CRISPR-based targeting of DNA methylation in Arabidopsis thaliana by a bacterial CG specific DNA methyltransferase.
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ABSTRACT:
PROVIDER: PRJNA630258 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR11685224.fastq.gz | Fastqsanger.gz | |||
| SRR11685225.fastq.gz | Fastqsanger.gz | |||
| SRR11685226.fastq.gz | Fastqsanger.gz | |||
| SRR11685227.fastq.gz | Fastqsanger.gz | |||
| SRR11685228.fastq.gz | Fastqsanger.gz |
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Project description:CRISPR-based targeted modification of epigenetic marks is an important strategy to regulate the expression of genes and their associated phenotypes in plants and animals. The manipulation of DNA cytosine methylation is particularly attractive in plants since DNA methylation changes are often heritable in subsequent plant generations. Although plants have DNA methylation in all sequence contexts (CG, CHG, CHH, where H can be any nucleotide but G), methylation at symmetrical CG sites is the most important for gene silencing, and is also the most efficiently maintained through miotic and meiotic cell divisions. Thus, DNA methylation targeting tools that directly install CG methylation are highly desirable. Here we have developed two CRISPR-based CG specific targeted DNA methylation systems for plants using the DNA methyltransferase MQ1 (SssI) from the bacterium Mollicutes spiroplasma. To reduce off-target effects, we used a variant of the enzyme with reduced catalytic activity. We demonstrate that methylation added by MQ1 is highly target-specific and can be heritably maintained in the absence of the effector. These tools should be valuable both in crop engineering and in plant genetic research.
2021-05-19 | GSE149840 | GEO
Project description:Targeting of epigenetic marks like DNA methylation to specific loci to manipulate gene expression is important in both basic research and in crop plant engineering. However, the extent to which targeted DNA methylation can be heritable is unknown. Here, we show that targeting the CG-specific methyltransferase M.SssI (SssI) from Spiroplasma sp. strain MQ1 with an artificial zinc finger (ZF) protein can establish heritable CG methylation and silencing of the targeted loci in Arabidopsis. This occurs even in the absence of a functional RNA-directed DNA methylation pathway, which is usually required for de novo methylation. In addition to the locus-specific targeted CG methylation, we observed widespread ectopic CG methylation throughout the genome that was highly heritable over generations, even in the absence of the effector transgene. A comparison with other epigenetic features showed that this ectopic methylation occurred preferentially at less open chromatin regions that were lacking in positive epigenetic histone marks. Although ectopic CG methylation was highly enriched over gene body regions, it showed little effect on transcription of these genes. However, we observed a mild but significant reduction in the histone variant H2A.Z and H3K27me3 over genes with ectopic CG methylation. These results outline general principals of the interaction of CG methylation with other epigenomic features that should help guide future efforts to engineer epigenomes.
2021-03-22 | GSE158027 | GEO
Project description:Ectopic targeting of CG DNA methylation in Arabidopsis with the bacterial SssI methyltransferase.
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Project description:CRISPR-based targeted modification of epigenetic marks such as DNA cytosine methylation is an important strategy to regulate the expression of genes and their associated phenotypes. Although plants have DNA methylation in all sequence contexts (CG, CHG, CHH, where H = A, T, C), methylation in the symmetric CG context is particularly important for gene silencing and is very efficiently maintained through mitotic and meiotic cell divisions. Tools that can directly add CG methylation to specific loci are therefore highly desirable but are currently lacking in plants. Here we have developed two CRISPR-based CG-specific targeted DNA methylation systems for plants using a variant of the bacterial CG-specific DNA methyltransferase MQ1 with reduced activity but high specificity. We demonstrate that the methylation added by MQ1 is highly target specific and can be heritably maintained in the absence of the effector. These tools should be valuable both in crop engineering and in plant genetic research.
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Project description:The ability to target epigenetic marks like DNA methylation to specific loci is important in both basic research and in crop plant engineering. However, heritability of targeted DNA methylation, how it impacts gene expression, and which epigenetic features are required for proper establishment are mostly unknown. Here, we show that targeting the CG-specific methyltransferase M.SssI with an artificial zinc finger protein can establish heritable CG methylation and silencing of a targeted locus in Arabidopsis. In addition, we observe highly heritable widespread ectopic CG methylation mainly over euchromatic regions. This hypermethylation shows little effect on transcription while it triggers a mild but significant reduction in the accumulation of H2A.Z and H3K27me3. Moreover, ectopic methylation occurs preferentially at less open chromatin that lacks positive histone marks. These results outline general principles of the heritability and interaction of CG methylation with other epigenomic features that should help guide future efforts to engineer epigenomes.
