BIRA FOR CHIP-CHIP · Optimisation of a non-antibody based technology to improve the efficiency of genome-wide analysis of DNA binding sites
FP6 — Marie Curie Actions (Human Resources and Mobility)
- Duration
- 2008-05-01 → 2010-04-30
- EU contribution
- €158,479
- Participants
- 2
- Scheme
- EIF
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Results in brief
Final Activity and Management Report Summary - BIRA FOR CHIP-CHIP (Optimisation of a non-antibody based technology to improve the efficiency of genome-wide analysis of DNA binding sites)
Now that the human genome is fully sequenced, a current goal is to study proteins important in controlling gene expression. These proteins ensure that genes are expressed in the right cell type, at the right level, and at the right time. Many of the proteins which control gene expression bind to DNA sequences in the genome - thus providing a framework for cellular responses which regulate the activity of genes nearby. Since current DNA-binding protein based strategies are dependent on specialised affinity reagents - antibodies - the present proposal aimed to overcome the requirement for specific antibodies for each and every different protein which are to be studied. This is accomplished by attaching a biotin "tag" onto the proteins which control gene expression. This is accomplished by altering the genes for these proteins so that they contain a biotin "tag". When these proteins are expressed, this "tag" became part of the proteins themselves. One can then isolate the "tagged" protein using existing protein purification techniques. This method allows the genome locations of these proteins and the genes that they regulate to be determined. During the Marie Curie post-doctoral training period, the applicant Serdar Kasakyan learned a state-of-the-art technique (Bacterial Artificial Chromosome recombineering) at the Technical University of Dresden and set up this approach in the host laboratory (Dr David Vetrie) at the University of Glasgow. By using this technology the applicant constructed biotin tagging vectors for four proteins which bind DNA and regulate gene expression (TAL1, LDB1, JHD1A, NSD1). These vectors will be placed into cells and will convert normal copies of the genes for these proteins into "tagged" versions. Cell lines containing these "tagged" proteins are currently being generated and will be a powerful set of tools for further studies in the host laboratory aimed at investigating gene regulation. Thus, the applicant has facilitated both his own training in this state-of-the-art field, as well as facilitating the research in the host laboratory.
Data: CORDIS, © European Union
Project objective
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.
Original text from CORDIS.
Participants
- UNIVERSITY OF GLASGOW · GLASGOWCoordinatorUnited Kingdom
- GENOME RESEARCH LTD. · LONDONCity levelUnited Kingdom
Links
Data: CORDIS, © European Union