The Development and Characterization of Antibodies to Site‐specific O‐GlcNAc modified Histones for Epigenetic Research

Abstract

O‐glycosylation of nuclear and cytoplasmic proteins by a single β‐N‐acetyl‐D‐glucosamine moiety (O‐GlcNAc) is a common post‐translational modification that is highly dynamic and fluctuates in response to cellular stimuli. This type of glycosylation has been found on approximately a thousand human proteins to date and is thought to be nearly as widespread and abundant as protein phosphorylation. Many of the first proteins identified carrying this modification were transcription factors, and it became apparent in the last several years that O‐GlcNAc plays a significant role in chromatin remodeling and gene expression.Given the complexity of the histone code, site‐specific antibodies are needed to begin to elucidate the hierarchy of modifications as well as the biological significance of single and multiple post‐translational modifications of the histones. We have developed and approach to provide high‐affinity site‐specific O‐GlcNAc antibodies that appear to overcome issues related to small carbohydrates being poor antigens and the O‐GlcNAc modified epitopes being self‐antigens. These site‐specific antibodies require a more elaborate characterization scheme than other antibodies as they need to demonstrate both a preference for the O‐GlcNAcylated species over the native peptide sequence and a specificity to the amino acid sequence. We use a multistep evaluation process to characterize our antibodies. 1) ELISA assay, to determine the preference of the antibody to the O‐GlcNAcylated peptide over the unmodified form; 2) Cross‐reactivity of the antibody to other peptide sequences modified with O‐GlcNAc; 3) Immunoprecipitation increasing wash stringencies and 4) Evaluation of the site‐specific O‐GlcNAc histone antibodies for their potential as ChIP‐Seq reagents. Using this process, we have authenticated our site‐specific O‐GlcNAc antibodies and have demonstrated that they possess the preference and specificity needed for epigenetic research.Support or Funding InformationResearch reported in this publication was supported by the National Institute of General Medical Sciences of the National Institutes of Health under Award Numbers 1R43GM110887‐01 and R44GM110887‐02.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.