INTRODUCTION

Top Abstract Introduction Materials & Methods Results Discussion References

The pericentric inversion of chromosome 16 [inv(16)(p13q22)] is a cytogenetic abnormality consistently associated with acute myeloid leukemia (AML) subtype M4Eo (2, 21), a variant of subtype M4 with abnormal eosinophils in the bone marrow and sometimes in the peripheral blood. The inversion results in the reciprocal fusions of two genes: the MYH11 gene (16p13), which encodes the smooth-muscle myosin heavy chain (SMMHC), and the CBFB gene (16q22), which encodes the  subunit of the core binding factor (CBF) (24). The chimeric gene CBFB-MYH11 fuses most of the 5' coding region of CBFB in frame with the 3' portion of MYH11, resulting in the production of the chimeric protein CBF-SMMHC. The reciprocal fusion, MYH11-CBFB, is not believed to be important since its expression is below detectable levels in leukemic cells and it is deleted in some patients with an unbalanced inversion (24, 25, 29).

CBF is the heterodimeric partner of CBF proteins, and together they constitute the core binding factors (CBF). CBF was initially identified as a transcriptional regulator of Moloney murine leukemia virus (50, 51) and polyomavirus (4, 18, 36, 37, 43) in mice, and it was subsequently shown to be an important transcriptional activator of genes involved in mammalian hematopoiesis and bone development (5, 12, 14, 15, 20, 32, 40, 46). Monomeric CBF proteins bind DNA, albeit weakly (3, 50). Although CBF does not make any detectable direct contact with DNA (50), it enhances the DNA binding affinity of the CBF proteins (4, 36). While CBF is expressed from a single gene in the human and mouse, there are three CBF genes, all of which encode the so-called runt domain (3, 22, 54), which is required for both DNA binding and interaction with CBF. One of the three genes, CBFA2, also known as AML1 or PEBP2B, is located on human chromosome 21 and is involved in several different leukemias as a result of translocations (16, 31, 34, 35, 41). Chromosomal inversions and translocations involving either CBFB or CBFA2 are the most frequent cytogenetic abnormalities in human AMLs (27).

Gene-targeting experiments in mice have demonstrated that the CBF2 and CBF subunits are likely to function together as a complex in vivo. Homozygous disruption of either Cbfa2 (39, 48) or Cbfb (42, 49) in mice produces an identical phenotype: both Cbfa2^/ and Cbfb^/ embryos demonstrate a failure of definitive hematopoiesis in the liver, and in both cases the embryos die at around day 12.5 due to extensive hemorrhages.

The inv(16) chimeric gene CBFB-MYH11 has been shown to exert a dominant negative effect in vivo by a mouse knock-in experiment (8). CBFB-MYH11 was introduced into the mouse genome to replace one copy of the Cbfb gene. The expression of this chimeric gene was controlled by the endogenous Cbfb promoter, thus simulating the condition in leukemic patients. CBF-SMMHC was found to dominantly suppress the function of the CBF2:CBF heterodimer, since mouse embryos heterozygous for the knock-in Cbfb-MYH11 gene (Cbfb^CBFB-MYH11/+) displayed a phenotype similar to that of Cbfb^/ and Cbfa2^/ embryos, i.e., failure of definitive hematopoiesis and midgestation lethality. In vitro, the chimeric protein was shown to retain its ability to interact with CBF proteins and participate in the formation of protein-DNA complexes (23). Although presence of the chimeric protein reduces CBF DNA-binding activity in cultured Ba/F3 lymphoid and 32D c13 myeloid cells (6), it is not clear how this reduction was achieved. Unlike wild-type CBF, CBF-SMMHC can potentially form dimers and multimers via the rod-like domain of the myosin chain (23, 25).

Two possible mechanisms could explain the dominant negative effect of the chimeric CBF-SMMHC protein. One is that CBF-SMMHC, via heterodimerization with CBF2, can assemble into a ternary complex at the core sites within promoters of target genes and interfere with the regulation of gene expression. The second possibility is that CBF-SMMHC, with its capacity to form multimers, can sequester CBF2 into nonfunctional complexes, thus preventing it from regulating transcription of target genes. Previous studies by our group demonstrated that CBF-SMMHC can form rod-like nuclear structures as well as cytoplasmic stress fibers in NIH 3T3 cells stably transfected with a CBFB-MYH11 cDNA construct (53). However, the effect on CBF2 subcellular localization by CBF-SMMHC has not been fully examined. In this study, we used transient-transfection assays in combination with immunofluorescence and green fluorescent protein (GFP) tags to demonstrate that CBF-SMMHC does, in fact, sequester CBF2 in abnormal locations. We also demonstrated that the sequestration requires the abilities of CBF-SMMHC to interact with CBF2 and to multimerize. This observed sequestration can at least partially explain the dominant negative effect of the CBF-SMMHC protein on CBF function in leukemogenesis.

