The hagfish genome and the evolution of vertebrates.

As the only surviving lineages of jawless fishes, hagfishes and lampreys provide a crucial window into early vertebrate evolution1-3. Here we investigate the complex history, timing and functional role of genome-wide duplications4-7 and programmed DNA elimination8,9 in vertebrates in the light of a chromosome-scale genome sequence for the brown hagfish Eptatretus atami. Combining evidence from syntenic and phylogenetic analyses, we establish a comprehensive picture of vertebrate genome evolution, including an auto-tetraploidization (1RV) that predates the early Cambrian cyclostome-gnathostome split, followed by a mid-late Cambrian allo-tetraploidization (2RJV) in gnathostomes and a prolonged Cambrian-Ordovician hexaploidization (2RCY) in cyclostomes. Subsequently, hagfishes underwent extensive genomic changes, with chromosomal fusions accompanied by the loss of genes that are essential for organ systems (for example, genes involved in the development of eyes and in the proliferation of osteoclasts); these changes account, in part, for the simplification of the hagfish body plan1,2. Finally, we characterize programmed DNA elimination in hagfish, identifying protein-coding genes and repetitive elements that are deleted from somatic cell lineages during early development. The elimination of these germline-specific genes provides a mechanism for resolving genetic conflict between soma and germline by repressing germline and pluripotency functions, paralleling findings in lampreys10,11. Reconstruction of the early genomic history of vertebrates provides a framework for further investigations of the evolution of cyclostomes and jawed vertebrates.

A novel molecular rotor facilitates detection of p53-DNA interactions using the Fluorescent Intercalator Displacement Assay.

We have investigated the use of fluorescent molecular rotors as probes for detection of p53 binding to DNA. These are a class of fluorophores that undergo twisted intramolecular charge transfer (TICT). They are non-fluorescent in a freely rotating conformation and experience a fluorescence increase when restricted in the planar conformation. We hypothesized that intercalation of a molecular rotor between DNA base pairs would result in a fluorescence turn-on signal. Upon displacement by a DNA binding protein, measurable loss of signal would facilitate use of the molecular rotor in the fluorescent intercalator displacement (FID) assay. A panel of probes was interrogated using the well-established p53 model system across various DNA response elements. A novel, readily synthesizable molecular rotor incorporating an acridine orange DNA intercalating group (AO-R) outperformed other conventional dyes in the FID assay. It enabled relative measurement of p53 sequence-specific DNA interactions and study of the dominant-negative effects of cancer-associated p53 mutants. In a further application, AO-R also proved useful for staining apoptotic cells in live zebrafish embryos.

The p53-Mdm2 interaction and the E3 ligase activity of Mdm2/Mdm4 are conserved from lampreys to humans.

The extant jawless vertebrates, represented by lampreys and hagfish, are the oldest group of vertebrates and provide an interesting genomic evolutionary pivot point between invertebrates and jawed vertebrates. Through genome analysis of one of these jawless vertebrates, the Japanese lamprey (Lethenteron japonicum), we identified all three members of the important p53 transcription factor family--Tp53, Tp63, and Tp73--as well as the Mdm2 and Mdm4 genes. These genes and their products are significant cellular regulators in human cancer, and further examination of their roles in this most distant vertebrate relative sheds light on their origin and coevolution. Their important role in response to DNA damage has been highlighted by the discovery of multiple copies of the Tp53 gene in elephants. Expression of lamprey p53, Mdm2, and Mdm4 proteins in mammalian cells reveals that the p53-Mdm2 interaction and the Mdm2/Mdm4 E3 ligase activity existed in the common ancestor of vertebrates and have been conserved for >500 million years of vertebrate evolution. Lamprey Mdm2 degrades human p53 with great efficiency, but this interaction is not blocked by currently available small molecule inhibitors of the human HDM2 protein, suggesting utility of lamprey Mdm2 in the study of the human p53 signaling pathway.

Characterization of the Runx gene family in a jawless vertebrate, the Japanese lamprey (Lethenteron japonicum).

