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.

SCL/TAL1 is a hematopoietic-specific transcription factor of the basic helix-loop-helix (bHLH) family that is essential for erythropoiesis. Here we identify the erythroid cell-specific glycophorin A gene (GPA) as a target of SCL in primary hematopoietic cells and show that SCL occupies the GPA locus in vivo. GPA promoter activation is dependent on the assembly of a multifactorial complex containing SCL as well as ubiquitous (E47, Sp1, and Ldb1) and tissue-specific (LMO2 and GATA-1) transcription factors. In addition, our observations suggest functional specialization within this complex, as SCL provides its HLH protein interaction motif, GATA-1 exerts a DNA-tethering function through its binding to a critical GATA element in the GPA promoter, and E47 requires its N-terminal moiety (most likely entailing a transactivation function). Finally, endogenous GPA expression is disrupted in hematopoietic cells through the dominant-inhibitory effect of a truncated form of E47 (E47-bHLH) on E-protein activity or of FOG (Friend of GATA) on GATA activity or when LMO2 or Ldb-1 protein levels are decreased. Together, these observations reveal the functional complementarities of transcription factors within the SCL complex and the essential role of SCL as a nucleation factor within a higher-order complex required to activate gene GPA expression.