Les oncogènes NUP98-PHF23 et NUP98-HOXD13 confèrent un potentiel aberrant d’auto-renouvellement aux progéniteurs thymiques.

L’initiation de la leucémogénèse dans la leucémie aigue lymphoblastique (LAL)-T résulte de l’activation aberrante de facteurs de transcription de la lignée lymphocytaire T. Nous démontrons que les gènes de fusion NUP98-PHF23 (NP23) et NUP98-HOXD13 (NHD13) reprogramment les thymocytes normaux en cellules souches pré-leucémiques (CS-préL) possédant un potentiel aberrant d’auto-renouvellement. Basé sur des essais de clonalité performés sur des thymocytes transplantés en série, nous avons découvert que cette population est hiérarchisée similairement aux cellules souches hématopoïétiques normales. Ces CS-préL dévoilent un enrichissement du compartiment de précurseurs thymiques immatures KIT+ où les deux oncogènes, NP23 et NHD13, activent des gènes impliqués dans l’autorenouvellement, incluant Hoxa9, Hoxa10, Lyl1 et Hhex. De plus, l’activité d’autorenouvellement est abrogée par les ARN interférents contre Lyl1 et Hhex, indiquant leur implication fonctionnelle en aval de NP23 et NHD13. Puisque ces gènes sont aussi activés en aval de trois autres oncogènes dans la LAL-T, SCL/TAL1, LMO1 et LMO2, nous concluons que les niveaux d’activation de Lyl1 et Hhex fixent le seuil de reprogrammation des thymocytes normaux en CS-préL. Malgré l'efficacité des traitements de chimiothérapie actuels à diminuer la masse tumorale, les CS-préL sont épargnées, pouvant mener à des rechutes. Nos résultats répondent à ce besoin et proposent de nouvelles avenues permettant de cibler les CS-préL du compartiment de thymocytes immatures dans la LAL-T.

Les oncogènes NUP98-PHF23 et NUP98-HOXD13 confèrent un potentiel aberrant d’auto-renouvellement aux progéniteurs thymiques

L’initiation de la leucémogénèse dans la leucémie aigue lymphoblastique (LAL)-T résulte de l’activation aberrante de facteurs de transcription de la lignée lymphocytaire T. Nous démontrons que les gènes de fusion NUP98-PHF23 (NP23) et NUP98-HOXD13 (NHD13) reprogramment les thymocytes normaux en cellules souches pré-leucémiques (CS-préL) possédant un potentiel aberrant d’auto-renouvellement. Basé sur des essais de clonalité performés sur des thymocytes transplantés en série, nous avons découvert que cette population est hiérarchisée similairement aux cellules souches hématopoïétiques normales. Ces CS-préL dévoilent un enrichissement du compartiment de précurseurs thymiques immatures KIT+ où les deux oncogènes, NP23 et NHD13, activent des gènes impliqués dans l’autorenouvellement, incluant Hoxa9, Hoxa10, Lyl1 et Hhex. De plus, l’activité d’autorenouvellement est abrogée par les ARN interférents contre Lyl1 et Hhex, indiquant leur implication fonctionnelle en aval de NP23 et NHD13. Puisque ces gènes sont aussi activés en aval de trois autres oncogènes dans la LAL-T, SCL/TAL1, LMO1 et LMO2, nous concluons que les niveaux d’activation de Lyl1 et Hhex fixent le seuil de reprogrammation des thymocytes normaux en CS-préL. Malgré l'efficacité des traitements de chimiothérapie actuels à diminuer la masse tumorale, les CS-préL sont épargnées, pouvant mener à des rechutes. Nos résultats répondent à ce besoin et proposent de nouvelles avenues permettant de cibler les CS-préL du compartiment de thymocytes immatures dans la LAL-T.

