1: Mol Cell Biol. 2004 Feb;24(4):1439-52. SCL assembles a multifactorial complex that determines glycophorin A expression. Lahlil R, Lecuyer E, Herblot S, Hoang T. Clinical Research Institute of Montreal, Montreal, Quebec H2W 1R7, Canada. 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. PMID: 14749362 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 2: Blood. 2002 Oct 1;100(7):2430-40. The SCL complex regulates c-kit expression in hematopoietic cells through functional interaction with Sp1. Lecuyer E, Herblot S, Saint-Denis M, Martin R, Begley CG, Porcher C, Orkin SH, Hoang T. Clinical Research Institute of Montreal and from the Departments of Pharmacology, Biochemistry, and Molecular Biology, Universite de Montreal, Quebec, Canada. 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 hematopoietic-specific (SCL, Lim-only 2 (LMO2), GATA-1/GATA-2) and ubiquitous (E2A, LIM- domain 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. PMID: 12239153 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 3: J Biol Chem. 1998 Oct 30;273(44):29032-42. Transcriptional regulation of the stem cell leukemia gene by PU.1 and Elf-1. Bockamp EO, Fordham JL, Gottgens B, Murrell AM, Sanchez MJ, Green AR. University of Cambridge, Department of Haematology, Medical Research Council Centre, Hills Road, Cambridge CB2 2QH, United Kingdom. The SCL gene, also known as tal-1, encodes a basic helix-loop-helix transcription factor that is pivotal for the normal development of all hematopoietic lineages. SCL is expressed in committed erythroid, mast, and megakaryocytic cells as well as in hematopoietic stem cells. Nothing is known about the regulation of SCL transcription in mast cells, and in other lineages GATA-1 is the only tissue-specific transcription factor recognized to regulate the SCL gene. We have therefore analyzed the molecular mechanisms underlying SCL expression in mast cells. In this paper, we demonstrate that SCL promoter 1a was regulated by GATA-1 together with Sp1 and Sp3 in a manner similar to the situation in erythroid cells. However, SCL promoter 1b was strongly active in mast cells, in marked contrast to the situation in erythroid cells. Full activity of promoter 1b was dependent on ETS and Sp1/3 motifs. Transcription factors PU.1, Elf-1, Sp1, and Sp3 were all present in mast cell extracts, bound to promoter 1b and transactivated promoter 1b reporter constructs. These data provide the first evidence that the SCL gene is a direct target for PU.1, Elf-1, and Sp3. PMID: 9786909 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 4: Blood. 1995 Aug 15;86(4):1502-14. Lineage-restricted regulation of the murine SCL/TAL-1 promoter. Bockamp EO, McLaughlin F, Murrell AM, Gottgens B, Robb L, Begley CG, Green AR. University of Cambridge, Department of Haematology, MRC Centre, UK. The SCL/TAL-1 gene encodes a basic helix-loop-helix transcription factor that is expressed in multipotent hematopoietic progenitors before lineage commitment. Its expression is maintained during differentiation along erythroid, mast, and megakaryocytic lineages, but is repressed after commitment to nonexpressing lineages. To begin to address the molecular mechanisms underlying this complex pattern of expression, we have studied the regulation of the murine SCL promoter in erythroid and T-cell lines. Analysis of the methylation and chromatin structure of the SCL promoter region showed that SCL mRNA expression correlated with DNase hypersensitive sites and methylation status of the promoter. Transient reporter assays showed that promoter 1a was active in erythroid cells but not in T cells. Sequences between -187 and +26 were sufficient for lineage-restricted activity of promoter 1a. A joint promoter construct containing both promoter 1a and promoter 1b also exhibited lineage-restricted activity. Conserved GATA (-37), MAZ (+242), and ETS (+264) motifs were all shown to contribute to SCL promoter activity in erythroid cells, but several other motifs were not required for full promoter activity. The pattern of complexes binding to the +242 MAZ and +264 ETS sites were the same in erythroid and T cells. However, GATA-1 bound the -37 GATA site in erythroid cells, whereas in T cells GATA-3 was only able to bind weakly, if at all. Moreover, GATA-1 but not GATA-2 or GATA-3 was able to transactivate SCL promoter 1a in a T-cell environment. These results suggest that inactivity of SCL promoter 1a in T cells reflected the absence of GATA-1 rather than the presence of trans-dominant negative regulators. PMID: 7632958 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 5: Oncogene. 1994 Sep;9(9):2623-32. GATA-and SP1-binding sites are required for the full activity of the tissue-specific promoter of the tal-1 gene. Lecointe N, Bernard O, Naert K, Joulin V, Larsen CJ, Romeo PH, Mathieu-Mahul D. CNRS UMR 9942, Institut de Genetique Moleculaire de Montpellier, France. The tal-1 gene, which is frequently activated in human T cell acute leukemias (T-ALLs), codes for a protein of the basic helix-loop-helix family (b-HLH) and potentially a transcription factor. In human and murine hematopoiesis tal-1 is expressed during the differentiation of the erythroid, megakaryocytic and mastocytic cell lineages. The expression of tal-1 appears to be comodulated with that of the transcription factor GATA-1 gene, suggesting that the GATA-1 protein may regulate the tal-1 gene activity in these hematopoietic lineages. To get further insights into the molecular mechanisms that control tal-1 expression, we have isolated 5' sequences of the murine gene and compared them to their human counterparts. The 5' flanking sequences from the two genes show several regions of high homology. The alignment of both sequences enabled us to predict that similarly, to the human, the mouse gene contains two alternative first exons (Ia and Ib). Remarkably, in both species, the proximal region of the tissue-specific exon Ia (i.e. gene segment -122 to +1) contains two GATA-motifs (at -65 and -33) and one SP-1 consensus binding site (-59). Mobility shift assays demonstrate that GATA proteins are able to interact with both GATA-motifs in a sequence specific fashion, but with different efficiencies. Moreover transfection studies show that the GATA-1 protein directly mediates tal-1 transcription by interacting with the -122/+1 fragment, defined as a minimal promoter in erythroid cells. Mutagenesis of the promoter establishes that the -33 GATA-binding site present in this fragment is critical for tal-1 expression in erythroid cells, but by itself does not lead to full promoter activity. Indeed, further mutations show that the second -65 GATA-binding site and the binding motif for SP1 (-59) significantly contribute to the overall activity of the proximal tal-1 promoter. Altogether, our data provide evidence that GATA-1 cooperates with the transcription factor SP1 to mediate the erythroid-specific expression of the tal-1 gene. PMID: 8058326 [PubMed - indexed for MEDLINE] ---------------------------------------------------------------