By utilizing the binding properties of two yeast proteins, researchers have now identified for the first time a method to determine which proteins bind to a certain DNA sequence.
Recently developed techniques such as chromatin immunoprecipitation coupled to DNA microarray analysis (ChIP–chip) or ChIPseq, in which immunoprecipitated DNA fragments are sequenced using a 'next-generation sequencing' technology, allow the identification of any DNA sequence that is bound by a specific protein. But is it possible to do the opposite and identify which proteins bind a certain sequence? A recent study in yeast offers a promising approach to tackle this question.
The retrotransposon Ty5 encodes an integrase that interacts with the silent information regulator 4 (Sir4) chromatin component and catalyses its insertion into the yeast genome, near Sir4-binding sites. The authors took advantage of the fact that fusing DNA-binding proteins to Sir4 directs Ty5 integration close to the binding sites of these proteins. Using this method, they were able to mark — through Ty5 integration — the DNA sequences that were 'visited' by transcription factors. For each transcription factor fused to Sir4, they used a Ty5 element that was engineered to contain a short sequence that could function as a 'barcode' (or 'calling card', as the authors propose) specific for that factor.
Following cell transformation (with a plasmid carrying the fusion gene and another carrying the corresponding Ty transposon) and induction of transposition, the DNA of the cells selected for the transposition events was harvested, amplified and analysed by DNA sequencing or DNA microarray hybridization. The authors tested the method first by using two transcription factors, Gal4 and Gcn4, independently, then together with five other transcription factors, each fused to Sir4 along with its corresponding Ty5 element. Analysis of three specific promoters revealed that each had only the barcode corresponding to the transcription factor that was known to bind it, indicating that the method is robust; moreover, it identified both known and unknown genomic targets.
Implementing this technology for all of the 200 yeast DNA-binding proteins could allow the whole yeast transcription factor network to be dissected in a single experiment.
Francesca Pentimalli
References
Wang, H., Johnston, M. & Mitra, R. D. Calling cards for DNA-binding proteins. Genome Res.17, 1202–1209 (2007) | Article | PubMed |
200 yeast DNA-binding proteins could allow the whole yeast transcription factor network to be dissected in a single experiment.