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AML1/RUNX1, a member of the core binding factor (CBF) family stimulates myelopoiesis and lymphopoiesis by activating lineage-specific genes. In addition, AML1 induces S phase entry in 32Dcl3 myeloid or Ba/F3 lymphoid cells via transactivation. We now found that AML1 levels are regulated during the cell cycle. 32Dcl3 and Ba/F3 cell cycle fractions were prepared using elutriation. Western blotting and a gel shift/supershift assay demonstrated that endogenous CBF DNA binding and AML1 levels were increased 2-4-fold in S and G(2)/M phase cells compared with G(1) cells. In addition, G(1) arrest induced by mimosine reduced AML1 protein levels. In contrast, AML1 RNA did not vary during cell cycle progression relative to actin RNA. Analysis of exogenous Myc-AML1 or AML1-ER demonstrated a significant reduction in G(1) phase cells, whereas levels of exogenous DNA binding domain alone were constant, lending support to the conclusion that regulation of AML1 protein stability contributes to cell cycle variation in endogenous AML1. However, cytokine-dependent AML1 phosphorylation was independent of cell cycle phase, and an AML1 mutant lacking two ERK phosphorylation sites was still cell cycle-regulated. Inhibition of AML1 activity with the CBFbeta-SMMHC or AML1-ETO oncoproteins reduced cyclin D3 RNA expression, and AML1 bound and activated the cyclin D3 promoter. Signals stimulating G(1) to S cell cycle progression or entry into the cell cycle in immature hematopoietic cells might do so in part by inducing AML1 expression, and mutations altering pathways regulating variation in AML1 stability potentially contribute to leukemic transformation.

Inversion(16) is one of the most frequent chromosomal translocations found in acute myeloid leukemia (AML), occurring in over 8% of AML cases. This translocation results in a protein product that fuses the first 165 amino acids of core binding factor beta to the coiled-coil region of a smooth muscle myosin heavy chain (CBFbeta/SMMHC). CBFbeta interacts with AML1 to form a heterodimer that binds DNA; this interaction increases the affinity of AML1 for DNA. The CBFbeta/SMMHC fusion protein cooperates with AML1 to repress the transcription of AML1-regulated genes. We show that CBFbeta/SMMHC contains a repression domain in the C-terminal 163 amino acids of the SMMHC region that is required for inv(16)-mediated transcriptional repression. This minimal repression domain is sufficient for the association of CBFbeta/SMMHC with the mSin3A corepressor. In addition, the inv(16) fusion protein specifically associates with histone deacetylase 8 (HDAC8). inv(16)-mediated repression is sensitive to HDAC inhibitors. We propose a model whereby the inv(16) fusion protein associates with AML1 to convert AML1 into a constitutive transcriptional repressor.

In CBFbeta-SMMHC, core binding factor beta (CBFbeta) is fused to the alpha-helical rod domain of smooth muscle myosin heavy chain (SMMHC). We generated Ba/F3 hematopoietic cells expressing a CBFbeta-SMMHC variant lacking 28 amino acids homologous to the assembly competence domain (ACD) required for multimerization of skeletal muscle myosin. CBFbeta-SMMHC(DeltaACD) multimerized less effectively than either wild-type protein or a variant lacking a different 28-residue segment. In contrast to the control proteins, the DeltaACD mutant did not inhibit CBF DNA binding, AML1-mediated reporter activation, or G(1) to S cell cycle progression, the last being dependent upon activation of CBF-regulated genes. We also linked the CBFbeta domain to 149 or 83 C-terminal CBFbeta-SMMHC residues, retaining 86 or 20 amino acids N-terminal to the ACD. CBFbeta-SMMHC(149C) multimerized and slowed Ba/F3 proliferation, whereas CBFbeta-SMMHC(83C) did not. The majority of CBFbeta-SMMHC and CBFbeta-SMMHC(149C) was detected in the nucleus, whereas the DeltaACD and 83C variants were predominantly cytoplasmic, indicating that multimerization facilitates nuclear retention of CBFbeta-SMMHC. When linked to the simian virus 40 nuclear localization signal (NLS), a significant fraction of CBFbeta-SMMHC(DeltaACD) entered the nucleus but only mildly inhibited CBF activities. As NLS-CBFbeta-SMMHC(83C) remained cytoplasmic, we directed the ACD to CBF target genes by linking it to the AML1 DNA binding domain or to full-length AML1. These AML1-ACD fusion proteins did not affect Ba/F3 proliferation, in contrast to AML1-ETO, which markedly slowed G(1) to S progression dependent upon the integrity of its DNA-binding domain. Thus, the ACD facilitates inhibition of CBF by mediating multimerization of CBFbeta-SMMHC in the nucleus. Therapeutics targeting the ACD may be effective in acute myeloid leukemia cases associated with CBFbeta-SMMHC expression.