	MATERIALS AND METHODS

Top Abstract Introduction Materials & Methods Results Discussion References

Construction of plasmids. pEGFP-C2, the vector expressing a modified GFP (EGFP) under the control of the cytomegalovirus (CMV) promoter, was obtained from Clontech. This vector allows for in-frame fusion of target genes at the COOH-terminal end of EGFP. Plasmids pCBFB, pCBFB-MYH11, and pCbfa2, each containing the entire coding region of the respective genes under the control of CMV promoter, have been described previously (23, 26, 45). pCBFB carries the CBF₁₈₇ isoform, pCBFB-MYH11 carries the CBF-SMMHC₂₀₄ isoform (26), and pCbfa2 carries the mouse CBF2₄₅₁ isoform. EGFP fusion constructs pEGFP-CBFB, pEGFP-CBFB-MYH11, and pEGFP-Cbfa2 were generated following standard subcloning procedures by using these reported full-length cDNA plasmids.

N2-11

N2-11

C390

C303

C95

C390

C303

C95

Antibodies. 3043, a rabbit polyclonal antibody specific for CBF2, and 141.2, a mouse monoclonal antibody against CBF, have been described previously (5, 49). Texas-red-labeled phalloidin was obtained from Molecular Probes for actin labeling. Fluorescein-labeled affinity purified secondary antibodies against mouse immunoglobulin G (IgG) and rabbit IgG were obtained from Kirkegaard & Perry Laboratories Inc. Texas-red-labeled affinity purified secondary antibodies against mouse IgG and rabbit IgG were obtained from Vector Laboratories. The primary antibodies were diluted in Triton buffer (phosphate-buffered saline [PBS], 0.3% Triton X-100, 2% bovine serum albumin [BSA]) at working dilutions of 1:10, and the secondary antibodies were diluted in the same buffer at the recommended dilutions.

Cell culture and DNA transfection. NIH 3T3 cells were maintained in Dulbecco's modified Eagle medium supplemented with 10% (vol/vol) heat-inactivated newborn calf serum. DNA transfections were performed with lipofectamine (GIBCO-BRL) according to the manufacturer's instructions. Fifty thousand cells were plated onto a 15-mm-diameter circular glass coverslip in a 12-well tissue culture dish for 12 h at 37°C before transfection. A maximum of 2.5 µg of DNA was used for each transfection. Twenty percent serum was added to cells at 5 h posttransfection, and the cells were cultured for 12 h at 37°C. The culture medium was replaced with complete medium at this time, and cells were collected for fixation or immunoprecipitation at times ranging from 12 to 48 h after addition of fresh media.

Cell fractionation. NIH 3T3 cells were plated on a 10-cm-diameter dish at 10⁶ cells per plate and transfected the next day with various expression constructs. Cells were harvested 24 h posttransfection, and cell pellets were resuspended in sucrose/Nonidet P-40 (NP-40) buffer containing 0.32 M sucrose, 3 mM CaCl₂, 2 mM magnesium acetate, 0.1 mM EDTA, 10 mM Tris-HCl (pH 8.0), 1 mM dithiothreitol, and 0.5% (vol/vol) NP-40 along with protease inhibitors (13). The lysates were microcentrifuged at 500 × g for 5 min at 4°C, and the supernatant containing the cytoplasm was transferred to a fresh tube. The nuclei were washed with 1 ml of sucrose buffer without NP-40, and then lysed in sodium dodecyl sulfate (SDS) sample buffer by boiling. The protein concentration in each sample was visually quantified on Coomassie-blue-stained SDS-polyacrylamide gel electrophoresis (SDS-PAGE) gel, and equivalent amounts of protein were then separated on an SDS-10% PAGE gel and visualized by Western blotting, as described below. GFP-specific polyclonal antibody from Clontech was used at recommended dilutions to visualize EGFP-CBF2 and EGFP-CBF in the presence and absence of CBF2. Anti-cytosolic phospholipase A2 (cPLA2) specific monoclonal antibody (Santa Cruz) and anti-retinoblastoma (Rb) specific monoclonal antibody (Pharmingen) were also used at recommended dilutions to probe the blots for ascertaining the purity of the cytoplasmic fraction and nuclear fraction, respectively.

Immunofluorescence. Coverslips with transfected cells were fixed in two different ways to avoid fixation artifacts. Cells were fixed either in cold (20°C) methanol for 7 min or in 4% formaldehyde for 30 min. In the latter case, the fixed cells were then permeabilized with 0.3% Triton buffer for 30 min. For experiments involving transfection with GFP fusion constructs, cells were fixed only in formaldehyde followed by permeabilization as described above, since methanol has been shown to inhibit GFP fluorescent activation. Detection of fluorescent signals was performed according to a modification of the procedure described by Cullen (11). Fixed cells were blocked overnight with a solution containing PBS, 10% goat or horse serum (same as that of secondary antibody), 2% milk, and 3% BSA. Transfected cells were incubated with primary antibodies diluted in Tween buffer (PBS, 2% BSA, 0.5% Tween 20) for 2 h at room temperature. Cells were then washed once with PBS for 5 min and twice with Triton buffer (PBS, 2% BSA, 0.3% Triton X-100) for 10 min each. The fluorescence-labeled secondary antibody was diluted in Triton buffer and added to transfected cells for 1 h at room temperature. Cells were then washed once with PBS for 5 min, twice with Triton buffer for 10 min each time, and once with PBS for 10 min before mounting with 4',6-diamidino-2-phenylindole (DAPI)-antifade on a glass slide for microscopy. Experiments were repeated three or more times.

