Chromatin Immunoprecipitation in Native Budding Yeast (Saccharomyces cerevisiae) Using Photo‐Crosslinking Unnatural Amino Acids

Abstract

Exploiting an expanded genetic code in yeast, we can site‐specifically encode unnatural amino acids into the native chromatin landscape of the living cell. Utilizing the UV‐activated photo‐crosslinking amino acid, p‐benzoylphenylalanine (pBPA), we can covalently trap protein‐protein interactions when contacts between proteins are made within binding distances of ~ 0.4 nm. We have mapped the RSC remodeler complex protein, Sth1, to the nucleosome and shown that its binding is regulated by posttranslational modifications, including acetylation and SUMOylation. RSC has been linked to promoter sites and we want to further our binding map with an understanding of its occupancy across the genome. To map the loci of binding sites we use a double IP chromatin immunoprecipitation (ChIP) technique that utilizes the power of in vivo protein‐protein crosslinking from histones. Histones are well associated with DNA and efficient targets for ChIP analysis; therefore, we propose that if the histone protein is first crosslinked to a nucleosomal protein target, that the histone can be initially precipitated in tandem with its crosslinked protein and associated DNA fragments. The target protein can then be further precipitated to purify DNA, via its interaction with the histone. Our first goal was to establish histone occupancy of pBPA‐containing histones. Using ChIP, we were able to isolate DNA from both wild type and mutant histones where we can achieve a quantitate analysis of the difference with qPCR. Building on this we will move to a double IP system to determine Sth1 occupancy. This work aims to establish a novel approach in ChIP targeting of proteins that are difficult to analyze due to more transient interactions at the nucleosomal core.Support or Funding InformationNIH R15 grant