We describe a new AML entity, occurring in 30% of de novo acute myeloid leukemia, due to structural and epigenetic deregulation of the UNCX homeobox (HB) gene. By molecular approaches, we identified a M5 AML patient with a t(7;10)(p22;p14) translocation as the sole cytogenetic anomaly and showing ectopic expression of UNCX (7p22.3), which encode for a transcription factor involved in somitogenesis and neurogenesis. Since UNCX was never reported in association with cancer but only with common myeloid cell proliferation and regulation of cell differentiation, we decided to investigate its contribution to leukemogenesis. We observed UNCX ectopic expression in 32.3% (20/62) and in 8% (6/75) of acute myeloid leukemia (AML) patients and cell lines, respectively. Notably, retroviral-mediated UNCX transfer in CD34+ HSCs induced a slow-down in their proliferation and differentiation and transduced cells showed a lower growth rate but a higher percentage of CD34+ stem cells in liquid culture than controls. Additionally, UNCX infected cells displayed a decrease of MAP2K1 proliferation marker but increase of KLF4, HOXA10, and CCNA1, associated with impaired differentiation and pluripotency. Similarly, UNCX-positive patients revealed alteration of gene pathways involved in proliferation, cell cycle control and hematopoiesis. Since HB genes encode for transcription factors showing a crucial role in normal hematopoiesis and in leukemogenesis, we focused our attention on the role of altered UNCX expression level. Of note, its murine ortholog, (Uncx) was previously described as embedded within a low-methylated regions (≤ 10%) called "canyon" and dysregulated in murine hematopoietic stem cells (HSCs) as a consequence of altered methylation at canyons edges (borders) due to Dnmt3a inactivation. In our hands, UNCX activation was accompanied by methylation changes at both its canyon borders, clearly indicating an epigenetic regulation of this gene, although not induced by DNMT3A mutations.Clinical parameters and correlation with response to therapy will be presented.Taken together, our results indicate that more than 30% of de novo AML have a novel entity with a putative leukemogenic role of UNCX, whose activation may be ascribed to epigenetic regulators.Acknowledgments: MG, CP, GS, and AP(2) and this work was supported by ELN, AIL, AIRC, progetto Regione-Università 2010-12 (L. Bolondi), Fondazione del Monte di Bologna e Ravenna, FP7 NGS-PTL project. CTS, GD and AL are supported by Associazione Italiana Ricerca sul Cancro (AIRC) funding.Disclosures Nadarajah: MLL Munich Leukemia Laboratory: Employment. Martinelli: MSD: Consultancy; Novartis: Consultancy, Speakers Bureau; Ariad: Consultancy; BMS: Consultancy, Speakers Bureau; Pfizer: Consultancy; AMGEN: Consultancy; ROCHE: Consultancy.↵* Asterisk with author names denotes non-ASH members.

The invention refers to 5'TIR nucleotide sequences derived from the "Bari" family of transposable elements for the use as promoter in the expression of a transgene in a recombinant genetic construct adapted to be transfected in a host cell system

Insulators or chromatin boundary are DNA elements that organize the genome into discrete regulatory domains by limiting the actions of enhancers and silencers through a “positional-blocking mechanism”. The role of these sequences, both in modulation of the enhancers range of action (enhancer–promoter selectivity) and in the organization of the chromatin in functional domains, is emerging strongly in these last years. There is a great interest in identifying new insulators because deeper knowledge of these elements can help understand how cis-regulatory elements coordinate the expression of the target genes. However, while insulators are critical in gene regulation and genome functioning, only a few have been reported so far. Here, we describe a new insulator sequence that is located in the 5' UTR of the Drosophila retrotransposon ZAM. We have used an “enhancer–blocking assay” to test its e V ects on the activity of the enhancer in transiently transfected Drosophila S2R+ cell line. Moreover, we show that the new insulator is able to a V ect signi W cantly the enhancer–promoter interaction in the human cell line HEK293. These results suggest the possibility of employing the ZAM insulator in gene transfer protocols from insects to mammals in order to counteract the transgene positional and genotoxic effects.

