A novel evolutionary conserved mechanism of RNA stability regulates synexpression of primordial germ cell-specific genes prior to the sex-determination stage in medaka.

Dmrt1 is a highly conserved transcription factor, which is critically involved in regulation of gonad development of vertebrates. In medaka, a duplicate of dmrt1-acting as master sex-determining gene-has a tightly timely and spatially controlled gonadal expression pattern. In addition to transcriptional regulation, a sequence motif in the 3' UTR (D3U-box) mediates transcript stability of dmrt1 mRNAs from medaka and other vertebrates. We show here that in medaka, two RNA-binding proteins with antagonizing properties target this D3U-box, promoting either RNA stabilization in germ cells or degradation in the soma. The D3U-box is also conserved in other germ-cell transcripts, making them responsive to the same RNA binding proteins. The evolutionary conservation of the D3U-box motif within dmrt1 genes of metazoans-together with preserved expression patterns of the targeting RNA binding proteins in subsets of germ cells-suggest that this new mechanism for controlling RNA stability is not restricted to fishes but might also apply to other vertebrates.

The identification of patient-specific mutations reveals dual pathway activation in most patients with melanoma and activated receptor tyrosine kinases in BRAF/NRAS wild-type melanomas.

BACKGROUND: Increasing knowledge of cancer genomes has triggered the development of specific targeted inhibitors, thus providing a valuable therapeutic pool. METHODS: In this report, the authors analyze the presence of targetable alterations in 136 tumor samples from 92 patients with melanoma using a comprehensive approach based on targeted DNA sequencing and supported by RNA and protein analysis. Three topics of high clinical relevance are addressed: the identification of rare, activating alterations; the detection of patient-specific, co-occurring single nucleotide variants (SNVs) and copy number variations (CNVs) in parallel pathways; and the presence of cancer-relevant germline mutations. RESULTS: The analysis of patient-matched blood and tumor samples was done with a custom-designed gene panel that was enriched for genes from clinically targetable pathways. To detect alterations with high therapeutic relevance for patients with unknown driver mutations, genes that are untypical for melanoma also were included. Among all patients, CNVs were identified in one-third of samples and contained amplifications of druggable kinases, such as CDK4, ERBB2, and KIT. Considering SNVs and CNVs, 60% of patients with metastases exhibited co-occurring activations of at least 2 pathways, thus providing a rationale for individualized combination therapies. Unexpectedly, 9% of patients carry potentially protumorigenic germline mutations frequently affecting receptor tyrosine kinases. Remarkably two-thirds of BRAF/NRAS wild-type melanomas harbor activating mutations or CNVs in receptor tyrosine kinases. CONCLUSIONS: The results indicate that the integrated analysis of SNVs, CNVs, and germline mutations reveals new druggable targets for combination tumor therapy.

Sox5 is involved in germ-cell regulation and sex determination in medaka following co-option of nested transposable elements.

BACKGROUND: Sex determination relies on a hierarchically structured network of genes, and is one of the most plastic processes in evolution. The evolution of sex-determining genes within a network, by neo- or sub-functionalization, also requires the regulatory landscape to be rewired to accommodate these novel gene functions. We previously showed that in medaka fish, the regulatory landscape of the master male-determining gene dmrt1bY underwent a profound rearrangement, concomitantly with acquiring a dominant position within the sex-determining network. This rewiring was brought about by the exaptation of a transposable element (TE) called Izanagi, which is co-opted to act as a silencer to turn off the dmrt1bY gene after it performed its function in sex determination. RESULTS: We now show that a second TE, Rex1, has been incorporated into Izanagi. The insertion of Rex1 brought in a preformed regulatory element for the transcription factor Sox5, which here functions in establishing the temporal and cell-type-specific expression pattern of dmrt1bY. Mutant analysis demonstrates the importance of Sox5 in the gonadal development of medaka, and possibly in mice, in a dmrt1bY-independent manner. Moreover, Sox5 medaka mutants have complete female-to-male sex reversal. CONCLUSIONS: Our work reveals an unexpected complexity in TE-mediated transcriptional rewiring, with the exaptation of a second TE into a network already rewired by a TE. We also show a dual role for Sox5 during sex determination: first, as an evolutionarily conserved regulator of germ-cell number in medaka, and second, by de novo regulation of dmrt1 transcriptional activity during primary sex determination due to exaptation of the Rex1 transposable element.

