Development of genome wide tools to study regulatory elements is particularly important to understand how gene expression is controlled in the human genome. The rational study of transcription factors and DNA-binding proteins in sequenced human genome is critical for making the genome sequences maximally useful.
Chromatin Immuno-Precipitation (ChIP) materials analysed on microarrays for specific interactions (ChIP-chip) is a rapidly emerging technology in the study of DNA binding regulation proteins. A major current limitation of this technology is the lack of high quality antibodies proven to work.
Therefore, the use of an appropriate approach to produce high specific functional binding agents for high throughput functional genomic studies by ChIP-chip is crucial. The present proposal implements an approach for single-step purification of transcription factor and DNA-binding protein complexes based on specific in vivo biotinylation.
For this purpose the humanized (codon optimized) bacterial BirA biotin ligase and the target proteins of interest tagged with a biotin acceptor domain of 13 amino acid residue will be co-expressed in mammalian cells. This will provide the high-specific immuno-precipitation of in vivo biotinylated proteins with their DNA binding sites by strong biotinstreptavidin interaction.
This approach can be scaled up and can serve as the basis for successful genome wide studies. Through the use of this powerful assay important biological questions will find response. By accelerating the ChIP-c hip studies, this approach will help to better understand the gene regulation.
By identifying the DNA sequences reacting with regulatory proteins this approach will help to make the genome sequences maximally useful for further studies and classification, which will accelerate the global genome analysis.
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