Histone Acid Extraction and High Throughput Mass Spectrometry to Profile Histone Modifications in Arabidopsis thaliana

Abstract

Histone post-translational modifications (PTMs) play important roles in many biological processes, including gene regulation and chromatin dynamics, and are thus of high interest across many fields of biological research. Chromatin immunoprecipitation coupled with sequencing (ChIP-seq) is a powerful tool to profile histone PTMs in vivo. This method, however, is largely dependent on the specificity and availability of suitable commercial antibodies. While mass spectrometry (MS)-based proteomic approaches to quantitatively measure histone PTMs have been developed in mammals and several other model organisms, such methods are currently not readily available in plants. One major challenge for the implementation of such methods in plants has been the difficulty in isolating sufficient amounts of pure, high-quality histones, a step rendered difficult by the presence of the cell wall. Here, we developed a high-yielding histone extraction and purification method optimized for Arabidopsis thaliana that can be used to obtain high-quality histones for MS. In contrast to other methods used in plants, this approach is relatively simple, and does not require membranes or additional specialized steps, such as gel excision or chromatography, to extract highly purified histones. We also describe methods for producing MS-ready histone peptides through chemical labeling and digestion. Finally, we describe an optimized method to quantify and analyze the resulting histone PTM data using a modified version of EpiProfile 2.0 for Arabidopsis. In all, the workflow described here can be used to measure changes to histone PTMs resulting from various treatments, stresses, and time courses, as well as in different mutant lines.

Detection and quantification of the histone code in the fungal genusAspergillus

“…For histone PTMs on histone proteins H3 and H4 in Aspergilli, we successfully detected methylations on histone H3 at lysines 4, 9, 36, and 79, acetylation on histone H3 at lysines 9, 14, 18, 23, 27, and 56, and histone H4 at lysines 5, 8, 12, 16, and 31 (Figure 3). All these modifications have previously been reported in human (38,51), Arabidopsis (52), S. cerevisiae (53), and in a few filamentous fungi (5,20,54). However, it is to our knowledge the first time that H3K79me1, H3K79me2, H4K5ac, H4K8ac, H4K12ac, and H4K31ac are detected in Aspergilli (Figure 3).…”

Atypical epigenetic and small RNA control of transposons in clonally reproducingSpirodela polyrhiza

“…This was unexpected as we anticipated in Spirodela a decrease of both marks, given their connection with DNA methylation and reduction upon loss of non-CG methylation in Arabidopsis [23,123]. To corroborate the observation, we investigated histone post-translational modifications (PTMs) by mass-spectrometry [124,125], which confirmed an approximately 8-10-times lower level of H3K9me2 but similar H3K9me1 levels in Spirodela relative to Arabidopsis ( Supplemental Fig. S22 ).…”