Immunoprecipitation. pCBFB, pCBFB-MYH11, and related deletion constructs (pCBFB-MYH11^N2-11, pCBFB-MYH11^C390, pCBFB-MYH11^C303, pCBFB-MYH11^C95) were translated in vitro with ³⁵S-labeled methionine by using the Promega reticulocyte lysate translation kit. pCbfa2 was translated in vitro with or without radioactive methionine. Protein A-Sepharose-plus (Pierce) was washed with PBS, then blocked with 2% BSA in PBS. Thirty microliters of the blocked protein A was incubated with antibody 3043 for 2 h at 4°C on a rotator and then washed twice with Triton buffer. The protein A beads were then incubated overnight at 4°C with ³⁵S-labeled CBF, CBF-SMMHC, and related deletion proteins, in the presence or absence of cold in vitro-translated CBF2. The Sepharose beads were then collected by centrifugation and washed with Triton buffer four times to remove any residual buffer. The beads were then resuspended and boiled for 5 min in 25 µl of 2× SDS buffer. Ten microliters of this buffer containing the eluted proteins was then analyzed by denaturing PAGE and autoradiography.

Chemical cross-linking. Proteins were translated in vitro in the presence of ³⁵S-labeled methionine by using the Promega reticulocyte lysate kit. They were then diluted fivefold with PBS and incubated at room temperature for 1 h with glutaraldehyde at a final concentration of 0.0025%. The reactions were stopped by addition of Tris (pH 8) and glycine at final concentrations of 25 and 192 mM, respectively, and analyzed by denaturing PAGE and autoradiography.

Western blot analysis. Whole-cell extracts in SDS gel loading buffer were prepared from NIH 3T3 cells (10⁶ cells per dish) transfected with the appropriate expression plasmids, separated by SDS-12% PAGE, and transferred to a nitrocellulose membrane. The membrane was blocked with Tris-buffered saline (TBS) buffer (135 mM NaCl, 20 mM Tris [pH 7.5], 5% milk, and 0.1% Tween 20), incubated for 2 h on a shaker at room temperature with an appropriate primary antibody, washed with TBS buffer, and then incubated with a peroxidase-labeled secondary antibody (KPL Inc.) at the recommended dilution in TBS for 1 h at room temperature. Proteins were visualized by developing chemiluminescent signal with reagents from Pierce and exposure to X-ray films.

Analysis of mouse embryonic fibroblasts. Generation of mice heterozygous for the Cbfa2^lz allele will be described elsewhere (33). Briefly, exons 7 and 8 of the Cbfa2 gene were replaced with lacZ-bearing sequences in such a way that a CBF2-gal fusion protein was synthesized from the modified Cbfa2 allele. Female mice heterozygous for the Cbfa2^lz allele (Cbfa2^lz/+) were mated to a male chimeric mouse generated from mouse embryonic stem cells heterozygous for a knock-in inv(16) allele (Cbfb^CBFB-MYH11/+) (8). Embryos from this cross were isolated at 11.5 days postcoitus (dpc), the yolk sacs and anterior portions (heads) of the embryos were removed, and the rest of each embryo was used to generate mouse embryonic fibroblasts. Embryos were suspended in 5 ml of HEPES-buffered saline and passed through an 18-gauge needle, and the cells were collected by centrifugation, resuspended in 5 ml of Dulbecco's modified Eagle medium-10% fetal calf serum, and then passed through a 21-gauge needle. Disaggregated cells from individual embryos were plated in one well (35-mm-diameter) of a six-well tissue culture plate and cultured at 37°C. The mouse embryonic fibroblasts were passed one additional time in duplicate plates, and the cells from one plate were processed for -galactosidase activity following standard protocols. Fibroblasts with both Cbfa2^lz/+ and Cbfb^CBFB-MYH11/+ alleles were further stained with DAPI for nuclear visualization by standard procedures. Inheritance of Cbfb^CBFB-MYH11 allele was demonstrated by the presence of the CBF-SMMHC fusion protein, assayed by Western blot analysis of the mouse embryonic fibroblasts, as described previously (8).

