Paul-Henri Romeo

A neuronal receptor, neuropilin-1, is essential for the initiation of the primary immune response.

The initiation of a primary immune response requires contact between dendritic cells (DCs) and resting T cells. However, little is known about the proteins that mediate this initial contact. We show here that neuropilin-1, a receptor involved in axon guidance, was expressed by human DCs and resting T cells both in vitro and in vivo. The initial contact between DCs and resting T cells led to neuropilin-1 polarization on T cells. DCs and resting T cells specifically bound soluble neuropilin-1, and resting T cells formed clusters with neuropilin-1–transfected COS-7 cells in a neuropilin-1–dependent manner. Functionally, preincubation of DCs or resting T cells with blocking neuropilin-1 antibodies inhibited DC-induced proliferation of resting T cells. These data suggest that neuropilin-1 mediates interactions between DCs and T cells that are essential for initiation of the primary immune response and show parallels between the nervous and immune systems.

GATA and Ets cis-acting sequences mediate megakaryocyte-specific expression.

The human glycoprotein IIB (GPIIB) gene is expressed only in megakaryocytes, and its promoter displays cell type specificity. We show that this specificity involved two cis-acting sequences. The first one, located at -55, contains a GATA binding site. Point mutations that abolish protein binding on this site decrease the activity of the GPIIB promoter but do not affect its tissue specificity. The second one, located at -40, contains an Ets consensus sequence, and we show that Ets-1 or Ets-2 protein can interact with this -40 GPIIB sequence. Point mutations that impair Ets binding decrease the activity of the GPIIB promoter to the same extent as do mutations that abolish GATA binding. A GPIIB 40-bp DNA fragment containing the GATA and Ets binding sites can confer activity to a heterologous promoter in megakaryocytic cells. This activity is independent of the GPIIB DNA fragment orientation, and mutations on each binding site result in decreased activity. Using cotransfection assays, we show that c-Ets-1 and human GATA1 can transactive the GPIIB promoter in HeLa cells and can act additively. Northern (RNA) blot analysis indicates that the ets-1 mRNA level is increased during megakaryocyte-induced differentiation of erythrocytic/megakaryocytic cell lines. Gel retardation assays show that the same GATA-Ets association is found in the human GPIIB enhancer and the rat platelet factor 4 promoter, the other two characterized regulatory regions of megakaryocyte-specific genes. These results indicate that GATA and Ets cis-acting sequences are an important determinant of megakaryocytic specific gene expression.

NOTCH is a key regulator of human T-cell acute leukemia initiating cell activity.

Understanding the pathways that regulate the human T-cell acute lymphoblastic leukemia (T-ALL) initiating cells (T-LiC) activity has been hampered by the lack of biologic assays in which this human disease can be studied. Here we show that coculture of primary human T-ALL with a mouse stromal cell line expressing the NOTCH ligand delta-like-1 (DL1) reproducibly allowed maintenance of T-LiC and long-term growth of blast cells. Human T-ALL mutated or not on the NOTCH receptor required sustained activation of the NOTCH pathway via receptor/ligand interaction for growth and T-LiC activity. On the reverse, inhibition of the NOTCH pathway during primary cultures abolished in vitro cell growth and in vivo T-LiC activity. Altogether, these results demonstrate the major role of the NOTCH pathway activation in human T-ALL development and in the maintenance of leukemia-initiating cells.

A T-cell specific TCR delta DNA binding protein is a member of the human GATA family.

The human GATA1, hGATA1 (previously called NF‐E1, GF‐1 or Eryf‐1), a major sequence‐specific DNA‐binding protein of the erythrocytic lineage, is a member of a zinc‐finger family of DNA‐binding proteins. We report here the cloning of a human cDNA for a new member of this family. This member, called hGATA3, has 85% amino acid homology with hGATA1 in the DNA‐binding domain and no homology elsewhere in the protein. Unlike hGATA1, hGATA3 is not localized on the X chromosome and we map it to the 10p15 band of the human genome. Northern blot analysis indicates that this factor is a T‐cell specific transcription factor, present before activation and up‐regulated during T‐cell activation. The encoded hGATA3 protein, made in an in vitro transcription‐translation assay, binds the WGATAR motif present in the human T‐cell receptor (TCR) delta gene enhancer and, by transfection in HeLa cells, we show that hGATA3 can transactivate this TCR delta gene enhancer. Interestingly this enhancer binds and is also transactivated by hGATA1. Conversely, the promoter of the human glycophorin B (GPB), which is erythroid‐specific and contains two WGATAR motifs, binds and is transactivated by hGATA1 and, to a lesser extent, by hGATA3. These results indicate that the activation of specific genes by hGATA1 or hGATA3 is partly governed by the lineage expression of these two factors during haematopoiesis and that, in the T‐cell lineage, hGATA3 binds the human TCR delta gene enhancer and is involved in its expression.

