The Core Binding Factor (CBF) α Interaction Domain and the Smooth Muscle Myosin Heavy Chain (SMMHC) Segment of CBFβ-SMMHC Are Both Required to Slow Cell Proliferation

Abstract

We have expressed several variants of core binding factor ␤ (CBF␤)-smooth muscle myosin heavy chain (SMMHC) from the metallothionein promoter in Ba/F3 cells. Deletion of amino acids 2-11 from the CBF␤ segment, required for interaction with CBF␣, prevented CBF␤-SMMHC from inhibiting CBF DNA binding and cell cycle progression. Deletion of 283 carboxyl-terminal residues from the SMMHC domain, required for multimerization, also inactivated CBF␤-SMMHC. Nuclear expression of CBF␤(⌬2-11)-SMMHC was decreased relative to CBF␤-SMMHC. CBF␤(⌬2-11)-SMMHC linked to a nuclear localization signal still did not slow cell growth. The ability of each CBF␤-SMMHC variant to inhibit CBF DNA binding and cell proliferation correlated with its ability to inhibit transactivation by an AML1-VP16 fusion protein. Thus, CBF␤-SMMHC slows cell cycle progression from G 1 to S phase by inhibiting CBF DNA binding and transactivation.The core binding factor (CBF) 1 family is made up of transcription factors that contain a common CBF␤ subunit and one of three CBF␣ subunits: CBF␣1, AML1 (CBF␣2), or CBF␣3 (1-7). CBF␤ increases the affinity of the CBF␣ subunits for DNA, but does not bind DNA directly (4, 7). The CBF␣ subunits contain a domain required for heterodimerization and DNA binding (3,8).Translocations involving subunits of CBF are common in acute leukemias (9). Inv(16) is present in 10% of acute myeloid leukemias (AMLs) and encodes CBF␤-SMMHC, in which CBF␤ is fused to the tail domain of SMMHC (10). t(8;21) is present in 12% of AMLs and encodes AML1-ETO, which includes the DNA-binding domain of AML1 (11). 25% of pediatric B-lineage acute lymphocytic leukemias contain t(12;21), which encodes TEL-AML1 (12, 13).Each of these "CBF oncoproteins" inhibits CBF activities (14 -20). Also, mice expressing CBF␤-SMMHC or AML1-ETO fail to develop definitive hematopoiesis, just as do mice lacking AML1 or CBF␤ (21-26).CBF activates the expression of several lymphoid and myeloid genes, suggesting that lack of differentiation accounts for the phenotypes of CBF null mice (27-30). We expressed CBF␤-SMMHC from the zinc-responsive MT promoter in 32D cl3 myeloid and Ba/F3 B-lymphoid cells (14). Induction of CBF␤-SMMHC resulted in decreased CBF DNA binding and slowed proliferation during G 1 phase. The differentiation of 32D cl3 cells in response to granulocyte colony-stimulating factor was unaffected. We proposed that initial genetic alterations occur during leukemogenesis that bypass the growth inhibitory effect of CBF␤-SMMHC, potentiating inhibition of differentiation.We have now expressed several CBF␤-SMMHC mutants in Ba/F3 cells. Amino acids 1-165 of the 182-amino acid CBF␤ protein are present in the majority of CBF␤-SMMHC fusion proteins, although a variant containing only residues 1-133 is present in rare patients (31). Amino acids 1-73 and 102-137 are highly conserved between Drosophila Brother and Bigbrother and murine CBF␤ (32). Segment 1-137 is sufficient to strongly increase the affinity of CBF␣ subunits for DNA, whereas segment 1-133 or 1-132 does so o...

The PEBP2β/CBFβ-SMMHC chimeric protein is localized both in the cell membrane and nuclear subfractions of leukemic cells carrying chromosomal inversion 16

“…For example, stable transformants of Ba/F3 (lymphoid) and 32Dc13 (myeloid) cell lines were reported to express PEBP2␤/CBF␤-SMMHC in the nucleus. 16,30 Interestingly, endogenous PEBP2/CBF DNA binding activity is greatly reduced in these transformants. In addition, in neutrophils from mice which express PEBP2␤/CBF␤-MYH11 as a transgene, the chimeric protein was detected in the nucleus.…”

“…In most reports, PEBP2␤/CBF␤-SMMHC inhibited PEBP2/CBF site dependent transcriptional activation as measured by reporter assays. [15][16][17] Furthermore, a dominant interfering effect of PEBP2␤/CBF␤-SMMHC on PEBP2/CBF function has been demonstrated in a remarkable fashion by a PEBP2␤/CBF␤-MYH11 knocking-in strategy. 18 Murine embryos that were heterozygous for the chimeric allele, eg PEBP2␤/CBF␤-MYH11/+, revealed phenotypes similar to those of PEBP2␤/CBF␤ (−/−) or AML1 (−/−) embryos.…”

Exogenous cdk4 overcomes reducedcdk4 RNA and inhibition of G1 progression in hematopoietic cells expressing a dominant-negative CBF – a model for overcoming inhibition of proliferation by CBF oncoproteins

“…In acute myeloid leukemia (AML), t(8;21), t(3;21), and inv(16) encode AML1-ETO, AML1-MDS1/EVI1, and CBFb-SMMHC, respectively, and in acute lymphocytic leukemia t(12;21) encodes TEL-AML1 (Miyoshi et al, 1993;Liu et al, 1993;Nucifora et al, 1993;Golub et al, 1995;Romana et al, 1995). Each of these CBF oncoproteins inhibit CBF trans-activation in cell lines (Liu et al, 1994;Meyers et al, 1995;Hiebert et al, 1996;Zent et al, 1996;Cao et al, 1998). Also, mice expressing AMLI-ETO or CBFb-SMMHC and mice lacking AML1 or CBFb each fail to develop de®nitive hematopoiesis, indicating that these CBF oncoproteins inhibit CBF activities in vivo as well (Castilla et al, 1996;Okuda et al, 1996;Sasaki et al, 1996;Wang et al, 1996a,b;Yergeau et al, 1997).…”

SUV39H1 interacts with AML1 and abrogates AML1 transactivity. AML1 is methylated in vivo

“…Genetic studies in the mouse showed that in the developing murine embryo, AML1 is essential for liver hematopoiesis (Okuda et al, 1996;Wang et al, 1996) and regulates the expression of the hematopoietic genes interleukin 3 (IL-3), macrophage colony-stimulating factor (G-CSF), granulocyte macrophage colony-stimulating factor (GM-CSF), and myeloperoxidase (MPO) (for a recent review on AML1, see Speck and Gilliland, 2002). In addition, AML1 has been implicated in the regulation of the cell cycle Cao et al, 1998). Given these diverse and critical cellular functions, it is not surprising that AML1 is one of the most frequent targets of chromosomal lesions associated with human lymphoblastic and myeloid leukemia.…”