Biological characteristics of the leukemia-associated transcriptional factor AML1 disclosed by hematopoietic rescue of AML1-deficient embryonic stem cells by using a knock-in strategy

AML1 is one of the most frequently mutated genes associated with human acut e leukemia and encodes the DNA-binding subunit of the heterodimering transc riptional factor complex, core-binding factor (CBF) (or polyoma enhancer bi nding protein 2 [PEBP2]). A null mutation in either AML1 or its dimerizing partner, CBF beta, results in embryonic lethality secondary to a complete b lock in fetal liver hematopoiesis, indicating an essential role of this tra nscription complex in the development of definitive hematopoiesis. The hema topoietic phenotype that results from the loss of AML1 can be replicated in vitro with a two-step culture system of murine embryonic stem (ES) cells. Using this experimental system, we now demonstrate that this hematopoietic defect can be rescued by expressing the PEBP2 alpha B1 (AML1b) isoform unde r the endogenous AML1-regulatory sequences through a knock-in (targeted ins ertion) approach. Moreover, we demonstrate that the rescued AML1(-/-) ES ce ll clones contribute to lymphohematopoiesis within the context of chimeric animals. Rescue requires the transcription activation domain of AML1 but do es not require the C-terminal VWRPY motif, which is conserved in all AML1 f amily members and has been shown to interact with the transcriptional corep ressor, Groucho/transducin-like Enhancer of split. Taken together, these da ta provide compelling evidence that the phenotype seen in AML1-deficient mi ce is due solely to the loss of transcriptionally active AML1.