Adya N, Stacy T, Speck N A, Liu P P
Oncogenesis and Development Section, Genetics and Molecular Biology Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, Maryland 20892, USA.
Mol Cell Biol. 1998 Dec;18(12):7432-43. doi: 10.1128/MCB.18.12.7432.
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 beta (CBFbeta) and the smooth-muscle myosin heavy chain (SMMHC). Mouse model studies suggest that this chimeric protein CBFbeta-SMMHC dominantly suppresses the function of CBF, a heterodimeric transcription factor composed of DNA binding subunits (CBFalpha1 to 3) and a non-DNA binding subunit (CBFbeta). 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 CBFbeta-SMMHC, CBFbeta, and CBFalpha2 (also known as AML1 or PEBP2alphaB). When expressed individually, CBFalpha2 was located in the nuclei of transfected cells, whereas CBFbeta was distributed throughout the cell. On the other hand, CBFbeta-SMMHC formed filament-like structures that colocalized with actin filaments. Upon cotransfection, CBFalpha2 was able to drive localization of CBFbeta into the nucleus in a dose-dependent manner. In contrast, CBFalpha2 colocalized with CBFbeta-SMMHC along the filaments instead of localizing to the nucleus. Deletion of the CBFalpha-interacting domain within CBFbeta-SMMHC abolished this CBFalpha2 sequestration, whereas truncation of the C-terminal-end SMMHC domain led to nuclear localization of CBFbeta-SMMHC when coexpressed with CBFalpha2. CBFalpha2 sequestration by CBFbeta-SMMHC was further confirmed in vivo in a knock-in mouse model. These observations suggest that CBFbeta-SMMHC plays a dominant negative role by sequestering CBFalpha2 into cytoskeletal filaments and aggregates, thereby disrupting CBFalpha2-mediated regulation of gene expression.
融合基因CBFB-MYH11由与急性髓系白血病相关的16号染色体倒位产生。该基因编码一种嵌合蛋白，涉及核心结合因子β（CBFβ）和平滑肌肌球蛋白重链（SMMHC）。小鼠模型研究表明，这种嵌合蛋白CBFβ-SMMHC主要抑制CBF的功能，CBF是一种由DNA结合亚基（CBFα1至3）和非DNA结合亚基（CBFβ）组成的异二聚体转录因子。这种显性抑制导致小鼠造血受阻，可能促进白血病发生。我们使用瞬时转染实验，结合免疫荧光和绿色荧光蛋白标记蛋白，来监测CBFβ-SMMHC、CBFβ和CBFα2（也称为AML1或PEBP2αB）的亚细胞定位。单独表达时，CBFα2位于转染细胞的细胞核中，而CBFβ分布于整个细胞。另一方面，CBFβ-SMMHC形成与肌动蛋白丝共定位的丝状结构。共转染时，CBFα2能够以剂量依赖的方式驱动CBFβ进入细胞核。相反，CBFα2与CBFβ-SMMHC沿细丝共定位，而不是定位于细胞核。删除CBFβ-SMMHC内的CBFα相互作用结构域消除了这种CBFα2隔离，而截断C末端SMMHC结构域导致与CBFα2共表达时CBFβ-SMMHC定位于细胞核。在敲入小鼠模型中进一步在体内证实了CBFβ-SMMHC对CBFα2的隔离。这些观察结果表明，CBFβ-SMMHC通过将CBFα2隔离到细胞骨架细丝和聚集体中发挥显性负性作用，从而破坏CBFα2介导的基因表达调控。
