Pontin is a critical regulator for AML1-ETO-induced leukemia.

The oncogenic fusion protein AML1-ETO, also known as RUNX1-RUNX1T1 is generated by the t(8;21)(q22;q22) translocation, one of the most frequent chromosomal rearrangements in acute myeloid leukemia (AML). Identifying the genes that cooperate with or are required for the oncogenic activity of this chimeric transcription factor remains a major challenge. Our previous studies showed that Drosophila provides a genuine model to study how AML1-ETO promotes leukemia. Here, using an in vivo RNA interference screen for suppressors of AML1-ETO activity, we identified pontin/RUVBL1 as a gene required for AML1-ETO-induced lethality and blood cell proliferation in Drosophila. We further show that PONTIN inhibition strongly impaired the growth of human t(8;21)(+) or AML1-ETO-expressing leukemic blood cells. Interestingly, AML1-ETO promoted the transcription of PONTIN. Moreover, transcriptome analysis in Kasumi-1 cells revealed a strong correlation between PONTIN and AML1-ETO gene signatures and demonstrated that PONTIN chiefly regulated the expression of genes implicated in cell cycle progression. Concordantly, PONTIN depletion inhibited leukemic self-renewal and caused cell cycle arrest. All together our data suggest that the upregulation of PONTIN by AML1-ETO participate in the oncogenic growth of t(8;21) cells.

New members of the Drosophila Myc transcription factor subfamily revealed by a genome-wide examination for basic helix-loop-helix genes.

The basic helix-loop-helix (bHLH) proteins represent an evolutionary conserved class of transcription factors that are known to play important roles in cell determination and differentiation during animal embryonic development. Following an exhaustive search of the complete Drosophila genome sequence using a PSI-BLAST strategy, we identified 19 new genes, bringing the total number of bHLH-encoding genes in the Drosophila genome to 56. These new genes belong to various subfamilies of bHLH transcription factors, such as the Daughterless, Hairy-Enhancer of split, bHLH-PAS or bHLHZip subfamilies. The embryonic expression pattern of each of these new genes has been analyzed by in situ hybridization. By looking for closely structurally related motifs, we found two genes that represent likely orthologues of vertebrate Mnt and Mlx. Together with previous reports, our data suggest that, similar to networks involved in neurogenesis and myogenesis, the network of Myc-related genes has been globally conserved throughout evolution.

Two different activities of Suppressor of Hairless during wing development in Drosophila.

The Notch pathway plays a crucial and universal role in the assignation of cell fates during development. In Drosophila, Notch is a transmembrane protein that acts as a receptor of two ligands Serrate and delta. The current model of Notch signal transduction proposes that Notch is activated upon binding its ligands and that this leads to the cleavage and release of its intracellular domain (also called Nintra). Nintra translocates to the nucleus where it forms a dimeric transcription activator with the Su(H) protein. In contrast with this activation model, experiments with the vertebrate homologue of Su(H), CBF1, suggest that, in vertebrates, Nintra converts CBF1 from a repressor into an activator. Here we have assessed the role of Su(H) in Notch signalling during the development of the wing of Drosophila. Our results show that, during this process, Su(H) can activate the expression of some Notch target genes and that it can do so without the activation of the Notch pathway or the presence of Nintra. In contrast, the activation of other Notch target genes requires both Su(H) and Nintra, and, in the absence of Nintra, Su(H) acts as a repressor. We also find that the Hairless protein interacts with Notch signalling during wing development and inhibits the activity of Su(H). Our results suggest that, in Drosophila, the activation of Su(H) by Notch involve the release of Su(H) from an inhibitory complex, which contains the Hairless protein. After its release Su(H) can activate gene expression in absence of Nintra.

u-shaped encodes a zinc finger protein that regulates the proneural genes achaete and scute during the formation of bristles in Drosophila.

The pattern of the large sensory bristles on the notum of Drosophila arises as a consequence of the expression of the achaete and scute genes. The gene u-shaped encodes a novel zinc finger that acts as a transregulator of achaete and scute in the dorsal region of the notum. Viable hypomorphic u-shaped mutants display additional dorsocentral and scutellar bristles that result from overexpression of achaete and scute. In contrast, overexpression of u-shaped causes a loss of achaete-scute expression and consequently a loss of dorsal bristles. The effects on the dorsocentral bristles appear to be mediated through the enhancer sequences that regulate achaete and scute at this site. The effects of u-shaped mutants are similar to those of a class of dominant alleles of the gene pannier with which they display allele-specific interactions, suggesting that the products of both genes cooperate in the regulation of achaete and scute. A study of the sites at which the dorsocentral bristles arise in mosaic u-shaped nota, suggests that the levels of the u-shaped protein are crucial for the precise positioning of the precursors of these bristles.

