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...

SUMMARY Retinoic acid (RA) triggers growth-suppressive effects in tumor cells and therefore RA has and its synthetic analogs have great potential as anti-carcinogenic agent. RA effects are mediated by Retinoic Acid Receptors (RARs), which regulate gene expression in an RA-dependent manner. To define the genetic network regulated by RARs in breast cancer, we identified RAR genomic targets using chromatin immunoprecipitation and expression analysis. We found that RAR binding throughout the genome is highly co-incident with estrogen receptor α (ERα) binding, and identified a widespread crosstalk of RA and estrogen signaling to antagonistically regulate breast cancer-associated genes. ERα and RAR binding sites appear to be co-evolved on a large scale throughout the human genome, allowing for competitive binding between these transcription factors via nearby or overlapping cis-regulatory elements. Together these data indicate the existence of a highly coordinated intersection between these two critical nuclear hormone receptor signaling pathways providing a global mechanism for balancing gene expression output via local regulatory interactions dispersed throughout the genome.

RNA interference (RNAi) is an evolutionarily conserved defence mechanism whereby genes are specifically silenced through degradation of messenger RNAs; this process is mediated by homologous double-stranded (ds)RNA molecules. In invertebrates, long dsRNAs have been used for genome-wide screens and have provided insights into gene functions. Because long dsRNA triggers a nonspecific interferon response in many vertebrates, short interfering (si)RNA or short hairpin (sh)RNAs must be used for these organisms to ensure specific gene silencing. Here we report the generation of a genome-scale library of endoribonuclease-prepared short interfering (esi)RNAs from a sequence-verified complementary DNA collection representing 15,497 human genes. We used 5,305 esiRNAs from this library to screen for genes required for cell division in HeLa cells. Using a primary high-throughput cell viability screen followed by a secondary high content videomicroscopy assay, we identified 37 genes required for cell division. These include several splicing factors for which knockdown generates mitotic spindle defects. In addition, a putative nuclear-export terminator was found to speed up cell proliferation and mitotic progression after knockdown. Thus, our study uncovers new aspects of cell division and establishes esiRNA as a versatile approach for genomic RNAi screens in mammalian cells.

The human Augmin complex (HAUS) is a critical and evolutionary conserved multisubunit protein complex that regulates centrosome and spindle integrity.