The techniques of chromatin immunoprecipitation coupled with DNA microarray (ChIP-chip) and sequencing (ChIP-seq) have gained widespread popularity among the greater scientific community. The data generated from such analyses have begun to provide valuable insight to the mechanisms of gene regulation, disease pathogenesis, embryonic stem (ES) cell pluripotency, and development. However, application of the technology to most transcription factors is hindered by the lack of ChIP-grade antibodies. To overcome this limitation, we have developed a method whereby recombinant adeno-associated virus (rAAV) is used to introduce epitope tag-encoding DNA into endogenous loci by homologous recombination-mediated """"""""knock-in"""""""". As proof of principle, we used this strategy to knock-in sequence encoding a triple FLAG epitope (3xFLAG) into the STAT3 and CHD7 loci in colorectal cancer cells. Using ChIP-chip analyses, we show that the FLAG tag facilitates genome wide identification of STAT3 and CHD7 binding sites with a commercially available anti-FLAG antibody. In this 3-year R01 application, we propose 3 aims.
In Aim 1, we will test the general applicability of the targeting approach to cell lines derived from different lineages.
In Aim 2, we will assess the fidelity of the 3xFLAG tagged transcription factors for genome wide ChIP analyses.
In Aim 3, we will develop methods for high-throughput tagging of a large number of transcription factors. The achievement of these aims should facilitate large-scale ChIP analysis of multiple transcription factors. Such studies will profoundly impact our knowledge of transcriptional networks and the biological processes they control.
Regulation of transcription is a highly coordinated process. Mutations in genes which lead to aberrant regulation of transcription can cause a host of human diseases, including developmental disorders, neurodegenerative conditions, cardiovascular disease, and cancer. The technology we propose to develop here is designed to accelerate the identification of functional DNA elements within the human genome. In turn, this should hasten our understanding of transcriptional regulation and the underlying mechanisms of human disease.
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