Transcriptional activity of pannier is regulated negatively by heterodimerization of the GATA DNA-binding domain with a cofactor encoded by the u-shaped gene of Drosophila.

The genes pannier (pnr) and u-shaped (ush) are required for the regulation of achaete-scute during establishment of the bristle pattern in Drosophila. pnr encodes a protein belonging to the GATA family of transcription factors, whereas ush encodes a novel zinc finger protein. Genetic interactions between dominant pnr mutants bearing lesions situated in the amino-terminal zinc finger of the GATA domain and ush mutants have been described. We show here that both wild-type Pannier and the dominant mutant form activate transcription from the heterologous alpha globin promoter when transfected into chicken embryonic fibroblasts. Furthermore, Pnr and Ush are found to heterodimerize through the amino-terminal zinc finger of Pnr and when associated with Ush, the transcriptional activity of Pnr is lost. In contrast, the mutant pnr protein with lesions in this finger associates only poorly with Ush and activates transcription even when cotransfected with Ush. These interactions have been investigated in vivo by overexpression of the mutant and wild-type proteins. The results suggest an antagonistic effect of Ush on Pnr function and reveal a new mode of regulation of GATA factors during development.

Transcriptional regulation of Notch and Delta: requirement for neuroblast segregation in Drosophila.

Segregation of a single neural precursor from each proneural cluster in Drosophila relies on Notch-mediated lateral signalling. Studies concerning the spacing of precursors for the microchaetes of the peripheral nervous system suggested the existence of a regulatory loop between Notch and its ligand Delta within each cell that is under transcriptional control. Activation of Notch leads to repression of the achaete-scute genes which themselves regulate transcription of Delta, perhaps directly. Here we have tested a requirement for transcriptional regulation of Notch and/or Delta during neuroblast segregation in embryos, by providing Notch and Delta ubiquitously at uniform levels. Neuroblast segregation occurs normally under conditions of uniform Notch expression. Under conditions of uniform Delta expression, a single neuroblast segregates from each proneural group in 80% of the cases, more than one in the remaining 20%. Thus transcriptional regulation of Delta is largely dispensable. We discuss the possibility that segregation of single precursors in the central nervous system may rely on a heterogeneous distribution of neural potential between different cells of the proneural group. Notch signalling would enable all cells to mutually repress each other and only a cell with an elevated neural potential could overcome this repression.

Requirement for dynamin during Notch signaling in Drosophila neurogenesis.

Singling out of a unique neural precursor from a group of equivalent cells, during Drosophila neurogenesis, involves Notch-mediated lateral signaling. During this process, activation of the Notch signaling pathway leads to repression of neural development. Disruption of this signaling pathway results in the development of an excess of neural cells. The loss of activity of dynamin, which is encoded by the gene shibire and is required for endocytosis, results in a similar phenotype. Here we have investigated the requirement of shibire function for Notch signaling during the segregation of sensory bristles on the notum of the fly. Overexpression of different constitutively active forms of Notch in shibire mutant flies indicates that shibire function is not necessary for transduction of the signal downstream of Notch, even when the receptor is integrated in the plasma membrane. However, when wild-type Notch is activated by its ligand Delta, dynamin is required in both signaling and receiving cells for normal singling out of precursors. This suggests an active role of the signaling cell for ligand-mediated receptor endocytosis in the case of transmembrane ligands. We discuss the possible implications of these results for normal functioning of Notch-mediated lateral signaling.

A genetic analysis of pannier, a gene necessary for viability of dorsal tissues and bristle positioning in Drosophila.

A genetic and phenotypic analysis of the gene pannier is described. Animals mutant for strong alleles die as embryos in which the cells of the amnioserosa are prematurely lost. This leads to a dorsal cuticular hole. The dorsal-most cells of the imagos are also affected: viable mutants exhibit a cleft along the dorsal midline. pannier mRNA accumulates specifically in the dorsal-most regions of the embryo and the imaginal discs. Viable mutants and mutant combinations also affect the thoracic and head bristle patterns in a complex fashion. Only those bristles within the area of expression of pannier are affected. A large number of alleles have been studied and reveal that pannier may have opposing effects on the expression of achaete and scute leading to a loss or a gain of bristles.

The angle of the dorsoventral axis with respect to the anteroposterior axis in the Drosophila embryo is controlled by the distribution of gurken mRNA in the oocyte.

