The interpretation of genome sequences requires reliable and standardized methods to assess protein function at high throughput. Here we describe a fast and reliable pipeline to study protein function in mammalian cells based on protein tagging in bacterial artificial chromosomes (BACs). The large size of the BAC transgenes ensures the presence of most, if not all, regulatory elements and results in expression that closely matches that of the endogenous gene. We show that BAC transgenes can be rapidly and reliably generated using 96-well-format recombineering. After stable transfection of these transgenes into human tissue culture cells or mouse embryonic stem URL.The BACFinder clone search and oligo design tool is available online at http://www.mitocheck.org/cgi-bin/BACfinder.Database accession codes. The ChIP/chip data has been submitted to the Gene Expression Omnibus database with accession number GSE10845. COMPETING INTERESTS STATEMENTThe authors declare competing financial interests: details accompany the full-text HTML version of the paper at http:// www.nature.com/naturemethods/. Europe PMC Funders GroupAuthor Manuscript Nat Methods. Author manuscript; available in PMC 2010 May 17. Europe PMC Funders Author ManuscriptsEurope PMC Funders Author Manuscripts cells, the localization, protein-protein and/or protein-DNA interactions of the tagged protein are studied using generic, tag-based assays. The same high-throughput approach will be generally applicable to other model systems.At a time when the 'thousand-dollar genome' seems a realistic goal for the near future, methods for dissecting the functions of the encoded genetic information lag far behind the genome sequence, both in throughput and in quality of the produced data. Genome sequencing and subsequent bioinformatics analysis have made it possible to study the function of genes in mammalian tissue culture cells using systematic reverse-genetic approaches1-3 and have radically improved researchers' ability to identify human disease genes. Such studies typically identify single genes, whose biological function has often not yet been described. In order to place the proteins these genes encode in pathways, these studies must be followed by detailed molecular-level analysis, of which the most powerful types are protein localization and protein-protein interaction. The power of protein localization and protein-protein interaction studies can be seen from the genome-wide application of GFP localization and tandem affinity tag-based complex purification in the yeast Saccharomyces cerevisiae, which has produced a comprehensive picture of the core proteome of a simple, well-studied model system4-8. The key advantage of yeast for these studies was their efficient intrinsic homologous recombination, which allowed the same tagcoding sequence to be introduced at the endogenous locus of nearly every gene of the genome. The tagged proteins were then systematically analyzed through standardized, generic, tag-based assays.To transfer this approach to mammali...

Chromosome segregation and cell division are essential, highly ordered processes that depend on numerous protein complexes. Results from recent RNA interference (RNAi) screens indicate that the identity and composition of these protein complexes is incompletely understood. Using gene tagging on bacterial artificial chromosomes, protein localization and tandem affinity purificationmass spectrometry, the MitoCheck consortium has analyzed about 100 human protein complexes, many of which had not or only incompletely been characterized. This work has led to the discovery of previously unknown, evolutionarily conserved subunits of the anaphase-promoting complex (APC/C) and the γ-tubulin ring complex (γ-TuRC), large complexes which are essential †

In Drosophila, PIWI proteins and bound PIWI-interacting RNAs (piRNAs) form the core of a small RNA-mediated defense system against selfish genetic elements. Within germline cells, piRNAs are processed from piRNA clusters and transposons to be loaded into Piwi/Aubergine/AGO3 and a subset of piRNAs undergoes target-dependent amplification. In contrast, gonadal somatic support cells express only Piwi, lack signs of piRNA amplification and exhibit primary piRNA biogenesis from piRNA clusters. Neither piRNA processing/loading nor Piwi-mediated target silencing is understood at the genetic, cellular or molecular level. We developed an in vivo RNAi assay for the somatic piRNA pathway and identified the RNA helicase Armitage, the Tudor domain containing RNA helicase Yb and the putative nuclease Zucchini as essential factors for primary piRNA biogenesis. Lack of any of these proteins leads to transposon de-silencing, to a collapse in piRNA levels and to a failure in Piwi-nuclear accumulation. We show that Armitage and Yb interact physically and co-localize in cytoplasmic Yb bodies, which flank P bodies. Loss of Zucchini leads to an accumulation of Piwi and Armitage in Yb bodies, indicating that Yb bodies are sites of primary piRNA biogenesis.

Sister chromatid cohesion depends on Sororin, a protein that stabilizes acetylated cohesin complexes on DNA by antagonizing the cohesin release factor Wings-apart like protein (Wapl). Cohesion is essential for chromosome biorientation but has to be dissolved to enable sister chromatid separation. To achieve this, the majority of cohesin is removed from chromosome arms in prophase and prometaphase in a manner that depends on Wapl and phosphorylation of cohesin's subunit stromal antigen 2 (SA2), whereas centromeric cohesin is cleaved in metaphase by the protease separase. Here we show that the mitotic kinases Aurora B and Cyclin-dependent kinase 1 (Cdk1) destabilize interactions between Sororin and the cohesin subunit precocious dissociation of sisters protein 5 (Pds5) by phosphorylating Sororin, leading to release of acetylated cohesin from chromosome arms and loss of cohesion. At centromeres, the cohesin protector shugoshin (Sgo1)-protein phosphatase 2A (PP2A) antagonizes Aurora B and Cdk1 partly by dephosphorylating Sororin and thus maintains cohesion until metaphase. We propose that the stepwise loss of cohesion between chromosome arms and centromeres is caused by local regulation of Wapl activity, which is controlled by the phosphorylation state of Sororin.cell cycle | chromosome segregation | prophase pathway R eplicated DNA molecules (sister chromatids) remain physically connected with each other from S phase until metaphase to enable biorientation of chromosomes on the mitotic spindle. This association, called sister chromatid cohesion, is mediated by cohesin, whose core subunits Structural maintenance of chromosomes 1 (Smc1), Smc3, Sister chromatid cohesion 1 (Scc1), and Scc3/stromal antigen (SA/STAG) form a molecular ring. This structure is crucial for cohesion, presumably because cohesin topologically embraces the two sister chromatids (reviewed in ref.