	RESULTS

Top Abstract Introduction Materials & Methods Results Discussion References

Coimmunoprecipitation of CBF and of CBF-SMMHC and its deletions with CBF2. The ability of the CBF-SMMHC deletion proteins to interact with CBF2 was characterized by immunoprecipitation of in vitro-translated proteins. Fig. 1A shows the in vitro-translated ³⁵S-labeled proteins used in the reactions. To assess binding to CBF2, a polyclonal antibody that recognizes the CBF2 runt domain (3043) was first bound to protein A-Sepharose beads. The matrix-bound antibody was then incubated with or without nonradioactive CBF2 (in vitro-translated from pCbfa2) in conjunction with radioactively labeled CBF, CBF-SMMHC, CBF-SMMHC^C390, CBF-SMMHC^C303, CBF-SMMHC^C95, and CBF-SMMHC^N2-11 (in vitro-translated from pCBFB, pCBFB-MYH11, pCBFB-MYH11^C390, pCBFB-MYH11^C303, pCBFB-MYH11^C95, and pCBFB-MYH11^N2-11, respectively). As seen in Fig. 1B, lanes 2 and 4, CBF and full-length CBF-SMMHC were immunoprecipitated with CBF2, as expected. The protein with the N-terminal deletion CBF-SMMHC^N2-11 failed to be immunoprecipitated with CBF2 (Fig. 1B, lane 12). Those with the C-terminal deletions of CBF-SMMHC, which retain the N-terminal end, interacted with CBF2 and were immunoprecipitated (Fig. 1B, lanes 6, 8, and 10). The ³⁵S-labeled CBF2 was immunoprecipitated in the presence of the antibody (lane 14) but not in its absence (lane 13), indicating the specificity of the interaction.

View larger version (32K): [in this window] [in a new window]   FIG. 1.   Characterization of CBF2, CBF, CBF-SMMHC, and the deletion mutants. (A) Generation of protein products by in vitro translation. [35S]methionine-labeled proteins were produced by in vitro translation, separated by gel electrophoresis, and detected by autoradiography. The intense bands at the bottom of the gel are the free [35S]methionine. (B) Interactions between CBF2 and the deletion mutants. The protein products shown in panel A were immunoprecipitated with polyclonal antibody 3043, in the presence or absence of cold in vitro-translated CBF2. (C) Expression of cDNA constructs after transient transfection into NIH 3T3 cells. The Western blot with extracts derived from transiently transfected NIH 3T3 cells was probed with 3043 to detect CBF2 and EGFP-CBF2 (lanes 1 to 4). The relevant proteins are shown by arrowheads in lanes 2 and 4. Similarly, the antibody 141.2 was used to identify CBF, CBF-SMMHC, the deletion proteins, and their GFP fusions by Western blotting of cellular extracts (lanes 5 to 17). Protein molecular mass markers are shown on the right sides of panels B and C.

Protein expression from plasmid constructs after transient transfection. The expression of CBF2, CBF, CBF-SMMHC, and the various deletion proteins after transient transfection of NIH 3T3 cells was tested by Western blotting. The polyclonal antibody against CBF2, 3043, was used to probe whole-cell extracts from untransfected NIH 3T3 cells and from NIH 3T3 cells transfected with pCbfa2, pEGFP-C2 (empty vector), and pEGFP-Cbfa2 (Fig. 1C, lanes 1 to 4). Proteins of expected sizes (49 kDa for CBF2 and 76 kDa for EGFP-CBF2) were detected in cells transfected with pCbfa2 and pEGFP-Cbfa2 (Fig. 1C, lanes 2 and 4, respectively). The ~50 kDa band present in all the samples (Fig. 1C, lanes 1 to 4) most likely results from nonspecific binding of the antibody to an endogenous protein. Cell extracts from NIH 3T3 cells transfected with pCBFB-MYH11 and the deletion constructs with or without EGFP fusion were probed with 141.2, a monoclonal antibody against CBF (Fig. 1C, lanes 5 to 17 for plasmids pCBFB-MYH11, pCBFB-MYH11^N2-11, pCBFB-MYH11^C390, pCBFB-MYH11^C303, pCBFB-MYH11^C95, pCBFB, pEGFP-CBFB-MYH11, pEGFP-CBFB-MYH11^N2-11, pEGFP-CBFB-MYH11^C390, pEGFP-CBFB-MYH11^C303, pEGFP-CBFB-MYH11^C95, pEGFP-CBFB, and pEGFP-C2, respectively). Again, proteins of expected sizes were visible in all the transfected cell samples. Protein bands corresponding to the endogenous CBF were detected in certain samples (Fig. 1C, lanes 7 to 9), although the variability suggests that these could be derived from degradation of the higher-molecular-weight proteins.

Subcellular localization of CBF2, CBF, and CBF-SMMHC after transient transfection. Plasmids containing full-length coding sequences (pCbfa2, pCBFB, and pCBFB-MYH11) were transiently transfected into NIH 3T3 cells to examine the subcellular localization of CBF2, CBF, and CBF-SMMHC. Expression and localization of these proteins were detected by primary antibodies 3043 and 141.2, followed by immunofluorescence labeling with fluorescein and Texas-red-conjugated secondary antibodies. The levels of endogenous CBF and CBF2 proteins, though detectable by gel shifts (unpublished data), were below the limits of detection by immunofluorescence. Transient transfection of GFP fusion constructs (pEGFP-Cbfa2, pEGFP-CBFB, and pEGFP-CBFB-MYH11) was used as an independent method of assessing subcellular locations, and the results were in agreement with those of immunofluorescence staining.

