Biologic characterization of leukemia-associated transcription factor AML1 (PEBP2αB) in hematopoiesis

AML1 （PEBP2α b）是与人类急性白血病相关的最常见突变基因之一，它编码异源二聚体转录因子复合物的dna结合亚基，核心结合因子：CBF（或多瘤增强子结合蛋白2:PEBP2）。AML1或其二聚体伙伴CBFβ (PEBP2β)的破坏都会导致胎儿肝脏造血完全阻断后的胚胎致死性，这表明该转录复合物在最终造血发育中起重要作用。AML1缺失导致的造血表型可以通过小鼠胚胎干细胞的两步培养系统在体外复制。利用这个实验系统，我们证明了通过敲入（靶向插入）方法，在内源性aml1调控序列下表达PEBP2αB1 （AML1b）亚型可以挽救这种造血缺陷。此外，我们发现这些获救的AML1-/- es细胞可以在嵌合动物的背景下促进淋巴造血。这些结果提供了令人信服的证据，证明AML1敲除表型仅仅是由于缺乏该基因，并且PEBP2αB1 （AML1b）亚型具有生物活性。有趣的是，当AML1b在异源启动子的控制下表达时，没有观察到造血拯救，这表明AML1的转录控制对其生物活性很重要。通过该实验，我们还证明AML1b的反活化结构域是其生物活性所必需的，而位于羧基末端的保守的vwrpy基序则不是。通过本研究建立的胚胎干细胞实验系统可以作为进一步确定AML1在正常和白血病造血中的生物学特性的独特工具。

AML1 (PEBP2αB) is one of the most frequently mutated genes associated with human acute leukemia, and it encodes the DNA-binding subunit of the heterodimering transcriptional factor complex, core binding factor : CBF (or polyoma enhancer binding protein 2 : PEBP2). Disruption of either AML1 or its dimerizing partner, CBFβ (PEBP2β), results in the embryonic-lethality secondary to a complete block in fetal liver hematopoiesis, indicating an essential role of this transcription complex in the development of definitive hematopoiesis. The hematopoietic phenotype that results from the loss of AML1 could be replicated in vitro using a two-step culture system of murine embryonic stem (ES) cells. Using this experimental system, we demonstrated that this hematopoietic defect could be rescued by expressing the PEBP2αB1 (AML1b) isoform under the endogenous AML1-regulatory sequences through a knock-in (targeted-insertion) approach. Moreover, we found that these rescued AML1-/-ES cells could contribute to lympho-hematopoiesis within the context of chimeric animals. These results provide compelling evidence that the AML1 knockout phenotype is due solely to the lack of this gene, and that the PEBP2αB1 (AML1b) isoform is biologically active. Interestingly, hematopoietic rescue was not observed when AML1b was expressed under the control of a heterologous promoter, suggesting that the transcriptional control of AML1 is important for its biological activity. Using this assay, we also demonstrated that the transactivation domain of AML1b is required for its biological activity, whereas the conserved VWRPY-motif at the carboxyl terminus is not. The ES cell experimental system developed through the present study should serve as a unique tool to further define the biological properties of AML1 in normal and leukemic hematopoiesis.