Infectious complications in patients with myelodysplastic syndromes: A review of the literature with emphasis on patients treated with 5-azacitidine.

Myelodysplastic Syndromes are oligo-clonal stem cell disorders that are associated with cytopenias in the peripheral blood. Major causes for morbidity and mortality in myelodysplastic syndromes (MDS) patients are infections mostly due to bacteria or fungi. Beside leucopenia per se in affected patients, function of white blood cells particularly that of neutrophils seems to be impaired. Here we summarize the available data on infections in MDS patients in general and particularly those treated with 5-azacitidine.

Phenotypic and functional characterization of neutrophils and monocytes from patients with myelodysplastic syndrome by flow cytometry.

Myelodysplastic syndrome (MDS) is a clonal stem cell disorder frequently associated with inefficient granulopoiesis showing dysplastic polymorphonuclear neutrophils (PMNs). To assess PMN functionality in MDS in a clinical routine setting, 30 MDS patients and ten healthy volunteers were analyzed for PMN and monocyte phenotype and function (degranulation, CD62L shedding, oxidative burst and phagocytosis) upon stimulation with lipopolysaccharide by multi-color flow cytometry (MCFC). Our data show a heterogeneous pattern for CD66, CD16 and CD64 expression on PMNs of MDS patients. CD62L shedding rate and CD66 degranulation were reduced. Interestingly, we detected correlations between the WHO adapted prognostic scoring system (WPSS) and CD16 expression on PMNs as well as the international prognostic scoring system (IPSS) and CD11b degranulation by MCFC, suggesting clinical relevance of MCFC based function testing. In conclusion, MCFC of myelodysplastic immunophenotypes and PMN functionality are applicable in clinical settings, but further prospective studies are needed to assess the practical clinical value of such analyses.

Divergent expression regulation of gonad development genes in medaka shows incomplete conservation of the downstream regulatory network of vertebrate sex determination.

Genetic control of male or female gonad development displays between different groups of organisms a remarkable diversity of "master sex-determining genes" at the top of the genetic hierarchies, whereas downstream components surprisingly appear to be evolutionarily more conserved. Without much further studies, conservation of sequence has been equalized to conservation of function. We have used the medaka fish to investigate the generality of this paradigm. In medaka, the master male sex-determining gene is dmrt1bY, a highly conserved downstream regulator of sex determination in vertebrates. To understand its function in orchestrating the complex gene regulatory network, we have identified targets genes and regulated pathways of Dmrt1bY. Monitoring gene expression and interactions by transgenic fluorescent reporter fish lines, in vivo tissue-chromatin immunoprecipitation and in vitro gene regulation assays revealed concordance but also major discrepancies between mammals and medaka, notably amongst spatial, temporal expression patterns and regulations of the canonical Hedgehog and R-spondin/Wnt/Follistatin signaling pathways. Examination of Foxl2 protein distribution in the medaka ovary defined a new subpopulation of theca cells, where ovarian-type aromatase transcriptional regulation appears to be independent of Foxl2. In summary, these data show that the regulation of the downstream regulatory network of sex determination is less conserved than previously thought.

Zisupton--a novel superfamily of DNA transposable elements recently active in fish.

