activator to target genes.

Dimerization with PEBP2β protects RUNX1/AML1 from ubiquitin–proteasome‐mediated degradation

The RUNX family genes are the mammalian homologs of the Drosophila genes runt and lozenge, and members of this family function as master regulators of definitive hematopoiesis and osteogenesis. The RUNX genes encode the α subunit of the transcription factor PEBP2/CBF. The β subunit consists of the non‐RUNX protein PEBP2β. We found that RUNX1/AML1, which is essential for hematopoiesis, is continuously subjected to proteolytic degradation mediated by the ubiquitin–proteasome pathway. When PEBP2β is present, however, the ubiquitylation of RUNX1 is abrogated and this causes a dramatic inhibition of RUNX1 proteolysis. Heterodimerization between PEBP2β and RUNX1 thus appears to be an essential step in the generation of transcriptionally competent RUNX1. Consistent with this notion, RUNX1 was barely detected in PEBP2β−/− mouse. CBF(PEBP2)β– SMMHC, the chimeric protein associated with inv(16) acute myeloid leukemia, was found to protect RUNX1 from proteolytic degradation more efficiently than PEBP2β. These results reveal a hitherto unknown and major role of PEBP2β, namely that it regulates RUNX1 by controlling its turnover. This has allowed us to gain new insights into the mechanism of leukemogenesis by CBFβ–SMMHC.

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.

Cloning, mapping and expression of PEBP2αC, a third gene encoding the mammalian Runt domain

Abstract PEBP2/CBF is a heterodimeric transcription factor composed of α and β subunits. Previously, we reported two distinct mouse genes, PEBP2αA and PEBP2αB, which encode the α subunit. PEBP2αB is the homologue of human AML1, encoding the acute myeloid leukemia 1 protein. AMLI and human PEBP2/CBFβ were detected independently at the breakpoints of two characteristic chromosome translocations observed frequently in two subtypes of acute myeloid leukemia. The PEBP2α 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, PEBP2αC, which maps to lp36.11-p36.13 in the human chromosome and encodes a 415-aa protein. PEBP2αC forms a heterodimer with PEBP2β, binds to the PEBP2 site and transactivates transcription, similar to PEBP2αA and PEBP2αB.

Functional Analysis of RUNX2 Mutations in Japanese Patients with Cleidocranial Dysplasia Demonstrates Novel Genotype-Phenotype Correlations

Cleidocranial dysplasia (CCD) is an autosomal dominant heritable skeletal disease caused by heterozygous mutations in the osteoblast-specific transcription factor RUNX2. We have performed mutational analysis of RUNX2 on 24 unrelated patients with CCD. In 17 patients, 16 distinct mutations were detected in the coding region of RUNX2: 4 frameshift, 3 nonsense, 6 missense, and 2 splicing mutations, in addition to 1 polymorphism. The missense mutations were all clustered within the Runt domain, and their protein products were severely impaired in DNA binding and transactivation. In contrast, two RUNX2 mutants had the Runt domain intact and remained partially competent for transactivation. One criterion of CCD, short stature, was much milder in the patients with the intact Runt domain than in those without. Furthermore, a significant correlation was found between short stature and the number of supernumerary teeth. On the one hand, these genotype-phenotype correlations highlight a general, quantitative dependency, by skeleto-dental developments, on the gene dosage of RUNX2, which has hitherto been obscured by extreme clinical diversities of CCD; this gene-dosage effect is presumed to manifest on small reductions in the total RUNX2 activity, by approximately one-fourth of the normal level at minimum. On the other hand, the classic CCD phenotype, hypoplastic clavicles or open fontanelles, was invariably observed in all patients, including those with normal height. Thus, the cleidocranial bone formation, as mediated by intramembranous ossification, may require a higher level of RUNX2 than does skeletogenesis (mediated by endochondral ossification), as well as odontogenesis (involving still different complex processes). Overall, these results suggest that CCD could result from much smaller losses in the RUNX2 function than has been envisioned on the basis of the conventional haploinsufficiency model.

Subcellular localization of the alpha and beta subunits of the acute myeloid leukemia-linked transcription factor PEBP2/CBF.

Each of the two human genes encoding the alpha and beta subunits of a heterodimeric transcription factor, PEBP2, has been found at the breakpoints of two characteristic chromosome translocations associated with acute myeloid leukemia, suggesting that they are candidate proto-oncogenes. Polyclonal antibodies against the alpha and beta subunits of PEBP2 were raised in rabbits and hamsters. Immunofluorescence labeling of NIH 3T3 cells transfected with PEBP2 alpha and -beta cDNAs revealed that the full-size alpha A1 and alpha B1 proteins, the products of two related but distinct genes, are located in the nucleus, while the beta subunit is localized to the cytoplasm. Deletion analysis demonstrated that there are two regions in alpha A1 responsible for nuclear accumulation of the protein: one mapped in the region between amino acids 221 and 513, and the other mapped in the Runt domain (amino acids 94 to 221) harboring the DNA-binding and the heterodimerizing activities. When the full-size alpha A1 and beta proteins are coexpressed in a single cell, the former is present in the nucleus and the latter still remains in the cytoplasm. However, the N- or C-terminally truncated alpha A1 proteins devoid of the region upstream or downstream of the Runt domain colocalized with the beta protein in the nucleus. In these cases, the beta protein appeared to be translocated into the nucleus passively by binding to alpha A1. The chimeric protein containing the beta protein at the N-terminal region generated as a result of the inversion of chromosome 16 colocalized with alpha A1 to the nucleus more readily than the normal beta protein. The implications of these results in relation to leukemogenesis are discussed.

