An enhanced workflow for variant interpretation in UniProtKB/Swiss-Prot improves consistency and reuse in ClinVar.

Personalized genomic medicine depends on integrated analyses that combine genetic and phenotypic data from individual patients with reference knowledge of the functional and clinical significance of sequence variants. Sources of this reference knowledge include the ClinVar repository of human genetic variants, a community resource that accepts submissions from external groups, and UniProtKB/Swiss-Prot, an expert-curated resource of protein sequences and functional annotation. UniProtKB/Swiss-Prot provides knowledge on the functional impact and clinical significance of over 30 000 human protein-coding sequence variants, curated from peer-reviewed literature reports. Here we present a pilot study that lays the groundwork for the integration of curated knowledge of protein sequence variation from UniProtKB/Swiss-Prot with ClinVar. We show that existing interpretations of variant pathogenicity in UniProtKB/Swiss-Prot and ClinVar are highly concordant, with 88% of variants that are common to the two resources having interpretations of clinical significance that agree. Re-curation of a subset of UniProtKB/Swiss-Prot variants according to American College of Medical Genetics and Genomics (ACMG) guidelines using ClinGen tools further increases this level of agreement, mainly due to the reclassification of supposedly pathogenic variants as benign, based on newly available population frequency data. We have now incorporated ACMG guidelines and ClinGen tools into the UniProt Knowledgebase (UniProtKB) curation workflow and routinely submit variant data from UniProtKB/Swiss-Prot to ClinVar. These efforts will increase the usability and utilization of UniProtKB variant data and will facilitate the continuing (re-)evaluation of clinical variant interpretations as data sets and knowledge evolve.

Representing virus-host interactions and other multi-organism processes in the Gene Ontology.

BACKGROUND: The Gene Ontology project is a collaborative effort to provide descriptions of gene products in a consistent and computable language, and in a species-independent manner. The Gene Ontology is designed to be applicable to all organisms but up to now has been largely under-utilized for prokaryotes and viruses, in part because of a lack of appropriate ontology terms. METHODS: To address this issue, we have developed a set of Gene Ontology classes that are applicable to microbes and their hosts, improving both coverage and quality in this area of the Gene Ontology. Describing microbial and viral gene products brings with it the additional challenge of capturing both the host and the microbe. Recognising this, we have worked closely with annotation groups to test and optimize the GO classes, and we describe here a set of annotation guidelines that allow the controlled description of two interacting organisms. CONCLUSIONS: Building on the microbial resources already in existence such as ViralZone, UniProtKB keywords and MeGO, this project provides an integrated ontology to describe interactions between microbial species and their hosts, with mappings to the external resources above. Housing this information within the freely-accessible Gene Ontology project allows the classes and annotation structure to be utilized by a large community of biologists and users.

Collaborative annotation of genes and proteins between UniProtKB/Swiss-Prot and dictyBase.

UniProtKB/Swiss-Prot, a curated protein database, and dictyBase, the Model Organism Database for Dictyostelium discoideum, have established a collaboration to improve data sharing. One of the major steps in this effort was the 'Dicty annotation marathon', a week-long exercise with 30 annotators aimed at achieving a major increase in the number of D. discoideum proteins represented in UniProtKB/Swiss-Prot. The marathon led to the annotation of over 1000 D. discoideum proteins in UniProtKB/Swiss-Prot. Concomitantly, there were a large number of updates in dictyBase concerning gene symbols, protein names and gene models. This exercise demonstrates how UniProtKB/Swiss-Prot can work in very close cooperation with model organism databases and how the annotation of proteins can be accelerated through those collaborations.

Establishment of Polycomb silencing requires a transient interaction between PC and ESC.

Two distinct types of Polycomb complexes have been identified in flies and in vertebrates, one containing ESC and one containing PC. Using LexA fusions, we show that PC and ESC can establish silencing of a reporter gene but that each requires the presence of the other. In early embryonic extracts, we find PC transiently associated with ESC in a complex that includes EZ, PHO, PH, GAGA, and RPD3 but not PSC. In older embryos, PC is found in a complex including PH, PSC, GAGA, and RPD3, whereas ESC is in a separate complex including EZ, PHO, and RPD3.

Recruitment of components of Polycomb Group chromatin complexes in Drosophila.

Polycomb Group complexes assemble at polycomb response elements (PREs) in vivo and silence genes in the surrounding chromatin. To study the recruitment of silencing complexes, we have targeted various Polycomb Group (PcG) proteins by fusing them to the LexA DNA binding domain. When LexA-PC, -PSC, -PH or -SU(Z)2 are targeted to a reporter gene, they recruit functional PcG-silencing complexes that recapitulate the silencing behavior of a PRE: silencing is sensitive to the state of activity of the target chromatin. When the target is transcriptionally active, silencing is not established but when the target is not active at syncytial blastoderm, it becomes silenced. The repressed state persists through embryonic development but cannot be maintained in larval imaginal discs even when the LexA-PcG fusion is constitutively expressed, suggesting a discontinuity in the mechanism of repression. These proteins also interact with other PC-containing complexes in embryonic nuclear extracts. In contrast LexA-PHO is neither able to silence nor to interact with PC-containing complexes. Analysis of pho mutant embryos and of PRE constructs whose PHO-binding sites are mutated suggests that, while PHO is important for silencing in imaginal discs, it is not necessary for embryonic PcG silencing.

Structure of a polycomb response element and in vitro binding of polycomb group complexes containing GAGA factor.

Polycomb response elements (PREs) are regulatory sites that mediate the silencing of homeotic and other genes. The bxd PRE region from the Drosophila Ultrabithorax gene can be subdivided into subfragments of 100 to 200 bp that retain different degrees of PRE activity in vivo. In vitro, embryonic nuclear extracts form complexes containing Polycomb group (PcG) proteins with these fragments. PcG binding to some fragments is dependent on consensus sequences for the GAGA factor. Other fragments lack GAGA binding sites but can still bind PcG complexes in vitro. We show that the GAGA factor is a component of at least some types of PcG complexes and may participate in the assembly of PcG complexes at PREs.

Hunchback-independent silencing of late Ubx enhancers by a Polycomb Group Response Element.

Drosophila homeotic genes are kept silent outside of their appropriate expression domains by a repressive chromatin complex formed by the Polycomb Group proteins. In the case of the Ubx gene, it has been proposed that the early repressor HB, binding at enhancers, recruits the Polycomb complex and specifies the domain of repression. We show that some Ubx enhancers are activated after blastoderm. If a Polycomb Response Element (PRE) is combined with such late enhancers, repression of a reporter gene can be established everywhere in the embryo, irrespective of the presence or absence of hunchback protein. If, however, these late enhancers are combined with a Ubx early enhancer, as well as a PRE, repression is established only where the reporter gene was inactive at early stages. These results imply that the Polycomb complex is not dependent on hunchback and suggest that the pattern of silencing reflects rather the state of activity of the gene at the time the Polycomb complex is formed.