Transposable elements are widespread mobile DNA sequences able to integrate into new locations within genomes. Through transposition and recombination, they significantly contribute to genome plasticity and evolution. They can also regulate gene expression and provide regulatory and coding sequences (CDSs) for the evolution of novel gene functions. We have identified a new superfamily of DNA transposon on the Y chromosome of the platyfish Xiphophorus maculatus. This element is 11 kb in length and carries a single CDS of 24 exons. The N-terminal part of the putative protein, which is expressed in all adult tissues tested, contains several nucleic acid- and protein-binding domains and might correspond to a novel type of transposase/integrase not described so far in any transposon. In addition, a testis-specific splice isoform encodes a C-terminal Ulp1 SUMO protease domain, suggesting a function in posttranslational protein modification mediated by SUMO and/or ubiquitin small peptides. Accordingly, this element was called Zisupton, for Zinc finger SUMO protease transposon. Beside the Y-chromosomal sequence, five other very similar copies were identified in the platyfish genome. All copies are delimited by 99-bp conserved subterminal inverted repeats and flanked by copy-specific 8-nt target site duplications reflecting their integration at different positions in the genome. Zisupton elements are inserted at different genomic locations in different poeciliid species but also in different populations of X. maculatus. Such insertion polymorphisms between related species and populations indicate relatively recent transposition activity, with a high degree of nucleotide identity between species suggesting possible implication of horizontal gene transfer. Zisupton sequences were detected in other fish species, in urochordates, cephalochordates, and hemichordates as well as in more distant organisms, such as basidiomycete fungi, filamentous brown algae, and green algae. Possible examples of nuclear genes derived from Zisupton have been identified. To conclude, our analysis has uncovered a new superfamily of DNA transposons with potential roles in genome diversity and evolutionary innovation in fish and other organisms.

Transcriptional rewiring of the sex determining dmrt1 gene duplicate by transposable elements.

Control and coordination of eukaryotic gene expression rely on transcriptional and posttranscriptional regulatory networks. Evolutionary innovations and adaptations often require rapid changes of such networks. It has long been hypothesized that transposable elements (TE) might contribute to the rewiring of regulatory interactions. More recently it emerged that TEs might bring in ready-to-use transcription factor binding sites to create alterations to the promoters by which they were captured. A process where the gene regulatory architecture is of remarkable plasticity is sex determination. While the more downstream components of the sex determination cascades are evolutionary conserved, the master regulators can switch between groups of organisms even on the interspecies level or between populations. In the medaka fish (Oryzias latipes) a duplicated copy of dmrt1, designated dmrt1bY or DMY, on the Y chromosome was shown to be the master regulator of male development, similar to Sry in mammals. We found that the dmrt1bY gene has acquired a new feedback downregulation of its expression. Additionally, the autosomal dmrt1a gene is also able to regulate transcription of its duplicated paralog by binding to a unique target Dmrt1 site nested within the dmrt1bY proximal promoter region. We could trace back this novel regulatory element to a highly conserved sequence within a new type of TE that inserted into the upstream region of dmrt1bY shortly after the duplication event. Our data provide functional evidence for a role of TEs in transcriptional network rewiring for sub- and/or neo-functionalization of duplicated genes. In the particular case of dmrt1bY, this contributed to create new hierarchies of sex-determining genes.

The Developmental and Genetic Architecture of the Sexually Selected Male Ornament of Swordtails.

Sexual selection results in sex-specific characters like the conspicuously pigmented extension of the ventral tip of the caudal fin-the "sword"-in males of several species of Xiphophorus fishes. To uncover the genetic architecture underlying sword formation and to identify genes that are associated with its development, we characterized the sword transcriptional profile and combined it with genetic mapping approaches. Results showed that the male ornament of swordtails develops from a sexually non-dimorphic prepattern of transcription factors in the caudal fin. Among genes that constitute the exclusive sword transcriptome and are located in the genomic region associated with this trait we identify the potassium channel, Kcnh8, as a sword development gene. In addition to its neural function kcnh8 performs a known role in fin growth. These findings indicate that during evolution of swordtails a brain gene has been co-opted for an additional novel function in establishing a male ornament.

The sterlet sturgeon genome sequence and the mechanisms of segmental rediploidization.

