CN113265449A - Method for researching regulation and control modes of DNA methylation and histone methylation of gene promoter region - Google Patents
Method for researching regulation and control modes of DNA methylation and histone methylation of gene promoter region Download PDFInfo
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- 230000033228 biological regulation Effects 0.000 title claims abstract description 33
- 108700039691 Genetic Promoter Regions Proteins 0.000 title claims abstract description 31
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- 230000011987 methylation Effects 0.000 title claims abstract description 28
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- 102000011787 Histone Methyltransferases Human genes 0.000 claims description 6
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Abstract
The invention relates to a method for researching the regulation and control mode of DNA methylation and histone methylation of a gene promoter region, which comprises the following steps: determining the regulation of DNA methylation on a target gene, determining the regulation of histone H3K4me2/3 on the target gene, and determining the expression regulation of DNA methylation and histone H3K4me2/3 on the target gene; determining which epigenetic modifications have a major regulatory effect on the gene of interest. The method is simple and feasible, has feasibility, simple and convenient operation, clear and reliable research thought and wide applicability, and can be used for researching the regulation and control mode of DNA methylation and histone methylation on gene expression in different species or different biological processes because DNA methylation and histone methylation have high conservation.
Description
Technical Field
The invention relates to a method for researching the regulation and control modes of DNA methylation and histone methylation of a gene promoter region, belonging to the technical field of biology.
Background
DNA methylation can transfer methyl through methyltransferase, and can cause changes of chromatin structure, DNA stability and conformation and the like under the condition of not changing a DNA sequence, thereby regulating and controlling the specific expression of genes. Methylation of CpG islands is generally considered to be a factor inhibiting gene expression because it inhibits the binding of transcription factors to DNA and inhibits activities such as transposon. While histone is a basic protein bound to DNA, the methylation state of its different sites affects the regulation of gene expression. The research shows that the histone H3K4me2 and H3K4me3 modified sites are mainly enriched on a promoter of a transcription initiation site to activate gene expression, and H3K27me2 and H3K27me3 inhibit the gene expression. Thus, there may be simultaneous common modifications of DNA methylation and histone methylation in the promoter region, whereas the dominant effects of inhibitory DNA methylation and activating H3K4me2/3 on gene expression regulation may differ by tissue, species and biological processes. Therefore, there is still a need to further explore how DNA methylation and histone H3K4me2/3 regulate gene expression.
Disclosure of Invention
The present invention aims at solving the above problems and provides a method for studying the regulation and control patterns of DNA methylation and histone methylation in the promoter region of genes.
The technical scheme of the invention is as follows: a method for researching the regulation and control modes of DNA methylation and histone methylation of a gene promoter region is characterized by comprising the following steps:
(1) determining a promoter of a target gene of a sample, performing bioinformatics analysis on the promoter region of the target gene, analyzing a CpG island of the promoter region of the target gene by using an http:// www.Urogene.Org/cgi-bin/methprimer/methprimer.cgi online website, and further analyzing the DNA methylation level of the promoter region of the gene in different samples by bisulfite sequencing;
(2) acquiring an H3K4me2/3 enriched gene fragment in a target sample in a CHIP online database http:// chromosome. org/db/#/collecting the target sample to perform a CHIP-seq sequencing experiment of histone methylation H3K4me2/3, analyzing a histone methylation enriched area, and verifying the enrichment of H3K4me2/3 in a target gene promoter area by CHIP-qPCR;
(3) after DNA methylation and H3K4me2/3 enrichment in a target gene promoter region are determined, after the target gene promoter region is treated by using a DNA methylation inhibitor 5' aza, dual-luciferase and qRT-PCR (quantitative reverse transcription-polymerase chain reaction) are used for respectively detecting the promoter activity and the transcription level of a target gene, and the expression regulation and control of the DNA methylation on the target gene are determined;
(4) respectively treating a target gene by using a histone methylase Mll 2/demethylase Lsd1 interference vector, respectively detecting the starting activity and the transcription level of the target gene by using dual-luciferase and qRT-PCR, and determining the expression regulation and control of H3K4me2/3 on the target gene;
(5) in order to further research the specific regulation and control modes of DNA methylation and H3K4me2/3 methylation, 5' aza and histone methylase Mll 2/demethylase Lsd1 interference vectors are respectively used for processing target genes together, and dual-luciferase and qRT-PCR are respectively used for detecting the starting activity and the transcription level of the target genes; thereby determining which epigenetic modification plays a main regulating role on the target gene.
The sample is chicken primordial germ cells PGCs or chicken spermatogonial stem cells.
In the step (2), the enrichment of H3K4me2/3 in the promoter region of the target gene, especially in the CpG island region, is verified by CHIP-qPCR.