Like most organisms, Drosophila embryos develop in relation to two orthogonal axes, the anteroposterior and dorsoventral. These two axes are established by four independent systems of maternal information. Mutations in any system disrupt either the anteroposterior or the dorsoventral patterning of the embryo but never affect the orthogonal orientation of the axes relative to each other. Here we show by analyzing their embryonic fate map, that K10 embryos still possess a dorsoventral polarity. However, instead of forming a 90 degrees angle, the dorsoventral and the anteroposterior axes lie parallel to each other. This axis misorientation was partially corrected by decreasing the wild-type grk gene copy number such that the embryos issued from K10/K10; grk/+ females showed a variability in their fate map which could be interpreted as a progressive rotation of dorsoventral axis relative to the unmodified anteroposterior axis. This rotation is maximal in the K10 embryos where it reaches 90 degrees and results in the congruence of the two axes. The alteration of the embryonic fate map could be traced back to oogenesis where it was shown to correlate with the mislocalization of the grk transcripts.

Lateral inhibition mediated by the Drosophila neurogenic gene delta is enhanced by proneural proteins.

Cells in the neuroectoderm of Drosophila become either neural or epidermal progenitors. A critical threshold concentration of proneural gene products in a given cell causes it to develop as a neuroblast. The proteins encoded by the genes Delta (Dl) and Notch (N) act as the source and the receptor, respectively, of inhibitory signals sent by the neuroblast to neighboring cells that prevent these cells from also adopting the neural fate. We show here that proneural gene products activate transcription of Delta in the neuroectoderm by binding to specific sites in its promoter. This transcriptional activation enhances lateral inhibition and thus helps ensure that cells in the vicinity of prospective neuroblasts will themselves become epidermoblasts.

Genomic regions regulating early embryonic expression of the Drosophila neurogenic gene Delta.

To identify genomic regions required for transcriptional regulation of Delta during early embryogenesis, constructs carrying promoter and gene fusions to the lacZ gene were used for germ line transformation. Most cis-regulatory sequences are dispersed throughout 6.6 kb of genomic DNA, 5' of the transcription start site; the first intron contains an enhancer element that increases the amount of RNA produced in several organs. A region was defined which drives Delta-lacZ RNA expression in clusters of neuroectodermal cells preceding and during SI neuroblast segregation. This pattern is regulated by genes of the AS-C (achaete-scute complex). To identify regulatory regions necessary for normal function of Delta during neural-epidermal lineage segregation, five minigenes consisting of fragments of the 5' genomic DNA fused to a cDNA encoding the entire protein sequence were tested for their ability to rescue the neural hyperplasia caused by a deletion of the Delta locus. Regulatory sequences required for this function are differentially distributed throughout 9 kb of genomic DNA upstream of the transcription start site. The possible significance of these findings with respect to the function of Delta during lineage segregation is discussed.

pannier, a negative regulator of achaete and scute in Drosophila, encodes a zinc finger protein with homology to the vertebrate transcription factor GATA-1.

The gene pannier acts as a repressor of achaete and scute, two transcription factors expressed in discrete subsets of cells at the sites where neural precursors develop. Molecular analysis of mutant alleles revealed the presence of two functional domains within the pannier protein: a zinc finger domain showing homology to the GATA-1 family of vertebrate transcription factors and a domain comprising two putative amphipathic helices. Mutants associated with lesions in the zinc finger domain display an overexpression of achaete and scute and the development of extra neural precursors. Mutant proteins in which the domain including the putative helices is deleted act as hyperactive repressor molecules causing a loss of achaete/scute expression and a loss of neural precursors. Other results suggest that the activity of pannier may be modulated by association with position-specific factors.

The snail gene required for mesoderm formation in Drosophila is expressed dynamically in derivatives of all three germ layers.

The zygotic effect gene snail (sna) encodes a zinc-finger protein required for mesoderm formation in Drosophila embryos. By in situ analysis, sna transcripts are first detected at syncytial blastoderm and persist until very late stages of embryogenesis. Expression of sna is transient and is observed in tissues derived from all three germ layers. Prior to germband elongation, sna RNA accumulation is consistent with its genetically determined role in mesoderm formation. Starting at germband elongation, a second phase of sna expression appears to be initiated, characterized by a highly dynamic accumulation of transcripts in the developing central and peripheral nervous systems. Translation of sna RNA is apparently delayed as the sna protein is not detected before the onset of gastrulation. Its regional distribution generally correlates with that of sna transcripts. The complex pattern of sna expression strongly suggests that the function of the gene is not restricted to mesoderm formation.

