Multimerization via Its Myosin Domain Facilitates Nuclear Localization and Inhibition of Core Binding Factor (CBF) Activities by the CBFβ-Smooth Muscle Myosin Heavy Chain Myeloid Leukemia Oncoprotein

The core binding factor (CBF) transcription factors contain one of three CBFα subunits, CBFα1/AML3/RUNX2, CBFα2/AML1/RUNX1, or CBFα3/AML2/RUNX3, and a common CBFβ subunit (3, 14, 21, 35, 50). The CBFα subunits contact the consensus DNA binding site, 5′-(Pu)ACCPuCA-3′, via their 127-amino-acid Runt homology domains (3, 30). CBFβ does not bind DNA but increases the DNA affinity of the CBFα subunits via allosteric interaction with the Runt domain (36, 47, 50). Amino acids 1 to 137 of the 182-residue CBFβ are sufficient for increasing the DNA affinity of CBFα subunits (16, 19). CBFβ is widely expressed, whereas AML1 is largely restricted to hematopoietic cells (43, 50). Mice lacking AML1 or CBFβ do not develop definitive hematopoiesis (33, 37, 42, 51). A role for AML1 during maturation of pluripotent stem cells along the lymphoid and myeloid lineages has been inferred from its ability to transactivate promoters of lineage-restricted genes (34, 41, 46, 58). AML1 possesses only weak intrinsic transactivating potential but cooperates with additional transcription factors to activate genes in hematopoietic cells (7, 40, 46). AML1 is found in the cell nucleus, whereas the large majority of CBFβ is cytoplasmic, reflecting its expression in excess of CBFα and its affinity for the actin cytoskeleton (26, 48). Chromosomal and mutational abnormalities affecting the genes encoding AML1 and CBFβ are common in acute leukemia cases (14). CBFβ-SMMHC, encoded by inv(16) (p13;q22) or t(16;16) (p13;q22) in 8% of acute myeloid leukemias, inhibits CBF activities via its interactions with AML1 (9, 10, 22, 23). In contrast, AML1-ETO, another CBF oncoprotein, directly represses AML1 target genes (31). The phenotypes of AML1-ETO and CBFβ-SMMHC knock-in mice are similar to those lacking AML1 or CBFβ, indicating that these oncoproteins interfere with CBF activities in vivo (11, 38, 57). A direct role for CBFβ-SMMHC in the formation of human acute myeloid leukemias can be inferred from the formation of acute myeloid leukemias in chimeric mice exposed to ethylnitrosourea (12). In addition to stimulating differentiation, CBF is require for the G1 to S transition in 32D cl3 myeloid and Ba/F3 lymphoid cells (9, 25). Deletion of 11 N-terminal CBFβ residues required for interaction with AML1 prevents CBFβ-SMMHC from slowing Ba/F3 proliferation, and exogenous AML1 accelerates G1 progression via transactivation (4, 10, 25, 45). Exogenous AML1, cdk4, cyclin D2, and c-Myc prevent CBFβ-SMMHC from slowing Ba/F3 proliferation (5, 25). During leukemogenesis, genetic alterations which accelerate G1 may potentiate expression of CBFβ-SMMHC, enabling increased blockade of differentiation. Consistent with this model, deletion of the overlapping p16INK4A and p19ARF genes or expression of E7 cooperates with CBFβ-SMMHC or TEL-AML1 to induce acute leukemia in mice (6, 56). In CBFβ-SMMHC, the majority of CBFβ is linked to the rod domain of smooth muscle myosin heavy chain (SMMHC) (Fig. ​(Fig.1A).1A). The SMMHC rod domain is α-helical and consists of 28 related amino acid regions. Each of these regions contains four heptads in which the a and d residues are often hydrophobic. As a result, the rod domain has a hydrophobic face which mediates dimerization. The other face of the α-helix has alternating positively and negatively charged zones and mediates staggered multimerization (55). Alternative RNA splicing leads to the expression of two CBFβ-SMMHC isoforms, which have either an 8- or a 42-amino-acid nonhelical, C-terminal tailpiece (32). FIG. 1. Diagrams of CBFβ-SMMHC (INV), the INV variants studied herein, and the ACD. (A) INV contains CBFβ residues 1 to 165, an α-helical SMMHC rod domain, and an 8-residue nonhelical tailpiece. An alternatively spliced form of INV, INVa, ... Deletion analysis of the rod domain of skeletal muscle myosin identified a 29-amino-acid segment, designated the assembly competence domain (ACD), that was required for multimerization and capable of conferring the ability to multimerize on a 391-residue rod domain segment which was otherwise incapable of multimerization (44). These authors noted that multiple rat and human sarcomeric myosins contain a segment near their C termini that is virtually identical to the human fast IId sarcomeric myosin heavy chain ACD and that nonmuscle and smooth muscle myosins from several species, including Drosophila, Xenopus, chicken, rabbit, rat, and human, contain segments in a similar location which are highly related to the sarcomeric myosin ACD and which are virtually identical to each other. The amino acid sequences of the human sarcomeric fast IId myosin heavy chain and SMMHC ACDs are compared in Fig. ​Fig.1B.1B. Deletion analysis of the sarcomeric myosin rod domain also demonstrated that a 194-residue α-helical segment containing the ACD multimerized in vitro, whereas a 145-amino-acid segment did not (44). Thus, even with the ACD intact, a minimal length of α-helical coiled coil is required for multimerization of the myosin rod. CBFβ-SMMHC multimers compete with CBFβ for interaction with AML1, potentially sequestering AML1 away from chromatin (9, 23). As AML1:CBFβ-SMMHC monomeric and multimeric complexes retain DNA binding activity, CBFβ-SMMHC may also be capable of directly repressing CBF-regulated genes. To ascertain whether multimerization is essential for inhibition of CBF activities, we expressed several CBFβ-SMMHC variants from the zinc-responsive metallothionein (MT) promoter in Ba/F3 cells. Herein we refer to the shorter isoform of CBFβ-SMMHC as INV and to the splice variant with a longer C-terminal, nonhelical tail as INVa. Deletion of the ACD homology domain from INV or INVa inhibited multimerization. In contrast to INV, INV(ΔACD) and INVa(ΔACD) were predominantly cytoplasmic and were defective for inhibition of CBF-mediated transactivation of a reporter gene and of endogenous genes required for G1 to S cell cycle progression. We also expressed internal deletion variants linking the CBFβ domain to either 149 or 83 C-terminal SMMHC residues. The former multimerized, entered the nucleus, and slowed proliferation, whereas the latter did not. When INV was directed to the nucleus with a simian virus 40 nuclear localization signal (NLS), deletion of the ACD still largely prevented inhibition of CBF activities. As the NLS did not localize CBFβ-ACD fusion proteins to the nucleus, we linked the ACD to AML1 or to the AML1 DNA binding domain. In contrast to AML1-ETO, AML1-ACD proteins did not inhibit CBF-regulated genes. These findings indicate that the ACD facilitates inhibition of CBF by enabling CBFβ-SMMHC multimerization in the nucleus.