An In Vivo role of Runx1 Subnuclear Targeting In Regulating the Gene Expression Program of Definitive Hematopoiesis

Abstract 3151

Runx1, a master regulator of hematopoiesis, is critical for the emergence of the hematopoietic stem cell (HSC), as observed by gene ablation or replacement studies where either the entire gene is disrupted or replaced with specific mutations that abolish DNA binding activity. Like many phenotypic transcription factors, endogenous Runx1 localizes to distinct foci within the nucleus through a unique subnuclear targeting signal. Clinically relevant is the observation that many Runx1 translocations that are associated with acute myeloid leukemias (e.g., AML1/ETO) retain Runx1 DNA binding activity but exhibit modified localization due to the loss of subnuclear targeting signal and transactivation domains. We hypothesized that altered subnuclear routing of translocation-encoded leukemic proteins plays a key role and provides a common mechanism in the onset and progression of human leukemias. We created a knock-in mouse with a C-terminal truncation by introducing a single nucleic acid substitution (Runx1 Q307X) in the native Runx1 locus. This mutation models genetic lesions observed in patients with leukemia and myeloproliferative disorders. The Runx1 Q307X homozygous mouse exhibited mid-gestation lethality at 12.5dpc due to central nervous system hemorrhage and a complete lack of HSC function. Importantly, Runx1 Q307X, which retains DNA binding activity, failed to activate target genes, resulting in deregulation of various hematopoietic markers. This phenocopy of the complete Runx1 ablation models highlights the critical importance of subnuclear targeting and transactivation activity for Runx1 function during development. We next examined specific contributions of subnuclear targeting in vivo to this phenotype by creating another knock-in mouse model replacing endogenous Runx1 with a mutant (Runx1 HTY350-352AAA). This mutation specifically abrogates subnuclear localization of the protein without altering other known functions. Embryos homozygous for Runx1 HTY350-352AAA bypass the mid-gestation lethality observed with the other Runx1 mutants and survive till birth. Thus, the precise subnuclear targeting of Runx1 does not appear to be essential for emergence of the HSC and the start of definitive hematopoiesis. However, expression of Runx1 target genes that mediate hematopoiesis is modified in mice with this mutant. These findings together suggest a development independent role for Runx1 subnuclear targeting in controlling the hematopoietic gene program that is often modified in human leukemia.