Document Type

Article

Publication Date

12-1-1998

Publication Title

Molecular and Cellular Biology

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 β (CBFβ) and the smooth-muscle myosin heavy chain (SMMHC). Mouse model studies suggest that this chimeric protein CBFβ-SMMHC dominantly suppresses the function of CBF, a heterodimeric transcription factor composed of DNA binding subunits (CBFα1 to 3) and a non-DNA binding subunit (CBFβ). 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 CBFβ-SMMHC, CBFβ, and CBFα2 (also known as AML1 or PEBP2αB). When expressed individually, CBFα2 was located in the nuclei of transfected cells, whereas CBFβ was distributed throughout the cell. On the other hand, CBFβ-SMMHC formed filament-like structures that colocalized with actin filaments. Upon cotransfection, CBFα2 was able to drive localization of CBFβ into the nucleus in a dose-dependent manner. In contrast, CBFα2 colocalized with CBFβ-SMMHC along the filaments instead of localizing to the nucleus. Deletion of the CBFα-interacting domain within CBFβ-SMMHC abolished this CBFα2 sequestration, whereas truncation of the C-terminal-end SMMHC domain led to nuclear localization of CBFβ-SMMHC when coexpressed with CBFα2. CBFα2 sequestration by CBFβ-SMMHC was further confirmed in vivo in a knock-in mouse model. These observations suggest that CBFβ-SMMHC plays a dominant negative role by sequestering CBFα2 into cytoskeletal filaments and aggregates, thereby disrupting CBFα2-mediated regulation of gene expression.

The pericentric inversion of chromosome 16 [inv(16)(p13q22)] is a cytogenetic abnormality consistently associated with acute myeloid leukemia (AML) subtype M4Eo (2, 21), a variant of subtype M4 with abnormal eosinophils in the bone marrow and sometimes in the peripheral blood. The inversion results in the reciprocal fusions of two genes: theMYH11 gene (16p13), which encodes the smooth-muscle myosin heavy chain (SMMHC), and the CBFB gene (16q22), which encodes the β subunit of the core binding factor (CBFβ) (24). The chimeric gene CBFB-MYH11 fuses most of the 5′ coding region of CBFB in frame with the 3′ portion of MYH11, resulting in the production of the chimeric protein CBFβ-SMMHC. The reciprocal fusion,MYH11-CBFB, is not believed to be important since its expression is below detectable levels in leukemic cells and it is deleted in some patients with an unbalanced inversion (24, 25,29).

CBFβ is the heterodimeric partner of CBFα proteins, and together they constitute the core binding factors (CBF). CBF was initially identified as a transcriptional regulator of Moloney murine leukemia virus (50, 51) and polyomavirus (4, 18, 36, 37,43) in mice, and it was subsequently shown to be an important transcriptional activator of genes involved in mammalian hematopoiesis and bone development (5, 12, 14, 15, 20, 32, 40, 46). Monomeric CBFα proteins bind DNA, albeit weakly (3, 50). Although CBFβ does not make any detectable direct contact with DNA (50), it enhances the DNA binding affinity of the CBFα proteins (4, 36). While CBFβ is expressed from a single gene in the human and mouse, there are three CBFα genes, all of which encode the so-called runt domain (3, 22,54), which is required for both DNA binding and interaction with CBFβ. One of the three genes, CBFA2, also known as AML1 or PEBP2αB, is located on human chromosome 21 and is involved in several different leukemias as a result of translocations (16, 31, 34, 35, 41). Chromosomal inversions and translocations involving either CBFB orCBFA2 are the most frequent cytogenetic abnormalities in human AMLs (27).

Gene-targeting experiments in mice have demonstrated that the CBFα2 and CBFβ subunits are likely to function together as a complex in vivo. Homozygous disruption of either Cbfa2 (39,48) or Cbfb (42, 49) in mice produces an identical phenotype: both Cbfa2 −/− andCbfb −/− embryos demonstrate a failure of definitive hematopoiesis in the liver, and in both cases the embryos die at around day 12.5 due to extensive hemorrhages.

The inv(16) chimeric gene CBFB-MYH11 has been shown to exert a dominant negative effect in vivo by a mouse knock-in experiment (8). CBFB-MYH11was introduced into the mouse genome to replace one copy of theCbfb gene. The expression of this chimeric gene was controlled by the endogenous Cbfb promoter, thus simulating the condition in leukemic patients. CBFβ-SMMHC was found to dominantly suppress the function of the CBFα2:CBFβ heterodimer, since mouse embryos heterozygous for the knock-inCbfb-MYH11 gene (CbfbCBFB-MYH11/+ ) displayed a phenotype similar to that of Cbfb−/− andCbfa2−/− embryos, i.e., failure of definitive hematopoiesis and midgestation lethality. In vitro, the chimeric protein was shown to retain its ability to interact with CBFα proteins and participate in the formation of protein-DNA complexes (23). Although presence of the chimeric protein reduces CBF DNA-binding activity in cultured Ba/F3 lymphoid and 32D c13 myeloid cells (6), it is not clear how this reduction was achieved. Unlike wild-type CBFβ, CBFβ-SMMHC can potentially form dimers and multimers via the rod-like domain of the myosin chain (23, 25).

Two possible mechanisms could explain the dominant negative effect of the chimeric CBFβ-SMMHC protein. One is that CBFβ-SMMHC, via heterodimerization with CBFα2, can assemble into a ternary complex at the core sites within promoters of target genes and interfere with the regulation of gene expression. The second possibility is that CBFβ-SMMHC, with its capacity to form multimers, can sequester CBFα2 into nonfunctional complexes, thus preventing it from regulating transcription of target genes. Previous studies by our group demonstrated that CBFβ-SMMHC can form rod-like nuclear structures as well as cytoplasmic stress fibers in NIH 3T3 cells stably transfected with a CBFB-MYH11cDNA construct (53). However, the effect on CBFα2 subcellular localization by CBFβ-SMMHC has not been fully examined. In this study, we used transient-transfection assays in combination with immunofluorescence and green fluorescent protein (GFP) tags to demonstrate that CBFβ-SMMHC does, in fact, sequester CBFα2 in abnormal locations. We also demonstrated that the sequestration requires the abilities of CBFβ-SMMHC to interact with CBFα2 and to multimerize. This observed sequestration can at least partially explain the dominant negative effect of the CBFβ-SMMHC protein on CBF function in leukemogenesis.

DOI

10.1128/MCB.18.12.7432

Included in

Microbiology Commons

Share

COinS