'Calling Cards' method for high-throughput identification of targets of yeast DNA-binding proteins.
We present a protocol for a novel method for identifying the targets of DNA-binding proteins in the genome of the yeast Saccharomyces cerevisiae. This is accomplished by engineering a DNA-binding protein so that it leaves behind in the genome a permanent mark -- a 'calling card' -- that provides a record of that protein's visit to that region of the genome. The calling card is the yeast Ty5 retrotransposon, whose integrase interacts with the Sir4 protein. If Sir4 is fused to a DNA-binding protein, it recruits the Ty5 integrase, which directs insertion of a Ty5 calling card into the genome. The calling card along with the flanking genomic DNA is harvested by inverse PCR and its genomic location is determined by hybridization of the product to a DNA microarray. This method provides a straightforward alternative to the 'ChIP-chip' method for determining the targets of DNA-binding proteins. This protocol takes approximately 2 weeks to complete.
Wang
Haoyi
H
Department of Genetics, Washington University, School of Medicine, Genome Sequencing Center, St Louis, Missouri 63108, USA.
Heinz
Michael E
ME
Crosby
Seth D
SD
Johnston
Mark
M
Mitra
Robi D
RD
D002874:Chromosome Mapping / Q000379:methods*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D016681:Genome, Fungal / Q000235:genetics*; D008957:Models, Genetic; D015202:Protein Engineering / Q000379:methods*; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism; D018626:Retroelements / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D038281:Silent Information Regulator Proteins, Saccharomyces cerevisiae / Q000378:metabolism
D004268:DNA-Binding Proteins; D018626:Retroelements; C061573:SIR4 protein, S cerevisiae; D038281:Silent Information Regulator Proteins, Saccharomyces cerevisiae; D012194:RNA-Directed DNA Polymerase