Sturgeons seem to be frozen in time. The archaic characteristics of this ancient fish lineage place it in a key phylogenetic position at the base of the ~30,000 modern teleost fish species. Moreover, sturgeons are notoriously polyploid, providing unique opportunities to investigate the evolution of polyploid genomes. We assembled a high-quality chromosome-level reference genome for the sterlet, Acipenser ruthenus. Our analysis revealed a very low protein evolution rate that is at least as slow as in other deep branches of the vertebrate tree, such as that of the coelacanth. We uncovered a whole-genome duplication that occurred in the Jurassic, early in the evolution of the entire sturgeon lineage. Following this polyploidization, the rediploidization of the genome included the loss of whole chromosomes in a segmental deduplication process. While known adaptive processes helped conserve a high degree of structural and functional tetraploidy over more than 180 million years, the reduction of redundancy of the polyploid genome seems to have been remarkably random.

Free iron ions decrease indoleamine 2,3-dioxygenase expression and reduce IFNgamma-induced inhibition of Chlamydia trachomatis infection.

Interferon-gamma (IFNgamma)-mediated indoleamine 2,3-dioxygenase (IDO) expression, important in innate immunity, immune suppression, and tolerance, can be counteracted by ferrous iron (FeSO(4)). Elevation of intracellular iron levels during stimulation with IFNgamma impeded IFNgamma-induced IDO mRNA and protein expression in HEp-2 cells. Decreased IDO expression was accompanied by decreased tryptophan degradation. Accordingly, IFNgamma-mediated suppressing effects on Chlamydia trachomatis (CT) infection were reduced or even abolished in the presence of FeSO(4). Conversely, lowering intracellular iron levels by deferoxamine (DFO) did not increase IFNgamma-induced IDO expression but potentiated Chlamydia-suppressing effects by lowering intracellular iron availability. Additionally, DFO led to a CT-induced IDO expression in HEp-2 cells not treated with IFNgamma. In summary, this study demonstrates that iron acts as a regulatory element for modulating IDO expression, in addition to its function as an essential element for chlamydial growth. This may represent an important control mechanism of IDO expression at the transcriptional level.

The Piranha Genome Provides Molecular Insight Associated to Its Unique Feeding Behavior.

The piranha enjoys notoriety due to its infamous predatory behavior but much is still not understood about its evolutionary origins and the underlying molecular mechanisms for its unusual feeding biology. We sequenced and assembled the red-bellied piranha (Pygocentrus nattereri) genome to aid future phenotypic and genetic investigations. The assembled draft genome is similar to other related fishes in repeat composition and gene count. Our evaluation of genes under positive selection suggests candidates for adaptations of piranhas' feeding behavior in neural functions, behavior, and regulation of energy metabolism. In the fasted brain, we find genes differentially expressed that are involved in lipid metabolism and appetite regulation as well as genes that may control the aggression/boldness behavior of hungry piranhas. Our first analysis of the piranha genome offers new insight and resources for the study of piranha biology and for feeding motivation and starvation in other organisms.

Evolution of melanocortin receptors in teleost fish: the melanocortin type 1 receptor.

The melanocortin type 1 receptor (Mc1r) belongs to a family of G-protein-coupled receptors involved in various physiological processes in vertebrates. Melanocortins, the Mcr natural agonists, are pituitary peptide hormones including adrenocorticotropin and melanocyte-stimulating hormones. In mammals and birds, Mc1r is involved in pigmentation and expressed in melanocytes and melanoma. Activation of Mc1r leads to eumelanin production as well as to proliferation and survival of melanocytes in the epidermis. Here we report the molecular and evolutionary analysis of mc1r from three major fish models, the zebrafish Danio rerio, the medaka Oryzias latipes and the platyfish Xiphophorus maculatus. In contrast to some other melanocortin receptor genes, mc1r has been conserved as a single copy gene in divergent fish species. Its expression was detected in all organs tested in platyfish and medaka but was restricted to eyes, skin, brain and testis in zebrafish, this possibly reflecting differences in the distribution of extracutaneous melanophores. The mc1r gene was found to be expressed during embryogenesis, as well as in Xiphophorus hybrid melanoma, similar to human tumours. Protein sequence comparison between fish and mammalian Mc1r revealed a remarkable concordance between evolutionary and functional analyses for the identification of residues and regions critical for receptor function.

