A yeast two-hybrid screen for proteins that interacted with an RD conserved between N-CoR and SMRT (SMRT RD and N-CoR RD3; Fig. a) led to the identification of ETO as a CoR-interacting protein (Fig. b). Full-length SMRT and N-CoR interacted with ETO in pulldown experiments using a GST-ETO fusion protein and in vitro-translated CoR proteins (Fig. c and data not shown). Conversely, GST fusions of the SMRT RD (or N-CoR RD3; data not shown) pulled down in vitro-translated ETO (Fig. c). The strength of the interaction between CoR and ETO was at least as great as that which we have observed for nuclear receptor interaction (72
). N-CoR RD1 (not present in SMRT, Fig. a) did not interact with ETO (Fig. b to d), indicating that the ETO-CoR interaction was not a general feature of the CoR RDs. In human 293T cells, a VP16-ETO fusion protein greatly enhanced luciferase activity from a GAL4-based reporter in the presence of the GAL4-SMRT RD, suggesting that the interaction between ETO and SMRT occurred in vivo (Fig. d). We also performed coimmunoprecipitation experiments with 293T cells transiently transfected with an epitope-tagged ETO (Flag-ETO) expression vector. Cell lysates were immunoprecipitated with anti-N-CoR antibodies, and the resulting immunocomplexes were analyzed by Western blotting using antibodies directed against the Flag epitope. As shown in Fig. e, anti-N-CoR antibodies specifically precipitated ETO, confirming the in vivo interaction between ETO and endogenous N-CoR.
FIG. 1 In vitro and in vivo interactions of ETO with CoRs. (a) Modular organization of SMRT, N-CoR, and ETO. ID, nuclear receptor interaction domains. TBF-associated factor (TAF)-like and Nervy homology domains of ETO (including Zn fingers) are indicated. (b) (more ...)
To map the ETO domain(s) required for CoR interaction, we performed yeast two-hybrid (data not shown) and GST pulldown experiments. Truncation of ETO at amino acid 416 (ETOΔC) abrogated the interaction with SMRT (Fig. a). Finer mapping of the interaction revealed that deletion of amino acids 488 to 525, containing the zinc fingers of ETO, also prevented CoR interaction. Point mutation of cysteine residues in either zinc finger abolished the interaction (C488S and C508S; Fig. a), indicating that both zinc fingers are critical for the interaction between ETO and a CoR. The C-terminal truncation (ETOΔC) that abolished in vitro interaction between ETO and SMRT similarly abolished the ETO–N-CoR interaction in vivo, as demonstrated in coimmunoprecipitation experiments (Fig. b).
FIG. 2 In vivo interaction of ETO with HDAC1 and mapping of the ETO CoR-binding region. (a) Mapping of the CoR interaction domain in ETO. In vitro-translated ETO or mutant ETOs were precipitated with GST or a GST-SMRT RD fusion protein. ETOΔC contains (more ...)
To investigate the possibility that ETO might recruit the HDAC component of the CoR/HDAC complex, anti-Flag immunoprecipitates were analyzed for the presence of HDAC activity and protein (Fig. c and d). HDAC activity (Fig. c) and HDAC1 protein (Fig. d) were undetectable in anti-Flag immunoprecipitates from cells transfected with the empty Flag control vector, although anti-N-CoR antibodies immunoprecipitated levels of HDAC activity comparable to those of Flag-ETO-transfected cells (Fig. c and data not shown). HDAC activity was specifically detected in the anti-Flag immunoprecipitates from Flag-ETO-transfected 293T cells (Fig. c); likewise, anti-Flag antibodies specifically precipitated significant levels of HDAC1 protein (Fig. d). These results confirmed the specific association of ETO with HDAC in vivo. ETO did not interact with HDAC in vitro (Fig. e), strongly suggesting that the interaction with HDAC in vivo was indirect and due to the interaction with an endogenous CoR. Moreover, ETOΔC did not recruit HDAC activity or protein in vivo (Fig. c and d), further suggesting that the ETO–N-CoR interaction is required for HDAC recruitment in vivo. The ETO point mutants that did not interact with SMRT similarly did not coimmunoprecipitate with N-CoR or HDAC1 in 293T cells (data not shown).