Disruption of the FG nucleoporin NUP98 causes selective changes in nuclear pore complex stoichiometry and function

The NUP98 gene encodes precursor proteins that generate two nucleoplasmically oriented nucleoporins, NUP98 and NUP96. By using gene targeting, we have selectively disrupted the murine NUP98 protein, leaving intact the expression and localization of NUP96. We show that NUP98 is essential for mouse gastrulation, a developmental stage that is associated with rapid cell proliferation, but dispensable for basal cell growth. NUP98−/− cells had an intact nuclear envelope with a normal number of embedded nuclear pore complexes. Typically, NUP98-deficient cells contained on average approximately 5-fold more cytoplasmic annulate lamellae than control cells. We found that a set of cytoplasmically oriented nucleoporins, including NUP358, NUP214, NUP88, and p62, assembled inefficiently into nuclear pores of NUP98−/− cells. Instead, these nucleoporins were prominently associated with the annulate lamellae. By contrast, a group of nucleoplasmically oriented nucleoporins, including NUP153, NUP50, NUP96, and NUP93, had no affinity for annulate lamellae and assembled normally into nuclear pores. Mutant pores were significantly impaired in transport receptor-mediated docking of proteins with a nuclear localization signal or M9 import signal and showed weak nuclear import of such substrates. In contrast, the ability of mutant pores to import ribosomal protein L23a and spliceosome protein U1A appeared intact. These observations show that NUP98 disruption selectively impairs discrete protein import pathways and support the idea that transport of distinct import complexes through the nuclear pore complex is mediated by specific subsets of nucleoporins.

The nucleoporin Nup98 associates with the intranuclear filamentous protein network of TPR

The Nup98 gene codes for several alternatively spliced protein precursors. Two in vitro translated and autoproteolytically cleaved precursors yielded heterodimers of Nup98-6kDa peptide and Nup98-Nup96. TPR (translocated promoter region) is a protein that forms filamentous structures extending from nuclear pore complexes (NPCs) to intranuclear sites. We found that in vitro translated TPR bound to in vitro translated Nup98 and, via Nup98, to Nup96. Double-immunofluorescence microscopy with antibodies to TPR and Nup98 showed colocalization. In confocal sections the nucleolus itself was only weakly stained but there was intensive perinucleolar staining. Striking spike-like structures emanated from this perinucleolar ring and attenuated into thinner structures as they extended to the nuclear periphery. This characteristic staining pattern of the TPR network was considerably enhanced when a myc-tagged pyruvate kinase-6kDa fusion protein was overexpressed in HeLa cells. Double-immunoelectron microscopy of these cells using anti-myc and anti-TPR antibodies and secondary gold-coupled antibodies yielded row-like arrangements of gold particles. Taken together, the immunolocalization data support previous electron microscopical data, suggesting that TPR forms filaments that extend from the NPC to the nucleolus. We discuss the possible implications of the association of Nup98 with this intranuclear TPR network for an intranuclear phase of transport.

The pathophysiology of HOX genes and their role in cancer

The HOM-C clustered prototype homeobox genes of Drosophila, and their counterparts, the HOX genes in humans, are highly conserved at the genomic level. These master regulators of development continue to be expressed throughout adulthood in various tissues and organs. The physiological and patho-physiological functions of this network of genes are being avidly pursued within the scientific community, but defined roles for them remain elusive. The order of expression of HOX genes within a cluster is co-ordinated during development, so that the 3' genes are expressed more anteriorly and earlier than the 5' genes. Mutations in HOXA13 and HOXD13 are associated with disorders of limb formation such as hand-foot-genital syndrome (HFGS), synpolydactyly (SPD), and brachydactyly. Haematopoietic progenitors express HOX genes in a pattern characteristic of the lineage and stage of differentiation of the cells. In leukaemia, dysregulated HOX gene expression can occur due to chromosomal translocations involving upstream regulators such as the MLL gene, or the fusion of a HOX gene to another gene such as the nucleoporin, NUP98. Recent investigations of HOX gene expression in leukaemia are providing important insights into disease classification and prediction of clinical outcome. Whereas the oncogenic potential of certain HOX genes in leukaemia has already been defined, their role in other neoplasms is currently being studied. Progress has been hampered by the experimental approach used in many studies in which the expression of small subsets of HOX genes was analysed, and complicated by the functional redundancy implicit in the HOX gene system. Attempts to elucidate the function of HOX genes in malignant transformation will be enhanced by a better understanding of their upstream regulators and downstream target genes.

