Most human acute myeloid leukaemia (AML) cells have limited proliferative capacity, suggesting that the leukaemic clone may be maintained by a rare population of stem cells. This putative leukaemic stem cell has not been characterized because the available in vitro assays can only detect progenitors with limited proliferative and replating potential. We have now identified an AML-initiating cell by transplantation into severe combined immune-deficient (SCID) mice. These cells homed to the bone marrow and proliferated extensively in response to in vivo cytokine treatment, resulting in a pattern of dissemination and leukaemic cell morphology similar to that seen in the original patients. Limiting dilution analysis showed that the frequency of these leukaemia-initiating cells in the peripheral blood of AML patients was one engraftment unit in 250,000 cells. We fractionated AML cells on the basis of cell-surface-marker expression and found that the leukaemia-initiating cells that could engraft SCID mice to produce large numbers of colony-forming progenitors were CD34+ CD38-; however, the CD34+ CD38+ and CD34- fractions contained no cells with these properties. This in vivo model replicates many aspects of human AML and defines a new leukaemia-initiating cell which is less mature than colony-forming cells.

Cooperation between the stem cell leukemia (SCL) transcription factor and its nuclear partners LMO1 or LMO2 induces aggressive T cell acute lymphoblastic leukemia when inappropriately expressed in T cells. This study examined the cellular and molecular targets of the SCL-LMO complex at the preleukemic stage. We show that SCL and its partners are coexpressed in the most primitive thymocytes. Maturation to the pre-T cell stage is associated with a down-regulation of SCL and LMO1 and LMO2, and a concomitant up-regulation of E2A and HEB expression. Moreover, enforced expression of SCL-LMO1 inhibits T cell differentiation and recapitulates a loss of HEB function, causing a deregulation of the transition checkpoint from the CD4-CD8- to CD4+CD8+ stages. Finally, we identify the gene encoding pT alpha as a downstream target of HEB that is specifically repressed by the SCL-LMO complex.

The combinatorial interaction among transcription factors is believed to determine hematopoietic cell fate. Stem cell leukemia (SCL, also known as TAL1 [T-cell acute lymphoblastic leukemia 1]) is a tissue-specific basic helix-loop-helix (bHLH) factor that plays a central function in hematopoietic development; however, its target genes and molecular mode of action remain to be elucidated. Here we show that SCL and the c-Kit receptor are coexpressed in hematopoietic progenitors at the single-cell level and that SCL induces c-kit in chromatin, as ectopic SCL expression in transgenic mice sustains c-kit transcription in developing B lymphocytes, in which both genes are normally down-regulated. Through transient transfection assays and coimmunoprecipitation of endogenous proteins, we define the role of SCL as a nucleation factor for a multifactorial complex (SCL complex) that specifically enhances c-kit promoter activity without affecting the activity of myelomonocytic promoters. This complex, containing hematopoieticspecific (SCL, Lim-only 2 (LMO2), GATA-1/ GATA-2) and ubiquitous (E2A, LIMdomain binding protein 1 [Ldb-1]) factors, is tethered to DNA via a specificity protein 1 (Sp1) motif, through direct interactions between elements of the SCL complex and the Sp1 zinc finger protein. Furthermore, we demonstrate by chromatin immunoprecipitation that SCL, E2A, and Sp1 specifically co-occupy the c-kit promoter in vivo. We therefore conclude that c-kit is a direct target of the SCL complex. Proper activation of the c-kit promoter depends on the combinatorial interaction of all members of the complex. Since SCL is down-regulated in maturing cells while its partners remain expressed, our observations suggest that loss of SCL inactivates the SCL complex, which may be an important event in the differentiation of pluripotent hematopoietic cells. Tissue-restricted members can regulate gene expression by binding to E-box DNA sequences (CANNTG) following heterodimerization with ubiquitously expressed E2A gene products (E12 and E47) or HEB. Different dimers exhibit preferential binding to specific E-boxes, and this selectivity is thought to be an important determinant in the spatio-temporal control of gene expression. SCL is a prototypic tissue-specific bHLH factor normally expressed in pluripotent hematopoietic precursors, vascular endothelial cells, and the central nervous system (reviewed in Begley and Green 2 ) and acts as a master regulator of hematopoietic development. Indeed, SCL Ϫ/Ϫ mice lack all primitive and definitive hematopoietic lineages and precursors. 3,4 Complementary-gain-of-function experiments in zebra fish and Xenopus have demonstrated that SCL plays a role in specifying the formation of hemangioblasts, the common precursors of vascular endothelial and hematopoietic stem cells. [5][6][7] As for many hematopoietic transcription regulators, the SCL gene was originally identified by virtue of its involvement in a tumor-specific translocation. 2 In fact, chromosomal rearrangements causing aberrant activation of...

The passage from proliferation to terminal differentiation is critical for normal development and is often perturbed in malignancies. To define the molecular mechanisms that govern this process during erythropoiesis, we have used tagging/proteomics approaches and characterized protein complexes nucleated by TAL-1/SCL, a basic helix-loophelix transcription factor that specifies the erythrocytic lineage. In addition to known TAL-1 partners, GATA-1, E2A, HEB, LMO2 and Ldb1, we identify the ETO2 repressor as a novel component recruited to TAL-1 complexes through interaction with E2A/HEB. Ectopic expression and siRNA knockdown experiments in hematopoietic progenitor cells show that ETO2 actively represses erythroid TAL-1 target genes and governs the expansion of erythroid progenitors. At the onset of erythroid differentiation, a change in the stoichiometry of ETO2 within the TAL-1 complex activates the expression of known erythroidspecific TAL-1 target genes and of Gfi-1b and p21 Cip , encoding two essential regulators of erythroid cell proliferation. These results suggest that the dynamics of ETO2 recruitment within nuclear complexes couple cell proliferation to cell differentiation and determine the onset of terminal erythroid maturation.