The transposons of the Bari family are mobile genetic elements widespread in the Drosophila genus. However, despite a broad diffusion, virtually no information is available on the mechanisms underlying their mobility. In this paper we report the functional characterization of the Bari elements transposition system. Using the Bari1 element as a model, we investigated the subcellular localization of the transposase, its physical interaction with the transposon, and its catalytic activity. The Bari1 transposase localized in the nucleus and interacted with the terminal sequences of the transposon both in vitro and in vivo, however, no transposition activity was detected in transposition assays. Profiling of mRNAs expressed by the transposase gene revealed the expression of abnormal, internally processed transposase transcripts encoding truncated, catalytically inactive transposase polypeptides. We hypothesize that a post-transcriptional control mechanism produces transposase-derived polypeptides that effectively repress transposition. Our findings suggest further clues towards understanding the mechanisms that control transposition of an important class of mobile elements, which are both an endogenous source of genomic variability and widely used as transformation vectors/biotechnological tools.

Transposable elements are ubiquitous component of eukaryotic genomes and, besides their mutagenic role, they are considered as the major source of variability that can change genomes and their expression, either considering short term or large evolutionary scale time. The post-genomic era offers a great opportunity to shed light on the evolution of mobile genetic elements with respect to eukaryotic genome. In the last years a special interest in the field of mosquitoes’ genomics is highlighted by the completion of three genomic sequences (i.e. Anopheles gambiae, Aedes aegypti and Culex quinquefasciatus); this interest come from their role to function as vectors of virus-borne diseases. Few transposon families have been described in the Culex genus before the sequencing of C. quinquefasciatus genome. The genomic sequence analysis recently performed by Arensburger et al. (1) has revealed that nearly 30% of this genome is composed of TEs. The TE-related sequences described in Arensburger et al. were deposited in the TEfam database. The genome of C. quinquefasciatus has been analyzed using the LTR_STRUC program. Thirty novel families of LTR retrotransposons have been identified. Furthermore a group of non- autonomous elements has been identified, featured by tandem repeated sequences between the LTRs and apparently unrelated to any known Culex retrotransposon family. The potential role of the LTR-retrotransposon insertions on the host gene structure has been studied, and several insertions that may potentially contribute to the mature transcripts of endogenous genes have been identified. These results integrate the existing data on the genomics of an important disease vector.

The mitochondrial carriers are members of a family of transport proteins that mediate solute transport across the inner mitochondrial membrane. Two isoforms of the glutamate carriers, GC1 and GC2 (encoded by the SLC25A22 and SLC25A18 genes, respectively), have been identified in humans. Two independent mutations in SLC25A22 are associated with severe epileptic encephalopathy. In the present study we show that two genes (CG18347 and CG12201) phylogenetically related to the human GCs encoding genes are present in the D. melanogaster genome. We have functionally characterized the proteins encoded by CG18347 and CG12201, designated as DmGC1p and DmGC2p respectively, by overexpression in Escherichia coli and reconstitution into liposomes. Their transport properties demonstrate that DmGC1p and DmGC2p both catalyze the transport of glutamate across the inner mitochondrial membrane. Computational approaches have been used in order to highlight residues of DmGC1p and DmGC2p involved in substrate binding. Furthermore, gene expression analysis during development and in various adult tissues reveals that CG18347 is ubiquitously expressed in all examined D. melanogaster tissues, while the expression of CG12201 is strongly testis-biased. Finally, we identified mitochondrial glutamate carrier orthologs in 49 eukariotic species in order to attempt the reconstruction of the evolutionary history of the glutamate carrier function. Comparison of the exon/intron structure and other key features of the analyzed orthologs suggest that eukaryotic glutamate carrier genes descend from an intron-rich ancestral gene already present in the common ancestor of lineages that diverged as early as bilateria and radiata.

