Genes encoding subunits of the core binding factor CBF are frequently disrupted by chromosome rearrangements in human leukemias. Translocation t(8;21), found in AML subtype M2, generates a fusion protein, AML1-ETO. This protein has been shown to inhibit both AML1/CBFα2- and C/EBPα-dependent transcriptional activation, although the mechanism for this inhibition remains unknown (14
). AML1-ETO can also dominantly suppress CBF function, as shown in a mouse knock-in model (38
). The AML1-Evi-1 chimeric protein, produced in t(3;21) associated with t-MDS, t-AML, and CML-BC, can suppress transactivation by intact AML1/CBFα2 (56
). Again, the mechanism for such suppression is not fully understood. In this study we have addressed the mechanism by which the inv(16) chimeric protein CBFβ-SMMHC plays a dominant negative role. Our results suggest that CBFβ-SMMHC plays a dominant negative role by sequestering CBFα2/AML1 to cytoskeletal structures, thus initiating a process towards leukemia by disrupting transcriptional regulation of genes controlled by CBFα2/AML1.
Upon transient transfection into NIH 3T3 cells, CBFα2 was localized in the nucleus whereas CBFβ was distributed throughout the cell. CBFβ-SMMHC was located predominantly in the cytoplasm, forming filamentous or aggregated structures. We did observe some CBFβ-SMMHC protein in the nuclei of the transfected cells, in addition to the filaments and aggregates in the cytoplasm, but we did not observe the nuclear rod-like structure that we previously found in clonal NIH 3T3 cell lines overexpressing CBFβ-SMMHC (53
). This is probably due to the difference in expression level and/or the difference between transient and stable transfections. In fact, the rod-like structures were found only in cell lines expressing high levels of the CBFβ-SMMHC protein and only in a small percentage (10 to 40%) of cells within a cell population (1
). On the other hand, the stress fibers formed by CBFβ-SMMHC were evident in most of the cells stably expressing CBFβ-SMMHC, regardless of expression level (1
In the present study, we demonstrated that CBFβ localized to the nucleus when cotransfected with full-length CBFα2. The likely mechanism for this nuclear localization of CBFβ is that CBFα2 heterodimerizes with CBFβ in the cytoplasm and mediates the transport of the heterodimer into the nucleus. Nuclear localization of CBFα2 can be overridden by CBFβ-SMMHC, which forms homodimers and multimers and interacts with cytoskeletal molecules. The sequestration of CBFα2 by CBFβ-SMMHC is dependent on the heterodimerization domain in CBFβ that mediates interaction with the CBFα subunits. The truncated protein with a deletion of the CBFα2-interacting domain, CBFβ-SMMHCΔN2-11, lost the ability to sequester CBFα2, even though it retained the ability to assemble into cytoskeletal structures (Fig. 5B1 to B3). Sequestration of CBFα2 by CBFβ-SMMHC is also dependent on the presence of the C-terminal SMMHC domain. Deletions of the C-terminal SMMHC domain disrupted the ability of the chimeric protein to dimerize and to interact with other cytoskeletal proteins, most likely other myosin family members such as NMMHC, and compromised its ability to sequester CBFα2. In fact, once the C-terminal SMMHC domain was lost, the truncated CBFβ-SMMHC proteins behaved like wild-type CBFβ in that they were no longer localized along the cytoskeletal structures and localized to the nucleus when coexpressed with CBFα2 (Fig. E and F).
Our efforts to confirm these results in the samples from patients with inv(16) yielded equivocal results, most likely because the endogenous levels of the proteins were too low to be identified by immunofluorescence (1
). However, CBFα2 sequestration was observed in vivo, in a mouse gene-targeting model. This result not only confirmed the findings obtained with transient transfections in cultured cells which could potentially result from overexpression, but also demonstrated the dominant nature of this sequestration, since both wild-type CBFβ and CBFβ-SMMHC proteins are expressed in the Cbfb-MYH11
knock-in mice (8
The mechanism for the dominant nature of the CBFα2 sequestration in vivo is still not clear. At least two possibilities exist: one is that CBFβ-SMMHC is more stable than the wild-type CBFβ and therefore has higher concentration in the cells, and the other is that CBFβ-SMMHC binds CBFα2 better than does CBFβ. Results from our previous study (8
) showed that CBFβ-SMMHC was expressed at a level similar to that of the wild-type CBFβ in the Cbfb-MYH11
knock-in embryos. CBFβ-SMMHC and CBFβ were found to be expressed at comparable levels in leukemia cells from a number of patients as well (9
). However, Western blotting is not very quantitative, and variations up to fivefold have been observed. Therefore, it is possible that the fusion protein is more stable than the wild-type CBFβ. On the other hand, the binding property of CBFβ-SMMHC to CBFα2 has not been studied in detail, since purified full-length CBFβ-SMMHC in large enough quantity for such experiments is not available. Future investigations are needed to find out if either or both of these two factors contribute to the dominant sequestration of CBFα2 by CBFβ-SMMHC.
