Hematopoietic stem cells (HSCs) are thought to arise in the aorta-gonad-mesonephros (AGM) region of embryo proper, although HSC activity can be detected in yolk sac (YS) and paraaortic splanchnopleura (P-Sp) when transplanted in newborn mice. We examined the role of Notch signaling in embryonic hematopoiesis. The activity of colony-forming cells in the YS from Notch1(-/-) embryos was comparable to that of wild-type embryos. However, in vitro and in vivo definitive hematopoietic activities from YS and P-Sp were severely impaired in Notch1(-/-) embryos. The population representing hemogenic endothelial cells, however, did not decrease. In contrast, Notch2(-/-) embryos showed no hematopoietic deficiency. These data indicate that Notch1, but not Notch2, is essential for generating hematopoietic stem cells from endothelial cells.

Vertebrates have greatly elaborated the basic chordate body plan and evolved highly distinctive genomes that have been sculpted by two whole-genome duplications. Here we sequence the genome of the Mediterranean amphioxus ( Branchiostoma lanceolatum ) and characterize DNA methylation, chromatin accessibility, histone modifications and transcriptomes across multiple developmental stages and adult tissues to investigate the evolution of the regulation of the chordate genome. Comparisons with vertebrates identify an intermediate stage in the evolution of differentially methylated enhancers, and a high conservation of gene expression and its cis -regulatory logic between amphioxus and vertebrates that occurs maximally at an earlier mid-embryonic phylotypic period. We analyse regulatory evolution after whole-genome duplications, and find that—in vertebrates—over 80% of broadly expressed gene families with multiple paralogues derived from whole-genome duplications have members that restricted their ancestral expression, and underwent specialization rather than subfunctionalization. Counter-intuitively, paralogues that restricted their expression increased the complexity of their regulatory landscapes. These data pave the way for a better understanding of the regulatory principles that underlie key vertebrate innovations.

When growth hormone binds to its receptor, which belongs to the cytokine receptor superfamily, it activates the Janus kinase Jak2 which has tyrosine-kinase activity and initiates an activation of several key intracellular proteins (for example, mitogen-activated protein (MAP) kinases) that eventually execute the biological actions induced by growth hormone, including the expression of particular genes. In contrast to receptors that themselves have tyrosine kinase activity, the signalling pathways leading to MAP kinase activation that are triggered by growth hormone are poorly understood, but appear to be mediated by the proteins Grb2 and Shc. We now show that growth hormone stimulates tyrosine phosphorylation of the receptor for epidermal growth factor (EGFR) and its association with Grb2 and at the same time stimulates MAP kinase activity in liver, an important target tissue of growth hormone. Expression of EGFR and its mutants revealed that growth-hormone-induced activation of MAP kinase and expression of the transcription factor c-fos requires phosphorylation of tyrosines on EGFR, but not its own intrinsic tyrosine-kinase activity. Moreover, tyrosine at residue 1,068 of the EGFR is proposed to be one of the principal phosphorylation sites and Grb2-binding sites stimulated by growth hormone via Jak2. Our results indicate that the role of EGFR in signalling by growth hormone is to be phosphorylated by Jak2, thereby providing docking sites for Grb2 and activating MAP kinases and gene expression, independently of the intrinsic tyrosine kinase activity of EGFR. This may represent a novel cross-talk pathway between the cytokine receptor superfamily and growth factor receptor.

The Delta/Serrate/LAG-2 (DSL) domain containing proteins are considered to be ligands for Notch receptors. However, the physical interaction between DSL proteins and Notch receptors is poorly understood. In this study, we cloned a cDNA for mouse Jagged1 (mJagged1). To identify the receptor interacting with mJagged1 and to gain insight into its binding characteristics, we established two experimental systems using fusion proteins comprising various extracellular parts of mJagged1, a "cell" binding assay and a "solid-phase" binding assay. mJagged1 physically bound to mouse Notch2 (mNotch2) on the cell surface and to a purified extracellular portion of mNotch2, respectively, in a Ca 2؉ -dependent manner. Scatchard analysis of mJagged1 binding to BaF3 cells and to the soluble Notch2 protein demonstrated dissociation constants of 0.4 and 0.7 nM, respectively, and that the number of mJagged1-binding sites on BaF3 is 5,548 per cell. Furthermore, deletion mutant analyses showed that the DSL domain of mJagged1 is a minimal binding unit and is indispensable for binding to mNotch2. The epidermal growth factor-like repeats of mJagged1 modulate the affinity of the interaction, with the first and second repeats playing a major role. Finally, solid-phase binding assay showed that Jagged1 binds to Notch1 and Notch3 in addition to Notch2, suggesting that mJagged1 is a ligand for multiple Notch receptors.

