Genome-wide Analysis of Histone Modifications Distribution using the Chromatin Immunoprecipitation Sequencing Method in Magnaporthe oryzae

Summary

Here, we present a protocol to analyze the genome-wide distribution of histone modifications, which can identify new target genes in the pathogenesis of M. oryzae and other filamentous fungi.

Abstract

Chromatin immunoprecipitation sequencing (ChIP-seq) is a powerful and widely used molecular technique for mapping whole genome locations of transcription factors (TFs), chromatin regulators, and histone modifications, as well as detecting entire genomes for uncovering TF binding patterns and histone posttranslational modifications. Chromatin-modifying activities, such as histone methylation, are often recruited to specific gene regulatory sequences, causing localized changes in chromatin structures and resulting in specific transcriptional effects. The rice blast is a devastating fungal disease on rice throughout the world and is a model system for studying fungus-plant interaction. However, the molecular mechanisms in how the histone modifications regulate their virulence genes in Magnaporthe oryzae remain elusive. More researchers need to use ChIP-seq to study how histone epigenetic modification regulates their target genes. ChIP-seq is also widely used to study the interaction between protein and DNA in animals and plants, but it is less used in the field of plant pathology and has not been well developed. In this paper, we describe the experimental process and operation method of ChIP-seq to identify the genome-wide distribution of histone methylation (such as H3K4me3) that binds to the functional target genes in M. oryzae. Here, we present a protocol to analyze the genome-wide distribution of histone modifications, which can identify new target genes in the pathogenesis of M. oryzae and other filamentous fungi.

Introduction

Epigenetics is a branch of genetic research that refers to the heritable change of gene expression without changing the nucleotide sequence of genes. An increasing number of studies have shown that epigenetic regulation plays an important role in the growth and development of eukaryotic cells, including chromatin that regulates and affects gene expression through the dynamic process of folding and assembly into higher-order structures^1,2. Chromatin remodeling and covalent histone modification affect and regulate the function and structure of chromatin through the variation of chromatin polymers, thereby achiev....

Protocol

1. Preparation of protoplasts from M. oryzae

1. Prepare the oatmeal-tomato agar (OTA).
   1. Weigh 30-50 g of oatmeal and boil it in 800 mL of water (ddH₂O) for 20 min. Filter through two layers of gauze and take the filtrate.
   2. Pick ripe tomatoes and peel them. Squeeze the juice, and filter through two layers of gauze to collect 150 mL of the filtered juice.
   3. Mix all the tomato juice and the prepared oat filtrate thoroughly and add ddH₂O up to 1000 mL.
   4. Add 250 mL of the OTA and 2.5 g of agar powder to a 500 mL conical flask, and autoclave for 20 min. Store at 25 °C.
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Results

The whole flow chart of the ChIP-seq method is shown in Figure 1. ChIP-seq experiments were performed using antibodies against H3K4me3 in the wild-type strain P131 and three null mutant strains that were devoid of mobre2, mospp1, and moswd2 gene to verify the whole genome-wide profile of histone H3K4me3 distribution in M. oryzae. The protoplasts of the wild-type strain, Δmobre2, Δmospp1, and Δmoswd2, were prepared and sonicated a.......

Discussion

Recently, ChIP-seq has become a widely used genomic analysis method for determining the binding sites of TFs or enrichment sites modified by specific histones. Compared to previous ChIP-seq technology, new ChIP-seq technology is highly sensitive and flexible. Results are provided in high resolution without negative effects, such as the noise signal caused by the non-specific hybridization of nucleic acids. Although this is a common gene expression analysis, many computational methods have been validated, and the complexi.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
acidic casein hydrolysate	WAKO	65072-00-6	Medium configuration
agar powder	scientan	9002-18-0	Medium configuration
deoxycholic acid	MedChemExpress	83-44-3	protein and dissolution
DNA End-Repair kit	NovoBiotec	ER81050	Repair DNA or cDNA damaged by enzymatic or mechanical shearing
Dynabeads	Invitrogen	no.100.02D	Binding target
EB buffer	JIMEI	JC2174	Membrane and liquid
EDTA	ThermoFisher	AM9912	protease inhibitor
enzymatic casein hydrolysate	Sigma	91079-40-2	Medium configuration
glucose	Sigma	50-99-7	Medium configuration
glycogen	ThermoFisher	AM9510	Precipitant action
H3K4me3 antibodies	Abcam	ab8580	Immune response to H3K4me3 protein
illumina Genome Analyzer	illumina	illumina Hiseq 2000	Large configuration
Illumina PCR primers	illumina	CleanPlex	Random universal primer
isoamyl alcohol	chemical book	30899-19-5	Purified DNA
LiCl	ThermoFisher	AM9480	specific removal RNA
lysing enzymes	Sigma	L1412-10G	cell lysis buffer
Mouse IgG	Yeasen	36111ES10	Animal normal immunoglobulin
NaCl solution	ThermoFisher	7647-14-5	Medium configuration
NaHCO3	Seebio	SH30173.08*	preparation of protein complex eluent
NP-40	ThermoFisher	85124	cell lysate to promote cell lysis
PCR Purification kit	Qiagen	28004	The purification procedure removes primers from DNA samples
protease inhibitors	ThermoFisher	A32965	A protein inhibitor that decreases protein activity
Proteinase K	ThermoFisher	AM2546	DNA Extraction Reagent
Qubit 4.0	ThermoFisher	Q33226	Medium configuration
RIPA buffer	ThermoFisher	9806S	cell lysis buffer
RNase A	ThermoFisher	AM2271	Purified DNA
SDS	ThermoFisher	AM9820	cover up the charge differences
sodium acetate solution	ThermoFisher	R1181	Acetic acid buffer
sodium deoxycholate	ThermoFisher	89904	inhibition of protease degradation
T4 DNA ligase	ThermoFisher	EL0011	Under the condition of ATP as coenzyme, DNA ligase
T4 DNA ligase buffer	ThermoFisher	B69	DNA ligase buffer
Tris-HCl	ThermoFisher	1185-53-1	buffer action
Triton X-100	ThermoFisher	HFH10	keep the membrane protein stable
yeast extract	OXOID	LP0021	Medium configuration