The use of biotin tagging in Saccharomyces cerevisiae improves the sensitivity of chromatin immunoprecipitation.
Affinity tagging has been used in many global studies towards protein function. We describe a highly efficient system for in vivo biotinylation of transcription factors in the yeast Saccharomyces cerevisiae, which is based on the bacterial BirA biotin ligase. The strength of the biotin-streptavidin interaction was exploited to improve detection of in vivo protein-DNA complexes in chromatin immunoprecipitation (ChIP) experiments. In a test system using the biotin-tagged LexA DNA-binding protein, we found that stringent washing conditions resulted in a strong improvement of the signal-to-noise ratios. Yeast strains with chromosomally integrated versions of tagged transcription factor genes were generated using N- or C-terminal biotin-tagging cassettes. ChIP experiments with biotinylated Rbp3p, a RNA polymerase II subunit, showed that Rbp3p-binding could even be detected at weakly expressed genes. Other methods failed to detect RNA polymerase II binding at such genes. Our results show that biotinylation of yeast transcription factors improves the detection of in vivo protein-DNA complexes.
van Werven
Folkert J
FJ
Department of Physiological Chemistry, Division of Biomedical Genetics, University Medical Centre Utrecht, Universiteitsweg 100, 3584 CG Utrecht, The Netherlands.
Timmers
H Th Marc
HT
D001426:Bacterial Proteins / Q000378:metabolism; D001665:Binding Sites; D019921:Biotinylation / Q000379:methods*; D019731:Carbon-Nitrogen Ligases / Q000378:metabolism; D047369:Chromatin Immunoprecipitation / Q000379:methods*; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000032:analysis / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000378:metabolism; D016366:Open Reading Frames; D012319:RNA Polymerase II / Q000378:metabolism; D012097:Repressor Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000032:analysis* / Q000378:metabolism; D012697:Serine Endopeptidases / Q000378:metabolism; D014157:Transcription Factors / Q000032:analysis* / Q000378:metabolism; D000099160:LexA Repressor Protein
D001426:Bacterial Proteins; D019731:Carbon-Nitrogen Ligases; D004247:DNA; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D012319:RNA Polymerase II; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D012697:Serine Endopeptidases; D014157:Transcription Factors; D000099160:LexA Repressor Protein; C058125:birA protein, E coli