A Novel Method For Developing Recombinant Histone Ptm Antibodies Specific For Chip

Histones are decorated by post translational modifications (PTMs) that serve as epigenetic signatures for gene expression. These marks are essential for organizing the genome into active regions of euchromatin where the DNA is accessible and silenced regions of heterochromatin where the DNA is more tightly compact. Histone PTMs provide a unique challenge for antibody validation as these antibodies need to recognize the difference between very similar modifications such as a mono, di or tri-methyl on a single residue. Traditional screening methods in developing histone PTM antibodies have used direct ELISA and peptide array to identify candidates and validate their specificity. These screening methods recognize the epitope in a denatured format which may be a poor predictor of their performance and specificity in applications such as ChIP that requires the antibody to recognize the modification within the context of an assembled nucleosome. Indeed, recent evidence demonstrates that peptide arrays do not recapitulate antibody specificity performance in immunoprecipitation-based assays including ChIP [1-3]. This lack of specificity can have the consequence of misleading our understanding of the biology and function of a histone modification. We have thus altered our screening workflow to better select recombinant antibodies specific for the intended modification for ChIP. We have employed a Luminex based assay utilizing beads coupled to nucleosomes expressing specific PTMs is used to screen candidate supernatants. This assay has been found to be a good predictor for antibodies that will be specific in SNAP-ChIP (Sample Normalization and Antibody Profiling Chromatin ImmunoPrecipitation). SNAP-ChIP is a panel of spike-in recombinant semi-synthetic modified nucleosomes that allows one to determine if an antibody is enriching the target of interest relative to other histone PTMs (i.e. off-target) in the panel. Positive candidates from the Luminex assay are purified and then challenged for specificity using SNAP-ChIP. This screening approach has resulted in identifying highly specific histone PTM antibodies for ChIP. 1. Nishikori S, Hattori T, Fucks SM et al. (2012) J Mol Biol 424:391-399. 2. Grzbowski AT, Chen Z, Ruthenburg AJ (2015) Mol Cell 58:886-899. 3. Shah RN, Grzbowski AT, Cornett EM et al. (2018) Mol Cell 72:162-177. Citation Format: Eliza C. Small, Afzal Husain, Alhad A. Ketkar, Andrea Johnstone, Mathew Marunde, Keli Rodriguez, Danielle Maryanski, Aparna Chandraekaran, Sudha Balasubramanian, Nora Marbaniang, Michael-Christopher Keogh, Kevin J. Harvey. A novel method for developing recombinant histone PTM antibodies specific for ChIP [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3644.