The cyclostomes (jawless vertebrates), comprising lampreys and hagfishes, are the sister group of jawed vertebrates (gnathostomes) and are hence an important group for the study of vertebrate evolution. In mammals, three Runx genes, Runx1, Runx2 and Runx3, encode transcription factors that are essential for cell proliferation and differentiation in major developmental pathways such as haematopoiesis, skeletogenesis and neurogenesis and are frequently associated with diseases. We describe here the characterization of Runx gene family members from a cyclostome, the Japanese lamprey (Lethenteron japonicum). The Japanese lamprey contains three Runx genes, RunxA, RunxB, and RunxC. However, phylogenetic and synteny analyses suggest that they are not one-to-one orthologs of gnathostome Runx1, Runx2 and Runx3. The major protein domains and motifs found in gnathostome Runx proteins are highly conserved in the lamprey Runx proteins. Although all gnathostome Runx genes each contain two alternative promoters, P1 (distal) and P2 (proximal), only lamprey RunxB possesses the alternative promoters; lamprey RunxA and RunxC contain only P2 and P1 promoter, respectively. Furthermore, the three lamprey Runx genes give rise to fewer alternative isoforms than the three gnathostome Runx genes. The promoters of the lamprey Runx genes lack the tandem Runx-binding motifs that are highly conserved among the P1 promoters of gnathostome Runx1, Runx2 and Runx3 genes; instead these promoters contain dispersed single Runx-binding motifs. The 3'UTR of lamprey RunxB contains binding sites for miR-27 and miR-130b/301ab, which are conserved in mammalian Runx1 and Runx3, respectively. Overall, the Runx genes in lamprey seem to have experienced a different evolutionary trajectory from that of gnathostome Runx genes which are highly conserved all the way from cartilaginous fishes to mammals.

Molecular rotors as conditionally fluorescent labels for rapid detection of biomolecular interactions.

We demonstrate the use of fluorescent molecular rotors as probes for detecting biomolecular interactions, specifically peptide-protein interactions. Molecular rotors undergo twisted intramolecular charge transfer upon irradiation, relax via the nonradiative torsional relaxation pathway, and have been typically used as viscosity probes. Their utility as a tool for detecting specific biomolecular interactions has not been explored. Using the well characterized p53-Mdm2 interaction as a model system, we designed a 9-(2-carboxy-2-cyanovinyl) julolidine-based p53 peptide reporter, JP1-R, which fluoresces conditionally only upon Mdm2 binding. The reporter was used in a rapid, homogeneous assay to screen a fragment library for antagonists of the p53-Mdm2 interaction, and several inhibitors were identified. Subsequent validation of these hits using established secondary assays suggests increased sensitivity afforded by JP1-R. The fluorescence of molecular rotors contingent upon target binding makes them a versatile tool for detecting specific biomolecular interactions.

Integrative genome-wide analysis reveals cooperative regulation of alternative splicing by hnRNP proteins.

Understanding how RNA binding proteins control the splicing code is fundamental to human biology and disease. Here, we present a comprehensive study to elucidate how heterogeneous nuclear ribonucleoparticle (hnRNP) proteins, among the most abundant RNA binding proteins, coordinate to regulate alternative pre-mRNA splicing (AS) in human cells. Using splicing-sensitive microarrays, crosslinking and immunoprecipitation coupled with high-throughput sequencing (CLIP-seq), and cDNA sequencing, we find that more than half of all AS events are regulated by multiple hnRNP proteins and that some combinations of hnRNP proteins exhibit significant synergy, whereas others act antagonistically. Our analyses reveal position-dependent RNA splicing maps, in vivo consensus binding sites, a surprising level of cross- and autoregulation among hnRNP proteins, and the coordinated regulation by hnRNP proteins of dozens of other RNA binding proteins and genes associated with cancer. Our findings define an unprecedented degree of complexity and compensatory relationships among hnRNP proteins and their splicing targets that likely confer robustness to cells.

Conservation of all three p53 family members and Mdm2 and Mdm4 in the cartilaginous fish.