Evi-1 encodes a zinc-finger protein that may be involved in leukaemic transformation of haematopoietic cells. Evi-1 has two zinc-finger domains, one with seven repeats of a zinc-finger motif and one with three repeats, and it has characteristics of a transcriptional regulator. Although Evi-1 is thought to be able to promote growth and to block differentiation in some cell types, its biological functions are poorly understood. Here we study the mechanisms that underlie oncogenesis induced by Evi-1 by investigating whether Evi-1 perturbs signalling through transforming growth factor-beta (TGF-beta), one of the most studied growth-regulatory factors, which inhibits proliferation of a wide range of cell types. We show that Evi-1 represses TGF-beta signalling and antagonizes the growth-inhibitory effects of TGF-beta. Two separate regions of Evi-1 are responsible for this repression; one of these regions is the first zinc-finger domain. Through this domain, Evi-1 interacts with Smad3, an intracellular mediator of TGF-beta signalling, thereby suppressing the transcriptional activity of Smad3. These results define a new function of Evi-1 as a repressor of signalling through TGF-beta.

Evi-1 encodes a nuclear protein involved in leukemic transformation of hematopoietic cells. Evi-1 possesses two sets of zinc ®nger motifs separated into two domains, and its characteristics as a transcriptional regulator have been described. Here we show that Evi-1 acts as an inhibitor of c-Jun N-terminal kinase (JNK), a class of mitogen-activated protein kinases implicated in stress responses of cells. Evi-1 physically interacts with JNK, although it does not affect its phosphorylation. This interaction is required for inhibition of JNK. Evi-1 protects cells from stressinduced cell death with dependence on the ability to inhibit JNK. These results reveal a novel function of Evi-1, which provides evidence for inhibition of JNK by a nuclear oncogene product. Evi-1 blocks cell death by selectively inhibiting JNK, thereby contributing to oncogenic transformation of cells. Keywords: apoptosis/Evi-1/JNK/zinc ®ngers Introduction Evi-1 was ®rst identi®ed as a gene existing in a common locus of retroviral integration in myeloid tumors in AKXD mice . This gene encodes a 145 kDa nuclear-localized protein, which possesses seven and three repeats of zinc ®nger motifs separated into two clusters (Morishita et al., 1988(Morishita et al., , 1990. The human Evi-1 gene is located on chromosome 3q26, and rearrangements involving this region often activate Evi-1 expression in myeloid leukemia and myelodysplasia (Morishita et al., 1992b;Levy et al., 1994;Ogawa et al., 1996a; Peeters et al., 1997), although its expression is barely detectable in normal bone marrow and peripheral blood. In t(3;21)(q26;q22), found in cases with blastic crisis of chronic myelogenous leukemia, we have reported that Evi-1 is fused to the AML1 gene and is transcriptionally activated as the AML1±Evi-1 chimera . Many lines of evidence suggest a critical role for Evi-1 in t(3;21) leukemogenesis Kurokawa et al., 1995Kurokawa et al., , 1998a. Elevated expression of Evi-1 also occurs without cytogenetically evident translocations in some myeloid malignancies (Russell et al., 1994;Ogawa et al., 1996b). These facts indicate that Evi-1 has a pivotal role in malignant transformation of hematopoietic cells as a dominant oncogene.Thus far, characteristics of Evi-1 as a transcriptional regulator have been described (Lopingco and Perkins, 1996;Bartholomew et al., 1997). We reported that Evi-1 elevates intracellular AP-1 activity and stimulates the c-fos promoter with dependence on the second zinc ®nger domain . With regard to the biological effects of Evi-1, it is known that overexpressed Evi-1 can disturb hematopoietic cell differentiation (Morishita et al., 1992a;Kreider et al., 1993). We have reported that Evi-1 causes cellular transformation when overexpressed in Rat-1 ®broblast cells and that it antagonizes the growth-inhibitory effects of transforming growth factor-b (TGF-b) by inhibiting Smad3 (Kurokawa et al., 1998a,b). Available evidence suggests that Evi-1 potentially possesses abilities for growth promotion and differentiation block in some types of cells...

