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Previous Article | Next Article 
Molecular and Cellular Biology, January 2003, p. 607-619, Vol. 23, No. 2
0270-7306/03/$08.00+0 DOI: 10.1128/MCB.23.2.607-619.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.
Tanawan Kummalue,2 Alan D. Friedman,2 and Scott W. Hiebert1,3*
Department of Biochemistry,1 Vanderbilt-Ingram Cancer Center, Vanderbilt University School of Medicine, Nashville, Tennessee 37232,3 Department of Oncology, Johns Hopkins University, Baltimore, Maryland 212312
Received 22 April 2002/ Returned for modification 6 June 2002/ Accepted 14 October 2002
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ABSTRACT |
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INTRODUCTION |
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The CBFß-AML1 complex regulates genes encoding cytokines and their receptors, genes involved in differentiation such as T-cell receptors, neutrophil peptide 3, and myeloperoxidase as well as the p14ARF tumor suppressor (4, 12, 14, 27, 36, 39, 43, 46, 52). Furthermore, CBFß/SMMHC slows the cell cycle transition from G1 to S in hematopoietic cells; thus, cell cycle regulatory genes such as cdk4 may also be targeted by this complex (5).
Animal studies indicate that CBFß and AML1 are required for hematopoiesis. CBFß and AML-1 knockout mice share a phenotype; these mice lack fetal liver hematopoiesis and are embryonic lethal at embryonic day 12.5 (e12.5) to e13.5 (41, 49). A similar phenotype is observed in mice expressing CBFß/SMMHC from a "knocked-in" CBFB-MYH11 gene (7), suggesting that inv(16) creates a dominant repressor of AML1- and CBFß-regulated genes. Chimeric mice created using Cbfß+/cbfß-MYH11 embryonic stem cells fail to develop leukemia at high frequency within the first year of life. However, 4- to 16-week-old chimeras that are treated with N-ethyl-N-nitrosourea develop leukemia 2 to 6 months after treatment (6). Coexpression of CBFß/SMMHC with the human papillomavirus E7 oncogene or expression of the fusion protein in the absence of the tumor suppressors p16INK4a and p19ARF also leads to acute leukemia in mice (51). Thus, CBFß/SMMHC predisposes mice to leukemia, and secondary mutations are necessary for the onset of leukemia in inv(16)-mediated cases.
CBFß and CBFß/SMMHC lack a nuclear localization signal; thus, they must complex with AML1 in the cytoplasm in order to be transported into the nucleus. Although a significant amount of CBFß/SMMHC is retained in the cytoplasm in overexpression studies, CBFß/SMMHC is both nuclear and cytoplasmic (1, 5, 29, 31); furthermore, the fusion protein cooperates with AML1 to repress transcription (31). AML1 binds the mSin3A and Groucho corepressors, and the inv(16) fusion protein can form a trimeric complex with AML1 and mSin3A (31). In addition, the C terminus of the CBFß/SMMHC fusion protein is required for repression (31), suggesting that the fusion protein may cooperate with AML1 to recruit corepressors.
Other leukemogenic fusion proteins that affect AML1-dependent transcriptional control, such as AML1/ETO and TEL/AML1, repress transcription through interactions with the mSin3A, N-CoR, and SMRT corepressors and histone deacetylases (HDACs) (2, 11, 13, 18, 33, 48). We demonstrate here that the inv(16) fusion protein interacts with mSin3A and HDAC8. The C-terminal SMMHC portion of inv(16) is both necessary and sufficient for transcriptional repression and is sufficient for association with these corepressors. This region contains an assembly competence domain (ACD) that is required for multimerization of the SMMHC (24, 44). The 28-aa ACD contributes to both transcriptional repression and association with mSin3A and HDAC8. Identification of the repression domain of inv(16) and its interacting proteins may stimulate the development of novel treatments for inv(16)-mediated AML.