A set of 67 novel LTR-retrotransposon has been identified by in silico analyses of the Culex quinquefasciatus genome using the LTR_STRUC program. The phylogenetic analysis shows that 29 novel and putatively functional LTR-retrotransposons detected belong to the Ty3/gypsy group. Our results demonstrate that, by considering only families containing potentially autonomous LTR-retrotransposons, they account for about 1% of the genome of C. quinquefasciatus. In previous studies it has been estimated that 29% of the genome of C. quinquefasciatus is occupied by mobile genetic elements. The potential role of retrotransposon insertions strictly associated with host genes is described and discussed along with the possible origin of a retrotransposon with peculiar Primer Binding Site region. Finally, we report the presence of a group of 38 retrotransposons, carrying tandem repeated sequences but lacking coding potential, and apparently lacking ‘‘master copy’’ elements from which they could have originated. The features of the repetitive sequences found in these non- autonomous LTR retrotransposons are described, and their possible role discussed. These results integrate the existing data on the genomics of an important virus-borne disease vector.

The exact mechanism by which ectonucleotide pyrophosphatase phosphodiesterase 1 (ENPP1) inhibits insulin signaling is not known. ENPP1 contains two somatomedin-B-like domains (i.e. SMB 1 and 2) involved in ENPP1 dimerization in animal cells. The aim of the present study was to investigate if these domains modulate ENPP1 inhibitory activity on insulin signaling in human insulin target cells (HepG2). ENPP1 (ENPP1-3'myc), ENPP1 deleted of SMB 1 (ENPP1-ΔI-3'myc) or of SMB 2 (ENPP1-ΔII-3'myc) domain were cloned in frame with myc tag in mammalian expression vector pRK5. Plasmids were transiently transfected in human liver HepG2 cells. ENPP1 inhibitory activity on insulin signaling, dimerization and protein-protein interaction with insulin receptor (IR), reported to mediate the modulation of ENPP1 inhibitory activity, were studied. As compared to untransfected cells, a progressive increase of ENPP1 inhibitory activity on insulin-induced IR β-subunit autophosphorylation and on Akt-S473 phosphorylation was observed in ENPP1-3'myc, ENPP1-ΔI-3'myc and ENPP1-ΔII-3'myc cells. Under non reducing conditions a 260 kDa homodimer, indicating ENPP1 dimerization, was observed. The ratio of non reduced (260 kDa) to reduced (130 kDa) ENPP1 was significantly decreased by two thirds in ENPP1-ΔII-3'myc vs. ENPP1-3'myc but not in ENPP1-ΔI-3'myc. A similar ENPP1/ IR interaction was detectable by co-immunoprecipitation in ENPP1-3'myc, ENPP1-ΔI-3'myc and ENPP1-ΔII-3'myc cells. In conclusion, SMB 1 and SMB 2 are negative modulators of ENPP1 inhibitory activity on insulin signaling. For SMB 2 such effect might be mediated by a positive role on protein dimerization.

A peculiar form of hepatocerebral mtDNA depletion syndrome is caused by mutations in the MPV17 gene, which encodes a small hydrophobic protein of unknown function located in the mitochondrial inner membrane. In order to define the molecular basis of MPV17 variants associated with the human disorder, we have previously taken advantage of S. cerevisiae as a model system thanks to the presence of an MPV17 ortholog gene, SYM1. We demonstrate here that the SYM1 gene product is essential to maintain OXPHOS, glycogen storage, mitochondrial morphology and mtDNA stability in stressing conditions such as high temperature and ethanol-dependent growth. To gain insight into the molecular basis of the Sym1-less phenotype, we identified and characterized multicopy suppressor genes and metabolic suppressor compounds. Our results suggest that (i) metabolic impairment and mtDNA instability occur independently from each other as a consequence of SYM1 ablation; (ii) ablation of Sym1 causes depletion of glycogen storage, possibly due to defective anaplerotic flux of tricarboxylic acid (TCA) cycle intermediates to the cytosol; (iii) flattening of mitochondrial cristae in Sym1-defective organelles suggests a role for Sym1 in the structural preservation of the inner mitochondrial membrane, which could in turn control mtDNA maintenance and stability.

