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.

Using reference-free compressed data structures to analyze sequencing reads from thousands of human genomes.

We are rapidly approaching the point where we have sequenced millions of human genomes. There is a pressing need for new data structures to store raw sequencing data and efficient algorithms for population scale analysis. Current reference-based data formats do not fully exploit the redundancy in population sequencing nor take advantage of shared genetic variation. In recent years, the Burrows-Wheeler transform (BWT) and FM-index have been widely employed as a full-text searchable index for read alignment and de novo assembly. We introduce the concept of a population BWT and use it to store and index the sequencing reads of 2705 samples from the 1000 Genomes Project. A key feature is that, as more genomes are added, identical read sequences are increasingly observed, and compression becomes more efficient. We assess the support in the 1000 Genomes read data for every base position of two human reference assembly versions, identifying that 3.2 Mbp with population support was lost in the transition from GRCh37 with 13.7 Mbp added to GRCh38. We show that the vast majority of variant alleles can be uniquely described by overlapping 31-mers and show how rapid and accurate SNP and indel genotyping can be carried out across the genomes in the population BWT. We use the population BWT to carry out nonreference queries to search for the presence of all known viral genomes and discover human T-lymphotropic virus 1 integrations in six samples in a recognized epidemiological distribution.

A novel function of the proneural factor Ascl1 in progenitor proliferation identified by genome-wide characterization of its targets.

Proneural genes such as Ascl1 are known to promote cell cycle exit and neuronal differentiation when expressed in neural progenitor cells. The mechanisms by which proneural genes activate neurogenesis--and, in particular, the genes that they regulate--however, are mostly unknown. We performed a genome-wide characterization of the transcriptional targets of Ascl1 in the embryonic brain and in neural stem cell cultures by location analysis and expression profiling of embryos overexpressing or mutant for Ascl1. The wide range of molecular and cellular functions represented among these targets suggests that Ascl1 directly controls the specification of neural progenitors as well as the later steps of neuronal differentiation and neurite outgrowth. Surprisingly, Ascl1 also regulates the expression of a large number of genes involved in cell cycle progression, including canonical cell cycle regulators and oncogenic transcription factors. Mutational analysis in the embryonic brain and manipulation of Ascl1 activity in neural stem cell cultures revealed that Ascl1 is indeed required for normal proliferation of neural progenitors. This study identified a novel and unexpected activity of the proneural gene Ascl1, and revealed a direct molecular link between the phase of expansion of neural progenitors and the subsequent phases of cell cycle exit and neuronal differentiation.

NPC-db, a Niemann-Pick type C disease gene variation database.

Niemann-Pick type C (NPC) disease is a rare autosomal-recessive lysosomal storage disease typically accompanied by progressive impairment of nervous system and liver function. Biochemically, the disorder presents with an inhibited egress of cholesterol and glycosphingolipids from endosomal and lysosomal compartments in neuronal and nonneuronal cells. In the majority of NPC patients, mutations in the NPC1 gene can be identified. About 5% of patients show mutations in the NPC2 gene. Many different mutations can cause NPC disease and multiple variants not associated with the disease are known in both genes. A continuously updated collection of gene variants is lacking to date and only limited information is available on genotype-phenotype correlation. We have created the NPC disease gene variation database (NPC-db; http://npc.fzk.de; last accessed 24 October 2007). This database aims to provide a comprehensive overview of the sequence variants in NPC1 and NPC2, including information on their functional consequences and associated haplotypes. Moreover, genotype data and clinical information from individual NPC patients provide information on the impact of functional variants. NPC-db addresses professionals and nonprofessionals dealing with NPC disease on a clinical, diagnostic, research, or personal basis. The user is encouraged to search contents and submit novel information, thereby contributing to generate a valuable open-access tool that will allow a better understanding of the molecular and clinical details of NPC disease.

Sixteen diverse laboratory mouse reference genomes define strain-specific haplotypes and novel functional loci.

We report full-length draft de novo genome assemblies for 16 widely used inbred mouse strains and find extensive strain-specific haplotype variation. We identify and characterize 2,567 regions on the current mouse reference genome exhibiting the greatest sequence diversity. These regions are enriched for genes involved in pathogen defence and immunity and exhibit enrichment of transposable elements and signatures of recent retrotransposition events. Combinations of alleles and genes unique to an individual strain are commonly observed at these loci, reflecting distinct strain phenotypes. We used these genomes to improve the mouse reference genome, resulting in the completion of 10 new gene structures. Also, 62 new coding loci were added to the reference genome annotation. These genomes identified a large, previously unannotated, gene (Efcab3-like) encoding 5,874 amino acids. Mutant Efcab3-like mice display anomalies in multiple brain regions, suggesting a possible role for this gene in the regulation of brain development.

Handling Permutation in Sequence Comparison: Genome-Wide Enhancer Prediction in Vertebrates by a Novel Non-Linear Alignment Scoring Principle.

Enhancers have been described to evolve by permutation without changing function. This has posed the problem of how to predict enhancer elements that are hidden from alignment-based approaches due to the loss of co-linearity. Alignment-free algorithms have been proposed as one possible solution. However, this approach is hampered by several problems inherent to its underlying working principle. Here we present a new approach, which combines the power of alignment and alignment-free techniques into one algorithm. It allows the prediction of enhancers based on the query and target sequence only, no matter whether the regulatory logic is co-linear or reshuffled. To test our novel approach, we employ it for the prediction of enhancers across the evolutionary distance of ~450Myr between human and medaka. We demonstrate its efficacy by subsequent in vivo validation resulting in 82% (9/11) of the predicted medaka regions showing reporter activity. These include five candidates with partially co-linear and four with reshuffled motif patterns. Orthology in flanking genes and conservation of the detected co-linear motifs indicates that those candidates are likely functionally equivalent enhancers. In sum, our results demonstrate that the proposed principle successfully predicts mutated as well as permuted enhancer regions at an encouragingly high rate.

