AML1-ETO reprograms hematopoietic cell fate by downregulating scl expression

Jing-Ruey J. Yeh Kathleen M. Munson Yvonne L. Chao Quinn P. Peterson Calum A. MacRae Randall T. Peterson AML1-ETO is one of the most common chromosomal translocation products associated with acute myelogenous leukemia (AML). Patients carrying the AML1-ETO fusion gene exhibit an accumulation of granulocyte precursors in the bone marrow and the blood. Here, we describe a transgenic zebrafish line that enables inducible expression of the human AML1-ETO oncogene. Induced AML1ETO expression in embryonic zebrafish causes a phenotype that recapitulates some aspects of human AML. Using this highly tractable model, we show that AML1-ETO redirects myeloerythroid progenitor cells that are developmentally programmed to adopt the erythroid cell fate into the granulocytic cell fate. This fate change is characterized by a loss of gata1 expression and an increase in pu.1 expression in myeloerythroid progenitor cells. Moreover, we identify scl as an early and essential mediator of the effect of AML1-ETO on hematopoietic cell fate. AML1-ETO quickly shuts off scl expression, and restoration of scl expression rescues the effects of AML1-ETO on myeloerythroid progenitor cell fate. These results demonstrate that scl is an important mediator of the ability of AML1-ETO to reprogram hematopoietic cell fate decisions, suggesting that scl may be an important contributor to AML1ETO-associated leukemia. In addition, treatment of AML1-ETO transgenic zebrafish embryos with a histone deacetylase inhibitor, Trichostatin A, restores scl and gata1 expression, and ameliorates the accumulation of granulocytic cells caused by AML1-ETO. Thus, this zebrafish model facilitates in vivo dissection of AML1-ETO-mediated signaling, and will enable large-scale chemical screens to identify suppressors of the in vivo effects of AML1-ETO. INTRODUCTION Acute myelogenous leukemia (AML) is the most common form of leukemia. Each year, more than ten thousand new cases of AML are diagnosed in the United States and approximately a quarter of a million new cases are reported in the world (Stone et al., 2004). The (8;21)(q22;q22) chromosomal translocation can be found in 12-15% of AML patients. This translocation joins the AML1 (also known as CBF 2, RUNX1 and PEBP B) gene and the ETO (also known as MTG8) gene (Downing, 1999; Peterson and Zhang, 2004). A majority of the patients carrying the AML1ETO fusion gene have the M2 subtype of AML, according to the French-American-British classification, which is characterized by overproduction of granulocytic precursors. Granulocytes, like the cells of all blood lineages, are derived from multipotent hematopoietic stems cells (HSCs) through a series of cell fate decisions. It is thought that many leukemic oncogenes, including AML1-ETO, may contribute to the development of AML by affecting the specification and maturation of hematopoietic cells (Tenen, 2003). AML1 by itself plays an important role in hematopoiesis. Normally, AML1 forms a complex with CBF , called the corebinding factor (CBF) complex. This complex binds the enhancer core motif and activates tissue-specific expression of a number of hematopoietic genes (Borregaard et al., 2001; Lutterbach and *Authors for correspondence (e-mails: ; ) Hiebert, 2000). By contrast, ETO normally functions by recruiting the nuclear receptor co-repressor (N-CoR)/mSin3/histone deacetylase (HDAC) complex (Licht, 2001). The chromosomal translocation juxtaposes the region encoding the DNA-binding domain of the AML1 protein to the region encoding almost all of the ETO protein. Thus, the AML1-ETO fusion product is thought to antagonize the functions of AML1. In addition, previous studies indicate that this fusion protein has additional activities other than antagonizing AML1 function. For example, AML1-ETO knock-in mouse embryos contain abnormal hematopoietic progenitor cells that are not found in AML1-deficient mouse embryos (Okuda et al., 1998; Yergeau et al., 1997). Moreover, many of the AML1-ETO target genes that have been identified are not affected by AML1 (Shimada et al., 2000). At present, the full range of AML1-ETO target genes and their roles in AML pathogenesis remain poorly understood. Many lines of evidence indicate that the (8;21) chromosomal translocation is likely to occur in a primitive hematopoietic progenitor cell capable of generating all hematopoietic lineages. For example, transcripts of the AML1-ETO fusion gene can be found in hematopoietic cells of non-myeloid lineages in some patients (Miyamoto et al., 2000). Moreover, it has been demonstrated that the cells capable of initiating leukemia are CD34+CD38, a characteristic of non-committed primitive hematopoietic progenitor cells (Bonnet and Dick, 1997). Thus, it is important to know how AML1-ETO affects the specification of various hematopoietic lineages from multipotent progenitor cells. Unfortunately, AML1ETO knock-in mouse embryos die in early gestation, making it difficult to analyze the effect of AML1-ETO in these models (Okuda et al., 1998; Yergeau et al., 1997). Interestingly, it has been shown that AML1-ETO can promote myelopoiesis in adult mice (de Guzman et al., 2002; Fenske et al., 2004; Schwieger et al., 2002). However, these mouse models manifest phenotypes only after several months of latency. Thus, it is difficult to identify the immediate transcriptional and cytological changes that lead to the observed AML1-ETO effects. During embryonic development of mammals, hematopoiesis starts and continues as two successive waves. The first wave, named primitive hematopoiesis, begins in the blood islands of the yolk sac at embryonic day 7 (E7.0) in the mouse. Although it is generally thought that only erythrocytes and macrophages are produced during primitive hematopoiesis in mammals, multilineage precursors are detected in the later, but still precirculation, yolk sac (Palis et al., 2001). The second wave of hematopoiesis, named definitive hematopoiesis, begins at E8.5 in the aorta-gonad-mesonephros (AGM) region. Definitive hematopoiesis produces HSCs that not only give rise to all blood lineages, but also possess self-renewal capabilities. Studies of zebrafish embryonic development have also identified two waves of hematopoiesis, a primitive wave beginning at 12 hours postfertilization (hpf), and a definitive wave beginning at 24 hpf (Davidson and Zon, 2004). There is some evidence that the myeloerythoid progenitor cells (MPCs) arising from zebrafish primitive hematopoiesis are functionally equivalent to the common myeloid progenitors (CMPs) arising from mammalian definitive hematopoeisis, and many of the pathways governing hematopoietic cell fate decisions may be shared between these cells (Galloway et al., 2005; Rhodes et al., 2005). The MPCs of the primitive wave of hematopoiesis reside in two distinct embryonic locations that appear to influence their ultimate cell fates. MPCs of the rostral blood island (RBI) express the myeloidspecific transcription factor (...truncated)