RNA interference: PCR strategies for the quantification of stable degradation-fragments derived from siRNA-targeted mRNAs.

mRNA targeted by siRNA is endogeneously cleaved into a 5'- and a 3'-fragment and finally degraded in cells. Little is known about the relative stability and degradation kinetics of these 5'- and 3'-fragments after the siRNA mediated first cut. We present a qRT-PCR protocol which allows the determination of the optimal time point for mRNA analyses, helping to avoid the generation of false positive effects in downstream experiments, such as microarray analysis, which may be caused by undegraded fragments of a siRNA-targeted mRNA.

Gene amplification and functional diversification of melanocortin 4 receptor at an extremely polymorphic locus controlling sexual maturation in the platyfish.

In two swordtail species of the genus Xiphophorus, the onset of puberty has been shown to be modulated at the P locus by sequence polymorphism and gene copy-number variation affecting the type 4 melanocortin hormone receptor Mc4r. The system works through the interaction of two allelic types, one encoding wild type and the other dominant-negative receptors. We have analyzed the structure and evolution of the P locus in the platyfish Xiphophorus maculatus, where as many as nine alleles of P determining the onset of sexual maturity in males and females, fecundity in females, and adult size in males are located on both the X and Y chromosomes in a region linked to the master sex-determining locus. In this species, mc4r has been amplified to up to 10 copies on both the X and Y chromosomes through recent large serial duplications. Subsequently, mc4r paralogues have diverged considerably into many different subtypes. Certain copies have acquired new untranslated regions through genomic rearrangements, and transposable element insertions and other mutations have accumulated in promoter regions, possibly explaining observed deviations from the classical mc4r transcriptional pattern. In the mc4r-coding sequence, in-frame insertions and deletions as well as nonsense and missense mutations have generated a high diversity of Mc4r-predicted proteins. Most of these variants are expressed in embryos, adults, and/or tumors. Functional receptor characterization demonstrated major divergence in pharmacological behavior for Mc4r receptors encoded by different copies of platyfish mc4r, with differences in constitutive activity as well as binding and stimulation by hormones. The high degree of allelic and copy-number variation observed between individuals can explain the high level of polymorphism for sexual maturation, fecundity, and body size in the platyfish: multiple combinations of Mc4r variants with different biochemical properties might interact to modulate the melanocortin signaling that regulates the hypothalamus-pituitary-gonadal axis.

Liver hyperplasia after tamoxifen induction of Myc in a transgenic medaka model.

Myc is a global transcriptional regulator and one of the most frequently overexpressed oncoproteins in human tumors. It is well established that activation of Myc leads to enhanced cell proliferation but can also lead to increased apoptosis. The use of animal models expressing deregulated levels of Myc has helped to both elucidate its function in normal cells and give insight into how Myc initiates and maintains tumorigenesis. Analyses of the medaka (Oryzias latipes) genome uncovered the unexpected presence of two Myc gene copies in this teleost species. Comparison of these Myc versions to other vertebrate species revealed that one gene, myc17, differs by the loss of some conserved regulatory protein motifs present in all other known Myc genes. To investigate how such differences might affect the basic biological functions of Myc, we generated a tamoxifen-inducible in vivo model utilizing a natural, fish-specific Myc gene. Using this model we show that, when activated, Myc17 leads to increased proliferation and to apoptosis in a dose-dependent manner, similar to human Myc. We have also shown that long-term Myc17 activation triggers liver hyperplasia in adult fish, allowing this newly established transgenic medaka model to be used to study the transition from hyperplasia to liver cancer and to identify Myc-induced tumorigenesis modifiers.

Determination of onset of sexual maturation and mating behavior by melanocortin receptor 4 polymorphisms.

