Oncogene (2004) 23, 4297–4307 & 2004 Nature Publishing Group All rights reserved 0950-9232/04 $30.00 www.nature.com/onc Mechanism of leukemogenesis by the inv(16) chimeric gene CBFB/PEBP2B-MHY11 Katsuya Shigesada*,1, Bart van de Sluis2 and P Paul Liu*,2 1Department of Cell Biology, Institute for Virus Research, Kyoto University, Kyoto 606-8507, Japan; 2Oncogenesis and Development Section, National Human Genome Research Institute, NIH, Bethesda, MD 20892, USA Inv(16)(p13q22) is associated with acute myeloid leukemia subsequent to which a certain mechanism(s) should act subtype M4Eo that is characterized by the presence of to bring Runx1 into a functionally incompetent state in myelomonocytic blasts and atypical eosinophils. This the end (repression).If one of these steps were lacking or chromosomal rearrangement results in the fusion of CBFB impaired, CBFb-SMMHC would fail to elicit any and MYH11 genes. CBFb normally interacts with RUNX1 substantial dominant-negative effect regardless of how to form a transcriptionally active nuclear complex. well the other step might work. The MYH11 gene encodes the smooth muscle myosin Until recently, however, most functional studies of heavy chain. The CBFb-SMMHC fusion protein is CBFb-SMMHC have centered around the mechanism capable of binding to RUNX1 and form dimers and of repression, paying relatively little attention to the first multimers through its myosin tail. Previous results from heterodimerization step.Nevertheless, evidence sugges- transgenic mouse models show that Cbfb-MYH11 is able tive of its enhanced ability for heterodimerization to inhibit dominantly Runx1 function in hematopoiesis, (hyper-heterodimerization) has come from our previous and is a key player in the pathogenesis of leukemia. In finding that CBFb-SMMHC is predominantly localized recent years, molecular and cellular biological studies in the cytoplasm in association with the actin cytoske- have led to the proposal of several models to explain the leton, and simultaneously capable of sequestering function of CBFb-SMMHC. In this review, we will first RUNX proteins to the cytoplasm in a manner over- focus our attention on the molecular mechanisms proposed riding the intrinsic or artificially modulated ability of in the recent publications. We will next examine recent Runx to localize in the nucleus (Lu et al., 1995; Adya gene expression profiling studies on inv(16) leukemia cells. et al., 1998; Kanno et al., 1998). In sharp contrast, Cbfb Finally, we will describe a recent study from one of our tends to localize in the cytoplasm in a diffuse pattern by labs on the identification of cooperating genes for itself, and can be partly, although not completely, leukemogenesis with CBFB-MYH11. translocated into the nucleus only through heterodimer- Oncogene (2004) 23, 4297–4307. doi:10.1038/sj.onc.1207748 ization with Runx protein.Furthermore, CBF b- SMMHC can stabilize RUNX1 against intracellular Keywords: CBFb-MYH11; CBFb-SMMHC; inv(16); proteasome-mediated degradation much more strongly leukemia; hematopoiesis than Cbfb (Huang et al., 2001a). To characterize the hyper-heterodimerization activity of CBFb-SMMHC more directly, we conducted ex- tensive functional analyses using a series of CBFb- SMMHC deletions as truncated exon by exon either C- Molecular basis for the dominant inhibition of terminally or internally (Huang et al., 2003). Through in RUNX1-dependent transcription by CBFb-SMMHC vitro binding experiments by means of co-immunopre- cipitation and GST-pulldown assays as well as an Identification of dual functional domains in intracellular hyperprotection assay, it was confirmed CBFb-SMMHC that CBFb-SMMHC can heterodimerize with RUNX1 at an affinity higher than that of CBFb by one order of It has been shown that the phenotype of heterozygous magnitude or more.Parallel analyses with RUNX1 Cbfb-MYH11 knockin mice is very similar to that of deletions revealed that the region of RUNX1 required Runx1 knockout mice (Castilla et al., 1996). The results for hyper-heterodimerization is the Runt domain. imply that CBFb-SMMHC inactivates the function of Further, a minimum region of the myosin tail respon- Runx1 nearly completely despite the presence of a sible for hyper-heterodimerization was further mapped residual normal Cbfb/PEBP2b.In order for this to to exons 33–36 (residues 166–363).The myosin tail occur, CBFb-SMMHC would have to outcompete Cbfb alone, of course, did not show any detectable binding to at the step of heterodimerization with Runx1 first of all, the Runt domain.However, appreciable hetrodimeriza- tion activities became detectable when the myosin tail *Correspondence: K Shigesada; E-mail: [email protected]; was fused to PEBP2b proteins made heterodimerization- P Liu; E-mail: [email protected] defective