BACKGROUND: Bari-like transposons belong to the Tc1-mariner superfamily, and they have been identified in several genomes of the Drosophila genus. This transposon's family has been used as paradigm to investigate the complex dynamics underlying the persistence and structural evolution of transposable elements (TEs) within a genome. Three structural Bari variants have been identified so far and can be distinguished based on the organization of their terminal inverted repeats. Bari3 is the last discovered member of this family identified in Drosophila mojavensis, a recently emerged species of the Repleta group of the genus Drosophila. RESULTS: We studied the insertion pattern of Bari3 in different D. mojavensis populations and found evidence of recent transposition activity. Analysis of the transposase domains unveiled the presence of a functional nuclear localization signal, as well as a functional binding domain. Using luciferase-based assays, we investigated the promoter activity of Bari3 as well as the interaction of its transposase with its left terminus. The results suggest that Bari3 is transposition-competent. Finally we demonstrated transposase transcript processing when the transposase gene is overexpressed in vivo and in vitro. CONCLUSIONS: Bari3 displays very similar structural and functional features with its close relative, Bari1. Our results strongly suggest that Bari3 is an independent element that has generated genomic diversity in D. mojavensis. It can autonomously transcribe its transposase gene, which in turn can localize in the nucleus and bind the terminal inverted repeats of the transposon. Nevertheless, the identification of an unpredicted spliced form of the Bari3 transposase transcript allows us to hypothesize a control mechanism of its mobility based on mRNA processing. These results will aid the studies on the Bari family of transposons, which is intriguing for its widespread diffusion in Drosophilids coupled with a structural diversity generated during the evolution of Bari-like elements in their host genomes.

An intriguing gene necessary for the maintenance of mtDNA is human MPV17, mutation of which leads to a peculiar form of hepatocerebral mtDNA depletion syndrome (MDS). Even though Mpv17 mutations are one of the causes of MDS in humans and the discovery of this protein has been reported more than 20 years ago, its function is not yet understood. Originally considered as a peroxisomal membrane protein, it was later demonstrated that Mpv17 is localized to the inner mitochondrial membrane, as also previously demonstrated for the yeast orthologue Sym1, identified as a heat shock protein with a role in metabolism and/or tolerance to ethanol. With the aim of clarifying the role of MPV17 pathological alleles in MDS, we took advantage of S. cerevisiae as a model system. These studies in yeast have shed some light on the function of Sym1. The sym1 mutant mitochondria are morphologically abnormal, with flattened mitochondrial cristae and accumulation of electron-dense particles, suggesting a role for Sym1 in the structural preservation of the inner mitochondrial membrane. This defect is not a consequence of the mtDNA instability because it has been observed under cultural conditions where no defect of mtDNA was observed, indicating that the morphogenetic effects of Sym1 are likely to precede and possibly determine its effects on mtDNA stability. The phenotypes of double mutants (cit1 sym1, cit2 sym1) and the nature of multicopy suppressors (ODC1, YMC1) suggest for sym1 null mutant a defect in Krebs cycle confirmed by an enzymatic analysis that clearly indicates a heavy reduction of succinate dehydrogenase activity. Accordingly, sym1Δ displays a significant reduction in the amount of glycogen that is dependent on gluconeogenesis, which is in turn regulated by the anaplerotic flux of tricarboxylic acid intermediates from mitochondria to the cytosol. Interestingly, patients with Mpv17 mutations suffer from drastic, often fatal, hypoglycaemic crises, which are likely due to glycogen shortage in liver. Moreover blue-native gel electrophoresis immunovisualization clearly demonstrated that Sym1 is part of a high-molecular weight complex. While further work is necessary to identify the primary role of Sym1, including the molecular dissection and characterization of the Sym1-containing protein complex, these results indicate that Sym1 is involved in the structural and functional stability of the inner mitochondrial membrane, thus controlling crucial mechanisms related to this compartment, including respiratory chain complexes activity, mitochondria morphology and mtDNA maintenance.

The invention refers to 5'TIR nucleotide sequences derived from the "Bari" family of transposable elements for the use as promoter in the expression of a transgene in a recombinant genetic construct adapted to be transfected in a host cell system.