Elia Stupka

The Ensembl genome database project

The Ensembl (http://www.ensembl.org/) database project provides a bioinformatics framework to organise biology around the sequences of large genomes. It is a comprehensive source of stable automatic annotation of the human genome sequence, with confirmed gene predictions that have been integrated with external data sources, and is available as either an interactive web site or as flat files. It is also an open source software engineering project to develop a portable system able to handle very large genomes and associated requirements from sequence analysis to data storage and visualisation. The Ensembl site is one of the leading sources of human genome sequence annotation and provided much of the analysis for publication by the international human genome project of the draft genome. The Ensembl system is being installed around the world in both companies and academic sites on machines ranging from supercomputers to laptops.

Long non-coding antisense RNA controls Uchl1 translation through an embedded SINEB2 repeat

Most of the mammalian genome is transcribed. This generates a vast repertoire of transcripts that includes protein-coding messenger RNAs, long non-coding RNAs (lncRNAs) and repetitive sequences, such as SINEs (short interspersed nuclear elements). A large percentage of ncRNAs are nuclear-enriched with unknown function. Antisense lncRNAs may form sense–antisense pairs by pairing with a protein-coding gene on the opposite strand to regulate epigenetic silencing, transcription and mRNA stability. Here we identify a nuclear-enriched lncRNA antisense to mouse ubiquitin carboxy-terminal hydrolase L1 (Uchl1), a gene involved in brain function and neurodegenerative diseases. Antisense Uchl1 increases UCHL1 protein synthesis at a post-transcriptional level, hereby identifying a new functional class of lncRNAs. Antisense Uchl1 activity depends on the presence of a 5′ overlapping sequence and an embedded inverted SINEB2 element. These features are shared by other natural antisense transcripts and can confer regulatory activity to an artificial antisense to green fluorescent protein. Antisense Uchl1 function is under the control of stress signalling pathways, as mTORC1 inhibition by rapamycin causes an increase in UCHL1 protein that is associated to the shuttling of antisense Uchl1 RNA from the nucleus to the cytoplasm. Antisense Uchl1 RNA is then required for the association of the overlapping sense protein-coding mRNA to active polysomes for translation. These data reveal another layer of gene expression control at the post-transcriptional level.

Ensembl 2002: accommodating comparative genomics

The Ensembl (http://www.ensembl.org/) database project provides a bioinformatics framework to organise biology around the sequences of large genomes. It is a comprehensive source of stable automatic annotation of human, mouse and other genome sequences, available as either an interactive web site or as flat files. Ensembl also integrates manually annotated gene structures from external sources where available. As well as being one of the leading sources of genome annotation, Ensembl is an open source software engineering project to develop a portable system able to handle very large genomes and associated requirements. These range from sequence analysis to data storage and visualisation and installations exist around the world in both companies and at academic sites. With both human and mouse genome sequences available and more vertebrate sequences to follow, many of the recent developments in Ensembl have focusing on developing automatic comparative genome analysis and visualisation.

Integrated Genetic and Epigenetic Analysis Identifies Haplotype-Specific Methylation in the FTO Type 2 Diabetes and Obesity Susceptibility Locus

Recent multi-dimensional approaches to the study of complex disease have revealed powerful insights into how genetic and epigenetic factors may underlie their aetiopathogenesis. We examined genotype-epigenotype interactions in the context of Type 2 Diabetes (T2D), focussing on known regions of genomic susceptibility. We assayed DNA methylation in 60 females, stratified according to disease susceptibility haplotype using previously identified association loci. CpG methylation was assessed using methylated DNA immunoprecipitation on a targeted array (MeDIP-chip) and absolute methylation values were estimated using a Bayesian algorithm (BATMAN). Absolute methylation levels were quantified across LD blocks, and we identified increased DNA methylation on the FTO obesity susceptibility haplotype, tagged by the rs8050136 risk allele A (p = 9.40×10−4, permutation p = 1.0×10−3). Further analysis across the 46 kb LD block using sliding windows localised the most significant difference to be within a 7.7 kb region (p = 1.13×10−7). Sequence level analysis, followed by pyrosequencing validation, revealed that the methylation difference was driven by the co-ordinated phase of CpG-creating SNPs across the risk haplotype. This 7.7 kb region of haplotype-specific methylation (HSM), encapsulates a Highly Conserved Non-Coding Element (HCNE) that has previously been validated as a long-range enhancer, supported by the histone H3K4me1 enhancer signature. This study demonstrates that integration of Genome-Wide Association (GWA) SNP and epigenomic DNA methylation data can identify potential novel genotype-epigenotype interactions within disease-associated loci, thus providing a novel route to aid unravelling common complex diseases.