In the AML1/ETO fusion protein, the transcriptional activation domain of AML1 has been replaced with ETO (41
). Therefore, we tested whether AML1/ETO retained the ability of ETO to interact with CoRs and HDACs. Both GST-NCoR RD3 and GST-SMRT RD fusion proteins (Fig. a and data not shown) interacted with in vitro-translated AML1/ETO. A specific AML1/ETO–N-CoR/HDAC complex was detected in vivo in coimmunoprecipitation experiments performed with 293T cells cotransfected with a Myc-tagged AML1/ETO expression vector. Cell lysates were immunoprecipitated with anti-Myc tag antibodies, and the resulting immunocomplexes were analyzed for the presence of N-CoR protein and HDAC activity. As shown in Fig. b and c, N-CoR was specifically detected by Western blotting and anti-Myc tag antibodies precipitated significant levels of HDAC activity from AML1/ETO-transfected cells. In contrast, N-CoR did not interact with AML1/ETOΔC in vitro (Fig. a) and it was absent in immunoprecipitates from AML1/ETOΔC-transfected cells (Fig. b). Likewise, no detectable HDAC activity was found in the AML1/ETOΔC immunoprecipitates (Fig. c).
FIG. 3 In vitro and in vivo interactions of AML1/ETO and AML1/ETOΔC with CoRs and HDAC. (a) Pulldown experiments. In vitro translated AML1/ETO or AML1/ETOΔC was precipitated with GST or A GST-SMRT RD fusion protein. (b) Coimmunoprecipitation (more ...)
It has been previously demonstrated that the ectopic expression of AML1/ETO into hematopoietic precursor cell lines blocks terminal differentiation (48
). To explore the biological relevance of the interaction between AML1/ETO and the N-CoR–HDAC complex, we compared the abilities of AML1/ETO and the AML1/ETOΔC mutant to block terminal differentiation of human promonocytic U937 cells after vitamin D3
and TGF-β treatment. To facilitate the monitoring of ectopic protein expression, the two fusion proteins were fused to the GFP. The parental GFP, GFP-AML1/ETO, and GFP-AML1/ETOΔC cDNAs were cloned under the control of the 5′ long terminal repeat of a derivative of the hybrid Epstein-Barr virus–retroviral PINCO vector (see Materials and Methods) (14
). Efficiency of infection, as evaluated by the frequency of GFP-positive cells, varied from 70 to 90% (PINCO control) to 50 to 75% (GFP-AML1/ETO and GFP-AML1/ETOΔC) (Fig. a). The intensities of the fluorescence signals were similar in AML1/ETO and AML1/ETOΔC cells, indicating comparable levels of expression that were confirmed by Western analysis (Fig. a and data not shown). Evaluation of vitamin D3
-induced differentiation in cells infected with either the control, GFP-AML1/ETO, or GFP-AML1/ETOΔC retrovirus was performed by double-fluorescence fluorescence-activated cell sorter (FACS) analysis (see Materials and Methods) of the CD14 differentiation antigen in GFP-positive and -negative cells. In cells infected with the control retrovirus, CD14 expression was low or absent without stimulation but increased progressively during vitamin D3
-induced differentiation in both the GFP-positive and -negative cell populations (Fig. b). Comparable up-regulation of CD14 expression was also detected in the GFP-negative cells of both the AML1/ETO- and AML1/ETOΔC-infected populations. Differentiation was, instead, inhibited in the GFP-AML1/ETO GFP-positive cells, while it was almost complete in the GFP-AML1/ETOΔC GFP-positive cells (Fig. b). The ability of the GFP-AML1/ETO fusion protein to inhibit differentiation was similar to what we have observed for the parental AML1/ETO when it is expressed in U937 or 32D cells (unpublished results). It therefore appears that the integrity of the N-CoR binding region is critical for the capacity of AML1/ETO to block differentiation by vitamin D3
and TGF-β, suggesting that recruitment of the N-CoR–HDAC complex is critical to the biological activity of AML1/ETO.
FIG. 4 Effects of AML1/ETO and AML/ETOΔC on differentiation. (a) The parental GFP or the GFP-AML1/ETO and GFP-AML1/ETOΔC fusion proteins were expressed in U937 cells by using a derivative of the PINCO Epstein-Barr virus–retroviral vector. (more ...)