The method is advanced and scientific, and by the method, after the promoter of the target gene is determined, the bioinformatics analysis is firstly carried out on the promoter region of the target gene, the CpG island of the promoter region of the target gene is analyzed by using an http:// www.Urogene.Org/cgi-bin/methprimer/methprimer.cgi online website, and the DNA methylation level of the promoter region of the gene in different samples is further analyzed by bisulfite sequencing. Meanwhile, obtaining an H3K4me2/3 enriched gene fragment in a target sample from a CHIP online database (http:// genome. org/db/# /), collecting the target sample to perform a CHIP-seq sequencing experiment of histone methylation H3K4me2/3, analyzing a histone methylation enriched area, and verifying the enrichment of H3K4me2/3 in a target gene promoter area (particularly a CpG island) by CHIP-qPCR. After DNA methylation and H3K4me2/3 enrichment of a target gene promoter region are determined to exist at the same time, after the target gene promoter region is treated by using a DNA methylation inhibitor 5' aza, dual-luciferase and qRT-PCR are used for respectively detecting the promoter activity and the transcription level of a target gene, and the expression regulation and control of the DNA methylation on the target gene are determined. Meanwhile, a histone methylase Mll 2/demethylase Lsd1 interference vector is used for processing a target gene respectively, dual-luciferase and qRT-PCR are used for detecting the starting activity and the transcription level of the target gene respectively, and the expression regulation and control of H3K4me2/3 on the target gene are determined. In order to further research specific regulation and control modes of DNA methylation and H3K4me2/3 methylation, 5' aza and histone methylase Mll 2/demethylase Lsd1 interference vectors are respectively used for processing target genes, and dual-luciferase and qRT-PCR are respectively used for detecting the starting activity and the transcription level of the target genes. Thereby determining which epigenetic modification plays a main regulating role on the target gene.
The regulation of gene expression is affected at various levels by a variety of factors, including the level of transcription, mRNA, and translation. Regulation at the gene transcription level is achieved primarily by transcription factors and chromatin conformation changes. It is well known that transcription factors can form a transcription initiation complex together with RNA polymerase II, and participate in the transcription initiation process. This process also typically requires the involvement of epigenetic modifications. DNA methylation serves as an epigenetic factor that suppresses gene expression, usually by acting on the regulation of gene expression through methylation at CpG. However, the epigenetic modifications present during transcription are not only DNA methylation. Histone proteins serve as the core of nucleosomes, and methylation modifications at different sites of the histone proteins also have regulation on gene expression. However, if the CpG island is subjected to DNA methylation and activated histone methylation (such as H3K4me 2/3), the expression regulation and control patterns of the genes by the CpG island and the histone methylation are not researched. The method combines bioinformatics analysis, CHIP-seq and other experiments, systematically studies the regulation and control mode of double recombinant protein modification of the gene promoter region, and provides theoretical basis and operation method for exploring the gene expression regulation and control mechanism.
Has the advantages that:
the method is simple and feasible, has feasibility, simple and convenient operation, clear and reliable research thought and wide applicability, and can be used for researching the regulation and control mode of DNA methylation and histone methylation on gene expression in different species or different biological processes because DNA methylation and histone methylation have high conservation.
Detailed description of the invention
The invention is further described with reference to specific examples. The scope of the invention is not limited thereto:
1. determining the regulation and control of DNA methylation on a target gene;
according to the method, a chicken Primordial Germ Cell (PGCs) sample is taken as an example, and the DNA methylation of a target gene promoter region and the regulation and control mode of histone H3K4me2/3 in the sample are researched. First, using an online site
The CpG islands of the promoter region of the obtained gene were analyzed by http:// www.Urogene.Org/cgi-bin/methprimer. cgi, and the region enriched in DNA methylation was determined. Then designing an amplification primer according to the region; the genome of the PGC cells was extracted, transformed and purified using a bisulfite kit (Tiangen), and DNA methylated fragments were amplified using this DNA as a template. And (3) carrying out agarose gel electrophoresis and gel cutting recovery on the PCR product to obtain a target fragment, carrying out TA cloning and transforming competent cells, and selecting 10 positive colonies. And comparing the sequencing result with the DNA methylation fragment by utilizing a QUMA online website, and determining the DNA methylation level of the target gene promoter region in the PGCs. Transfecting a chicken fibroblast line/293T cell by using a dual-luciferase report vector of a target gene, treating the chicken fibroblast line/293T cell by using 5' aza of a DNA methylase inhibitor, culturing for 48 hours, and collecting cells for dual-luciferase report detection. Treating PGCs and chicken fibroblast lines/293T cells by using 5' aza of a DNA methylase inhibitor, culturing for 48 hours, collecting the cells, extracting RNA, carrying out reverse transcription to obtain cDNA, and carrying out qRT-PCR detection. Determining the transcriptional regulation of DNA methylation on the target gene.