due to double point mutations (residues 64 and Mechanism of leukemogenesis by CBFB/PEBP2B-MHY11 K Shigesada et al 4298 104) (Tang et al., 2000) or a short N-terminal truncation (D2-11) (Adya et al., 1998). Presumably, the myosin tail and a part of the PEBP2b protein may physically or conformationally cooperate to create a new RUNX- binding interface that functions independent of and in synergy with the original heterodimerization interface on CBFb.A minimum region of PEBP2 b involved in this second putative heterodimerization interface was narrowed down to residues 134–165. We next investigated whether and how much the hyper-heterodimerization domain could contribute to the dominant repression of RUNX1-mediated transcrip- tion by CBFb-SMMHC against PEBP2b.Upon a transcription assay using an M-CSFR promoter-based reporter system, coexpressions of CBFb-SMMHC and Figure 1 Diagrammatic summary of functional domain analyses. PEBP2b at equimolar nonsaturating doses resulted in a The top diagram represents CBFb/PEBP2b-MYH11.Red-colored segments (bE1–bE5) and blue-colored segments (E33–42) represent strong repression of transcription to a level several-fold exons encoding Cbfb/PEBP2b and SMMHC, respectively.Double- less than the control obtained by transfection of headed arrows above and below the diagram indicate the mapped PEBP2b alone.When the hyperdimerization domain locations of respective functional and structural domains as (exons 33–36) was removed from CBFb-SMMHC by annotated.ACD and ACD2: assembly competence domains internal deletion, this repression was considerably identified by Sohn et al.(1997) and Ikebe et al.(2001), respectively. The dotted red line represents an intermediate zone that was weakened to twofold or less.This confirmed that the suggested to be unpaired upon deletion of the C-terminally flanking hyperdimerization domain does have an important coiled coil region, but could be integrated at least partly into the positive impact in augmenting repression.Curiously, coiled coil domain in the intact CBFb/PEBP2b -MYH11.The blue however, a converse deletion construct retaining the double-headed arrow represents the terminal nonhelical tail that is hyper-heterodimerization domain but lacking the rest of implicated in the regulation of multimerization (Ikebe et al., 2001) the C-terminal region caused a moderate stimulation of transcription, rather than repression.A simple explana- tion for these seemingly paradoxical effects of the hyper- (Huang et al., 2003). In EMSA experiments, the intact heterodimerization domain may be that the hyperdi- CBFb-SMMHC mixed with RUNX1 forms large DNA- merization domain itself has no or little inhibitory bound complexes that were unable to penetrate into a influence on RUNX1-mediated transactivation, and polyacrylamide gel.When CBF b-SMMHC was deleted that another functional domain responsible for repres- from the C-terminus past exon 39 and further beyond, sion (repression domain) resides within the C-terminal the resulting DNA–protein complexes started to migrate proximal region centering around exons 39–40.In into the gel in increasing fractions at accelerated apparent coincidence with this explanation, other mobilities.Judged from their mobilities, these complexes groups previously reported that C-terminal segments represented heterodimers consisting of single RUNX1 of SMMHC overlapping exons 39–42 could show and CBFb-SMMHC molecules.On the other hand, repressive activities when artificially fused to the Gal4 internally truncated constructs lacking the hyperdimer- DNA-binding domain and assayed using a Gal4-TK luc ization domain with or without additional C-terminally reporter system (Lutterbach et al., 1999). With this extending deletions up to exon 38 still produced low- bipartite functional domain model (Figure 1), we can mobility complexes in much higher proportions than did readily predict that CBFb-SMMHC deleted by the pure C-terminal deletion constructs.Chemical cross- hyperdimerization should no longer be able to compete linking experiments using glutaraldehyde also confirmed dominantly with the normal CBFb and hence would fail that CBFb-SMMHC C-terminally truncated beyond to display its maximal possible repression potential. exon 39 tended to dissociate into monomers in a manner proportionate to their degrees of deletion.Previous glutaraldehyde crosslinking studies with a fewer vari- Molecular conformation and higher order assembly eties of C-terminal deletions also indicated similar of CBFb-SMMHC trends (Adya et al., 1998; Cao et al., 1998). In contrast, Ever since the discovery of CBFb-SMMHC (Liu et al., the above-noted internal deletions largely remained as 1993, 1995), it has implicitly been assumed that the dimers and multimers. myosin tail is