E4bp4 is required for commitment to the NK lineage and promotes NK development by directly regulating the expression of Eomes and Id2.

No abstract

NK cells contribute to antitumor and antiviral immunosurveillance. Their development in the bone marrow (BM) requires the transcription factor E4BP4/NFIL3, but requirements in other organs are less well defined. In this study, we show that CD3−NK1.1+NKp46+CD122+ NK cells of immature phenotype and expressing low eomesodermin levels are found in thymus, spleen, and liver of E4BP4-deficient mice, whereas numbers of mature, eomesoderminhigh conventional NK cells are drastically reduced. E4BP4-deficient CD44+CD25− double-negative 1 thymocytes efficiently develop in vitro into NK cells with kinetics, phenotype, and functionality similar to wild-type controls, whereas no NK cells develop from E4BP4-deficient BM precursors. In E4BP4/Rag-1 double-deficient (DKO) mice, NK cells resembling those in Rag-1–deficient controls are found in similar numbers in the thymus and liver. However, NK precursors are reduced in DKO BM, and no NK cells develop from DKO BM progenitors in vitro. DKO thymocyte precursors readily develop into NK cells, but DKO BM transfers into nude recipients and NK cells in E4BP4/Rag-1/IL-7 triple-KO mice indicated thymus-independent NK cell development. In the presence of T cells or E4BP4-sufficient NK cells, DKO NK cells have a selective disadvantage, and thymic and hepatic DKO NK cells show reduced survival when adoptively transferred into lymphopenic hosts. This correlates with higher apoptosis rates and lower responsiveness to IL-15 in vitro. In conclusion, we demonstrate E4BP4-independent development of NK cells of immature phenotype, reduced fitness, short t1/2, and potential extramedullary origin. Our data identify E4BP4-independent NK cell developmental pathways and a role for E4BP4 in NK cell homeostasis.

Polyglutamine pathologies are neurodegenerative diseases that manifest both general polyglutamine toxicity and mutant protein-specific effects. Dentatorubral-pallidoluysian Atrophy (DRPLA) is one of these disorders caused by mutations in the Atrophin-1 protein. We have generated several models for DRPLA in Drosophila and analysed the mechanisms of cellular and organism toxicity. Our genetic and ultrastructural analysis of neurodegeneration suggests that autophagy may have a role in cellular degeneration when polyglutamine proteins are overexpressed in neuronal and glial cells. Clearance of autophagic organelles is blocked at the lysosomal level after correct fusion between autophagosomes and lysosomes. This leads to accumulation of autofluorescent pigments and proteinaceous residues usually degraded by the autophagy-lysosome system. Under these circumstances, further pharmacological and genetic induction of autophagy does not rescue neurodegeneration by polyglutamine Atrophins, in contrast to many other neurodegenerative conditions. Our data thus provide a crucial insight into the specific mechanism of a polyglutamine disease and reveal important differences in the role of autophagy with respect to other diseases of the same family. PolyQ-expanded proteins misfold and accumulate in large aggregates, which have been initially described as toxic, 1 and more recently as a positive prognosis factor in neuronal survival.2 The molecular and cellular mechanisms of toxicity due to polyQ proteins are not yet fully characterised and many of the principal aspects are under intense scrutiny. In particular, many reports have described autophagy as a protective cellular mechanism in neurodegeneration, 3 although its full contribution to the pathogenesis, including cell killing, is still poorly understood. The full cycle of autophagic degradation of cellular components and recycle of simpler constituents is not involved in cell killing. However, many dysfunctions at different steps of this cycle that have been reported upon expression of toxic proteins, including polyQ proteins, can lead to cell degeneration and death. 4 Over the past few years a growing number of studies have focused on identifying the interplay between polyQ effects and protein-specific misfunctions, 5-7 with the assumption that polyQ pathologies combine general polyQ toxicity with disease-specific effects due to the proteins affected. The fruitfly Drosophila melanogaster has proved to be a valuable model organism for polyQ diseases and neurodegeneration. New models have also moved on from initial basic observations, uncovering the complex interplay between polyQ effects and RNA or protein-specific misfunction. 5,7,9 We generated several Drosophila models of DRPLA ( Figure 1a and Supplementary Figure 1), a polyQ disease caused by mutations in atrophin-1.10,11 Atrophins are transcriptional cofactors conserved from Drosophila to mammals, 12-15 providing an ideal background for the dissection of polyQ effects and specific Atrophin functions through Drosophila ...

Retroviral and lentiviral vectors often use the envelope G protein from the vesicular stomatitis virus Indiana strain (VSVind.G). However, lentivector producer cell lines that stably express VSVind.G have not been reported, presumably because of its cytotoxicity, preventing simple scale-up of vector production. Interestingly, we showed that VSVind.G and other vesiculovirus G from the VSV New Jersey strain (VSVnj), Cocal virus (COCV), and Piry virus (PIRYV) could be constitutively expressed and supported lentivector production for up to 10 weeks. All G-enveloped particles were robust, allowing concentration and freeze-thawing. COCV.G and PIRYV.G were resistant to complement inactivation, and, using chimeras between VSVind.G and COCV.G, the determinant for complement inactivation of VSVind.G was mapped to amino acid residues 136–370. Clonal packaging cell lines using COCV.G could be generated; however, during attempts to establish LV producer cells, vector superinfection was observed following the introduction of a lentivector genome. This could be prevented by culturing the cells with the antiviral drug nevirapine. As an alternative countermeasure, we demonstrated that functional lentivectors could be reconstituted by admixing supernatant from stable cells producing unenveloped virus with supernatant containing envelopes harvested from cells stably expressing VSVind.G, COCV.G, or PIRYV.G.

Mutations in human Atrophin1, a transcriptional corepressor, cause dentatorubral-pallidoluysian atrophy, a neurodegenerative disease. Drosophila Atrophin (Atro) mutants display many phenotypes, including neurodegeneration, segmentation, patterning and planar polarity defects. Despite Atro’s critical role in development and disease, relatively little is known about Atro’s binding partners and downstream targets. We present the first genomic analysis of Atro using ChIP-seq against endogenous Atro. ChIP-seq identified 1300 potential direct targets of Atro including engrailed, and components of the Dpp and Notch signaling pathways. We show that Atro regulates Dpp and Notch signaling in larval imaginal discs, at least partially via regulation of thickveins and fringe. In addition, bioinformatics analyses, sequential ChIP and coimmunoprecipitation experiments reveal that Atro interacts with the Drosophila GAGA Factor, Trithorax-like (Trl), and they bind to the same loci simultaneously. Phenotypic analyses of Trl and Atro clones suggest that Atro is required to modulate the transcription activation by Trl in larval imaginal discs. Taken together, these data indicate that Atro is a major Trl cofactor that functions to moderate developmental gene transcription.DOI: http://dx.doi.org/10.7554/eLife.23084.001

Mutations in human Atrophin1, a transcriptional corepressor, cause dentatorubral-