Polymorphisms in reproductive strategies are among the most extreme and complex in nature. A prominent example is male body size and the correlated reproductive strategies in some species of platyfish and swordtails of the genus Xiphophorus. This polymorphism is controlled by a single Mendelian locus (P) that determines the onset of sexual maturity of males. Because males cease growth after reaching puberty, this results in a marked size polymorphism. The different male size classes show pronounced behavioral differences (e.g., courtship versus sneak mating), and females prefer large over small males. We show that sequence polymorphisms of the melanocortin receptor 4 gene (mc4r) comprise both functional and non-signal-transducing versions and that variation in copy number of mc4r genes on the Y chromosome underlies the P locus polymorphism. Nonfunctional Y-linked mc4r copies in larger males act as dominant-negative mutations and delay the onset of puberty. Copy number variation, as a regulating mechanism, endows this system with extreme genetic flexibility that generates extreme variation in phenotype. Because Mc4r is critically involved in regulation of body weight and appetite, a novel link between the physiological system controlling energy balance and the regulation of reproduction becomes apparent.

Molecular analysis of the sex chromosomes of the platyfish Xiphophorus maculatus: Towards the identification of a new type of master sexual regulator in vertebrates.

In contrast to mammals and birds, fish display an amazing diversity of genetic sex determination systems, with frequent changes during evolution possibly associated with the emergence of new sex chromosomes and sex-determining genes. To better understand the molecular and evolutionary mechanisms driving this diversity, several fish models are studied in parallel. Besides the medaka (Oryzias latipes Temminck and Schlegel, 1846) for which the master sex-determination gene has been identified, one of the most advanced models for studying sex determination is the Southern platyfish (Xiphophorus maculatus, Günther 1966). Xiphophorus maculatus belongs to the Poeciliids, a family of live-bearing freshwater fish, including platyfish, swordtails and guppies that perfectly illustrates the diversity of genetic sex-determination mechanisms observed in teleosts. For X. maculatus, bacterial artificial chromosome contigs covering the sex-determination region of the X and Y sex chromosomes have been constructed. Initial molecular analysis demonstrated that the sex-determination region is very unstable and frequently undergoes duplications, deletions, inversions and other rearrangements. Eleven gene candidates linked to the master sex-determining gene have been identified, some of them corresponding to pseudogenes. All putative genes are present on both the X and the Y chromosomes, suggesting a poor degree of differentiation and a young evolutionary age for platyfish sex chromosomes. When compared with other fish and tetrapod genomes, syntenies were detected only with autosomes. This observation supports an independent origin of sex chromosomes, not only in different vertebrate lineages but also between different fish species.

Peptidomic analysis of human peripheral monocytes persistently infected by Chlamydia trachomatis.

Peptidomic analysis using Differential Peptide Display (DPD) of human peripheral blood mononuclear cells (PBMC) mock-infected or persistently infected by Chlamydia trachomatis (CT) revealed 10 peptides, expressed upon CT infection. Analysis of these 10 candidates by tandem mass spectrometry enabled the determination of seven candidates as fragments from the precursors (I) ferritin heavy chain subunit, (II) HLA class II histocompatibility antigen, (III) vimentin, (IV) indoleamine 2,3-dioxygenase, (V and VI) pre-B cell enhancing factor (PBEF), and (VII) Interleukin-8 (CXCL8). The identified candidates proved the presence of anti-bacterial and immunologically active monocytic proteins after CT infection.

Male-biased expression of X-chromosomal DM domain-less Dmrt8 genes in the mouse.

The vertebrate DMRT gene family encodes putative transcription factors related to the sexual regulators Doublesex (Drosophila melanogaster) and MAB-3 (Caenorhabditis elegans). They share a highly conserved DNA binding motif, the DM domain. In human and mouse seven DMRT genes (DMRT1-DMRT7) have been analyzed. DMRT8, a gene related to DMRT7, is located on the X chromosome in placental mammals. While DMRT8 is single copy in most mammals, three copies are present in mouse, rat, and rabbit. Despite the loss of the DM domain, DMRT8 genes have been maintained in the mammalian lineage, suggesting a DM domain-independent function. In adult mouse, two Dmrt8 genes are expressed exclusively in testis. Dmrt8.1 mRNA was detected in Sertoli cells by in situ hybridization. In embryos, Dmrt8.2 shows a dynamic expression restricted to male and female gonads and might therefore be involved in sexual development in the mouse.