Identification of novel genetic alterations in pediatric cytogenetically normal acute myeloid leukemia by next-generation sequencing

Pediatric acute myeloid leukemia (AML) is a molecularly heterogeneous disease that arises from genetic alterations in pathways that regulate self-renewal and myeloid differentiation. While the majority of patients carry recurrent chromosomal translocations, almost 20% of childhood AML do not show any recognizable cytogenetic alteration and are defined as cytogenetically normal (CN)-AML. CN-AML patients have always showed a great variability in response to therapy and overall outcome, underlining the presence of unknown genetic changes, not detectable by conventional analyses, but relevant for pathogenesis, and outcome of AML. The development of novel genome-wide techniques such as next-generation sequencing, have tremendously improved our ability to interrogate the cancer genome. Based on this background, the aim of this research study was to investigate the mutational landscape of pediatric CN-AML patients negative for all the currently known somatic mutations reported in AML through whole-transcriptome sequencing (RNA-seq). RNA-seq performed on diagnostic leukemic blasts from 19 pediatric CN-AML cases revealed a considerable incidence of cryptic chromosomal rearrangements, with the identification of 21 putative fusion genes. Several of the fusion genes that were identified in this study are recurrent and might have a prognostic and/or therapeutic relevance. A paradigm of that is the CBFA2T3-GLIS2 fusion, which has been demonstrated to be a common alteration in pediatric CN-AML, predicting poor outcome. Important findings have been also obtained in the identification of novel therapeutic targets. On one side, the identification of NUP98-JARID1A fusion suggests the use of disulfiram; on the other, here we describe alteration-activating tyrosine kinases, providing functional data supporting the use of tyrosine kinase inhibitors to specifically inhibit leukemia cells. This study provides new insights in the knowledge of genetic alterations underlying pediatric AML, defines novel prognostic markers and putative therapeutic targets, and prospectively ensures a correct risk stratification and risk-adapted therapy also for the “all-neg” AML subgroup.

Carbon and oxygen stable isotope composition of bulk sediment and abundance of Braarudospahaera in ODP Site 122-762 (Table 1)

Nannofossil assemblages enriched in Braarudosphaera occur in lower Oligocene to lower Miocene sediments at Ocean Drilling Program Sites 762 and 763 on the central Exmouth Plateau. Braarudosphaerids appear here rather abruptly in the lower Oligocene (in Zone NP21). They reach their greatest numbers in the lower Oligocene (in Zones NP22 and NP23), where they comprise up to 10% of some samples. Braarudosphaera bigelowii is the overwhelmingly dominant species, occurring together with rare specimens of B. discula and Micrantholithus pinguis. The holococcoliths Peritrachelina joidesa and Lantemithus minutus are also associated with the Braarudosphaera enrichment. There are two populations of B. bigelowii: one of normal size (10-14 µm) and one of large specimens (20-22 µm). The larger braarudosphaerids are more common than the smaller forms. Braarudosphaera-rich sediments are absent at Wombat Plateau sites during the same time interval. We attribute this to latitudinal control, because the Wombat sites are about 4° north of the central Exmouth Plateau sites. We believe that the occurrence of braarudosphaerids is related to an Oligocene to early Miocene oceanographic event on the Exmouth Plateau. We suspect that mid-ocean up welling of cool, low-salinity, nutrient-rich water along a divergent zone created the Braarudosphaera-nch sediments in the South Atlantic and Indian oceans.