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MATERIALS AND METHODS |
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Deletion mutants.
inv(16) mutants were made by PCR amplification of inv(16) cDNA fragments, restriction digestion of the fragments, and ligation into the pCMV5 or pCMV5-GAL4(M1R) vector. The CBFß portion of inv(16) (aa 1 to 165) was PCR amplified using primers at the 5' start of the protein (5'-CGG AAT TCA TGC CGC GCG TCG TGC CCG AC-3') and at the fusion (aa 165) to create a BamHI site (5'-CGC GGA TCC CTC CAT TTC CTC CCG GTG AGA-3') (restriction sites are underlined). This PCR product was cloned into pCMV5-GAL4(M1R) using EcoRI and BamHI to make pCMV5-GAL4-CBFß. Internal deletion mutants were made by amplification of the SMMHC region using a primer at the 3' end of the cDNA (5'-GTC AAG CTT TTA TTC ACT GGC CTT GGT TCC AT-3') with one the following primers: 
496-720, 5'-CAG GGA TCC GAG ACG GAA CTG GAA GAC GAG-3'; 496-945, 5'-CAG GGA TCC GAG AT GAG AAG AAA GCC AAG AG-3'; 496-1170, 5'-CAG GGA TCC ATG GAG GCC ATG AGC GAC CGG-3'; 496-1347, 5'-CAG GGA TCC AAG TTC AAA TCC ACC ATC GCG-3'; and 496-1665, 5'-CAG GGA TCC ATG GGC CGC GAG GTG AAC GCA-3'. The fragments were cloned into pCMV5-GAL4-CBFß after digestion with BamHI and HindIII. C-terminal fragments of inv(16) were PCR amplified using 5'-CGG GAT CCT TAT TCA CTG GCC TTG GTT CC-3' with the following primers: 449-611, 5'-CGG AAT TCA AGT TCA AAT CCA CCA TCG CG-3'; 502-611, 5'-CGG AAT TCA TGG CCG AGC AGT ACA AGG AG-3'; 532-611, 5'-CGG AAT TCC AGC GCA TCA ACG CCA ACC GC-3'; and 572-611, 5'-CGG AAT TCA CCT CTT TCG TTC CTT CTA GA-3'. The fragments were cloned into pCMV5-GAL4(M1R) using EcoRI and BamHI sites. Mutants were confirmed by sequencing.
The inv(16)ACD mutant (24) was cloned into pCMV5-GAL4(M1R) by the following strategy. pGEM-inv(16)ACD was digested with XbaI, and 3' recessed termini were filled in using Klenow fragment, followed by digestion with EcoRI. The vector was digested with HindIII, 3' recessed termini were filled in with Klenow fragment, and the linearized vector was digested with EcoRI. These fragments were ligated in order to obtain the construct used in subsequent experiments, pCMV-GAL4-inv(16)ACD.
The inv(16)1-449 mutants were made by PCR amplification of these amino acids (aa 1 to 449) using the following primers: 5'-AG GGA TTC ATG CCG CGC GTC GTG CCC-3' was used with 5'-GTC AAG CTT TTA GGA CTT GAC GGC CCC CTC-3' or 5'-AGT GGA TCC GGA CTT GAC GGC CCC CTC-3'. These fragments were digested with EcoRI and HindIII (with stop codon) or EcoRI and BamHI (to be fused to the p53 tetramerization domain) and ligated into linearized pCMV5-M1R. The p53 tetramerization domain (aa 340 to 393) was amplified using the following primers to create restriction sites for cloning: 5'-CCC AAG CTT TCA GTC TGA GTC AGG CCC-3' was used with 5'-CGG GAT CCA TGT TCC GAG AGC TGA AT-3' or 5'-CGG AAT TCA TGT TCC GAG AGC TGA AT-3'. These fragments were digested with BamHI or EcoRI and HindIII and cloned into pCMV5-M1R-inv(16)1-449 and pCMV5-M1R, respectively.
