Transcriptional repression by chromosomal translocation fusion proteins is a common theme in AML. The t(15;17), t(8;21), and t(11;17) fusion proteins contact corepressors that recruit HDACs or directly bind HDACs to repress transcription (17
). Here we demonstrate that the inv(16) fusion protein has the ability to interact with mSin3A and HDAC8 through an unexpected repression domain within the SMMHC portion of the fusion protein. The mSin3A and HDAC8 binding domains cosegregate with the ability of the inv(16) fusion protein to repress transcription, implying that these factors contribute to inv(16)-mediated repression. In addition, inv(16)-mediated repression was sensitive to TSA, a potent, broad-spectrum HDAC inhibitor. Taken together, these data suggest that HDACs, including HDAC8 and HDACs recruited by mSin3A, contribute to active repression mediated by this chromosomal translocation fusion protein.
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. ]), deletion of these domains did impair mSin3A binding because ionic detergents could not be used to coimmunoprecipitate these deletion mutants (Fig. ). Nevertheless, the C-terminal 163 aa were sufficient for interaction with mSin3A and HDAC8 (Fig. and ). 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. ).
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
). 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. ). In addition, replacement of this domain with the p53 tetramerization domain failed to complement the loss of repression or corepressor binding (Fig. and C). Previous studies also predict that the 502-611 and 532-611 C-terminal fragments of CBFβ/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. ). 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.