GATA1 and YY1 are developmental repressors of the human epsilon-globin gene.

The human epsilon‐globin gene is transcribed in erythroid cells only during the embryonic stages of development. Expression of epsilon‐globin gene, however, can be maintained in adult transgenic mice following removal of DNA positioned between ‐467 and ‐182 bp upstream of the epsilon‐globin cap site. We have identified three protein binding regions within this silencer; a CCACC motif around ‐379, two overlapping motifs for YY1 and GATA around ‐269 and a GATA motif around ‐208 and we have analyzed their function during development by studying several mutants in transgenic mice. Mutation of the ‐208 GATA motif allows high epsilon‐globin transgene expression in the adult suggesting that, in addition to its positive effects on transcription, GATA‐1 also plays a negative role in the regulation of globin gene expression during development. Repression of epsilon gene expression in the adult also requires a functional YY1 binding site at position ‐269. Finally, mutation of the ‐379 CCACC site results in a small but detectable level of epsilon expression in adult erythroid cells. Thus, multiple proteins, including GATA‐1, participate in the formation of the epsilon gene repressor complex that may disrupt the interaction between the proximal epsilon‐promoter and the locus control region (LCR) in definitive erythroid cells.

In vivo cellular imaging pinpoints the role of reactive oxygen species in the early steps of adult hematopoietic reconstitution

Few techniques are available to characterize in vivo the early cellular dynamics of long-term reconstitution of hematopoiesis after transplantation of hematopoietic stem cells (HSCs) after lethal irradiation. Using a fiber-optic imaging system, we track the early steps of in vivo recruitment and proliferation of Lin(-)Sca-1(+)c-Kit(+)CD34(-) (LSKCD34(-)) HSCs highly enriched in HSCs and transplanted into lethally irradiated mice. Recruitment of the transplanted LSKCD34(-) hematopoietic cells first occurs in the femoral head and is continuous during 24 hours. Quantification of the fluorescence emitted by the transplanted hematopoietic cells shows that proliferation of LSKCD34(-) hematopoietic cells in the femoral head was potent 3 days after transplantation. Using a development of this fiber-optic imaging system, we show that the transplanted LSKCD34(-) hematopoietic cells are associated with vascularized structures as early as 5 hours after transplantation. This early association is dependent on reactive oxygen species (ROS) partly through the regulation of vascular cell adhesion molecule-1 expression on endothelial cells and is followed by a ROS-dependent proliferation of LSKCD34(-) hematopoietic cells. This new in vivo imaging technique permits the observation of the early steps of hematopoietic reconstitution by HSCs in long bones and shows a new role of ROS in the recruitment of HSCs by bone marrow endothelial cells.

P45 NF-E2 REGULATES EXPRESSION OF THROMBOXANE SYNTHASE IN MEGAKARYOCYTES

Transcription factor p45 NF‐E2 is highly expressed in the erythroid and megakaryocytic lineages. Although p45 recognizes regulatory regions of several erythroid genes, mice deficient for this protein display only mild dyserythropoiesis but have abnormal megakaryocytes and lack circulating platelets. A number of megakaryocytic marker genes have been extensively studied, but none of them is regulated by NF‐E2. To find target genes for p45 NF‐E2 in megakaryopoiesis, we used an in vivo immunoselection assay: genomic fragments bound to p45 NF‐E2 in the chromatin of a megakaryocytic cell line were immunoprecipitated with an anti‐p45 antiserum and cloned. One of these fragments belongs to the second intron of the thromboxane synthase gene (TXS). We demonstrate that the TXS gene, which is mainly expressed in megakaryocytes, is indeed directly regulated by p45 NF‐E2. First, its promoter contains a functional NF‐E2 binding site; second, the intronic NF‐E2 binding site is located within a chromatin‐dependent enhancer element; third, p45‐null murine megakaryocytes do not express detectable TXS mRNA, although TXS expression can be detected in other cells. These data, and the structure of the TXS promoter and enhancer, suggest that TXS belongs to a distinct subgroup of genes involved in platelet formation and function.

