Published: June 2nd, 2021
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.
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.
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 structures1,2. Chromatin remodeling and covalent histone modification affect and regulate the function and structure of chromatin through the variation of chromatin polymers, thereby achiev....
1. Preparation of protoplasts from M. oryzae
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 .......
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.......
This work was supported by National Natural Science Foundation of China (Grant no. 31871638), the Special Scientific Research Project of Beijing Agriculture University (YQ201603), the Scientific Project of Beijing Educational Committee (KM201610020005), the High-level scientific research cultivation project of BUA (GJB2021005).....
|acidic casein hydrolysate
|protein and dissolution
|DNA End-Repair kit
|Repair DNA or cDNA damaged by enzymatic or mechanical shearing
|Membrane and liquid
|enzymatic casein hydrolysate
|Immune response to H3K4me3 protein
|illumina Genome Analyzer
|illumina Hiseq 2000
|Illumina PCR primers
|Random universal primer
|specific removal RNA
|cell lysis buffer
|Animal normal immunoglobulin
|preparation of protein complex eluent
|cell lysate to promote cell lysis
|PCR Purification kit
|The purification procedure removes primers from DNA samples
|A protein inhibitor that decreases protein activity
|DNA Extraction Reagent
|cell lysis buffer
|cover up the charge differences
|sodium acetate solution
|Acetic acid buffer
|inhibition of protease degradation
|T4 DNA ligase
|Under the condition of ATP as coenzyme, DNA ligase
|T4 DNA ligase buffer
|DNA ligase buffer
|keep the membrane protein stable
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