Genome-wide signatures of convergent evolution in echolocating mammals

Evolution is typically thought to proceed through divergence of genes, proteins and ultimately phenotypes. However, similar traits might also evolve convergently in unrelated taxa owing to similar selection pressures. Adaptive phenotypic convergence is widespread in nature, and recent results from several genes have suggested that this phenomenon is powerful enough to also drive recurrent evolution at the sequence level. Where homoplasious substitutions do occur these have long been considered the result of neutral processes. However, recent studies have demonstrated that adaptive convergent sequence evolution can be detected in vertebrates using statistical methods that model parallel evolution, although the extent to which sequence convergence between genera occurs across genomes is unknown. Here we analyse genomic sequence data in mammals that have independently evolved echolocation and show that convergence is not a rare process restricted to several loci but is instead widespread, continuously distributed and commonly driven by natural selection acting on a small number of sites per locus. Systematic analyses of convergent sequence evolution in 805,053 amino acids within 2,326 orthologous coding gene sequences compared across 22 mammals (including four newly sequenced bat genomes) revealed signatures consistent with convergence in nearly 200 loci. Strong and significant support for convergence among bats and the bottlenose dolphin was seen in numerous genes linked to hearing or deafness, consistent with an involvement in echolocation. Unexpectedly, we also found convergence in many genes linked to vision: the convergent signal of many sensory genes was robustly correlated with the strength of natural selection. This first attempt to detect genome-wide convergent sequence evolution across divergent taxa reveals the phenomenon to be much more pervasive than previously recognized.

Germline CDH1 deletions in hereditary diffuse gastric cancer families

Germline CDH1 point or small frameshift mutations can be identified in 30–50% of hereditary diffuse gastric cancer (HDGC) families. We hypothesized that CDH1 genomic rearrangements would be found in HDGC and identified 160 families with either two gastric cancers in first-degree relatives and with at least one diffuse gastric cancer (DGC) diagnosed before age 50, or three or more DGC in close relatives diagnosed at any age. Sixty-seven carried germline CDH1 point or small frameshift mutations. We screened germline DNA from the 93 mutation negative probands for large genomic rearrangements by Multiplex Ligation-Dependent Probe Amplification. Potential deletions were validated by RT–PCR and breakpoints cloned using a combination of oligo-CGH-arrays and long-range-PCR. In-silico analysis of the CDH1 locus was used to determine a potential mechanism for these rearrangements. Six of 93 (6.5%) previously described mutation negative HDGC probands, from low GC incidence populations (UK and North America), carried genomic deletions (UK and North America). Two families carried an identical deletion spanning 193 593 bp, encompassing the full CDH3 sequence and CDH1 exons 1 and 2. Other deletions affecting exons 1, 2, 15 and/or 16 were identified. The statistically significant over-representation of Alus around breakpoints indicates it as a likely mechanism for these deletions. When all mutations and deletions are considered, the overall frequency of CDH1 alterations in HDGC is ∼46% (73/160). CDH1 large deletions occur in 4% of HDGC families by mechanisms involving mainly non-allelic homologous recombination in Alu repeat sequences. As the finding of pathogenic CDH1 mutations is useful for management of HDGC families, screening for deletions should be offered to at-risk families.

Comparative methylome analysis of benign and malignant peripheral nerve sheath tumors