Regulation of embryonic/fetal globin genes by nuclear hormone receptors: a novel perspective on hemoglobin switching

The CCAAT box is one of the conserved motifs found in globin promoters. It binds the CP1 protein. We noticed that the CCAAT‐box region of embryonic/fetal, but not adult, globin promoters also contains one or two direct repeats of a short motif analogous to DR‐1 binding sites for non‐steroid nuclear hormone receptors. We show that a complex previously named NF‐E3 binds to these repeats. In transgenic mice, destruction of the CCAAT motif within the human ϵ‐globin promoter leads to substantial reduction in ϵ expression in embryonic erythroid cells, indicating that CP1 activates ϵ expression; in contrast, destruction of the DR‐1 elements yields striking ϵ expression in definitive erythropoiesis, indicating that the NF‐E3 complex acts as a developmental repressor of the ϵ gene. We also show that NF‐E3 is immunologically related to COUP‐TF orphan nuclear receptors. One of these, COUP‐TF II, is expressed in embryonic/fetal erythroid cell lines, murine yolk sac, intra‐embryonic splanchnopleura and fetal liver. In addition, the structure and abundance of NF‐E3/COUP‐TF complexes vary during fetal liver development. These results elucidate the structure as well as the role of NF‐E3 in globin gene expression and provide evidence that nuclear hormone receptors are involved in the control of globin gene switching.

Pathophysiology of sickle cell disease is mirrored by the red blood cell metabolome

Emerging metabolomic tools can now be used to establish metabolic signatures of specialized circulating hematopoietic cells in physiologic or pathologic conditions and in human hematologic diseases. To determine metabolomes of normal and sickle cell erythrocytes, we used an extraction method of erythrocytes metabolites coupled with a liquid chromatography-mass spectrometry-based metabolite profiling method. Comparison of these 2 metabolomes identified major changes in metabolites produced by (1) endogenous glycolysis characterized by accumulation of many glycolytic intermediates; (2) endogenous glutathione and ascorbate metabolisms characterized by accumulation of ascorbate metabolism intermediates, such as diketogulonic acid and decreased levels of both glutathione and glutathione disulfide; (3) membrane turnover, such as carnitine, or membrane transport characteristics, such as amino acids; and (4) exogenous arginine and NO metabolisms, such as spermine, spermidine, or citrulline. Finally, metabolomic analysis of young and old normal red blood cells indicates metabolites whose levels are directly related to sickle cell disease. These results show the relevance of metabolic profiling for the follow-up of sickle cell patients or other red blood cell diseases and pinpoint the importance of metabolomics to further depict the pathophysiology of human hematologic diseases.

Chromatin immunoselection defines a TAL-1 target gene

Despite the major functions of the basic helix–loop–helix transcription factor TAL‐1 in hematopoiesis and T‐cell leukemogenesis, no TAL‐1 target gene has been identified. Using immunoprecipitation of genomic fragments bound to TAL‐1 in the chromatin of murine erythro‐leukemia (MEL) cells, we found that 10% of the immunoselected fragments contained a CAGATG or a CAGGTG E‐box, followed by a GATA site. We studied one of these fragments containing two E‐boxes, CAGATG and CAGGTC, followed by a GATA motif, and showed that TAL‐1 binds to the CAGGTG E‐box with an affinity modulated by the CAGATG or the GATA site, and that the CAGGTG–GATA motif exhibits positive transcriptional activity in MEL but not in HeLa cells. This immunoselected sequence is located within an intron of a new gene co‐expressed with TAL‐1 in endothelial and erythroid cells, but not expressed in fibroblasts or adult liver where no TAL‐1 mRNA was detected. Finally, in vitro differentiation of embryonic stem cells towards the erythro/megakaryocytic pathways showed that the TAL‐1 target gene expression followed TAL‐1 and GATA‐1 expression. These results establish that TAL‐1 is likely to activate its target genes through a complex that binds an E‐box–GATA motif and define the first gene regulated by TAL‐1.