View larger version (39K): [in this window] [in a new window]   FIG. 2.   Subcellular localization of CBF2, CBF, and CBF-SMMHC in transiently transfected cells. NIH 3T3 cells were transfected with pCbfa2 (A1 and A2), pEGFP-Cbfa2 (B1 and B2), pCBFB (C1 and C2), pEGFP-CBFB (D1 and D2), pCBFB-MYH11 (E1 and E2), and pEGFP-CBFB-MYH11 (F1 and F2). Odd-numbered panels show fluorescent signals, while even-numbered panels show DAPI staining of the same fields. Panel A1 shows signal detected with 3043 and a Texas-red-labeled secondary antibody, panels C1 and E1 show signals detected with 141.2 and a fluorescein-labeled secondary antibody, and panels B1, D1, and F1 show the autofluorescent signal of GFP fused to the respective proteins. The antibodies failed to detect the endogenous proteins (compare the untransfected cell in panel A1 with panel A2, as well as that in panel C1 with panel C2).

Full-length CBF2 can bring CBF into the nucleus. pCBFB and pCbfa2 were cotransfected into NIH 3T3 cells and immunofluorescence labeling was carried out as described above. Two of the four cells shown in Fig. 3A expressed the transiently transfected pCbfa2, as shown by the green signal in Fig. 3A1, while one of these two cells also expressed the transiently transfected pCBFB as shown by the red signal in Fig. 3A2. As shown in Fig. 3A1, CBF2 was localized completely in the nucleus, similar to the pattern observed when it was expressed alone. Intriguingly, CBF also was localized to the nucleus, and it was distributed in a pattern similar to that of CBF2 (Fig. 3A2). This suggests that full-length CBF2 is able to bring CBF into the nucleus. This nuclear localization of CBF by CBF2 was dose dependent. When equal molar amounts of both plasmid DNAs were used for transfection, as in the cells shown in Fig. 3, approximately 88% of the cells expressing both CBF and CBF2 showed nuclear localization of CBF. Increasing the amount of pCbfa2 DNA relative to that of pCBFB increased the proportion of cells in which CBF localized to the nucleus. When the amount of pCbfa2 DNA was fivefold molar excess relative to that of pCBFB DNA, in approximately 93% of the cotransfected cells CBF and CBF2 colocalized in the nucleus. Increasing the pCbfa2 DNA amount to ninefold molar excess relative to that of pCBFB DNA resulted in almost 100% of the doubly transfected cells having CBF in nuclei along with CBF2. Cotransfections with pEGFP-CBFB and pCbfa2 gave comparable results (Fig. 3B1, B2, and B3).

View larger version (26K): [in this window] [in a new window]   View larger version (34K): [in this window] [in a new window]   FIG. 3.   CBF2 brings CBF into the nucleus when coexpressed. Cells were cotransfected with pCbfa2 and pCBFB (A1, A2, and A3) or with pCbfa2 and pEGFP-CBFB (B1, B2, and B3). Panels A1, A2, and A3 represent images of the same field seen through different filters, as do panels B1, B2, and B3. CBF2 was detected with antibody 3043 and a fluorescein-labeled (A1) or a Texas-red-labeled (B1) secondary antibody. CBF localization was detected with 141.2 and a Texas-red-labeled secondary antibody (A2). Panel B2 shows the autofluorescence of EGFP-CBF. Cells in panels A3 and B3 were stained by DAPI. (C) Cytoplasmic and nuclear fractionation of the transfected cells is shown. NIH 3T3 cells transfected with pEGFP (lanes 1 and 2), pEGFP-CBFB (lanes 3 and 4), pEGFP-CBFB plus pCbfa2 (lanes 5 and 6), and pEGFP-Cbfa2 alone (lanes 7 and 8) were fractionated into cytoplasmic and nuclear fractions, which were separated on SDS-PAGE gel and probed by Western blotting with a GFP-specific antibody. The quality of cell fractionation was analyzed by probing with antibodies for cPLA2 (cytoplasmic) and Rb (nuclear). C, cytoplasmic fraction; N, nuclear fraction. Molecular mass markers are shown to the right in panel C.

CBF-SMMHC sequesters CBF2 along filaments and aggregates. When CBF2 and CBF-SMMHC were coexpressed in NIH 3T3 cells through transient cotransfection (plasmids pCbfa2 and pCBFB-MYH11) and analyzed by immunofluorescence labeling, CBF-SMMHC assembled into the same filamentous or aggregated structures as it did when expressed alone (Fig. 4A1). However, CBF2 colocalized with CBF-SMMHC along the filaments and aggregates (Fig. 4A2). Similar results were obtained when pEGFP-Cbfa2 was cotransfected with pCBFB-MYH11 (Fig. 4B1, B2, and B3) and when pCbfa2 was cotransfected with pEGFP-CBFB-MYH11 (Fig. 4C1, C2, and C3). Occasionally, CBF2 and CBF-SMMHC proteins were found in both the nucleus and cytoplasm (Fig. 4C1 and C2). In more than 95% of the cells that expressed both transfected genes, CBF2 colocalized with CBF-SMMHC along the filaments and aggregates. In cells where the concentration of CBF-SMMHC was much lower than that of CBF2 (based on immunofluorescence intensity), while some CBF2 colocalized with CBF-SMMHC outside the nucleus, the remaining excess of CBF2 protein localized to the nucleus.

