An Unnatural Amino Acid-based Chromatin Isolation Method (UChIMe) for the Study of Cancer-associated Protein-DNA Interactions

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PROJECT SUMMARY/ABSTRACT Protein-DNA interactions play key roles in many essential biological processes of all known organisms. Many human diseases, such as cancers, result from the misregulation of protein-DNA interactions. The long-term goal is to accurately map genome-wide and disease-associated protein-DNA interactions in single cells. Chromatin immunoprecipitation (ChIP) coupled to high-throughput sequencing (ChIP-Seq) has been widely used to map DNA sites of protein occupancy. Like most immunoprecipitation experiments, the specificity and affinity of antibodies is the most critical factor in ChIP analysis. It is estimated that fewer than 20% of tested antibodies are suitable for ChIP analyses. More importantly, it is still challenging to minimize random background noises from non-specific binding of antibodies. A novel method to elucidate genome-wide DNA binding sites of proteins with improved accuracy and sensitivity is critically needed. As such, the goal of this project is to develop a novel Unnatural Amino Acid (unAA)-based Chromatin Isolation Method (UChIMe) to analyze genome-wide protein-DNA interactions with improved accuracy and sensitivity. More specifically, two aims will be achieved: 1) develop UChIMe to probe Pol II binding sites and 2) analyze global binding sites of MEIS (myeloid ectopic viral integration site) proteins using UChIMe-seq. An unAA containing an alkyne functional group will be site-specifically incorporated into the target protein in live cells. This nonproteinogenic alkyne group will serve as a chemical handle to react with an azide-containing biotin molecule. The target protein will therefore be labeled with biotin and be ready for isolation using streptavidin resin. In comparison to the antibody-dependent ChIP method, UChIMe will not require the use of antibody, leading to major reductions in experimental cost and time. UChIMe will also enable the incorporation of unAA at any site of the target protein and will therefore be more flexible than the epitope tagging CHIP-seq method. Furthermore, the precision of an unAA-initiated chemical reaction will minimize background caused by non-specific binding of the antibody, and the proposed method will improve the detection limit by taking advantage of the high-affinity biotin-streptavidin interaction, which is 103-106 times greater than interactions between epitopes and their specific antibodies. Overall, UChIMe will enable the analysis of smaller samples (cell numbers) with improved accuracy and sensitivity and therefore will broaden the types of samples that can be studied. Successful development of the method will have broad applications to the studies of genome-wide DNA binding sites of target proteins. This project fits the Nebraska Center for Integrated Biomolecular Communication’s thematic focus on unraveling signal pathway shifts in complex diseases by developing novel biomolecular strategies for genome-wide mapping of DNA binding sites of transcription factors. This multidisciplinary project will contribute to and be facilitated by the Center’s collaborative environment and core services.