2. Determining the regulation and control of the histone H3K4me2/3 on a target gene;
the H3K4me 2/3-enriched gene fragment in the target sample (e.g., chicken PGCs) was obtained in the CHIP online database (http:// cistome. org/db/# /). Meanwhile, chicken PGCs are collected, CHIP-seq sequencing of H3K4me2/3 is carried out, H3K4me2/3 enriched genes and sequences are obtained, existence of target genes is searched, and whether H3K4me2/3 enrichment exists in CpG island positions is searched. Designing CHIP-qPCR primers according to the fragment sequences, and verifying the sequencing result in PGCs. And determining that the promoter region of the target gene is enriched in H3K4me 2/3. Simultaneously transfecting a chicken fibroblast line/293T cell by using an Mll2/Lsd1 interference vector and a target gene dual-fluorescence report vector, culturing for 48h, and collecting cells for dual-luciferase report detection. Meanwhile, transfecting PGCs and chicken fibroblast cell line/293T cells with Mll2/Lsd1 interference vectors, culturing for 48H, collecting cells, extracting RNA, performing reverse transcription to obtain cDNA, detecting target gene expression by qRT-PCR, and determining the regulation and control of the histone H3K4me2/3 on the target gene.
3. Determining the expression regulation of DNA methylation and histone H3K4me2/3 on a target gene;
after separately determining the regulation of the expression of the gene by DNA methylation and H3K4me2/3, the regulation mode of the gene expression in the presence of the DNA methylation and the H3K4me2/3 is verified. Simultaneously transfecting a chicken fibroblast line/293T cell by using an Mll2/Lsd1 interference vector and a target gene dual-fluorescence report vector, carrying out 5' aza treatment on a DNA methylase inhibitor on the basis, culturing for 48h, and collecting cells for dual-luciferase report detection. Transfecting PGCs and chicken fibroblast cell lines/293T cells by using Mll2/Lsd1 interference vectors, simultaneously performing 5' aza treatment, culturing for 48H, collecting cells, extracting RNA, performing reverse transcription to obtain cDNA, detecting target gene expression by qRT-PCR, and determining the regulation and control modes of DNA methylation and histone H3K4me2/3 on the target gene.
In the above embodiments, chicken Primordial Germ Cells (PGCs) are used as the sample, and spermatogonial stem cells may also be used as the sample, and the procedure is the same as that for the chicken Primordial Germ Cells (PGCs).
Claims (3)
1. A method for researching the regulation and control modes of DNA methylation and histone methylation of a gene promoter region is characterized by comprising the following steps:
(1) determining a promoter of a target gene of a sample, performing bioinformatics analysis on the promoter region of the target gene, analyzing a CpG island of the promoter region of the target gene by using an http:// www.Urogene.Org/cgi-bin/methprimer/methprimer.cgi online website, and further analyzing the DNA methylation level of the promoter region of the gene in different samples by bisulfite sequencing;
(2) acquiring an H3K4me2/3 enriched gene fragment in a target sample in a CHIP online database http:// chromosome. org/db/#/collecting the target sample to perform a CHIP-seq sequencing experiment of histone methylation H3K4me2/3, analyzing a histone methylation enriched area, and verifying the enrichment of H3K4me2/3 in a target gene promoter area by CHIP-qPCR;
(3) after DNA methylation and H3K4me2/3 enrichment in a target gene promoter region are determined, after the target gene promoter region is treated by using a DNA methylation inhibitor 5' aza, dual-luciferase and qRT-PCR (quantitative reverse transcription-polymerase chain reaction) are used for respectively detecting the promoter activity and the transcription level of a target gene, and the expression regulation and control of the DNA methylation on the target gene are determined;
(4) respectively treating a target gene by using a histone methylase Mll 2/demethylase Lsd1 interference vector, respectively detecting the starting activity and the transcription level of the target gene by using dual-luciferase and qRT-PCR, and determining the expression regulation and control of H3K4me2/3 on the target gene;
(5) in order to further research the specific regulation and control modes of DNA methylation and H3K4me2/3 methylation, 5' aza and histone methylase Mll 2/demethylase Lsd1 interference vectors are respectively used for processing target genes together, and dual-luciferase and qRT-PCR are respectively used for detecting the starting activity and the transcription level of the target genes; thereby determining which epigenetic modification plays a main regulating role on the target gene.
2. The method of claim 1, wherein the sample is chicken Primordial Germ Cells (PGCs) or chicken spermatogonial stem cells.
3. The method for researching on the DNA methylation and histone methylation regulation patterns of the gene promoter region according to claim 1, wherein in the step (2), enrichment of H3K4me2/3 in the promoter region of the target gene, especially in the CpG island region, is verified by CHIP-qPCR.
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| CN111849918A (en) * | 2020-07-24 | 2020-10-30 | 扬州大学 | Method for researching target gene regulated and controlled by histone methylation in chicken SSCs |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103160573A (en) * | 2011-12-09 | 2013-06-19 | 上海聚类生物科技有限公司 | Model for researching genetic transcription regulation mechanism by multi-factor combination method |
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| CN103160573A (en) * | 2011-12-09 | 2013-06-19 | 上海聚类生物科技有限公司 | Model for researching genetic transcription regulation mechanism by multi-factor combination method |
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| SHUJIAN ZHOU等: "Epigenetic modification cooperates with Zeb1 transcription factor to regulate Bmp4 to promote chicken PGCs formation", GENE, no. 794, 9 June 2021 (2021-06-09), pages 7 * |
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| CN111849918A (en) * | 2020-07-24 | 2020-10-30 | 扬州大学 | Method for researching target gene regulated and controlled by histone methylation in chicken SSCs |
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