Resolution of pluripotential intermediates in murine hematopoietic differentiation by global complementary DNA amplification from single cells: confirmation of assignments by expression profiling of cytokine receptor transcripts

Billia, Filio; Barbara, Mary; McEwen, Jon; Trevisan, Maryanne; Iscove, Norman N.

doi:10.1182/blood.v97.8.2257

Cited by 52 publications

(39 citation statements)

References 89 publications

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“…The majority (95%) of colony-forming cells were found in the c-Kit ϩ /␤-gal ϩ fraction (data not shown). This population included multipotent (granulocyte, erythrocyte, megakaryocyte, macrophage colony-forming unit [CFU-GEMM]), erythroid (erythroid burstforming unit [BFU-E]), and granulocyte/macrophage (granulocyte/ macrophage CFU [CFU-GM]) precursors (data not shown), shown previously to express c-Kit 29 and SCL. 21,30 Within the LIN Ϫ fraction, c-Kit levels were found, strikingly, to be proportional to the activity of the SCL locus ( Figure 1B), as shown by the mean fluorescence intensities (MFIs) of FDG staining for c-Kit high , c-Kit int , and c-Kit neg populations, which were 9.5, 2.8, and 1.6, respectively.…”

Section: Colinearity Of C-kit Receptor and Scl Levelsmentioning

confidence: 99%

“…30,60,61 These seemingly contradictory roles can be reconciled by considering that SCL and GATA-1 are part of dynamically evolving protein complexes during red-cell differentiation. 62,63 In early progenitors, SCL, GATA-1/GATA-2, and LMO2 are coexpressed 29,30,64 and collaborate to maintain cells in an undifferentiated state, by activating target genes such as c-kit. When the cells receive the proper differentiation signals, induction of modulatory cofactors may inhibit the function of the SCL complex, allowing SCL and GATA-1 to exert other regulatory functions.…”

Section: Diversification Through Protein-protein Interactionmentioning

confidence: 99%

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The SCL complex regulates c-kit expression in hematopoietic cells through functional interaction with Sp1

Lécuyer

Herblot²,

Saint-Denis³

et al. 2002

Blood

153

188

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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...

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Section: Colinearity Of C-kit Receptor and Scl Levelsmentioning

confidence: 99%

Section: Diversification Through Protein-protein Interactionmentioning

confidence: 99%

The SCL complex regulates c-kit expression in hematopoietic cells through functional interaction with Sp1

Lécuyer

Herblot²,

Saint-Denis³

et al. 2002

Blood

153

188

View full text Add to dashboard Cite

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“…cDNA was generated from single hematopoietic precursor and terminally differentiating cells, or mouse tissues and dotblotted onto Hybond nylon membrane (Amersham), as described (Billia et al, 2001). A 329-bp probe from the 3 0 -UTR of mu-MLF1IP was prepared as a PCR fragment from mouse testis cDNA (Clontech), labeled by random-priming with [a-32 P]dCTP, and used for hybridization.…”

Section: Dot Blot Analysismentioning

confidence: 99%

“…cDNA was globally amplified from mouse bone marrow precursor cells sampled homogeneously from defined stages of the differentiation hierarchy (Billia et al, 2001). cDNA dot blots ( Figure 2c) included samples from multipotent in vivo reconstituting stem cells (sample 35), multipotent in vitro colony-forming cells with erythroid and other potentials (samples 1-4), CFU-GM (sample 5), primitive committed erythroid precursors (BFU-E, sample 6) and more advanced erythropoietin-responsive precursors (CFU-E, sample 7) close to the proerythroblast stage (Iscove, 1977).…”

Section: Mlf1ip Expression In Hematopoietic Precursor Cellsmentioning

confidence: 99%

cDNA cloning and characterization of a novel gene encoding the MLF1-interacting protein MLF1IP

et al. 2004

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Myelodysplasia/acute myeloid leukemia (MDS/AML) is characterized by a t(3;5)(q25.1;q34) chromosomal translocation that forms a fusion gene between nucleophosmin (NPM) and MDS/myeloid leukemia factor 1 (MLF1). We identified a novel protein, MLF1-interacting protein (MLF1IP), that specifically associates with MLF1 by yeast two-hybrid analysis and in pulldown assays, and colocalizes with it in both the nuclei and cytoplasm of cells. The MLF1IP gene locus is at chromosome 4q35.1 and is composed of 14 exons spanning 75.8 kb of genomic DNA. The MLF1IP cDNA encodes a 46-kDa protein that contains two bipartite and two classical nuclear localization signals, two nuclear receptor-binding motifs (LXXLL), two leucine zippers, two PEST residues and several potential phosphorylation sites. MLF1IP transcripts are expressed in a variety of tissues (e.g. fetal liver, bone marrow, thymus and testis). MLF1IP appears to be a lineage-specific gene whose expression is confined exclusively to the CFU-E erythroid precursor cells, but not in mature erythrocytes. These observations, together with previous data demonstrating a role for MLF1 in suppressing red cell maturation, suggest a possible role for MLF1IP and MLF1 deregulation in the genesis of erythroleukemias.

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“…This approach revealed that multipotent progenitors express myeloid receptors such as c-fms, the G-CSF receptor and the alpha chain of the GM-CSF receptor, normally expressed in committed granulocyte and/or macrophage precursors (Billia et al, 2001). In parallel, a third line of investigation monitored gene expression in populations of HSCs and later progenitors that are purified by flow cytometry.…”

Section: A Preview Of Things To Comementioning

confidence: 99%

The origin of hematopoietic cell type diversity

Hoang

2004

Oncogene

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Resolution of pluripotential intermediates in murine hematopoietic differentiation by global complementary DNA amplification from single cells: confirmation of assignments by expression profiling of cytokine receptor transcripts

Cited by 52 publications

References 89 publications

The SCL complex regulates c-kit expression in hematopoietic cells through functional interaction with Sp1

The SCL complex regulates c-kit expression in hematopoietic cells through functional interaction with Sp1

cDNA cloning and characterization of a novel gene encoding the MLF1-interacting protein MLF1IP

The origin of hematopoietic cell type diversity

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