Isolation of Histone from Sorghum Leaf Tissue for Top Down Mass Spectrometry Profiling of Potential Epigenetic Markers

Summary

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.

Abstract

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.

Introduction

The increasing severity and frequency of drought is expected to affect productivity of cereal crops^1,2. Sorghum is a cereal food and energy crop known for its exceptional ability to withstand water-limiting conditions^3,4. 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

Protocol

1. Preparing sorghum leaf material

NOTE: The sorghum plants were grown in soil in the field in Parlier, CA.

1. Collect sorghum leaves from plants into 50 mL centrifuge tubes and immediately freeze the tube in liquid nitrogen. Collect leaf tissue by tearing off the third and fourth fully emerged leaf from the primary tiller.
   NOTE: More details of field condition, sample growth, and collection can be found in the published report¹⁸.
2. Grind leaves with liquid nitrogen and immediately transfer to a centrifuge tube.
3. Store the ground leaf at -80 °C until use. Take about 4 g of cryo-g....

Results

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.......

Discussion

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.......

Materials

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