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

Abstract

AML1 is one of the most frequently mutated genes associated with human acute leukemia and encodes the DNA-binding subunit of the heterodimering transcriptional factor complex, core-binding factor (CBF) (or polyoma enhancer binding protein 2 [PEBP2]). A null mutation in either AML1 or its dimerizing partner, CBFβ, results in 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 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 PEBP2αB1 (AML1b) isoform under the endogenousAML1-regulatory sequences through a knock-in (targeted insertion) approach. Moreover, we demonstrate that the rescuedAML1^−/− ES cell clones contribute to lymphohematopoiesis within the context of chimeric animals. Rescue requires the transcription activation domain of AML1 but does not require the C-terminal VWRPY motif, which is conserved in all AML1 family members and has been shown to interact with the transcriptional corepressor, Groucho/transducin-like Enhancer of split. Taken together, these data provide compelling evidence that the phenotype seen in AML1-deficient mice is due solely to the loss of transcriptionally active AML1.

ACKNOWLEDGMENTS

We thank Shuji Takao, Katsuya Ashida, Yoshio Yanase, and Nobuhisa Onoda for excellent technical assistance. We are also grateful to Tomoko Asada for secretarial assistance in preparing figures.

This work was supported by grants-in-aid from the Ministry of Education, Science, Sports, and Culture, Japan; by grants-in-aid from the Ministry of Health and Welfare, Japan; by The Kowa Life Science Foundation; by The Shimizu Foundation for the Promotion of Immunology Research; by The Naito Foundation; by grants from CREST of Japan Science and Technology Corporation; by National Institute of Health (NIH) grant P01 CA71907-03; by NIH Cancer Center CORE grant CA-21765; and by the American Lebanese Syrian Associated Charities (ALSAC), St. Jude Children's Research Hospital.