Analysis of the genome of the elephant shark (Callorhinchus milii), a member of the cartilaginous fishes (Class Chondrichthyes), reveals that it encodes all three members of the p53 gene family, p53, p63 and p73, each with clear homology to the equivalent gene in bony vertebrates (Class Osteichthyes). Thus, the gene duplication events that lead to the presence of three family members in the vertebrates dates to before the Silurian era. It also encodes Mdm2 and Mdm4 genes but does not encode the p19(Arf) gene. Detailed comparison of the amino acid sequences of these proteins in the vertebrates reveals that they are evolving at highly distinctive rates, and this variation occurs not only between the three family members but extends to distinct domains in each protein.

A chemical genetic screen in Mycobacterium tuberculosis identifies carbon-source-dependent growth inhibitors devoid of in vivo efficacy.

Candidate antibacterials are usually identified on the basis of their in vitro activity. However, the apparent inhibitory activity of new leads can be misleading because most culture media do not reproduce an environment relevant to infection in vivo. In this study, while screening for novel anti-tuberculars, we uncovered how carbon metabolism can affect antimicrobial activity. Novel pyrimidine-imidazoles (PIs) were identified in a whole-cell screen against Mycobacterium tuberculosis. Lead optimization generated in vitro potent derivatives with desirable pharmacokinetic properties, yet without in vivo efficacy. Mechanism of action studies linked the PI activity to glycerol metabolism, which is not relevant for M. tuberculosis during infection. PIs induced self-poisoning of M. tuberculosis by promoting the accumulation of glycerol phosphate and rapid ATP depletion. This study underlines the importance of understanding central bacterial metabolism in vivo and of developing predictive in vitro culture conditions as a prerequisite for the rational discovery of new antibiotics.

Parathyroid hormone gene family in a cartilaginous fish, the elephant shark (Callorhinchus milii).

The development of bone was a major step in the evolution of vertebrates. A bony skeleton provided structural support and a calcium reservoir essential for the movement from an aquatic to a terrestrial environment. Cartilaginous fishes are the oldest living group of jawed vertebrates. In this study we have identified three members of the parathyroid hormone (Pth) gene family in a cartilaginous fish, the elephant shark (Callorhinchus milii). The three genes include two Pth genes, designated as Pth1 and Pth2, and a Pthrp gene. Phylogenetic analysis suggested that elephant shark Pth2 is an ancient gene whose orthologue is lost in bony vertebrates. The Pth1 and Pth2 genes have the same structure as the Pth gene in bony vertebrates, whereas the structure of the Pthrp gene is more complex in tetrapods compared with elephant shark. The three elephant shark genes showed distinct patterns of expression, with Pth2 being expressed only in the brain and spleen. This contrasts with localization of the corresponding proteins, which showed considerable overlap in their distribution. There were conserved sites of localization for Pthrp between elephant shark and mammals, including tissues such as kidney, skin, skeletal and cardiac muscle, pancreas, and cartilage. The elephant shark Pth1(1-34) and Pthrp(1-34) peptides were able to stimulate cAMP accumulation in mammalian UMR106.01 cells. However, Pth2(1-34) peptide did not show such PTH-like biologic activity. The presence of Pth and Pthrp genes in the elephant shark indicates that these genes played fundamental roles before their recruitment to bone development in bony jawed vertebrates.

Characterization of a hypoxia-response element in the Epo locus of the pufferfish, Takifugu rubripes.

Animals respond to hypoxia by increasing synthesis of the glycoprotein hormone erythropoietin (Epo) which in turn stimulates the production of red blood cells. The gene encoding Epo has been recently cloned in teleost fishes such as the pufferfish Takifugu rubripes (fugu) and zebrafish (Danio rerio). It has been shown that the transcription levels of Epo in teleost fishes increase in response to anemia or hypoxia in a manner similar to its human ortholog. However, the cis-regulatory element(s) mediating the hypoxia response of Epo gene in fishes has not been identified. In the present study, using the human hepatoma cell line (Hep3B), we have identified and characterized a hypoxia response element (HRE) in the fugu Epo locus. The sequence of the fugu HRE (ACGTGCTG) is identical to that of the HRE in the human EPO locus. However, unlike the HRE in the mammalian Epo locus, which is located in the 3' region of the gene, the fugu HRE is located in the 5' flanking region and on the opposite strand of DNA. This HRE is conserved in other teleosts such as Tetraodon and zebrafish in a similar location. A 365-bp fragment containing the fugu HRE was able to drive GFP expression in the liver of transgenic zebrafish. However, we could not ascertain if the expression of transgene is induced by hypoxia in vivo due to the low and variable levels of GFP expression in transgenic zebrafish. Our investigations also revealed that the Epo locus has experienced extensive rearrangements during vertebrate evolution.