The pattern of transcription of the neurogenic gene Delta of Drosophila melanogaster.

The function of the Delta locus of Drosophila melanogaster is required for the correct separation of neural and epidermal cell lineages. We describe here the transcriptional organization of this locus and the spatial pattern of mRNA accumulation during embryogenesis. Delta produces three mRNAs with protein-coding capacity, which differ only at their untranslated 3' ends and thus encode the same protein; other minor RNAs from the locus are shown not to have any protein-coding capacity and to correspond to introns. No indications were obtained for multiple translational products of the locus. In situ hybridization using digoxigenin-labelled probes confirms that Delta RNA is present at high concentration in all presumptive neurogenic territories of the embryo. Since all the constituent cells of these territories contain Delta RNA, a differential distribution of the protein among the derivatives of the neuroectodermal cells is improbable. Some time after segregation of lineages, Delta RNA reappears in neuroblasts. The possible significance of these observations with respect to the function of the Delta product during lineage segregation is discussed.

Role of the oocyte nucleus in determination of the dorsoventral polarity of Drosophila as revealed by molecular analysis of the K10 gene.

In Drosophila, the establishment of dorsoventral polarity of the developing embryo depends on the expression of at least 11 maternally acting genes. Mutant females that lack any of these gene activities produce normally shaped eggs that develop into dorsalized embryos. The female sterile K10 mutation differs from these mutants, because in addition to the dorsalized development of the embryo, it causes a dorsalization of the egg shape. During oogenesis, the K10 gene is specifically expressed in the oocyte. Antibodies raised against a beta-galactosidase-K10 fusion protein were used to visualize the K10 product in ovaries by indirect immunofluorescence. The protein, which contains a putative DNA recognition helix, accumulates in the nucleus of the oocyte, where it is assumed to have a regulatory function. Our results thus indicate that the controlled expression of some of the genes of the oocyte nucleus is essential for the determination of the dorsoventral polarity of the oocyte and possibility of the developing embryo.

DNA sequences homologous to the Drosophila opa repeat are present in murine mRNAs that are differentially expressed in fetuses and adult tissues.

A mouse embryonic cDNA containing two opa-like (CAX)n repeats was isolated on the basis of its cross-hybridization with a Drosophila K10 cDNA. Such repeated sequences were present in different murine mRNAs, some of which were specifically expressed during fetal life or in different adult tissues. This suggests that, as already described for Drosophila, opa-like sequences are parts of proteins involved in ontogenic or cell-type-specific functions in vertebrates. However, unlike Drosophila, such repeated sequences were not found within the murine homeo-boxes containing genes of the Hox-1 complex.

Oocyte-specific transcription of fs(1)K10: a Drosophila gene affecting dorsal-ventral developmental polarity.

The expression of the fs(1)K10 gene is required in early oogenesis for the establishment of the dorsal-ventral polarity of the oocyte, and later in the embryo. P-element-mediated transformation shows that the K10 function is located within a fragment of DNA of 5 kb, which encodes four RNA species. A major transcript of 3.1 kb is likely to be responsible for the K10 function. It is abundant in ovaries and in early developing embryos. Thus its expression profile corresponds closely to that which could be anticipated from the biological characteristics of the mutation. In situ hybridization on ovary sections shows that the gene is not only specifically transcribed in the germ line (which is consistent with the germ-line dependence of the mutation), but that its expression is also cell-specific since it is apparently restricted to the oocyte.

A 43 kilobase cosmid P transposon rescues the fs(1)K10 morphogenetic locus and three adjacent Drosophila developmental mutants.

The K10 female sterility locus involved in establishment of the embryonic dorsoventral axis maps genetically to the 2E2-2F1 interval of the Drosophila X chromosome. We microdissected the 2E2-2F3 region from salivary gland chromosomes and used clones obtained from the microdissected fragments to establish a chromosomal walk covering more than 200 kb. To identify the K10 gene we used P-mediated transformation with cosmid clones constructed in cos-P, a cosmid vector incorporating the terminal repeats of the P element. Clone cos9, containing a 43 kb insert, transformed the germ line of homozygous K10 females and allowed production of normal progeny. It also rescued three genes, crooked neck, pecanex, and kurz, which map genetically near K10. Transformation experiments using smaller fragments of cos9 localize the K10+ function within 11 kb. Northern blots hybridized with probes from this region indicate the presence of several mRNA species. Each transcript has been assigned to a complementation group.