Correction: Handling Permutation in Sequence Comparison: Genome-Wide Enhancer Prediction in Vertebrates by a Novel Non-Linear Alignment Scoring Principle.

[This corrects the article DOI: 10.1371/journal.pone.0141487.].

Ascl1 Coordinately Regulates Gene Expression and the Chromatin Landscape during Neurogenesis.

The proneural transcription factor Ascl1 coordinates gene expression in both proliferating and differentiating progenitors along the neuronal lineage. Here, we used a cellular model of neurogenesis to investigate how Ascl1 interacts with the chromatin landscape to regulate gene expression when promoting neuronal differentiation. We find that Ascl1 binding occurs mostly at distal enhancers and is associated with activation of gene transcription. Surprisingly, the accessibility of Ascl1 to its binding sites in neural stem/progenitor cells remains largely unchanged throughout their differentiation, as Ascl1 targets regions of both readily accessible and closed chromatin in proliferating cells. Moreover, binding of Ascl1 often precedes an increase in chromatin accessibility and the appearance of new regions of open chromatin, associated with de novo gene expression during differentiation. Our results reveal a function of Ascl1 in promoting chromatin accessibility during neurogenesis, linking the chromatin landscape at Ascl1 target regions with the temporal progression of its transcriptional program.

Genomic and phenotypic characterization of a wild medaka population: towards the establishment of an isogenic population genetic resource in fish.

Oryzias latipes (medaka) has been established as a vertebrate genetic model for more than a century and recently has been rediscovered outside its native Japan. The power of new sequencing methods now makes it possible to reinvigorate medaka genetics, in particular by establishing a near-isogenic panel derived from a single wild population. Here we characterize the genomes of wild medaka catches obtained from a single Southern Japanese population in Kiyosu as a precursor for the establishment of a near-isogenic panel of wild lines. The population is free of significant detrimental population structure and has advantageous linkage disequilibrium properties suitable for the establishment of the proposed panel. Analysis of morphometric traits in five representative inbred strains suggests phenotypic mapping will be feasible in the panel. In addition, high-throughput genome sequencing of these medaka strains confirms their evolutionary relationships on lines of geographic separation and provides further evidence that there has been little significant interbreeding between the Southern and Northern medaka population since the Southern/Northern population split. The sequence data suggest that the Southern Japanese medaka existed as a larger older population that went through a relatively recent bottleneck approximately 10,000 years ago. In addition, we detect patterns of recent positive selection in the Southern population. These data indicate that the genetic structure of the Kiyosu medaka samples is suitable for the establishment of a vertebrate near-isogenic panel and therefore inbreeding of 200 lines based on this population has commenced. Progress of this project can be tracked at http://www.ebi.ac.uk/birney-srv/medaka-ref-panel.

Large-scale analysis of the regulatory architecture of the mouse genome with a transposon-associated sensor.

We present here a Sleeping Beauty-based transposition system that offers a simple and efficient way to investigate the regulatory architecture of mammalian chromosomes in vivo. With this system, we generated several hundred mice and embryos, each with a regulatory sensor inserted at a random genomic position. This large sampling of the genome revealed the widespread presence of long-range regulatory activities along chromosomes, forming overlapping blocks with distinct tissue-specific expression potentials. The presence of tissue-restricted regulatory activities around genes with widespread expression patterns challenges the gene-centric view of genome regulation and suggests that most genes are modulated in a tissue-specific manner. The local hopping property of Sleeping Beauty provides a dynamic approach to map these regulatory domains at high resolution and, combined with Cre-mediated recombination, allows for the determination of their functions by engineering mice with specific chromosomal rearrangements.

The light responsive transcriptome of the zebrafish: function and regulation.

Most organisms possess circadian clocks that are able to anticipate the day/night cycle and are reset or "entrained" by the ambient light. In the zebrafish, many organs and even cultured cell lines are directly light responsive, allowing for direct entrainment of the clock by light. Here, we have characterized light induced gene transcription in the zebrafish at several organizational levels. Larvae, heart organ cultures and cell cultures were exposed to 1- or 3-hour light pulses, and changes in gene expression were compared with controls kept in the dark. We identified 117 light regulated genes, with the majority being induced and some repressed by light. Cluster analysis groups the genes into five major classes that show regulation at all levels of organization or in different subset combinations. The regulated genes cover a variety of functions, and the analysis of gene ontology categories reveals an enrichment of genes involved in circadian rhythms, stress response and DNA repair, consistent with the exposure to visible wavelengths of light priming cells for UV-induced damage repair. Promoter analysis of the induced genes shows an enrichment of various short sequence motifs, including E- and D-box enhancers that have previously been implicated in light regulation of the zebrafish period2 gene. Heterologous reporter constructs with sequences matching these motifs reveal light regulation of D-box elements in both cells and larvae. Morpholino-mediated knock-down studies of two homologues of the D-box binding factor Tef indicate that these are differentially involved in the cell autonomous light induction in a gene-specific manner. These findings suggest that the mechanisms involved in period2 regulation might represent a more general pathway leading to light induced gene expression.