Short Promoters in Viral Vectors Drive Selective Expression in Mammalian Inhibitory Neurons, but do not Restrict Activity to Specific Inhibitory Cell-Types.

Short cell-type specific promoter sequences are important for targeted gene therapy and studies of brain circuitry. We report on the ability of short promoter sequences to drive fluorescent protein expression in specific types of mammalian cortical inhibitory neurons using adeno-associated virus (AAV) and lentivirus (LV) vectors. We tested many gene regulatory sequences derived from fugu (Takifugu rubripes), mouse, human, and synthetic composite regulatory elements. All fugu compact promoters expressed in mouse cortex, with only the somatostatin (SST) and the neuropeptide Y (NPY) promoters largely restricting expression to GABAergic neurons. However these promoters did not control expression in inhibitory cells in a subtype specific manner. We also tested mammalian promoter sequences derived from genes putatively coexpressed or coregulated within three major inhibitory interneuron classes (PV, SST, VIP). In contrast to the fugu promoters, many of the mammalian sequences failed to express, and only the promoter from gene A930038C07Rik conferred restricted expression, although as in the case of the fugu sequences, this too was not inhibitory neuron subtype specific. Lastly and more promisingly, a synthetic sequence consisting of a composite regulatory element assembled with PAX6 E1.1 binding sites, NRSE and a minimal CMV promoter showed markedly restricted expression to a small subset of mostly inhibitory neurons, but whose commonalities are unknown.

Neuropeptide Y-family peptides and receptors in the elephant shark, Callorhinchus milii confirm gene duplications before the gnathostome radiation.

We describe here the repertoire of neuropeptide Y (NPY) peptides and receptors in the elephant shark Callorhinchus milii, belonging to the chondrichthyans that diverged from the rest of the gnathostome (jawed vertebrate) lineage about 450 million years ago and the first chondrichthyan with a genome project. We have identified two peptide genes that are orthologous to NPY and PYY (peptide YY) in other vertebrates, and seven receptor genes orthologous to the Y1, Y2, Y4, Y5, Y6, Y7 and Y8 subtypes found in tetrapods and teleost fishes. The repertoire of peptides and receptors seems to reflect the ancestral configuration in the predecessor of all gnathostomes, whereas other lineages such as mammals and teleosts have lost one or more receptor genes or have acquired 1-2 additional peptide genes. Both the peptides and receptors showed broad and overlapping mRNA expression which may explain why some receptor gene losses could take place in some lineages, but leaves open the question why all the known ancestral receptors have been retained in the elephant shark.

Genetic alterations in the tyrosine kinase transcriptome of human cancer cell lines.

Protein tyrosine kinases (PTKs) play a critical role in the manifestation of cancer cell properties, and respective signaling mechanisms have been studied extensively on immortalized tumor cells. To characterize and analyze commonly used cancer cell lines with regard to variations in the primary structure of all expressed PTKs, we conducted a cDNA-based sequence analysis of the entire tyrosine kinase transcriptome of 254 established tumor cell lines. The profiles of cell line intrinsic PTK transcript alterations and the evaluation of 155 identified polymorphisms and 234 somatic mutations are made available in a database designated "Tykiva" (tyrosine kinome variant). Tissue distribution analysis and/or the localization within defined protein domains indicate functional relevance of several genetic alterations. The cysteine replacement of the highly conserved Y367 residue in fibroblast growth factor receptor 4 or the Q26X nonsense mutation in the tumor-suppressor kinase CSK are examples, and may contribute to cell line-specific signaling characteristics and tumor progression. Moreover, known variants, such as epidermal growth factor receptor G719S, that were shown to mediate anticancer drug sensitivity could be detected in other than the previously reported tumor types. Our data therefore provide extensive system information for the design and interpretation of cell line-based cancer research, and may stimulate further investigations into broader clinical applications of current cancer therapeutics.

TFCONES: a database of vertebrate transcription factor-encoding genes and their associated conserved noncoding elements.

