In response to the ENCODE initiative, we propose to develop a novel ChiP-on-Chip technology to map functional DNA elements in the human genome. The proposed technology is to couple standard chromatin immunoprecipition with a new way to amplify and array the enriched DNA. The proposed technology aims to improve the sensitivity of an existing ChiP-on-Chip technology with new experimental design to permit future large-scale applications. ? ? We plan to systematically develop the technology in four steps. (1) We will start with the existing promoter array platform already in hand and determine whether we can use signature oligos to replace PCR-amplified promoter fragments in the array. This may improve hybridization efficiency and specificity of the existing system. The oligo-based array would be easier for future large scale mapping studies. More importantly, the experiments in the first aim will set the stage for the next phase of technology development. (2) We will implement a new strategy to amplify and label immunoprecipitated DNA. Our procedure will incorporate a number of strategies successfully used in other genomics approaches, including oligo ligation, solid phase selection, and signal amplification in a relatively unbiased way. According to the design, the technology has the potential to become a general platform with high specificity and sensitivity for a variety of functional genomics applications. (3) We will in the third aim to document the power of the technology under development by conducting three lines of experiments that can not be addressed with any existing technologies. Because each set of the experiments will be carried out in parallel by individual team labs, we will have extensive functional validation of the technology in the end. The experimental design also aims to create synergies among participating labs such that our array system will be progressively enlarged, which will prepare us for larger projects in the future. (4) Finally, we will test a general strategy for unbiased discovery of functional DNA elements involved in transcriptional regulation and transcription-splicing coupling in a chromosomal region defined by the ENCODE project. The proposed studies would be a power ? way to illustrate the utility of the technology in addressing a wide range of important biological questions. ? ?
| Garcia-Bassets, Ivan; Kwon, Young-Soo; Telese, Francesca et al. (2007) Histone methylation-dependent mechanisms impose ligand dependency for gene activation by nuclear receptors. Cell 128:505-18 |
| Kwon, Young-Soo; Garcia-Bassets, Ivan; Hutt, Kasey R et al. (2007) Sensitive ChIP-DSL technology reveals an extensive estrogen receptor alpha-binding program on human gene promoters. Proc Natl Acad Sci U S A 104:4852-7 |