Aberrant DNA methylation (DNAm) was first linked to cancer over 25 yr ago. Since then, many studies have associated hypermethylation of tumor suppressor genes and hypomethylation of oncogenes to the tumorigenic process. However, most of these studies have been limited to the analysis of promoters and CpG islands (CGIs). Recently, new technologies for whole-genome DNAm (methylome) analysis have been developed, enabling unbiased analysis of cancer methylomes. By using MeDIP-seq, we report a sequencing-based comparative methylome analysis of malignant peripheral nerve sheath tumors (MPNSTs), benign neurofibromas, and normal Schwann cells. Analysis of these methylomes revealed a complex landscape of DNAm alterations. In contrast to what has been reported for other tumor types, no significant global hypomethylation was observed in MPNSTs using methylome analysis by MeDIP-seq. However, a highly significant (P < 10(-100)) directional difference in DNAm was found in satellite repeats, suggesting these repeats to be the main target for hypomethylation in MPNSTs. Comparative analysis of the MPNST and Schwann cell methylomes identified 101,466 cancer-associated differentially methylated regions (cDMRs). Analysis showed these cDMRs to be significantly enriched for two satellite repeat types (SATR1 and ARLα) and suggests an association between aberrant DNAm of these sequences and transition from healthy cells to malignant disease. Significant enrichment of hypermethylated cDMRs in CGI shores (P < 10(-60)), non-CGI-associated promoters (P < 10(-4)) and hypomethylated cDMRs in SINE repeats (P < 10(-100)) was also identified. Integration of DNAm and gene expression data showed that the expression pattern of genes associated with CGI shore cDMRs was able to discriminate between disease phenotypes. This study establishes MeDIP-seq as an effective method to analyze cancer methylomes.

PRGdb: a bioinformatics platform for plant resistance gene analysis

PRGdb is a web accessible open-source (http://www.prgdb.org) database that represents the first bioinformatic resource providing a comprehensive overview of resistance genes (R-genes) in plants. PRGdb holds more than 16 000 known and putative R-genes belonging to 192 plant species challenged by 115 different pathogens and linked with useful biological information. The complete database includes a set of 73 manually curated reference R-genes, 6308 putative R-genes collected from NCBI and 10463 computationally predicted putative R-genes. Thanks to a user-friendly interface, data can be examined using different query tools. A home-made prediction pipeline called Disease Resistance Analysis and Gene Orthology (DRAGO), based on reference R-gene sequence data, was developed to search for plant resistance genes in public datasets such as Unigene and Genbank. New putative R-gene classes containing unknown domain combinations were discovered and characterized. The development of the PRG platform represents an important starting point to conduct various experimental tasks. The inferred cross-link between genomic and phenotypic information allows access to a large body of information to find answers to several biological questions. The database structure also permits easy integration with other data types and opens up prospects for future implementations.

The IKMC web portal: a central point of entry to data and resources from the International Knockout Mouse Consortium.

The International Knockout Mouse Consortium (IKMC) aims to mutate all protein-coding genes in the mouse using a combination of gene targeting and gene trapping in mouse embryonic stem (ES) cells and to make the generated resources readily available to the research community. The IKMC database and web portal (www.knockoutmouse.org) serves as the central public web site for IKMC data and facilitates the coordination and prioritization of work within the consortium. Researchers can access up-to-date information on IKMC knockout vectors, ES cells and mice for specific genes, and follow links to the respective repositories from which corresponding IKMC products can be ordered. Researchers can also use the web site to nominate genes for targeting, or to indicate that targeting of a gene should receive high priority. The IKMC database provides data to, and features extensive interconnections with, other community databases.

Genome-wide mapping of Myc binding and gene regulation in serum-stimulated fibroblasts

The transition from quiescence to proliferation is a key regulatory step that can be induced by serum stimulation in cultured fibroblasts. The transcription factor Myc is directly induced by serum mitogens and drives a secondary gene expression program that remains largely unknown. Using mRNA profiling, we identify close to 300 Myc-dependent serum response (MDSR) genes, which are induced by serum in a Myc-dependent manner in mouse fibroblasts. Mapping of genomic Myc-binding sites by ChIP-seq technology revealed that most MDSR genes were directly targeted by Myc, but represented a minor fraction (5.5%) of all Myc-bound promoters (which were 22.4% of all promoters). Other target loci were either induced by serum in a Myc-independent manner, were not significantly regulated or were negatively regulated. MDSR gene products were involved in a variety of processes, including nucleotide biosynthesis, ribosome biogenesis, DNA replication and RNA control. Of the 29 MDSR genes targeted by RNA interference, three showed a requirement for cell-cycle entry upon serum stimulation and 11 for long-term proliferation and/or survival. Hence, proper coordination of key regulatory and biosynthetic pathways following mitogenic stimulation relies upon the concerted regulation of multiple Myc-dependent genes.