Transcription assays. GAL4 reporter assays were performed in 3T3 cells transiently transfected with the indicated amounts of pCMV5-GAL4-inv(16) or plasmid expressing mutant inv(16) proteins, 1 µg of GAL4-thymidine kinase-luciferase (GAL-TK-Luc) reporter construct, and 200 ng of pCMV5-SEAP plasmid. The GAL-TK-Luc reporter contains four GAL4 DNA-binding sites in front of the thymidine kinase promoter that drives expression of the luciferase gene. Cells were lysed in 1x reporter lysis buffer (Promega, Madison, Wis.) and centrifuged at 16,000 x g for 20 s, and the supernatant was used in luciferase assays. Luciferase activity was measured using the luciferase assay system from Promega and normalized to secreted alkaline phosphatase (SEAP) activity.
For trichostatin A (TSA) experiments, cells were transfected with 100 ng of pCMV5-GAL4-inv(16) or empty expression vector, 1 µg of GAL-TK-Luc, and 100 ng of pRL-TK plasmid (thymidine kinase promoter driving expression of Renilla luciferase). Cells were treated with TSA (Biomol, Plymouth Meeting, Pa) for 48 h posttransfection and lysed as described above. Luciferase activity of the experimental (GAL-TK-Luc) and internal control (pRL-TK) reporters was measured using the Dual Luciferase Reporter Assay System from Promega, and GAL-TK-Luc activity was normalized to that of pRL-TK.
co-IP, immunoblotting, and cell fractionation. For mSin3A coimmunoprecipitations (co-IPs), Cos7 cells were transiently transfected with GAL4-inv(16) or the GAL4-tagged inv(16) mutants using Lipofectamine (Gibco BRL, Carlsbad, Calif.). Forty-eight hours posttransfection, cells were lysed in 300 µl of phosphate-buffered saline (PBS) containing 0.5% Triton X-100, 0.1% sodium deoxycholate (DOC), 0.1% sodium dodecyl sulfate (SDS), and protease inhibitors (0.5% Triton X-100 for co-IPs with internal deletion mutants; 0.5% Triton X-100 and 0.1% DOC for co-IPs with C-terminal fragments). ME-1 cells were lysed in 400 µl of PBS containing 0.5% Triton X-100, 0.3% DOC, and 0.2% SDS. Lysates were sonicated and precleared with pansorbin (Calbiochem, La Jolla, Calif.) and protein A (Amersham Pharmacia, Uppsala, Sweden) or protein G (Sigma) beads. Endogenous mSin3A was immunoprecipitated with anti-mSin3A antibody (K-20; Santa Cruz Biotechnology, Santa Cruz, Calif.). For ME-1 lysates, endogenous HDAC2 was precipitated with anti-HDAC2 antibody (H-54; Santa Cruz Biotechnology). The proteins were separated by SDS-10% polyacrylamide gel electrophoresis (PAGE) (SDS-7.5% PAGE for ME-1 IPs) and transferred to nitrocellulose.
For HDAC co-IPs, Cos7 cells were cotransfected with pCMV5-GAL4-inv(16), GAL4-tagged inv(16) mutants, or AML-1B and FLAG-, hemagglutinin (HA)-, or Myc-tagged HDACs. Forty-eight hours posttransfection, cells were lysed in 300 µl of PBS containing 0.5% Triton X-100 and protease inhibitors (0.5% Triton X-100 and 0.3% DOC for co-IPs with C-terminal fragments; 0.5% Triton X-100, 0.1% DOC, and 0.1% SDS for co-IPs with AML-1B). Lysates were sonicated and precleared with protein G. HDACs were immunoprecipitated with antibodies to their tags (anti-Flag M2 [Sigma]; anti-Myc 9E10 and anti-HA [both from Covance, Richmond, Calif.]). Proteins were separated by SDS-PAGE and transferred to nitrocellulose. Membranes were blocked in PBS containing 5% nonfat dry milk for 1 h at room temperature and immunoblotted with primary antibodies to the tagged proteins overnight at 4°C. Membranes were washed in PBS and blotted with secondary antibodies. Proteins were visualized by enhanced chemiluminescence (Pierce, Rockford, Ill.).