Three mammalian fringe proteins are implicated in controlling Notch activation by Delta/Serrate/Lag2 ligands during tissue boundary formation. It was proved recently that they are glycosyltransferases that initiate elongation of O-linked fucose residues attached to epidermal growth factor-like sequence repeats in the extracellular domain of Notch molecules. Here we demonstrate the existence of functional diversity among the mammalian fringe proteins. Although both manic fringe (mFng) and lunatic fringe (lFng) decreased the binding of Jagged1 to Notch2 and not that of Delta1, the decrease by mFng was greater in degree than that by lFng. We also found that both fringe proteins reduced Jagged1-triggered Notch2 signaling, whereas neither affected Delta1-triggered Notch2 signaling. However, the decrease in Jagged1-triggered Notch2 signaling by mFng was again greater than that by lFng. Furthermore, we observed that each fringe protein acted on a different site of the extracellular region of Notch2. Taking these findings together, we propose that the difference in modulatory function of multiple fringe proteins may result from the distinct amino acid sequence specificity targeted by these glycosyltransferases.The Notch family of proteins consists of transmembrane receptors that play a critical role in cell fate choices and the formation of compartment border, preventing distinct cell populations from intermixing, during the development of both vertebrates and invertebrates (1-4). In higher vertebrates, four Notch genes, Notch1 through Notch4, have been identified (5-10). An important structural characteristic of these is the common presence of multiple repeats of epidermal growth factor (EGF 1 )-like sequence in the extracellular domain. Ligands for the Notch receptors are designated Delta/Serrate/Lag2 (DSL) proteins (or DSL ligands), five of which have been identified thus far in mammals (Jagged1/Serrate1 (11-13), Jagged2/Serrate2 (14 -16), Delta1 (17), , and Delta4 (19)).Drosophila and mammalian fringe proteins modulate the formation of compartment border in the developing embryo through affecting Notch activation by the ligands (20 -25). Drosophila fringe inhibits a group of cells from responding to the ligand Serrate and potentiates them to respond to another ligand, Delta (20). In higher vertebrates, three fringe proteins, manic fringe (mFng), lunatic fringe (lFng), and radical fringe (rFng) have been identified (22,26). The mammalian fringe proteins modulate Notch signaling when expressed in Drosophila (26), and lFng null mouse phenotypes are similar to those described for mice deficient in components of the Notch signaling pathway (27,28). A weak sequence similarity shared by Drosophila fringe and a class of bacterial glycosyltransferases predicted that fringe proteins might be a glycosyltransferase (29). In a recent work, it was proven experimentally that Drosophila and mammalian fringe proteins have a fucose-specific ␤1,3-N-acetylglucosaminyltransferase activity that catalyzes the elongation of O-linked fucose...

Notch signaling is involved in cell fate decisions in many systems including hematopoiesis. It has been shown that expression of an activated form of Notch1 (aNotch1) in 32D mouse myeloid progenitor cells inhibits the granulocytic differentiation induced by granulocyte colonystimulating factor (G-CSF). Results of the current study show that aNotch1, when expressed in F5-5 mouse erythroleukemia cells, also inhibits erythroid differentiation. Comparison of the expression levels of several transcription factors after stimulation for myeloid and erythroid differentiation, in the presence or absence of aNotch1, revealed that aNotch1 did not change its regulation pattern with any of the transcription factors examined, except for GATA-2, despite its inhibitory effect on differentiation. GATA-2 was down-regulated when the parental 32D and F5-5 were induced to differentiate into granulocytic and erythroid lineages, respectively. In these induction procedures, however, the level of GATA-2 expression was sustained when aNotch1 was expressed. To ascertain whether maintenance of GATA-2 is required for the Notch-induced inhibition of differentiation, the dominant-negative form of GATA-3 (DN-GATA), which acted also against GATA-2, or transcription factor PU.1, which was recently shown to be the repressor of GATA-2, was introduced into