Calcareous nannoplankton from the Southwest Pacific Ocean

The positions of all cores recovered during Leg 90 in the southwest Pacific are shown within the standard calcareous nannoplankton zonation. The stratigraphic and regional occurrences and preservation of Paleogene calcareous nannoplankton found at Sites 588, 592, and 593 are discussed, and fossil lists are given for selected samples. Data on the Eocene/Oligocene boundary found in Holes 592 and 593 and on the Oligocene/Miocene boundary in Hole 588C are presented. Regional unconformities are noted in Hole 588C, where the upper Eocene to middle Oligocene interval (Zones NP17 to NP23) is missing, and in Hole 592, in which the middle Oligocene to lowest Miocene interval (Zones NP23 to NN1) is not represented.

Hydrochemical Atlas of the Arctic Ocean

Introduction: Chemical composition of water determines its physical properties and character of processes proceeding in it: freezing temperature, volume of evaporation, density, color, transparency, filtration capacity, etc. Presence of chemical elements in water solution confers waters special physical properties exerting significant influence on their circulation, creates necessary conditions for development and inhabitance of flora and fauna, and imparts to the ocean waters some chemical features that radically differ them from the land waters (Alekin & Liakhin, 1984). Hydrochemical information helps to determine elements of water circulation, convection depth, makes it easier to distinguish water masses and gives additional knowledge of climatic variability of ocean conditions. Hydrochemical information is a necessary part of biological research. Water chemical composition can be the governing characteristics determining possibility and limits of use of marine objects, both stationary and moving in sea water. Subject of investigation of hydrochemistry is study of dynamics of chemical composition, i.e. processes of its formation and hydrochemical conditions of water bodies (Alekin & Liakhin 1984). The hydrochemical processes in the Arctic Ocean are the least known. Some information on these processes can be obtained in odd publications. A generalizing study of hydrochemical conditions in the Arctic Ocean based on expeditions conducted in the years 1948-1975 has been carried out by Rusanov et al. (1979). The "Atlas of the World Ocean: the Arctic Ocean" contains a special section "Hydrochemistry" (Gorshkov, 1980). Typical vertical profiles, transects and maps for different depths - 0, 100, 300, 500, 1000, 2000, 3000 m are given in this section for the following parameters: dissolved oxygen, phosphate, silicate, pH and alkaline-chlorine coefficient. The maps were constructed using the data of expeditions conducted in the years 1948-1975. The illustrations reflect main features of distribution of the hydrochemical elements for multi-year period and represent a static image of hydrochemical conditions. Distribution of the hydrochemical elements on the ocean surface is given for two seasons - winter and summer, for the other depths are given mean annual fields. Aim of the present Atlas is description of hydrochemical conditions in the Arctic Ocean on the basis of a greater body of hydrochemical information for the years 1948-2000 and using the up-to-date methods of analysis and electronic forms of presentation of hydrochemical information. The most wide-spread characteristics determined in water samples were used as hydrochemical indices. They are: dissolved oxygen, phosphate, silicate, pH, total alkalinity, nitrite and nitrate. An important characteristics of water salt composition - "salinity" has been considered in the Oceanographic Atlas of the Arctic Ocean (1997, 1998). Presentation of the hydrochemical characteristics in this Hydrochemical Atlas is wider if compared with that of the former Atlas (Gorshkov, 1980). Maps of climatic distribution of the hydrochemical elements were constructed for all the standard depths, and seasonal variability of the hydrochemical parameters is given not only for the surface, but also for the underlying standard depths up to 400 m and including. Statistical characteristics of the hydrochemical elements are given for the first time. Detailed accuracy estimates of initial data and map construction are also given in the Atlas. Calculated values of mean-root deviations, maximum and minimum values of the parameters demonstrate limits of their variability for the analyzed period of observations. Therefore, not only investigations of chemical statics are summarized in the Atlas, but also some elements of chemical dynamics are demonstrated. Digital arrays of the hydrochemical elements obtained in nodes of a regular grid are the new form of characteristics presentation in the Atlas. It should be mentioned that the same grid and the same boxes were used in the Atlas, as those that had been used by creation of the US-Russian climatic Oceanographic Atlas. It allows to combine hydrochemical and oceanographic information of these Atlases. The first block of the digital arrays contains climatic characteristics calculated using direct observational data. These climatic characteristics were not calculated in the regions without observations, and the information arrays for these regions have gaps. The other block of climatic information in a gridded form was obtained with the help of objective analysis of observational data. Procedure of the objective analysis allowed us to obtain climatic estimates of the hydrochemical characteristics for the whole water area of the Arctic Ocean including the regions not covered by observations. Data of the objective analysis can be widely used, in particular, in hydrobiological investigations and in modeling of hydrochemical conditions of the Arctic Ocean. Array of initial measurements is a separate block. It includes all the available materials of hydrochemical observations in the form, as they were presented in different sources. While keeping in mind that this array contains some amount of perverted information, the authors of the Atlas assumed it necessary to store this information in its primary form. Methods of data quality control can be developed in future in the process of hydrochemical information accumulation. It can be supposed that attitude can vary in future to the data that were rejected according to the procedure accepted in the Atlas. The hydrochemical Atlas of the Arctic Ocean is the first specialized and electronic generalization of hydrochemical observations in the Arctic Ocean and finishes the program of joint efforts of Russian and US specialists in preparation of a number of atlases for the Arctic. The published Oceanographic Atlas (1997, 1998), Atlas of Arctic Meteorology and Climate (2000), Ice Atlas of the Arctic Ocean prepared for publication and Hydrochemical Atlas of the Arctic Ocean represent a united series of fundamental generalizations of empirical knowledge of Arctic Ocean nature at climatic level. The Hydrochemical Atlas of the Arctic Ocean was elaborated in the result of joint efforts of the SRC of the RF AARI and IARC. Dr. Ye. Nikiforov was scientific supervisor of the Atlas, Dr. R. Colony was manager on behalf of the USA and Dr. L. Timokhov - on behalf of Russia.

