Isolation of Histone from Sorghum Leaf Tissue for Top Down Mass Spectrometry Profiling of Potential Epigenetic Markers
Journal Article
·
· Journal of Visualized Experiments
- BATTELLE (PACIFIC NW LAB)
- Joint Genome Institute
- Lawrence Berkeley National Laboratory
- Private contributor; former PNNL post-masters
- University of California
- Kearney Agricultural Research & Extension Center
- UC Kearney Agricultural Research and Extension Center
- University of California, Davis
- University of California, Berkeley
Histones belong to a family of highly conserved proteins in eukaryotes. They pack DNA into nucleosomes as functional units of chromatin. Post-translational modifications (PTMs) of histones, which are highly dynamic and can be added or removed by enzymes, play critical roles in regulating gene expression. In plants, epigenetic factors including histone PTMs are related to their adaptive responses to the environment. Understanding the molecular mechanisms of epigenetic control can bring unprecedented opportunities for engineering solutions to increase the resilience of crops to climate change. Herein, we describe a protocol to isolate the nuclei and purify histones from sorghum leaf tissue. The extracted histones can be analyzed as their intact forms by top-down mass spectrometry (MS) coupled to online reversed-phase (RP) liquid chromatography (LC). Combinations and stoichiometry of multiple PTMs on the same histone proteoform can be readily identified. In addition, histone tail clipping can be detected using the top-down LC-MS workflow thus yielding the global PTM profile of core histones (H4, H2A, H2B, H3). By comparing the PTM profile among samples corresponding to different conditions (e.g. drought vs. control), potential epigenetic marks can be discovered as targets for further characterization using approaches such as chromatin immunoprecipitation – sequencing (ChIP-seq).
- Research Organization:
- Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States); Pacific Northwest National Laboratory (PNNL), Richland, WA (United States)
- Sponsoring Organization:
- USDOE Office of Science (SC)
- Grant/Contract Number:
- AC02-05CH11231; AC05-76RL01830
- OSTI ID:
- 1788205
- Alternate ID(s):
- OSTI ID: 1814619
- Report Number(s):
- PNNL-SA--153694
- Journal Information:
- Journal of Visualized Experiments, Journal Name: Journal of Visualized Experiments Journal Issue: 169 Vol. 2021; ISSN 1940-087X
- Publisher:
- MyJoVE Corp.Copyright Statement
- Country of Publication:
- United States
- Language:
- English
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