Cellular fractionation was performed in isotonic buffer (0.01 M Tris-HCl [pH 8.4], 0.14 M NaCl, 0.0015 M MgCl2) containing 0.5% Nonidet P-40 (NP-40) by low-speed centrifugation as described previously (31). Equal proportions of the cytoplasmic and nuclear fractions were analyzed by immunoblotting with antibodies to phospholipase C
1 (Robert Abraham, The Scripps Research Institute, San Diego, Calif.), HDAC2 (Santa Cruz Biotechnology), and the GAL4 DNA-binding domain (Santa Cruz Biotechnology).
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RESULTS |
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166-390 [Fig. 1B]). However, further deletion past residue 449 eliminated repression. The mutants were expressed at levels similar to that of the full-length fusion protein (Fig. 1C). Note that these proteins migrated as multiple bands, possibly due to cryptic alternative splicing with 3' vector sequences (K. L. Durst, unpublished data). Thus, the slowest migrating band is the full-length form. The transcription assay results indicate that CBFß sequences alone are not sufficient and the C-terminal 163 aa of SMMHC are required for inv(16)-mediated repression.
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Having defined a transcriptional repression domain within the myosin heavy chain portion of the fusion protein, we asked whether mSin3A binding cosegregated with the ability to repress transcription. The GAL4-inv(16) deletion mutants (Fig. 1A and 2A) were expressed in Cos7 cells and assayed for mSin3A association by co-IP, followed by immunoblot analysis. In contrast to full-length CBFß/SMMHC, all of the deletion mutants failed to bind mSin3A (Fig. 4A) in buffer containing 0.5% Triton X-100, 0.1% DOC, and 0.1% SDS, even though several mutants retained the ability to repress transcription (e.g., 166-240 [Fig. 1B]). Therefore, we repeated the assay using only 0.5% Triton X-100 in PBS to detect weaker interactions. Under these conditions, GAL4-inv(16) as well as the GAL4-inv(16)166-315 and GAL4-inv(16)166-390 mutants coimmunoprecipitated with mSin3A (Fig. 4B). The association was blocked when the antigenic peptide was included in the assay (Fig. 4B, lanes labeled C). In contrast, GAL4-inv(16)166-555, which fails to repress transcription (Fig. 1B), also failed to bind mSin3A (Fig. 4B). However, under this milder condition, we also observed some level of nonspecific association of GAL4-inv(16) with the protein G-Sepharose beads. By these criteria, mutants that retained the ability to repress transcription also associated with mSin3A (Fig. 1B and 4B).
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166-449, which contains the minimal repression domain, displayed too much background binding to protein G-Sepharose in PBS containing 0.5% Triton X-100 to make a clear determination as to whether it retained the ability to bind mSin3A (data not shown). Therefore, we surveyed a range of lysis conditions to determine whether residues 449 to 611 are sufficient to bind mSin3A. GAL4-inv(16)449-611 coimmunoprecipitated with mSin3A when cells were prepared in PBS containing 0.5% Triton X-100 and 0.1% DOC (Fig. 4C, left blot) but did not copurify in the presence of the antigenic peptide as a control (lanes labeled C). Under the same conditions, GAL4-inv(16)532-611, which retained some ability to repress transcription, also weakly associated with mSin3A (Fig. 4C, right blot). The C-terminal fragment (572-611) that had no activity in the GAL4 repression assay (Fig. 2B) failed to bind mSin3A, while the larger fragment, 502-611, showed nonspecific binding in the control immunoprecipitation (data not shown). inv(16) interacts with HDAC8. Corepressors recruit HDACs to promoters where HDACs alter chromatin structure by deacetylating histones in order to repress transcription. They are divided into two classes. The class I HDACs (HDAC1, HDAC2, HDAC3, and HDAC8) are mainly composed of the catalytic domain, while the larger, class II HDACs (HDAC4 to HDAC6, HDAC7, and HDAC9) include a noncatalytic domain (3, 9). Because HDACs interact with other leukemogenic fusion proteins that disrupt AML1, we determined whether the inv(16) fusion protein could bind HDACs. GAL4-inv(16) was coexpressed with FLAG-tagged HDAC1 to HDAC6 and a large fragment of HDAC9, also known as HDAC-related protein (HDRP) (53), as well as HA-tagged HDAC7 and Myc-tagged HDAC8 (Myc-HDAC8). The inv(16) fusion protein coimmunoprecipitated with Myc-HDAC8 but failed to bind the class II HDACs and HDAC1 to HDAC3 (Fig. 5A). Unfortunately, we were unable to detect HDAC8 protein in ME-1 cells, although HDAC8 mRNA is expressed in these cells (Durst, unpublished).