We observed that transfected CBFβ was localized throughout cells, with some cytoplasmic predominance. In addition, the transfected CBFβ did not colocalize with actin stress fibers (Fig. C and D). This differs from a recent study which showed that CBFβ colocalizes with actin by immunofluorescent staining and fractionation (47
). The reason for this difference is not clear, but it could be attributed to the differences in the CBFβ isoforms used (CBFβ187
), in the cell types used (REF52 cells versus NIH 3T3 cells), and the detergent treatments employed.
A previous study showed that truncated CBFα1/PEBP2αA can bring CBFβ into the nucleus whereas full-length CBFα1/PEBP2αA cannot (28
). This leaves open the question as to how CBFβ localizes to the nucleus to enable formation of a nuclear heterodimer with full-length PEBP2αA. One possible explanation for the differences between our data and those reported by Lu et al. (28
) is that different CBFα proteins and CBFβ isoforms were used by the two groups. While we used CBFα2, or AML1, the protein product of the gene involved in human leukemias, Lu et al. used PEBP2αA, or CBFα1, which differs from CBFα2 in sequence and has been shown to be important for osteogenesis (12
). These two CBFα proteins may have different affinities for CBFβ. In the case of CBFβ, we used the CBFβ187
isoform, while the CBFβ182
isoform was used by Lu et al. (28
). These two isoforms have two different C-terminal ends that are the result of alternative splicing, which may be responsible for the differences between the observations made by the two groups. Finally, the concentrations of CBFα1, CBFα2, and CBFβ achieved in the two studies may have differed, which would have influenced the extent of CBFα:CBFβ heterodimerization.
CBFβ-SMMHC-mediated CBFα2 sequestration can at least partially explain the dominant negative suppression of CBF function by CBFβ-SMMHC in hematopoiesis and leukemogenesis. Due to the limited sensitivities of the assays and the nature of transient transfection, we cannot rule out the possibility that free CBFα2 was present in the nuclei of some cells. Therefore, additional mechanisms, such as reduced binding of target DNA sequences by CBFβ-SMMHC:CBFα heterodimers, may also contribute to the dominant negative effect on CBF function of CBFβ-SMMHC. However, it has been postulated that CBFα2 is quantitatively limiting in vivo, since hematopoietic progenitor cells are either reduced in number or have decreased capacity to differentiate in Cbfa2+/−
). Therefore, substantial reduction of functional CBFα2 protein in the nucleus resulting from CBFβ-SMMHC sequestration is probably sufficient to cause defective CBF function, leading to dysregulation of downstream genes and contributing to leukemogenesis.
The functional significance of CBFα2 sequestration by CBFβ-SMMHC is evident in the following two sets of studies. First, it was recently demonstrated that CBFβ-SMMHC can reduce CBF DNA-binding and inhibit the G1
-to-S cell cycle transition in cultured myeloid and lymphoid cells (6
). These effects were demonstrated to require the ability of CBFβ-SMMHC to interact with CBFα2 and the presence of the C-terminal SMMHC domain, using deletion constructs similar to those described here (7
). These effects on CBF DNA-binding and cell cycle could therefore be explained by CBFα2 sequestration. Secondly, CBFα2 sequestration by CBFβ-SMMHC in mouse embryonic cells as described in this study correlates with the ability of CBFβ-SMMHC to block CBF function and disrupt hematopoiesis in mouse embryos. Future efforts will be aimed at confirming whether the sequestration of CBFα2 in vivo does in fact result in pathogenesis. This could potentially be demonstrated by generating knock-in mice expressing chimeric proteins with abrogated ability to dimerize or by substituting for the myosin chain an unrelated cytoskeletal protein. Targeted strategies to overcome or eliminate CBFα2 sequestration will be tested in cell culture and whole-animal models to see if CBFβ-SMMHC pathologic function can be subverted; the results will have clinical implications for leukemia management in the future.