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The protocol has been developed to effectively extract intact histones from sorghum leaf materials for profiling of histone post-translational modifications that can serve as potential epigenetic markers to aid engineering drought resistant crops.
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 innovative bioengineering solutions. Herein, we describe a protocol to isolate the nuclei and purify histones from sorghum leaf tissue. The extracted histones can be analyzed in their intact forms by top-down mass spectrometry (MS) coupled with 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). We have applied this protocol previously to profile histone PTMs from sorghum leaf tissue collected from a large-scale field study, aimed at identifying epigenetic markers of drought resistance. The protocol could potentially be adapted and optimized for chromatin immunoprecipitation-sequencing (ChIP-seq), or for studying histone PTMs in similar plants.
The increasing severity and frequency of drought is expected to affect productivity of cereal crops1,2. Sorghum is a cereal food and energy crop known for its exceptional ability to withstand water-limiting conditions3,4. We are pursuing mechanistic understanding of the interplay between drought stress, plant development, and epigenetics of sorghum [Sorghum bicolor (L.) Moench] plants. Our previous work has demonstrated strong connections between plant and rhizosphere microbiome in drought acclimation and responses at the molecular level
1. Preparing sorghum leaf material
NOTE: The sorghum plants were grown in soil in the field in Parlier, CA.
Following the protocol, the histones can be extracted and identified using the LC-MS analysis. The raw data and processed results are available at MassIVE (https://massive.ucsd.edu/) via accession: MSV000085770. Based on the TopPIC results from the representative sample (available also from MassIVE), we identified 303 histone proteoforms (106 H2A, 72 H2B, 103 H3, and 22 H4 proteoforms). Co-purified ribosomal proteoforms have also been detected, typically eluting early in the LC. They usually consist of ~20% of the identi.......
The presented protocol describes how to extract histones from sorghum leaf (or more generally plant leaf) samples. The average histone yield is expected to be 2–20 µg per 4–5 g sorghum leaf material. The materials are sufficiently pure for the downstream histone analysis by LC-MS (mostly histones with ~20% ribosomal protein contamination). Lower yield may be obtained due to sample variations, or potential mishandling/failures throughout the protocol. Maintaining the integrity of the nuclei before the nuc.......
We thank Ronald Moore and Thomas Fillmore for helping with mass spectrometry experiments, and Matthew Monroe for data deposition. This research was funded by grants from US Department of Energy (DOE) Biological and Environmental Research through the Epigenetic Control of Drought Response in Sorghum (EPICON) project under award number DE-SC0014081, from the US Department of Agriculture (USDA; CRIS 2030-21430-008-00D), and through the Joint BioEnergy Institute (JBEI), a facility sponsored by DOE (Contract DE-AC02-05CH11231) between Lawrence Berkeley National Laboratory and DOE. The research was performed using Environmental Molecular Sciences Laboratory (EMSL) (grid.436....
Name | Company | Catalog Number | Comments |
Acetonitrile | Fisher Chemical | A955-4L | |
Dithiothreitol (DTT) | Sigma | 43815-5G | |
EDTA, 500mM Solution, pH 8.0 | EMD Millipore Corp | 324504-500mL | |
Formic Acid | Thermo Scientific | 28905 | |
Guanidine Hydrochloride | Sigma | G3272-100G | |
MgCl2 | Sigma | M8266-100G | |
Potassium phosphate, dibasic | Sigma | P3786-100G | |
Protease Inhibitor Cocktail, cOmplete tablets | Roche | 5892791001 | |
Sodium butyrate | Sigma | 303410-5G | Used for histone deacetylase inhibitor |
Sodium Chloride (NaCl) | Sigma | S1888 | |
Sodium Fluoride | Sigma | S7020-100G | Used for phosphatase inhibitor |
Sodium Orthovanadate | Sigma | 450243-10G | Used for phosphatase inhibitor |
Sucrose | Sigma | S7903-5KG | |
Tris-HCl | Fisher Scientific | BP153-500 g | |
Triton X-100 | Sigma | T9284-100ML | |
Weak cation exchange resin, mesh 100-200 analytical (BioRex70) | Bio-Rad | 142-5842 | |
Disposables | |||
Chromatography column (Bio-Spin) | BIO-RAD | 732-6008 | |
Mesh 100 filter cloth | Millipore Sigma | NY1H09000 | This is part of the Sigma kit (catalog # CELLYTPN1) for plant nuclei extraction. Similar filters with the same mesh size can be used. |
Micropipette tips (P20, P200, P1000) | Sigma | ||
Tube, 50mL/15mL, Centrifuge, Conical | Genesee Scientific | 28-103 | |
Tube, Microcentrifuge, 1.5/2 mL | Sigma | ||
Equipment | |||
Analytical Balance | Fisher Scientific | 01-912-401 | |
Beakers (50mL – 2L) | |||
Microcentrifuge with cooling | Fisher Scientific | 13-690-006 | |
Micropipettes | |||
Swinging-bucket centrifuge with cooling | Fisher Scientific | ||
Vortex | Fisher Scientific | 50-728-002 | |
Water bath Sonicator | Fisher Scientific | 15-336-120 |
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