The C-terminus of CBFbeta-SMMHC is required to induce embryonic hematopoietic defects and leukemogenesis

The C-terminus of CBFbeta-SMMHC, the fusion protein produced by a chromosome 16 inversion in acute myeloid leukemia subtype M4Eo, contains domains for self-mulimerization and transcriptional repression, both of which have been proposed to be important for leukemogenesis by CBFbeta-SMMHC. To test the role of the fusion protein's C-terminus in vivo, we generated knock-in mice expressing a C-terminally truncated CBFbeta-SMMHC (CBFbeta-SMMHCdeltaC95). Embryos with a single copy of CBFbeta-SMMHCDC95 were viable and showed no defects in hematopoiesis, while embryos homozygous for the CBFbeta-SMMHCdeltaC95 allele had hematopoietic defects and died in mid-gestation, similar to embryos with a single-copy of the full-length CBFbeta-SMMHCdeltaC95. To identify gene expression changes induced by deltaC95-SMMHCDC95, we compared the gene expression profile in the blood cells of Cbfb+/+, Cbfb+/deltaC95, and deltaC95/deltaC95 embryonic day 12.5 (E12.5) mice. Cbfb+/deltaC95 were mated together to generate Cbfb+/+, Cbfb+/deltaC95, and deltaC95/deltaC95 embryos. Blood from 8-10 E12.5 embryos of the same genotype was pooled, and RNA was isolated, labeled, and hybridized to Affymetrix Genechip mouse microarray (430 2.0) chips. 3 chips were used for each genotype.由急性髓系白血病M4Eo亚型的16号染色体倒位所产生的融合蛋白CBFbeta-SMMHC，其C端包含自身多聚化与转录抑制结构域，二者均被认为对CBFbeta-SMMHC的致白血病功能具有重要作用。为验证该融合蛋白C端的体内功能，我们构建了表达C端截短型CBFbeta-SMMHC（CBFbeta-SMMHCΔC95）的敲入小鼠（knock-in mice）。携带单拷贝CBFbeta-SMMHCΔC95的胚胎可正常存活，造血功能无异常；而携带纯合子CBFbeta-SMMHCΔC95等位基因的胚胎则出现造血缺陷，并于妊娠中期死亡，表型与携带单拷贝全长CBFbeta-SMMHC的胚胎相似。为鉴定CBFbeta-SMMHCΔC95诱导的基因表达变化，我们对Cbfb+/+、Cbfb+/ΔC95及ΔC95/ΔC95基因型胚胎第12.5天（E12.5）小鼠的血细胞进行基因表达谱对比分析。通过将Cbfb+/ΔC95小鼠互交，获得上述三种基因型的胚胎。将同基因型的8~10只E12.5胚胎的血液混合，提取RNA并进行标记，随后与Affymetrix Genechip小鼠微阵列（430 2.0）芯片进行杂交；每种基因型使用3张芯片。