RanGTP-mediated nuclear export of karyopherin α involves its interaction with the nucleoporin Nup153

Using binding assays, we discovered an interaction between karyopherin α2 and the nucleoporin Nup153 and mapped their interacting domains. We also isolated a 15-kDa tryptic fragment of karyopherin β1, termed β1*, that contains a determinant for binding to the peptide repeat containing nucleoporin Nup98. In an in vitro assay in which export of endogenous nuclear karyopherin α from nuclei of digitonin-permeabilized cells was quantitatively monitored by indirect immunofluorescence with anti-karyopherin α antibodies, we found that karyopherin α export was stimulated by added GTPase Ran, required GTP hydrolysis, and was inhibited by wheat germ agglutinin. RanGTP-mediated export of karyopherin α was inhibited by peptides representing the interacting domains of Nup153 and karyopherin α2, indicating that the binding reactions detected in vitro are physiologically relevant and verifying our mapping data. Moreover, β1*, although it inhibited import, did not inhibit export of karyopherin α. Hence, karyopherin α import into and export from nuclei are asymmetric processes.

Karyopherin β2 mediates nuclear import of a mRNA binding protein

We have cloned and sequenced cDNA for human karyopherin β2, also known as transportin. In a solution binding assay, recombinant β2 bound directly to recombinant nuclear mRNA-binding protein A1. Binding was inhibited by a peptide representing A1’s previously characterized M9 nuclear localization sequence (NLS), but not by a peptide representing a classical NLS. As previously shown for karyopherin β1, karyopherin β2 bound to several nucleoporins containing characteristic peptide repeat motifs. In a solution binding assay, both β1 and β2 competed with each other for binding to immobilized repeat nucleoporin Nup98. In digitonin-permeabilized cells, β2 was able to dock A1 at the nuclear rim and to import it into the nucleoplasm. At low concentrations of β2, there was no stimulation of import by the exogenous addition of the GTPase Ran. However, at higher concentrations of β2 there was marked stimulation of import by Ran. Import was inhibited by the nonhydrolyzable GTP analog guanylyl imidodiphosphate by a Ran mutant that is unable to hydrolyze GTP and also by wheat germ agglutinin. Consistent with the solution binding results, karyopherin β2 inhibited karyopherin α/β1-mediated import of a classical NLS containing substrate and, vice versa, β1 inhibited β2-mediated import of A1 substrate, suggesting that the two import pathways merge at the level of docking of β1 and β2 to repeat nucleoporins.