BACKGROUND: Transcription factors (TFs) regulate gene transcription and play pivotal roles in various biological processes such as development, cell cycle progression, cell differentiation and tumor suppression. Identifying cis-regulatory elements associated with TF-encoding genes is a crucial step in understanding gene regulatory networks. To this end, we have used a comparative genomics approach to identify putative cis-regulatory elements associated with TF-encoding genes in vertebrates. DESCRIPTION: We have created a database named TFCONES (Transcription Factor Genes & Associated COnserved Noncoding ElementS) (http://tfcones.fugu-sg.org) which contains all human, mouse and fugu TF-encoding genes and conserved noncoding elements (CNEs) associated with them. The CNEs were identified by gene-by-gene alignments of orthologous TF-encoding gene loci using MLAGAN. We also predicted putative transcription factor binding sites within the CNEs. A significant proportion of human-fugu CNEs contain experimentally defined binding sites for transcriptional activators and repressors, indicating that a majority of the CNEs may function as transcriptional regulatory elements. The TF-encoding genes that are involved in nervous system development are generally enriched for human-fugu CNEs. Users can retrieve TF-encoding genes and their associated CNEs by conducting a keyword search or by selecting a family of DNA-binding proteins. CONCLUSION: The conserved noncoding elements identified in TFCONES represent a catalog of highly prioritized putative cis-regulatory elements of TF-encoding genes and are candidates for functional assay.

STAT4 is a target of the hematopoietic zinc-finger transcription factor Ikaros in T cells.

STAT4 is a transcription factor activated in response to IL-12, and is involved in Th1 cell development. The molecular mechanisms controlling the transcription of the STAT4 gene are however, unclear. Sequence comparison of the 5' flanking regions of human, mouse and pufferfish (Fugu rubripes) Stat4 genes revealed a high frequency of Ikaros (Ik) binding elements in all three species. We then investigated the role of Ik binding elements in the human STAT4 promoter using Jurkat T cells. Transactivation, electrophoretic mobility shift assay and RNA interference-mediated gene knockdown experiments revealed that Ik is involved in the regulation of STAT4 in human T cells.

Cloning and expression of the reverse transcriptase component of pufferfish (Fugu rubripes) telomerase.

The enzyme telomerase is essential for maintaining the ends of linear chromosomes. It plays an important role in cell proliferation, differentiation, tumorigenesis and aging. Telomerase is composed of an RNA subunit (TR) and a reverse transcriptase catalytic subunit (TERT). We report here the cloning and characterization of the gene encoding the TERT subunit from a teleost fish, Fugu rubripes. This is the first fish TERT gene to be cloned. The fugu TERT (fTERT) gene comprises of 16 exons and 15 introns similar to the human TERT (hTERT), and encodes a 1074 amino acid protein. The fTERT protein showed 33% to 35% sequence identity to other vertebrate TERTs, and contained all the signature motifs of the TERT family. Analysis of the promoter region of fTERT showed the presence of several transcription factor binding sites (E2F-1, E-box, ER, Sp1 and USF sites) in common with the hTERT promoter, and whose binding factors are known to regulate hTERT. The fTERT gene is expressed in a variety of tissues, with high expression detected in the gill, testis, and ovary. fTERT expression was detected in an immortalized fugu eye-derived cell line. The level of expression was found to be higher in actively dividing cells and reduced at quiescence, suggesting cell cycle regulation of TERT and possibly telomerase activity, in this cell line.

Ray-fin fish tetraploidization gave rise to pufferfish duplicates of NPY and PYY, but zebrafish NPY duplicate was lost.

We have used sequence information and gene location to identify NPY family genes in the pufferfish, Takifugu rubripes (fugu), and zebrafish. Fugu has two copies of NPY, presumably resulting from the ray-fin fish tetraploidization. Zebrafish has probably lost one of the copies. Both species have two copies of PYY, the second of which was previously named PY. The two fugu NPY genes are predominantly expressed in brain. The two PYY genes are expressed in a broad range of tissues including brain and gonads. Thus, the NPY system appears to be more complex in teleosts than in tetrapods.

Erythropoietin gene from a teleost fish, Fugu rubripes.

