Post-translational modifications (PTMs) regulate gene expression by governing the recruitment, binding and activity of a number of regulatory proteins. These epigenetic factors have become compelling candidates for therapeutic manipulation of gene expression in cancer, neurodegeneration and other conditions. Thus far however, high-throughput methods are not available to comprehensively evaluate the interaction between PTMs and their regulatory protein partners, rendering the identification of epigenetic drug targets extremely challenging and slow. Next generation sequencing is an invaluable tool for epigenetics studies, however most protein-PTM interaction experiments are limited to a single PTM target and do not make full use of NGS systems' throughput. In addition, to enable these studies, PTM targets are often positioned on a synthetic peptide backbone. While generally cost-effective to produce a high number of PTM targets, this method is unlikely to closely recapitulate the complex chromatin structure found in eukaryotic nuclei. To address both of these issues, EpiCypher, Inc. is developing a barcoded nucleosome method for PTM-protein interaction studies. EpiCypher has pioneered a proprietary method for the production of recombinant histones which can be customized with a broad range of single and combinatorial PTMs. Coupling this technology with the nucleosome barcoding method recently reported by our collaborator Dr. Alex Ruthenburg of the University of Chicago, a library of nucleosomes will be constructed for high-throughput protein-PTM interaction studies. Briefly, the method employs dozens of DNA barcode sequences, appended to the standard 601 sequence known to tightly associate with recombinant histone octamers. After adding proteins-of-interest or nuclear extract, immunoprecipitation is used to isolate specific regulatory proteins and bound PTM-customized nucleosomes carrying PTM-identifying DNA barcodes. Finally, the sample is analyzed using standard NGS methods, whereby quantification of PTM-identifying barcode reads reveals interactions between a given regulatory protein and a specific PTM. The final assay will allow nuclear extract or specific proteins-of-interest to be combined with the PTM-customized nucleosome library to identify new interactions and compare the relative strength of known protein-PTM interactions. EpiCypher envisions a platform assay, allowing investigators to rapidly test hundreds of PTM combinations.
Several pharmaceuticals have been developed which manipulate post-translational modification such as methylation or acetylation of chromatin, a key feature of epigenetic gene regulation. Identifying new protein- chromatin relationships that could serve as new drug targets is cumbersome and time-consuming. This proposal focuses on the development of a high-throughput method, allowing post-translational modifications to be incorporated in uniquely-barcoded nucleosomes and subsequently tested for protein interactions.
