Analysis of the transcriptional regulatory activity of the t(8;21) fusion protein led us to propose that AML-1/ETO interacts with a corepressor (24
). We have demonstrated that in mammalian cells, ETO associates with N-CoR, mSin3A, and HDACs. We have demonstrated that N-CoR interacts with ETO in yeast two-hybrid assays, in vitro, and in mammalian cells. ETO deletion mutants that have lost the ability to interact with N-CoR still bind mSin3A, indicating that N-CoR does not associate with ETO through mSin3A. Thus, the cumulative evidence suggests that the N-CoR interaction with ETO is direct. As well, the interaction of endogenous ETO with endogenous mSin3A under stringent conditions and the association of mSin3A with AML-1/ETO mutants that fail to bind N-CoR suggest that this interaction is also direct. However, these data do not preclude the possibility that an unidentified protein (that is conserved from yeast to humans) mediates the association of ETO and these corepressors. Based on the association of endogenous ETO with endogenous mSin3A at high stoichiometry and the observation that ETO is found only in high-molecular-weight complexes by sucrose gradient sedimentation analysis, we propose that ETO is a component of one or more complexes containing mSin3A, N-CoR, and HDACs in vivo.
The four domains of ETO that are conserved in its Drosophila
homologue Nervy appear to be protein interaction motifs (e.g., the HHR [27
] and MYND [Fig. ] regions). We have been unable to demonstrate that wild-type ETO binds DNA cellulose or that it binds DNA specifically (28
). Therefore, we propose that ETO functions in a corepressor complex as an adapter protein, perhaps linking N-CoR, mSin3, and other proteins. These interactions could occur within the complex, for instance, to stabilize N-CoR/mSin3A complexes, or ETO may link the corepressors to site-specific DNA binding proteins to regulate transcription. In the latter case, ETO would be analogous to the retinoblastoma protein, which represses transcription by linking an HDAC complex to DNA binding proteins (4
). t(8;21) takes advantage of this activity to create an AML-1 repressor by fusing the DNA binding domain of AML-1 to ETO.
The MYND domain of ETO interacts with the central portion of N-CoR, including repression domain 3, in yeast two-hybrid assays (Fig. and ). However, when the MYND domain was deleted, the mutant ETO retained the ability to interact with both N-CoR and mSin3A in mammalian cells. This result suggests the presence of a second N-CoR binding domain on ETO. Moreover, because an ETO protein lacking the HHR, Nervy, and MYND domains retained the ability to interact with mSin3A, we conclude that mSin3A can bind ETO in the absence of an ETO–N-CoR interaction. Because deletion of the TAF110 domain also did not affect mSin3A interactions (data not shown), mSin3A may contact ETO through more than one domain or the interaction site may be outside of the conserved domains. However, deletion of the MYND and HHR domains did impair the ability of the fusion protein to repress transcription. Therefore, the interaction of the fusion protein with mSin3A and/or N-CoR is not sufficient for repression, and specific interactions, such as the MYND domain contacting the central portion of N-CoR, may be required for full activity.
Although AML-1/ETO represses the transcription of most of the promoters tested, in two cases, transactivation has been observed. AML-1/ETO synergized with wild-type AML-1 to activate the M-CSF1 receptor promoter (46
). Because the cooperativity was mediated by a single AML-1 binding site, it was proposed that the fusion protein was acting indirectly, perhaps by titrating a corepressor, to active transcription (46
). Our current results are consistent with this interpretation. In the second report, AML-1/ETO was demonstrated to activate transcription of the BCL-2 promoter through an AML-1 binding site that resides within a negative regulatory region of the promoter (21
). While AML-1/ETO appears to strongly bind mSin3A, N-CoR, and HDACs, we cannot rule out the possibility that the fusion protein also can act to activate transcription through an undefined mechanism.
The observation that AML-1/ETO functions by interacting with an HDAC-containing complex(es) may also have therapeutic implications. In acute promyelocytic leukemia, t(11;17) and t(15;17) target the gene for retinoic acid receptor alpha. Both of these translocation fusion proteins interact with the N-CoR and SMRT corepressors and use HDACs to inhibit transcription. Leukemic blasts or cell lines containing t(15;17) differentiate in response to all-trans
-retinoic acid, but cells expressing the t(11;17) fusion protein differentiated only when all-trans
-retinoic acid was supplemented with the HDAC inhibitors (11
). Recently, we have observed that high-level expression of AML-1/ETO disrupts normal cell cycle control in hematopoietic cells. TSA completely ablated AML-1/ETO function in this biological system (41
). The association of t(8;21) with N-CoR, mSin3 corepressors, and HDACs, coupled with the ability of TSA to transcriptionally impair AML-1/ETO (Fig. ), indicates that HDAC inhibitors may have more general application for chemotherapeutic intervention in acute myeloid leukemia.