View larger version (47K): [in this window] [in a new window]   FIG. 4.   CBF-SMMHC sequesters CBF2 along filaments and aggregates. (A1, A2, and A3) NIH 3T3 cells were cotransfected with pCbfa2 and pCBFB-MYH11. CBF-SMMHC localization was detected with antibody 141.2 and a fluorescein-labeled secondary antibody (panel A1), and CBF2 localization was detected with antibody 3043 and a Texas-red-labeled secondary antibody (panel A2). (B1, B2, and B3) Localization of EGFP-CBF2 and CBF-SMMHC when cotransfected. CBF-SMMHC localization is shown by the red signal (141.2 and a Texas-red-labeled secondary antibody) in panel B1, while EGFP-CBF2 localization is shown by the green autofluorescent signal in panel B2. (C1, C2, and C3) Cotransfected EGFP-CBF-SMMHC (panel C1) and CBF2 (panel C2), which were detected in a fashion similar to that described for panel B. (A3, B3, and C3) DAPI staining of the respective fields.

Sequestration of CBF2 results from direct interaction between CBF2 and CBF-SMMHC. The first 11 amino acids of the CBF protein are required for the binding to CBF2 (44). A CBF-SMMHC protein missing this region (CBF-SMMHC^N2-11) failed to interact with CBF2, as seen by its inability to be coimmunoprecipitated (Fig. 1B). When pCBFB-MYH11^N2-11 was transfected into NIH 3T3 cells, the expressed protein localized along the filaments in the cytoplasm, similarly to the full-length protein (Fig. 5A1). However, when coexpressed with CBF2, the deletion protein did not change the nuclear location of CBF2. As shown in Fig. 5B, CBF-SMMHC^N2-11 and CBF2 behaved independently of each other: CBF-SMMHC^N2-11 was distributed mainly in the cytoplasm along filament-like structures, whereas CBF2 remained exclusively in the nucleus. This observation was confirmed by using EGFP-CBF-SMMHC^N2-11 (1).

View larger version (39K): [in this window] [in a new window]   FIG. 5.   Immunofluorescence of CBF-SMMHC deletion proteins and coexpressed CBF2. CBF-SMMHC deletion proteins and CBF2 were detected with 141.2 and 3043, respectively, plus appropriate secondary antibodies. (A and B) Immunofluorescence labeling of NIH 3T3 cells transfected with pCBFB-MYH11N2-11, either alone (panels A1 and A2) or with pCbfa2 (panels B1, B2, and B3). Panels A1 and A2 show the same field with the CBF-SMMHCN2-11 localization in green and DAPI-stained nuclei in blue, respectively. Localization of CBF-SMMHCN2-11 is again shown in green in panel B1, while cotransfected CBF2 is shown in red in panel B2. (C1 and C2) Immunofluorescence labeling of cells transfected with only pCBFB-MYH11C95. (D) Cells cotransfected with pCBFB-MYH11C95 (panel D1) and pCbfa2 (panel D2). CBF-SMMHCC303 was labeled in green when transfected alone (E1) or cotransfected (F1) with CBF2, which was labeled in red (F2). The panels labeled with the same letter are views of the same fields. (A2, B3, C2, D3, E2, and F3) DAPI staining showing the nuclei of cells in the respective fields.

C-terminal SMMHC domain of CBF-SMMHC is required for CBF2 sequestration. We next determined whether CBF2 sequestration was dependent on the myosin part of the chimeric protein CBF-SMMHC. Constructs with deletions at the C-terminal end of CBF-SMMHC (Fig. 1) were used for in vitro cross-linking analysis to test the ability of their protein products to dimerize and multimerize. In vitro-translated proteins were incubated under conditions allowing dimerization in the presence or absence of glutaraldehyde, which covalently cross-links the dimerized proteins. Full-length CBF-SMMHC was capable of forming dimers and tetramers as seen by the presence of more-slowly migrating complexes (Fig. 6, lane 6). CBF-SMMHC^C95 was able to dimerize (Fig. 6, lane 8), although with somewhat less efficiency than CBF-SMMHC. Removing 175 amino acids from the C-terminal end further decreased the efficiency of dimerization (1). Any further deletion in the C-terminal end abrogated the ability of CBF-SMMHC to assemble into dimers, as seen for CBF-SMMHC^C303 (lane 10) and CBF-SMMHC^C390 (lane 12). CBF2 (lane 2) and CBF (lane 4) did not dimerize under these conditions, as expected from previous studies (17, 50).

View larger version (50K): [in this window] [in a new window]   FIG. 6.   Glutaraldehyde cross-linking of CBF, CBF-SMMHC, and the deletion proteins. Proteins as labeled at the top of the lanes were translated in vitro in the presence of [35S]methionine. Cross-linking of each protein was accomplished by incubation in the presence (even-numbered lanes) or absence (odd-numbered lanes) of glutaraldehyde. Slow mobility bands were observed for CBF-SMMHC and CBF-SMMHCC95 but not for CBF2, CBF, CBF-SMMHCC303, and CBF-SMMHCC390. Protein molecular mass markers are shown on the right.

