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 achieving the function of regulating gene expression3,4,5,6. Posttranslational modifications of histone include acetylation, phosphorylation, methylation, monoubiquitination, sumoylation, and ADP ribosylation7,8,9. Histone H3K4 methylation, particularly trimethylation, has been mapped to transcription start sites where it is associated with transcription replication, recombination, repair, and RNA processing in eukaryotes10,11.
ChIP-seq technology was introduced in 2007 and has become the experimental standard for the genome-wide analysis of transcriptional regulation and epigenetic mechanisms12,13. This method is suitable at the genome-wide scale and for obtaining histone or transcription factor interaction information, including DNA segments of DNA binding proteins. Any DNA sequences crosslinked to proteins of interest will coprecipitate as a part of the chromatin complex. New-generation sequencing techniques are also used to sequence 36-100 bp of DNA, which are then matched to the corresponding target genome.
In phytopathogenic fungi, research has recently begun to study how histone methylation modifications regulate their target genes in the process of pathogenicity. Some previous studies proved that the regulation of histone methylase-related genes is mainly reflected in gene silencing and catalyzing the production of Secondary Metabolites (SM). MoSet1 is the H3K4 methylase in M. oryzae. Knockout of this gene results in the complete deletion of H3K4me3 modification14. Compared with the wild-type strain, the expression of the gene MoCEL7C in the mutant is inhibited in the CMC-induced state and in the non-induced state (glucose or cellobiose), the expression of MoCEL7C increased15. In Fusarium graminearum, KMT6 can catalyze the methylation modification of H3K27me3, regulate the normal development of fungi, and help regulate the "cryptic genome" containing the SM gene cluster16,17,18,19. In 2013, Connolly reported that H3K9 and H3K27 methylation regulates the pathogenic process of fungi through secondary metabolites and effector factors that regulate the inhibition of target genes20. In Aspergillus, the modification of histones H3K4me2 and H3K4me3 is related to gene activation and plays an important role in controlling the chromatin level regulation of SM gene clusters21. In M. oryzae, Tig1 (homologous to Tig1 in yeast and mammals) is an HADC (histone deacetylase)22. Knockout of the Tig1 gene leads to the complete loss of pathogenicity and spore production ability in the null mutant. It is more sensitive to a peroxygen environment, which cannot produce infective hyphae22.
The rice blast caused by M. oryzae. is one of the most serious rice diseases in most rice-growing areas in the world19. Due to its representative infection process, M. oryzae is similar to the infection process of many important pathogenic fungi. As it can easily carry out molecular genetic operations, the fungus has become a model organism for studying fungal-plant interactions23. Blocking every step of the infection process of M. oryzae may result in unsuccessful infection. The morphological changes during the infection process are strictly regulated by the entire genome function and gene transcription. Among them, epigenetic modifications such as histone methylation play an essential role in the transcriptional regulation of functional genes24,25. However, so far, few studies have been done on the molecular mechanism of epigenetic modifications such as histone methylation and histone acetylation in the transcription of pathogenesis genes in M. oryzae. Therefore, further developing the epigenetic regulation mechanism of the rice blast fungus while researchinh the upstream and downstream regulatory network of these pathogenic related genes will help develop rice blast prevention and control strategies.
With the development of functional genomics such as ChIP-seq, especially in epigenetics, this high-throughput data acquisition method has accelerated research on chromosomes. Using the ChIP-seq experimental technology, the genome-wide distribution of histone methylation (such as H3K4me3, H3K27me3, H3K9me3) in M. oryzae and other filamentous fungi can be identified. Therefore, this method can help elucidate the molecular mechanisms underlying how epigenetic modifications regulate their candidate target genes during fungal pathogenesis in plant pathology.
1. Preparation of protoplasts from M. oryzae
2. In vivo crosslinking and sonication
3. IP of crosslinked protein/DNA
4. Collecting and rinsing the IP products
5. Elution of protein/DNA complexes
6. Reverse crosslinking of protein/DNA complexes
7. Purification and recovery of DNA
8. DNA repair and Solexa library construction
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 at 25% W, output 3 s, stop 5 s, at 4 °C. Further, the chromatin was immunopurified with H3K4me3 antibody and Dynabeads protein A/G. Subsequently, DNA fragments were extracted using the phenol-chloroform method for constructing a sequencing library and sequenced with single ends on a high-throughput sequencing platform.
The representative results of the wild-type, Δmobre2, Δmospp1, and Δmoswd2 strains with ChIP-seq method using the H3K4me3 antibody are shown in Figure 2. The H3K4me3 signals of the Δmobre2, Δmospp1, and Δmoswd2 deletion mutants were significantly decreased at its functional target regions. As shown in Figure 2, some selected candidate target genes, including MGG_14897, MGG_04237, MGG_04236, and MGG_04235, were mapped for H3K4me3 distribution. Compared to the wild-type strain P131, the signals of enriched H3K4me3-ChIP-seq reads in the Δmobre2, Δmospp1, and Δmoswd2 deletion mutants were largely decreased (Figure 2)26. These results suggest that the H3K4me3 modification plays important roles in regulating target gene expression in M. oryzae.
Figure 1. The flow chart of the ChIP-seq method in M. oryzae. (A) The genomic DNA of M. oryzae wascrosslinked with 1% formaldehyde. (B) Lysed blast fungus cells, broken DNA, free DNA, and histone binding DNA were subsequently obtained. (C) DNA fragments bound to histones and were extracted by specific binding to the H3K4me3 antibody. (D) Through reverse crosslinking, purified DNA subsequently obtained DNA fragments modified by H3K4me3 histones. (E-F) DNA fragments were sequenced, the sequencing results were compared, and sequences were identified in the M. oryzae DNA group. (G) Specific genes and loci of H3K4me3 histones in M. oryzae were retrieved. Please click here to view a larger version of this figure.
