The Leukemic Protein Core Binding Factor β (CBFβ)–Smooth-Muscle Myosin Heavy Chain Sequesters CBFα2 into Cytoskeletal Filaments and Aggregates

Abstract

The fusion gene CBFB-MYH11 is generated by the chromosome 16 inversion associated with acute myeloid leukemias. This gene encodes a chimeric protein involving the core binding factor ␤ (CBF␤) and the smoothmuscle myosin heavy chain (SMMHC). Mouse model studies suggest that this chimeric protein CBF␤-SMMHC dominantly suppresses the function of CBF, a heterodimeric transcription factor composed of DNA binding subunits (CBF␣1 to 3) and a non-DNA binding subunit (CBF␤). This dominant suppression results in the blockage of hematopoiesis in mice and presumably contributes to leukemogenesis. We used transient-transfection assays, in combination with immunofluorescence and green fluorescent protein-tagged proteins, to monitor subcellular localization of CBF␤-SMMHC, CBF␤, and CBF␣2 (also known as AML1 or PEBP2␣B). When expressed individually, CBF␣2 was located in the nuclei of transfected cells, whereas CBF␤ was distributed throughout the cell. On the other hand, CBF␤-SMMHC formed filament-like structures that colocalized with actin filaments. Upon cotransfection, CBF␣2 was able to drive localization of CBF␤ into the nucleus in a dosedependent manner. In contrast, CBF␣2 colocalized with CBF␤-SMMHC along the filaments instead of localizing to the nucleus. Deletion of the CBF␣-interacting domain within CBF␤-SMMHC abolished this CBF␣2 sequestration, whereas truncation of the C-terminal-end SMMHC domain led to nuclear localization of CBF␤-SMMHC when coexpressed with CBF␣2. CBF␣2 sequestration by CBF␤-SMMHC was further confirmed in vivo in a knock-in mouse model. These observations suggest that CBF␤-SMMHC plays a dominant negative role by sequestering CBF␣2 into cytoskeletal filaments and aggregates, thereby disrupting CBF␣2-mediated regulation of gene expression.

A T Lymphocyte-Specific Transcription Complex Containing RUNX1 Activates MHC Class I Expression

“…AML1/ETO is thought to act as a dominant transcriptional repressor that deregulates the expression of RUNX1-responsive cytokine and/or tumor suppressor genes, severely impairing the normal differentiation process. Similarly, a mutated binding partner of RUNX1 identified in leukemias associated with Inv(16)(p13q22), CBF␤-MYH11, acts as a dominant negative inhibitor, although the mechanism of action is not clear (68,87). As we have shown, CBF␤-MYH11 inhibits MHC class I promoter activity in Jurkat T cells (which do not express CIITA constitutively).…”

“…Aberrations in RUNX1 expression and function have been linked to leukemias, which result as a consequence of somatic chromosomal translocations involving transcription factor genes (43). A number of translocation breakpoint products have been identified that block the normal function of the RUNX1/CBF␤ enhanceosome (27,68,87,88). The (8;21) translocation, found in ϳ10 -12% of acute myeloid leukemias, gives rise to AML1/ETO (new nomenclature: RUNX1-CBF2T1), consisting of the runt domain of RUNX1 in frame with almost the entire ETO gene (77,89).…”

Molecular basis of tissue‐specific gene expression mediated by the Runt domain transcription factor PEBP2/CBF

“…It is therefore thought that the ␣ subunit is synthesized in an inert form, in which the intramolecularly negative regulation of the Runt domain prevents it from interacting with DNA or PEBP2␤. The blocking of Runt domain interactions with PEBP2␤ and with DNA has been indirectly demonstrated by subcellular localization (Lu et al 1995;Adja et al 1998;Kanno et al 1998b) and electrophoretic mobility shift assay (EMSA) , respectively. The modular structure of AML1 as currently understood is shown in Figs 2 and 3.…”

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

“…17,19 This notion was based on the observation that, while the Runt homolog by itself was located in the nucleus, the presence of PEBP2␤/CBF␤-SMMHC resulted in retention of the Runt homolog in the cytoplasm. The latter situation appears plausible, since PEBP2␤/CBF␤-SMMHC can still bind the Runt homologs.…”

“…15 It must be noted, though, that these results were obtained by transfecting the PEBP2␤/CBF␤-SMMHC expression plasmid into NIH3T3 fibroblasts and REF52 cells or Jurkat cells of the T cell lineage. 17,19 Thus, we were interested to see if sequestration of Runt homologs by PEBP2␤/CBF␤-SMMHC could occur in the cytoplasm of leukemic cells carrying the inversion 16.…”