The Nucleoporin Nup153 Plays a Critical Role in Multiple Types of Nuclear Export

The fundamental process of nucleocytoplasmic transport takes place through the nuclear pore. Peripheral pore structures are presumably poised to interact with transport receptors and their cargo as these receptor complexes first encounter the pore. One such peripheral structure likely to play an important role in nuclear export is the basket structure located on the nuclear side of the pore. At present, Nup153 is the only nucleoporin known to localize to the surface of this basket, suggesting that Nup153 is potentially one of the first pore components an RNA or protein encounters during export. In this study, anti-Nup153 antibodies were used to probe the role of Nup153 in nuclear export in Xenopus oocytes. We found that Nup153 antibodies block three major classes of RNA export, that of snRNA, mRNA, and 5S rRNA. Nup153 antibodies also block the NES protein export pathway, specifically the export of the HIV Rev protein, as well as Rev-dependent RNA export. Not all export was blocked; Nup153 antibodies did not impede the export of tRNA or the recycling of importin β to the cytoplasm. The specific antibodies used here also did not affect nuclear import, whether mediated by importin α/β or by transportin. Overall, the results indicate that Nup153 is crucial to multiple classes of RNA and protein export, being involved at a vital juncture point in their export pathways. This juncture point appears to be one that is bypassed by tRNA during its export. We asked whether a physical interaction between RNA and Nup153 could be observed, using homoribopolymers as sequence-independent probes for interaction. Nup153, unlike four other nucleoporins including Nup98, associated strongly with poly(G) and significantly with poly(U). Thus, Nup153 is unique among the nucleoporins tested in its ability to interact with RNA and must do so either directly or indirectly through an adaptor protein. These results suggest a unique mechanistic role for Nup153 in the export of multiple cargos.

Mammalian karyopherin alpha 1 beta and alpha 2 beta heterodimers: alpha 1 or alpha 2 subunit binds nuclear localization signal and beta subunit interacts with peptide repeat-containing nucleoporins.

Although only 44% identical to human karyopherin alpha 1, human karyopherin alpha 2 (Rch1 protein) substituted for human karyopherin alpha 1 (hSRP-1/NPI-1) in recognizing a standard nuclear localization sequence and karyopherin beta-dependent targeting to the nuclear envelope of digitonin-permeabilized cells. By immunofluorescence microscopy of methanol-fixed cells, karyopherin beta was localized to the cytoplasm and the nuclear envelope and was absent from the nuclear interior. Digitonin permeabilization of buffalo rat liver cells depleted their endogenous karyopherin beta. Recombinant karyopherin beta can bind directly to the nuclear envelope of digitonin-permeabilized cells at 0 degree C (docking reaction). In contrast, recombinant karyopherin alpha 1 or alpha 2 did not bind unless karyopherin beta was present. Likewise, in an import reaction (at 20 degrees C) with all recombinant transport factors (karyopherin alpha 1 or alpha 2, karyopherin beta, Ran, and p10) import depended on karyopherin beta. Localization of the exogenously added transport factors after a 30-min import reaction showed karyopherin beta at the nuclear envelope and karyopherin alpha 1 or alpha 2, Ran, and p10 in the nuclear interior. In an overlay assay with SDS/PAGE-resolved and nitrocellulose-transferred proteins of the nuclear envelope, 35S-labeled karyopherin beta bound to at least four peptide repeat-containing nucleoporins--Nup358, Nup214, Nup153, and Nup98.

Mise au point d'un modèle de leucémie chez la Drosophile

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.