C95

C303

C390

C95

C95

C95

C95

C303

C390

CBF-SMMHC colocalizes with actin. The pattern of localization of the inv(16) fusion protein is similar to that of actin stress fibers. To determine if CBF-SMMHC colocalizes with actin, NIH 3T3 cells were transfected with pCBFB-MYH11 or pEGFP-CBFB-MYH11 and subjected to immunofluorescence labeling with 141.2 (for CBF-SMMHC) and Texas-red-labeled phalloidin, which specifically interacts with actin. The data in Fig. 7 clearly illustrate that CBF-SMMHC and EGFP-CBF-SMMHC colocalized with actin filaments (compare Fig. 7A1 with A2 and Fig. 7B1 with B2). Again, some of the EGFP-CBF-SMMHC protein can be seen in the nucleus in addition to the filamentous localization in the cytoplasm (Fig. 7B1). NIH 3T3 cells were also transfected with pCBFB or pEGFP-CBFB. In contrast to CBF-SMMHC, CBF and EGFP-CBF were distributed throughout the cell (Fig. 7C1 and D1), the localization showing no correlation with actin localization (Fig. 7C2 and D2).

View larger version (53K): [in this window] [in a new window]   FIG. 7.   CBF-SMMHC colocalizes with actin filaments, while CBF does not. NIH 3T3 cells were transfected with pCBFB-MYH11 (A1 and A2), pEGFP-CBFB-MYH11 (B1 and B2), pCBFB (C1 and C2), and pEGFP-CBFB (D1 and D2). Proteins were detected with 141.2 plus a fluorescein isothiocyanate-labeled secondary antibody (panels A1 and C1) and Texas-red-conjugated phalloidin (panels A2, B2, C2, and D2). Panels B1 and D1 show the autofluorescent signal from the GFP fusion proteins.

Colocalization of CBF2 and CBF-SMMHC in vivo. Through homologous recombination in mouse ES cells, the bacterial lacZ gene was fused in frame to the mouse Cbfa2 gene, downstream from the runt domain (33). The expression of the Cbfa2^lz allele is under the control of endogenous Cbfa2 promoter, resulting in near-physiological levels of Cbf2-gal fusion protein in the cells of Cbfa2^lz/+ mice. The chimeric Cbf2-gal protein produced retained its ability to interact with CBF through the runt domain, while the gal portion of the protein permitted detection of the protein by the relatively sensitive 5-bromo-4-chloro-3-indolyl--D-galactopyranoside assay. Matings were set up between Cbfa2^lz/+ mice and chimeric mice made with Cbfb^CBFB-MYH11/+ ES cells, which produce a CBF-SMMHC fusion protein (8). Mouse embryonic fibroblasts isolated from 11.5-dpc embryos derived from such matings contained either only the Cbfa2^lz allele or both the Cbfa2^lz and Cbfb^CBFB-MYH11/+ alleles. -Galactosidase activity was detected mainly in the nuclei of mouse embryonic fibroblasts derived from Cbfa2^lz/+ embryos (Fig. 8A) (92% of the cells had expression exclusively in the nucleus, and 8% had expression in both the cytoplasm and nucleus), whereas a significant amount of -galactosidase activity was found in the cytoplasm of fibroblasts containing the Cbfb^CBFB-MYH11/+ allele (Fig. 8B). Seventy percent of the cells had expression only in the cytoplasm (e.g., the cells shown on the right in Fig. 8B), 29% had it in both the cytoplasm and the nucleus (e.g., the cell shown on the left in Fig. 8B), and 1% had it only in the nucleus. The result demonstrates that cytoplasmic sequestration of CBF2 by the CBF-SMMHC fusion protein also occurs at normal physiological levels of the proteins.

View larger version (68K): [in this window] [in a new window]   FIG. 8.   In vivo sequestration of Cbf2 by Cbf-SMMHC. Embryonic fibroblasts were isolated from 11.5-dpc mouse embryos, cultured briefly in vitro, and stained for the Cbf2-gal protein. (A) A fibroblast from an embryo of Cbfa2lz/+ genotype stained for gal. (B1) Fibroblasts from an embryo of Cbfa2lz, CbfbCBFB-MYH11/+ genotype stained for gal. Arrows indicate the gal staining. (B2) DAPI staining of the same field as shown in panel B1.

	DISCUSSION

Top Abstract Introduction Materials & Methods Results Discussion References

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/CBF2- and C/EBP-dependent transcriptional activation, although the mechanism for this inhibition remains unknown (14, 30, 52). AML1-ETO can also dominantly suppress CBF function, as shown in a mouse knock-in model (38, 55). 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/CBF2 (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 CBF-SMMHC plays a dominant negative role. Our results suggest that CBF-SMMHC plays a dominant negative role by sequestering CBF2/AML1 to cytoskeletal structures, thus initiating a process towards leukemia by disrupting transcriptional regulation of genes controlled by CBF2/AML1.

Upon transient transfection into NIH 3T3 cells, CBF2 was localized in the nucleus whereas CBF was distributed throughout the cell. CBF-SMMHC was located predominantly in the cytoplasm, forming filamentous or aggregated structures. We did observe some CBF-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 CBF-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 CBF-SMMHC protein and only in a small percentage (10 to 40%) of cells within a cell population (1, 53). On the other hand, the stress fibers formed by CBF-SMMHC were evident in most of the cells stably expressing CBF-SMMHC, regardless of expression level (1, 53).

