Functional Genomics by Transposon-Tagging and ChIP-Chip Analysis in Yeast

Anuj Kumar, Mike Snyder
Dept. of MCD Biology
Yale University
P.O. Box 208103
New Haven, CT 06520-8103
telephone: 203-432-9949
fax: 203-432-6161
email: anuj.kumar@yale.edu
prestype: Platform
presenter: Anuj Kumar

Anuj Kumar & Mike Snyder

Here we present two novel approaches by which gene function may be investigated on a genome-wide scale. The first approach utilizes transposon-based insertional mutagenesis as a means of conveniently generating informative mutant alleles for subsequent functional analysis. Transposons employed for this purpose have been modified such that a single insertion is sufficient to derive a reporter gene fusion, gene disruption, and epitope-tagged allele, while also serving as a simple gene trap. As applied to the genome of Saccharomyces cerevisiae, these multi-functional transposons have been used in conjunction with high-throughput screening to generate a collection of yeast strains mutated for nearly 4,000 different annotated genes (roughly two-thirds of the yeast genome). The cumulatively unique data set resulting from analysis of these insertions is cataloged in the Web-accessible database TRIPLES (http://ygac.med.yale.edu). While transposable elements are powerful tools for functional genomics, microarray technologies provide an equally exciting avenue for large-scale biological discovery. Utilizing microarray-based methods, we have recently developed a genomic approach (in collaboration with Pat Brown's group) to globally map binding sites of yeast transcription factors. In this approach, protein-DNA interactions are "captured" in vivo by crosslinking proteins to their genomic binding sites; crosslinked DNA is subsequently extracted, sheared, and purified by immunoprecipitation with antibodies directed against the chosen transcription factor. Bound DNA (enriched for transcription factor binding sites) is subsequently prepared as probes against a microarray of yeast intergenic regions. By this ChIP-Chip strategy (Chromatin ImmunoPrecipitation and microarray analysis), we have defined over 200 previously unidentified targets of the transcription factors SBF and MBF, two sequence-specific activators regulating gene expression during the G1/S transition in yeast. As a means of globally identifying protein-DNA interactions, our ChIP-Chip approach can be used to map a variety of functional sites in the yeast genome as well as in DNA from other organisms.