Abstract
Histone post-translational modifications (PTMs) contribute to chromatin accessibility due to their chemical properties and their ability to recruit enzymes responsible for DNA readout and chromatin remodeling. To date, more than 400 different histone PTMs and thousands of combinations of PTMs have been identified, the vast majority with still unknown biological function. Identification and quantification of histone PTMs has become routine in mass spectrometry (MS) but, since raising antibodies for each PTM in a study can be prohibitive, lots of potential is lost from MS datasets when uncharacterized PTMs are found to be significantly regulated. We developed an assay that uses metabolic labeling and MS to associate chromatin accessibility with histone PTMs and their combinations. The labeling is achieved by spiking in the cell media a 5x concentration of stable isotope labeled arginine and allow cells to grow for at least one cell cycle. We quantified the labeling incorporation of about 200 histone peptides with a proteomics workflow, and we confirmed that peptides carrying PTMs with extensively characterized roles in active transcription or gene silencing were in highly or poorly labeled forms, respectively. Data were further validated using next-generation sequencing to assess the transcription rate of chromatin regions modified with five selected PTMs. Furthermore, we quantified the labeling rate of peptides carrying co-existing PTMs, proving that this method is suitable for combinatorial PTMs. We focus on the abundant bivalent mark H3K27me3K36me2, showing that H3K27me3 dominantly represses histone swapping rate even in the presence of the more permissive PTM H3K36me2. Together, we envision this method will help to generate hypotheses regarding histone PTM functions and, potentially, elucidate the role of combinatorial histone codes.
Highlights
Epigenetics is the study of heritable changes in gene regulation that are not coded in the DNA sequence, a phenomenon playing a fundamental role in a wide range of biological processes including cellular differentiation and disease pathogenesis[1]
We present a new workflow and data processing method to integrate in a single analysis the relative quantification of histone post-translational modifications (PTMs) and the turnover rate of nuclear histone proteins carrying the given PTM
Canonical histones are synthesized during the S-phase of the cell cycle[32], but they are incorporated into the chromatin and modified differently depending on the properties and biological function of the chromatin region
Summary
We considered the following: (i) one cell contains about 300 pg of proteins, 1,000,000 cells contain about 300 μg of proteins; (ii) arginine occurs in cellular proteins at 5.78% compared to total amino acids[33]; (iii) 3 days cell growth leads to a maximum of 4,000,000 cells at the end of the culture. Together, this corresponds to ~70 μg of unlabeled arginine within the cell proteome. This indicates that the histone carrying the acetylated form has a faster accumulation of heavy labeling than the
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