(A) The B2-HuN-CoR fragment was fused with GST. ³⁵S-labeled ETO protein generated by in vitro translation was specifically coprecipitated by GST-B2 but not by GST alone. (B) mRNA from various human tissues (CLONTECH) was subjected to Northern blot analysis using the B2 probe. The analysis revealed that the hybridizing mRNA (labeled HuNCoR) was approximately 8,000 bases in size, indicating that the B2 fragment was a partial cDNA. β-Actin mRNA controls are shown below.

(A) Using B2 as a probe, we screened a human fetal brain cDNA library to obtain the full-length sequence of the ETO-binding protein, HuN-CoR. Shown is the complete amino acid sequence (GenBank accession no. AF044209). (B) The structure of the murine N-CoR (Mu N-CoR) can be divided into domains that mediate repression (RI, RII, RIII) (17), interact with the nuclear receptor (NRI, NRII) (17), or interact with the Sin3 corepressor complex (SIDI, SIDII) (15). By comparison with the functional domains of murine N-CoR, the ETO-binding domain of HuN-CoR lies between SIDI and SIDII and roughly corresponds to RIII.

(A) ETO associates with the N-CoR/mSin3A/HDAC1 complex in vivo. Cells were transfected with ETO alone or with Flag, N-CoR/Flag, or HDAC1/Flag. Immunoprecipitation (IP) was performed with either the M2 anti-FLAG antibody (against Flag, N-CoR/Flag, or HDAC1/Flag) or antibody against mSin3A. Normal rabbit serum (NRS) was used as negative control for the IP antibody. After IP, ETO specifically associated with the N-CoR/mSin3A/HDAC1 complex as demonstrated by Western blotting with an anti-ETO antibody. Cell lysates from ETO-transfected and untransfected (mock) cells, precipitated by anti-ETO antibody, were used as positive and negative controls, respectively (1/4 amount of lysate used as for the other experiments). Molecular mass markers are shown in kilodaltons. IgH, immunoglobulin heavy chain. (B) AML1/ETO (abbreviated A/E) associates with the N-CoR/mSin3A/HDAC1 complex in vivo. Cells were transfected with A/E, alone or with Flag, N-CoR/Flag, or HDAC1/Flag. After IP, proteins were subjected to Western blotting using anti-AML1/RHD antibody. Proteins from lysates of A/E-transfected or mock-transfected cells precipitated by ETO antibody and blotted by AML1/RHD antibody were used as positive and negative controls for the AML1/ETO protein (1/4 amount of lysate used as for the other experiments). The doublet band (denoted by the double tick) seen in the figure may be due to translation at different ATG start codons.

To determine the HuN-CoR binding domain of ETO, a series of ETO truncation mutants were constructed and inserted into the pGBT9 vector. Yeast cells were cotransformed with the B2 plasmid and the ETO truncation mutants. β-Galactosidase assays were used to test for binding activity in vivo (scored from − to +++). Shown in the schematic is the structure of the ETO protein, with proline/serine/threonine (PST)- and proline (Pro)-rich domains as well as the two zinc finger motifs (Zn). Deletion of either one of the two zinc finger motifs in the ETO protein abrogated binding to the HuN-CoR, thus localizing the binding domain to these motifs.

In the mammalian expression plasmid GAL4 DBD/ETO, ETO is fused with the GAL4 DBD (amino acids 1–147). The firefly luciferase reporter gene is driven by the TK promoter with four copies of the GAL4 DNA-binding site upstream. (A) ETO exhibited potent, dose-dependent, transcriptional repression. (B) GAL4 DBD alone partially relieved repression by GAL4 DBD/ETO, due to competition for GAL4 binding sites. When AML1/ETO or RARα LBD, both driven by the cytomegalovirus promoter, were cotransfected with GAL4 DBD/ETO, the repressive effects of ETO were almost abrogated. The AML1/ETO fusion and RARα LBD may compete with ETO for HuN-CoR binding, leading to relief of repression.