Multimerization and Corepression Mediated by the CBFβ-SMMHC Assembly Competence Domain Are Partially Separable and Corepression Is Required to Inhibit Core Binding Factor Activities.

Core Binding Factor (CBF) is a family of heterodimeric transcription factors consisting of a CBFα subunit (AML1/RUNX1, AML2, or AML3) and CBFβ. The CBFα subunits bind DNA and regulate transcription. CBFβ increases the DNA affinity of the CBFα subunits. CBF induces lineage-specific genes and also accelerates the cell cycle via gene activation. CBFβ-SMMHC, encoded by the inv(16) or t(16;16) chromosomes in 8% of acute myeloid leukemia (AML) cases, is a fusion protein containing amino acids 1–165 of the 182 residue CBFβ and the α-helical rod domain of smooth muscle myosin heavy chain (SMMHC). The SMMHC domain mediates multimerization dependent upon a 28 residue, C-terminal Assembly Competence Domain (ACD). CBFβ-SMMHC potentially inhibits CBF by sequestering CBFα subunits in multimeric complexes in the nucleus. The CBFβ domain of CBFβ-SMMHC retains the ability to interact with AML1, and the resulting heterodimer can bind DNA. In addition, the SMMHC domain interacts directly with corepressors, including mSin3A, allowing AML1:CBFβ-SMMHC to directly repress CBF-regulated genes. Repression is obviated, however, upon deletion of the ACD. We have now carried out a mutagenic analysis of the ACD, in an effort to separate its contributions to multimerization and repression. We designated the 4 α-helices of the ACD as A, B, C, and D, and the subseqent helix as E. Each helix consists of 7 residues, abcdefg, with a and d hydrophobic and mediating dimerization, e and g often forming salt-bridges to stabilize this dimer, and b, c, and f available to mediate multimerization and corepressor binding. We mutated the bcf residues as a cluster within helix A, B, C, D, or E and also generated additional combinations: BC, CD, DE, BCD, ABCD, and ABCDE, the latter carrying alterations in 15 residues on the outer surface of the dimeric coiled-coil. Mutants DE, BCD, ABCD, and ABCDE were defective for multimerization, inhibition of CBF DNA-binding, nuclear localization, and inhibition of Ba/F3 proliferation. Even when directed to the nucleus using a nuclear localization signal, DE and ABCD did not inhibit CBF DNA-binding or CBF-mediated cell cycle progression. In addition, mutants B, C, or D did not inhibit Ba/F3 proliferation, and mutants A or E only mildly reduced proliferation. As each of these single helix mutants assembled into multimers, multimerization and sequestration of CBFα subunits is not sufficient for CBF inhibition. Mutation of helix C or E also prevented transrepression by a Gal4 DNA-binding domain-SMMHC fusion protein, in NIH 3T3 cells (other mutants remain to be evaluated in this assay). Of note, mutants C and E effectively coimmunoprecipitated with mSin3A from Ba/F3 extracts, suggesting that additional corepressors participate in suppression of CBF activities in these cells. In summary, we have identified CBFβ-SMMHC variants which multimerize and are defective for repression of CBF-regulated genes, but we have yet to identify mutants which cannot multimerize but still inhibit CBF. This may reflect overlap of the corepressor-binding site with residues required for multimerization. Nevertheless, we have identified amino acids on the outer surface of the ACD critical for inhibition of CBF. Drugs targeting the BCD or DE cluster of bcf residues may simultaneously prevent CBFβ-SMMHC multimerization and corepressor interaction and assist in the therapy of AMLs expressing this oncoprotein.