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2-11 mutant that no longer binds AML1 (1). This mutant also associated with HDAC8 (Fig. 5B), suggesting that the interaction is mediated by the SMMHC sequence. Furthermore, mSin3A and HDAC8 did not associate by in Cos7 cells (data not shown), indicating that the association between the fusion protein and HDAC8 is not mediated by mSin3A. We also tested whether AML1 could associate with HDAC1 to HDAC9 in order to confirm that the inv(16)-HDAC8 interaction was not mediated by AML1. Epitope-tagged forms of each HDAC were coexpressed with AML1, and the cell lysates were immunoprecipitated with antibodies directed to the epitope tag (FLAG, HA, or Myc); coimmunoprecipitating AML1 was detected by immunoblot analysis (Fig. 6, upper blot). AML1 associated with HDAC1, HDAC3, and HDAC9 and more weakly copurified with HDAC2, HDAC5, and HDAC6. However, we did not detect an association with HDAC8.
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166-555, which failed to repress transcription, was impaired in binding HDAC8 (Fig. 7A). Some of the mutants showed nonspecific binding to protein G-Sepharose. Therefore, 0.3% DOC was added to the lysis buffer for copurification with the C-terminal fragments in which GAL4-inv(16)449-611 and GAL4-inv(16)502-611 coimmunoprecipitated with Myc-HDAC8 with only modest background levels (Fig. 7B). However, GAL4-inv(16)532-611, which retains some ability to repress transcription and weakly associates with mSin3A, failed to bind HDAC8 under these conditions.
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166-555 [Fig. 4B]; 572-611 [data not shown]) eliminated mSin3A binding. These data suggest that the N-terminal boundaries of the HDAC8 and mSin3A binding motifs overlap with the ACD. Therefore, we tested whether this motif was required for inv(16)-mediated repression and corepressor binding. The inv(16) cDNA with a deletion of aa 514 to 542 (ACD) (24) was subcloned into the GAL4-expressing plasmid (Fig. 8A). We fused this mutant to GAL4 for use in the transcription assays; however, previous data showed that it may be retained in the cytoplasm to a greater extent than the wild-type protein (24). Cell fractionation indicated that a significant amount of the GAL4-inv(16)ACD did reach the nucleus (Fig. 8B). In transient-transfection assays using the GAL4-TK-Luc reporter, this mutant exhibited impaired transcriptional repression compared to the full-length protein, even at high levels of input inv(16)ACD plasmid (Fig. 8C). Deletion of the ACD also resulted in a loss of interaction between inv(16) and mSin3A (Fig. 8D) as well as HDAC8 (Fig. 8E) in co-IP assays. Thus, the ACD contributes to transcriptional repression and corepressor binding.
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-helical basic region that is sufficient for tetramerization (45). p53tet was fused to the GAL4 DNA-binding domain and a GAL4-inv(16)1-449 mutant that lacks both the ACD and the C-terminal repression domain (Fig. 9A). Once again, the wild-type inv(16) fusion protein associated with mSin3A, and the ACD mutant failed to bind to mSin3A (Fig. 9B). Addition of the p53 multimerization domain to the GAL4 DNA-binding domain did not induce an mSin3A association. Although GAL-inv(16)1-449 alone did display a weak association with mSin3A, the addition of the p53 sequences did not induce binding to mSin3A (Fig. 9B). In addition, these mutants did not significantly repress transcription compared to the full-length fusion protein (Fig. 9C). Thus, the addition of a multimerization domain to the coiled-coil domain of the inv(16) fusion protein is not sufficient for transcriptional repression or corepressor binding.
