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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 PTMsin 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 forArabidopsis thalianathat 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. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Nuclear isolation and histone acid extraction Basic Protocol 2: Peptide labeling, digestion, and desalting Basic Protocol 3: Histone HPLC‐MS/MS and data analysis
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High-throughput capture of transcription factor-driven epigenome dynamics using PHILO ChIP-seq
Abstract Assessing the dynamics of chromatin features and transcription factor (TF) binding at scale remains a significant challenge in plants. Here, we present PHILO (Plant HIgh-throughput LOw input) ChIP-seq, a high-throughput ChIP-seq platform that enables the cost-effective and extensive capture of TF binding and genome-wide distributions of histone modifications. The PHILO ChIP-seq pipeline is adaptable to many plant species, requires very little starting material (1mg), and provides the option to use MNase (micrococcal nuclease) for chromatin fragmentation. By employing H3K9ac PHILO ChIP-seq on eight Arabidopsis thaliana jasmonic acid (JA) pathway mutants, with the simultaneous processing of over 100 samples, we not only recapitulated but also expanded the current understanding of the intricate interplay between the master TFs MYC2/3/4 and various chromatin regulators. Additionally, our analyses brought to light previously unknown histone acetylation patterns within the regulatory regions of MYC2 target genes in Arabidopsis, which is also conserved in tomato (Solanum lycopersicum). In summary, our PHILO ChIP-seq platform demonstrates its high effectiveness in investigating TF binding and chromatin dynamics on a large scale in plants, paving the way for the cost-efficient realization of complex experimental setups.
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- Award ID(s):
- 2339927
- PAR ID:
- 10556746
- Publisher / Repository:
- Oxford University Press
- Date Published:
- Journal Name:
- Nucleic Acids Research
- Volume:
- 52
- Issue:
- 22
- ISSN:
- 0305-1048
- Format(s):
- Medium: X Size: p. e105-e105
- Size(s):
- p. e105-e105
- Sponsoring Org:
- National Science Foundation
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