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Project description:DNA methylation occurs in both CG and non-CG sequence contexts. Non-CG methylation is abundant in plants, and is mediated by CHROMOMETHYLASE (CMT) and DOMAINS REARRANGED METHYLTRANSFERASE (DRM) proteins; however its roles remain poorly understood. Here we characterize the roles of non-CG methylation in Arabidopsis thaliana. We show that a poorly characterized methyltransferase, CMT2, is a functional methyltransferase in vitro and in vivo. CMT2 specifically binds histone H3 lysine 9 (H3K9) dimethylation and methylates non-CG cytosines at sites that are also regulated by H3K9 dimethylation. By generating different combinations of non-CG methylation mutants, we reveal the contributions and redundancies between each methyltransferase in DNA methylation patterning and in regulating transposable elements (TEs) and protein-coding genes. We also demonstrate extensive dependencies of small RNA accumulation and H3K9 methylation patterning on non-CG methylation, suggesting self-reinforcing mechanisms between these epigenetic factors. The results suggest that non-CG methylation patterns are critical in shaping the histone modification and small non-coding RNA landscapes.
2013-12-15 | GSE51304 | GEO
Project description:DNA methylation occurs in both CG and non-CG sequence contexts. Non-CG methylation is abundant in plants, and is mediated by CHROMOMETHYLASE (CMT) and DOMAINS REARRANGED METHYLTRANSFERASE (DRM) proteins; however its roles remain poorly understood. Here we characterize the roles of non-CG methylation in Arabidopsis thaliana. We show that a poorly characterized methyltransferase, CMT2, is a functional methyltransferase in vitro and in vivo. CMT2 specifically binds histone H3 lysine 9 (H3K9) dimethylation and methylates non-CG cytosines at sites that are also regulated by H3K9 dimethylation. By generating different combinations of non-CG methylation mutants, we reveal the contributions and redundancies between each methyltransferase in DNA methylation patterning and in regulating transposable elements (TEs) and protein-coding genes. We also demonstrate extensive dependencies of small RNA accumulation and H3K9 methylation patterning on non-CG methylation, suggesting self-reinforcing mechanisms between these epigenetic factors. The results suggest that non-CG methylation patterns are critical in shaping the histone modification and small non-coding RNA landscapes. Eighteen mRNA-seq samples, five smRNA-seq samples, five bisulfite-seq samples, twenty ChIP-seq samples. Bisulfite-seq data for cmt2-7 single mutants, cmt3 single mutants, drm1/2 double mutants, drm1/2 cmt3 triple mutants are deposited in GSE39901. Processed wiggle format files for all datasets can be downloaded at http://genomes.mcdb.ucla.edu/AthBSseq/
2013-12-15 | E-GEOD-51304 | biostudies-arrayexpress
Project description:The CpG depleted Mycoplasma penetrans harbors a CpG specific C5 methyltransferase. The aim of this experiment was to confirm the specificity of the methyltransferase in vivo and in vitro. Genomic DNA from Mycoplasma penetrans and Escherichia coli genomic DNA that either was or was not methylated in vitro by M.MpeI were subjected to Illumina MiSeq bisulfite sequencing.
2012-11-29 | E-GEOD-41022 | biostudies-arrayexpress
Project description:Vertebrate genomes exhibit marked CG-suppression, that is lower than expected numbers of 5’-CG-3’ dinucleotides1. This feature is likely due to C-to-T mutations that have accumulated over hundreds of millions of years, driven by CG-specific DNA methyl transferases and spontaneous methyl-cytosine deamination. Remarkably, many RNA viruses of vertebrates that are not substrates for DNA methyl transferases mimic the CG-suppression of their hosts2-4. This striking property of viral genomes is unexplained4-6. In a synonymous mutagenesis experiment, we found that CG-suppression is essential for HIV-1 replication. The deleterious effect of CG dinucleotides on HIV-1 replication was cumulative, evident as cytoplasmic RNA depletion, and exerted by CG dinucleotides in both translated and non-translated exonic RNA sequences. A focused siRNA screen revealed that zinc finger antiviral protein (ZAP)7 inhibited virion production by cells infected with CG-enriched HIV-1. Crucially, HIV-1 mutants containing segments whose CG-content mimicked random sequence were defective in unmanipulated cells, but replicated normally in ZAP-deficient cells. Crosslinking-immunoprecipitation-sequencing assays demonstrated that ZAP binds directly and selectively to RNA sequences containing CG dinucleotides. These findings suggest that ZAP exploits host CG-suppression to discriminate non-self RNA. The dinucleotide composition of HIV-1, and perhaps other RNA viruses, appears to have adapted to evade this host defense.
2017-08-22 | GSE102843 | GEO
Project description:Genome wide inherited modifications of the tomato epigenome by trans-activated bacterial CG methyltransferase
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