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DISCUSSION |
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The contribution of a transcriptional repression domain by the myosin heavy chain portion of the inv(16) fusion protein was unexpected. This domain contains two known protein interaction motifs: the ACD, which regulates multimerization of the myosin heavy chain, and the coiled-coil domains that make extensive contacts between the chains. While the majority of the coiled-coil domains could be deleted with little or no effect on transcriptional repression (residues 166 to 449 [Fig. 1B]), deletion of these domains did impair mSin3A binding because ionic detergents could not be used to coimmunoprecipitate these deletion mutants (Fig. 4A). Nevertheless, the C-terminal 163 aa were sufficient for interaction with mSin3A and HDAC8 (Fig. 4C and 7B). Thus, the coiled-coil domain may contribute a mSin3A interaction motif, or oligomerization of these domains may yield a more stable association with corepressors binding the C-terminal domain. However, if the coiled-coil domains can contact mSin3A, this association alone is not sufficient to mediate repression (Fig. 8C).
The ability of a nucleus-localized myosin heavy chain to associate with corepressors could be a result of spurious homology to transcription factors. The Ski and Sno oncogenes that were transduced by avian leukemia viruses contain regions of homology to myosin heavy chains (8, 37, 42). Both of these oncogenes associate with mSin3A and are capable of repressing transcription and/or acting as corepressors (38). A comparison of the repression domain of the inv(16) fusion protein with the mSin3A binding domain of Ski (38) indicates 23% identity within these domains. We also considered the possibility that the inv(16) fusion protein may associate with Ski or Sno to repress transcription, given that they contain homologous protein interaction motifs. However, we did not observe an interaction between Ski or Sno and the inv(16) fusion protein (data not shown).
Our deletion mapping studies pinpointed a domain that was necessary and sufficient for active transcriptional repression. While this domain was sufficient for binding corepressors, it also contains the ACD that is required for multimerization of the inv(16) fusion protein. We were able to separate these two functions, as deletion of the first 18 aa of this domain (GAL4-532-611) impaired but did not eliminate mSin3A association (Fig. 4C). In addition, replacement of this domain with the p53 tetramerization domain failed to complement the loss of repression or corepressor binding (Fig. 9B and C). Previous studies also predict that the 502-611 and 532-611 C-terminal fragments of CBFß/SMMHC, which coimmunoprecipitate with mSin3A, will not multimerize (24). These data argue that the ACD contributes directly to association with the corepressors, rather than causing oligomerization of a distal corepressor binding domain. However, under our assay conditions, the fusion protein most likely retains its ability to dimerize (and GAL4 forms dimers). Thus, it is possible that dimerization of the fusion protein would amplify the ability of the fusion protein to bind mSin3A and HDAC8 and repress transcription.
The inv(16) fusion protein acts as a corepressor for AML1 to repress AML1-regulated genes (31). Because AML1 can also associate with mSin3A to repress transcription and this repression is sensitive to TSA, we screened the known class I and class II HDACs for binding to AML1. Although HDAC8 failed to bind AML1, HDAC1, HDAC3, and HDAC9 associated with AML1 (Fig. 6). Given that both the inv(16) fusion protein and AML1 bind mSin3A, we speculate that association of AML1 with mSin3A is stabilized by the inv(16) fusion protein to convert AML1 from a regulated transcription factor to a constitutive repressor.
inv(16) is one of the most frequently observed chromosomal translocations associated with AML. Our data imply that those HDACs associated with mSin3, and perhaps HDAC8, contribute to the biological actions of inv(16). The observation that TSA impairs inv(16)-mediated repression provides hope that directed therapies for this leukemia may be developed using rational drug design. However, it is likely that targeted therapeutic strategies must account for the HDACs that associate with AML1, the inv(16) fusion protein, and mSin3A.
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ACKNOWLEDGMENTS |
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This work was supported in part by National Institutes of Health (NIH) grants RO1-CA64140, RO1-CA77274, and RO1-CA87549 and a Center grant from the National Cancer Institute (CA68485) to the Vanderbilt-Ingram Cancer Center. A.D.F. is a Leukemia and Lymphoma Society Scholar.
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FOOTNOTES |
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Present address: Merck, Inc., West Point, Pa.
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