In the present study, we demonstrated that CBF localized to the nucleus when cotransfected with full-length CBF2. The likely mechanism for this nuclear localization of CBF is that CBF2 heterodimerizes with CBF in the cytoplasm and mediates the transport of the heterodimer into the nucleus. Nuclear localization of CBF2 can be overridden by CBF-SMMHC, which forms homodimers and multimers and interacts with cytoskeletal molecules. The sequestration of CBF2 by CBF-SMMHC is dependent on the heterodimerization domain in CBF that mediates interaction with the CBF subunits. The truncated protein with a deletion of the CBF2-interacting domain, CBF-SMMHC^N2-11, lost the ability to sequester CBF2, even though it retained the ability to assemble into cytoskeletal structures (Fig. 5B1 to B3). Sequestration of CBF2 by CBF-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 CBF2. In fact, once the C-terminal SMMHC domain was lost, the truncated CBF-SMMHC proteins behaved like wild-type CBF in that they were no longer localized along the cytoskeletal structures and localized to the nucleus when coexpressed with CBF2 (Fig. 5E 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, CBF2 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 CBF and CBF-SMMHC proteins are expressed in the Cbfb-MYH11 knock-in mice (8).

The mechanism for the dominant nature of the CBF2 sequestration in vivo is still not clear. At least two possibilities exist: one is that CBF-SMMHC is more stable than the wild-type CBF and therefore has higher concentration in the cells, and the other is that CBF-SMMHC binds CBF2 better than does CBF. Results from our previous study (8) showed that CBF-SMMHC was expressed at a level similar to that of the wild-type CBF in the Cbfb-MYH11 knock-in embryos. CBF-SMMHC and CBF were found to be expressed at comparable levels in leukemia cells from a number of patients as well (9, 10). 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 CBF. On the other hand, the binding property of CBF-SMMHC to CBF2 has not been studied in detail, since purified full-length CBF-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 CBF2 by CBF-SMMHC.

We observed that transfected CBF was localized throughout cells, with some cytoplasmic predominance. In addition, the transfected CBF did not colocalize with actin stress fibers (Fig. 7C and D). This differs from a recent study which showed that CBF 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 CBF isoforms used (CBF₁₈₇ versus CBF₁₈₂), in the cell types used (REF52 cells versus NIH 3T3 cells), and the detergent treatments employed.

A previous study showed that truncated CBF1/PEBP2A can bring CBF into the nucleus whereas full-length CBF1/PEBP2A cannot (28). This leaves open the question as to how CBF localizes to the nucleus to enable formation of a nuclear heterodimer with full-length PEBP2A. One possible explanation for the differences between our data and those reported by Lu et al. (28) is that different CBF proteins and CBF isoforms were used by the two groups. While we used CBF2, or AML1, the protein product of the gene involved in human leukemias, Lu et al. used PEBP2A, or CBF1, which differs from CBF2 in sequence and has been shown to be important for osteogenesis (12, 20, 32, 40). These two CBF proteins may have different affinities for CBF. In the case of CBF, we used the CBF₁₈₇ isoform, while the CBF₁₈₂ 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 CBF1, CBF2, and CBF achieved in the two studies may have differed, which would have influenced the extent of CBF:CBF heterodimerization.

CBF-SMMHC-mediated CBF2 sequestration can at least partially explain the dominant negative suppression of CBF function by CBF-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 CBF2 was present in the nuclei of some cells. Therefore, additional mechanisms, such as reduced binding of target DNA sequences by CBF-SMMHC:CBF heterodimers, may also contribute to the dominant negative effect on CBF function of CBF-SMMHC. However, it has been postulated that CBF2 is quantitatively limiting in vivo, since hematopoietic progenitor cells are either reduced in number or have decreased capacity to differentiate in Cbfa2^+/ mice (48). Therefore, substantial reduction of functional CBF2 protein in the nucleus resulting from CBF-SMMHC sequestration is probably sufficient to cause defective CBF function, leading to dysregulation of downstream genes and contributing to leukemogenesis.

The functional significance of CBF2 sequestration by CBF-SMMHC is evident in the following two sets of studies. First, it was recently demonstrated that CBF-SMMHC can reduce CBF DNA-binding and inhibit the G₁-to-S cell cycle transition in cultured myeloid and lymphoid cells (6, 7). These effects were demonstrated to require the ability of CBF-SMMHC to interact with CBF2 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 CBF2 sequestration. Secondly, CBF2 sequestration by CBF-SMMHC in mouse embryonic cells as described in this study correlates with the ability of CBF-SMMHC to block CBF function and disrupt hematopoiesis in mouse embryos. Future efforts will be aimed at confirming whether the sequestration of CBF2 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 CBF2 sequestration will be tested in cell culture and whole-animal models to see if CBF-SMMHC pathologic function can be subverted; the results will have clinical implications for leukemia management in the future.