In this paper we report the cloning and characterization of the erythropoietin (Epo) gene from the pufferfish, Fugu rubripes. This is the first nonmammalian Epo gene to be cloned. The Fugu Epo comprises 5 exons and 4 introns similar to the human EPO, and encodes a 185-amino acid protein that is 32% to 34% identical to Epo from various mammals. The synteny of genes at the Epo locus is conserved between the Fugu and humans. Unlike in mammals in which adult kidney is the primary Epo-producing organ, the heart is the main Epo-producing organ in adult Fugu. In addition to the heart, Fugu Epo is also expressed in the liver and brain similar to the human EPO. Interestingly, the transcripts in the Fugu brain are generated from a distal promoter and include an alternatively spliced first coding exon. No such brain-specific alternative splicing of Epo has been reported in mammals so far. Transient transfection studies in a fish hepatoma cell line (PLHC-1) and a human hepatoma cell line (HepG2) suggest that although the Fugu Epo promoter many not be hypoxia inducible, the gene may be regulated by hypoxia.

Molecular cloning of the pufferfish (Takifugu rubripes) Mx gene and functional characterization of its promoter.

Mx proteins are members of a family of interferon-inducible genes that are expressed by cells in response to viral infection. They are important determinants of innate immunity against viral infection in vertebrates. We cloned the pufferfish ( Takifugu rubripes) Mx gene and sequenced 80 kb from the Mx locus. The Fugu Mx gene spans 3.4 kb from the transcription start site to the polyadenylation signal, and is made up of 12 exons and 11 introns. The protein sequence encoded by the Fugu Mx gene is 77%, 48%, and 51% identical to that of trout Mx1, chicken Mx, and mouse Mx1 genes, respectively. The Fugu Mx gene is expressed in a variety of tissues, with high expression detected in the heart, gill, kidney, intestine, and brain. Analysis of the 5'-flanking sequence of the gene showed the presence of two interferon-stimulated response elements (ISRE) at positions -51 to 38 and -97 to 85, relative to the transcription start site. The Fugu Mx promoter was inducible by human IFN-beta in the human hepatoma (Huh7) cells and by polyinosinic: polycytidilic acid in the top minnow hepatoma (PLHC-1) cells. Deletion analysis of the promoter showed that both ISREs contributed to inducibility. These results demonstrate that the molecular mechanisms involved in Mx gene regulation are conserved between fish and mammals.

Inducible system in human hepatoma cell lines for hepatitis C virus production.

We cloned the complete complementary DNA of an isolate of the hepatitis C virus, HCV-S1, into a tetracycline-inducible expression vector and stably transfected it into two human hepatoma cell lines, Huh7 and HepG2. Twenty-six Huh7 and two HepG2-positive clones were obtained after preliminary screening. Two Huh7 (SH-7 and -9) and one HepG2 (G-19) clones were chosen for further characterisation. Expression of HCV proteins in these cells accumulated from 6 h to 4 days posttreatment. Full-length viral plus-strand RNA was detected by Northern analyses. Using RT-PCR and ribonuclease protection assay, we also detected the synthesis of minus-strand HCV RNA. Plus- and minus-strand viral RNA was still detected after treatment with actinomycin D. Indirect immunofluorescence staining with anti-E2, NS4B, and NS5A revealed that these proteins were mostly localised to the endoplasmic reticulum (ER). Culture media from tet-induced SH-9 cells was separated on sucrose density gradients and analysed for the presence of HCV RNA. Viral RNA levels peaked at two separate ranges, one with a buoyant density of 1.08 g/ml and another from 1.17 to 1.39 g/ml. Electron microscopy demonstrated the presence of subviral-like particles (approximately 20-25 nm in diameter) in the cytoplasm of SH-9 and G-19 cells, which were positively labelled by anti-HCV core antibodies. Anti-E2 antibodies strongly labelled cytoplasmic vesicular structures and some viral-like particles. Complete viral particles of about 50 nm which reacted with anti-E2 antibodies were observed in the culture media of tet-induced SH-9 cells following negative staining. Supernatant from tet-treated SH-9 cells was found to infect nai;ve Huh7 and stable Huh7-human CD81 cells.