FUNCTIONAL DISSECTION OF THE ALPHA AND BETA SUBUNITS OF TRANSCRIPTION FACTOR PEBP2 AND THE REDOX SUSCEPTIBILITY OF ITS DNA BINDING ACTIVITY

The mouse transcription factor PEBP2 is a heterodimer of two subunits: a DNA binding subunit α and its partner subunit β. The α subunit shares a region of high homology, termed the Runt domain, with the products of the Drosophila melanogaster segmentation gene runt and the human acute myeloid leukemia-related gene AML1. To study the molecular basis for the DNA binding and heterodimerization functions of this factor, we constructed series of deletions of the α and β subunits and examined their activities by electrophoretic mobility shift and affinity column assays. The minimal functional region of the α subunit for DNA binding and dimerization was shown to coincide with the Runt domain. On the other hand, the region of the β subunit required for heterodimerization was localized to the N-terminal 135 amino acids. Furthermore, it was found that the DNA binding activity of the Runt domain is regulated by a reduction/oxidization (redox) mechanism and that its reductively activated state, which is extremely labile, is stabilized by the β subunit. These findings add a new layer to the mechanism and significance of the regulatory interplay between the two subunits of PEBP2.

Immunoglobulin motif DNA recognition and heterodimerization of the PEBP2/CBF Runt domain

The polyomavirus enhancer binding protein 2 (PEBP2) or core binding factor (CBF) is a heterodimeric enhancer binding protein that is associated with genetic regulation of hematopoiesis and osteogenesis. Aberrant forms of PEBP2/CBF are implicated in the cause of the acute human leukemias and in a disorder of bone development known as cleidocranial dysplasia. The common denominator in the natural and mutant forms of this protein is a highly conserved domain of PEBP2/CBFα, termed the Runt domain (RD), which is responsible for both DNA binding and heterodimerization with the β subunit of PEBP2/CBF. The three-dimensional structure of the RD bound to DNA has been determined to be an S-type immunoglobulin fold, establishing a structural relationship between the RD and the core DNA binding domains of NF-κB, NFAT1, p53 and the STAT proteins. NMR spectroscopy of a 43.6 kD RD–β–DNA ternary complex identified the surface of the RD in contact with the β subunit, suggesting a mechanism for the enhancement of RD DNA binding by β. Analysis of leukemogenic mutants within the RD provides molecular insights into the role of this factor in leukemogenesis and cleidocranial dysplasia.

Serine phosphorylation of RUNX2 with novel potential functions as negative regulatory mechanisms

The RUNX family represents a small group of heterodimeric transcription factors that master‐regulate osteogenesis and hematopoiesis in mammals. Their genetic defects cause human diseases such as cleidocranial dysplasia (CCD) and acute myelogenous leukemia. However, the mechanism(s) regulating their functions are still poorly understood. Here, we report a novel observation that suggests that the osteogenesis‐associated homologue RUNX2 is negatively regulated by the phosphorylation of two conserved serines (S104 and S451) in two distinct functional aspects. The phosphorylation of S104 could abolish the heterodimerization of RUNX2 with the partner subunit, PEBP2β, which enhances the metabolic stability of RUNX2. On the other hand, the phosphorylation of S451 resides within the C‐terminal transcription inhibition domain of RUNX2 and hence is implicated in its functional mobilization. One CCD mutation, S104R of RUNX2, appears to mimic the phosphorylation‐dependent inhibition of heterodimerization, thereby rendering RUNX2 metabolically unstable.

Mechanism of leukemogenesis by the inv(16) chimeric gene CBFB/PEBP2B-MHY11

Inv(16)(p13q22) is associated with acute myeloid leukemia subtype M4Eo that is characterized by the presence of myelomonocytic blasts and atypical eosinophils. This chromosomal rearrangement results in the fusion of CBFB and MYH11 genes. CBFβ normally interacts with RUNX1 to form a transcriptionally active nuclear complex. The MYH11 gene encodes the smooth muscle myosin heavy chain. The CBFβ-SMMHC fusion protein is capable of binding to RUNX1 and form dimers and multimers through its myosin tail. Previous results from transgenic mouse models show that Cbfb-MYH11 is able to inhibit dominantly Runx1 function in hematopoiesis, and is a key player in the pathogenesis of leukemia. In recent years, molecular and cellular biological studies have led to the proposal of several models to explain the function of CBFβ-SMMHC. In this review, we will first focus our attention on the molecular mechanisms proposed in the recent publications. We will next examine recent gene expression profiling studies on inv(16) leukemia cells. Finally, we will describe a recent study from one of our labs on the identification of cooperating genes for leukemogenesis with CBFB-MYH11.