Figure 2. The Δmobre2, Δmospp1, and Δmoswd2 deletion mutants significantly decreased H3K4me3 profiles in their target regions. The H3K4me3-ChIP-seq distribution of enriched peaks around the coding regions of overlapped genes in Δmobre2, Δmospp1, and Δmoswd2 deletion mutants are decresased compared to the wild-type strain in the MGG_14897,MGG_04237, MGG_04236 and MGG_04235 genes26. The number in WT (input) labelled as [0-2074] signify means the results of ChIP in the range of genomic DNA [0-2074]. [0-2074] refers to 0-2074bp of Chromosome 6.The figure shows a random selection of the sequencing results, which only represents the DNA distribution on Chromosome 6. The complete sequencing results have been submitted to Genbank. (https//www.ncbi.nlm.nih.gov/bioproject/ accession 649321)26. Please click here to view a larger version of this figure.
Serial number | Sample name | Sample serial number | Number of tubes | Total (μg) | Fragment distribution | Database type | Remarks | ||||
1 | input | WH1703004169 | 1 | 2.7948 | The main peak is below 100bp, but there is DNA distribution between 100bp-500bp | ChIP-seq | Fragment is too small | ||||
P131(2) | |||||||||||
2 | Input | WH1703004170 | 1 | 2.4748 | The main peak is below 100bp, but there is DNA distribution between 100bp-500bp | ChIP-seq | Fragment is too small | ||||
Δmobre2(3) | |||||||||||
3 | input Δmospp1(4) | WH1703004171 | 1 | 3.22 | The main peak is below 100bp, but there is DNA distribution between 100bp-500bp | ChIP-seq | Fragment is too small | ||||
4 | input Δmoswd(5) | WH1703004172 | 1 | 3.97 | The main peak is below 100bp, but there is DNA distribution between 100bp-500bp | ChIP-seq | Fragment is too small | ||||
5 | P131(2) | WH1703004174 | 1 | 0.0735 | The main peak is between 100bp-500bp | ChIP-seq | |||||
6 | Δmobre2(3) | WH1703004175 | 1 | 0.0491 | The main peak is between 100bp-500bp | ChIP-seq | |||||
7 | Δmospp1(4) | WH1703004176 | 1 | 0.0288 | The main peak is between 100bp-500bp | ChIP-seq | |||||
8 | Δmoswd(5) | WH1703004177 | 1 | 0.0527 | The main peak is between 100bp-500bp | ChIP-seq |
Table 1. The total amount of DNA in this experiment. The total amount of input P131(2) is 2.7948 µg, the total amount of Δmobre2(3) (input) is 2.4748 µg, the total amount of Δmospp1(4) (input) is 3.22 µg, the total amount of Δmoswd2 (5) (input) is 3.97 µg, and the total amount of P131(2) is 0.0735 µg, the total amount of Δmobre2(3) is 0.0491µg, the total amount of Δmospp1(4) is 0.0288 µg, the total amount of Δmoswd2(5) is 0.0527 µg.
Figure 3. Electrophoresis detection of DNA after ultrasound. After ultrasound sonication, the DNA is subjected to a 1% agarose gel experiment to analyze the length of DNA fragments. The sonicated DNA fragment length is from 200-500 bp, and these DNA fragments can be used for the following steps of ChIP-seq. Please click here to view a larger version of this figure.
Figure 4. Bioanalyzer trace of Input and ChIP samples. The figure shows the fragment distribution of each sample, where the abscissa represents the fragment size, and the ordinate represents the peak size. The samples running on the bioanalyzer are input P131(2), Δmobre2(3) (input), Δmospp1(4) (input), Δmoswd2(5) (input), P131(2), Δmobre2(3), Δmospp1(4), Δmoswd(5). Among them, the distribution of input P131(2), Δmobre2(3) (input), Δmospp1(4) (input), Δmoswd2(5) (input) shows the main peak is below 100 bp, but there is DNA distribution at 100-500 bp. The true distribution of P131(2), Δmobre2(3), Δmospp1(4), and Δmoswd2(5) is between 100-500 bp as the main peak26. Please click here to view a larger version of this figure.
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 complexity of ChIP-seq data in terms of noise and variability makes this problem particularly difficult for ChIP-seq to overcome. In terms of data analysis, managing and analyzing the large amount of data generated by ChIP-seq experiments is also a challenge that has yet to be adequately addressed.
There are several key steps in the ChIP-seq experiment. First of all, the preparation of the protoplasts is very important. It is necessary to control the collapse time so that high-quality protoplasts can be collected. Ultrasound is also very important, the ultrasound time should be controlled, too long or too short will not work. Secondly, the amount of antibody added should be sufficient to facilitate the enrichment of more DNA fragments that bind to the protein. When verifying the quality and quantity of DNA precipitated in the ChIP-seq experiment Qubit Fluorometer was used. Agilent 2100 was used to detect the mass concentration and fragment distribution of DNA, which provides a basis for whether the sample can be used for subsequent library establishment and sequencing experiments.
Overall, this protocol enhances the understanding of the whole genome-wide distribution of epigenetic modifications that regulate pathogenic genes during pathogen infection. This method will contribute to identifying molecular mechanisms of epigenetic modifications and identify new target genes during fungi development and pathogen-induced pathogenesis in M. oryzae and other filamentous fungi.
The authors have nothing to disclose.
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 | 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 |