Virus-Mediated Targeted DNA Methylation Illuminates the Dynamics of Methylation in an Endogenous Plant Gene.
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ABSTRACT:
SUBMITTER: Atsumi G
PROVIDER: S-EPMC8073618 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Accurate analysis of DNA methylation by bisulphite sequencing depends on the complete conversion of all cytosines into uracil. Until now there has been no standard or universal gene identified as an endogenous control to monitor the conversion frequency in plants. Here, we report the development of PCR based assays for one nuclear gene IND (INDEHISCENT) and two mitochondrial genes, NAD (NICOTINAMIDE ADENINE DINUCLEOTIDE) and ATP1 (ATPase SUBUNIT 1). We demonstrated their efficacy as bisulphite conversion controls in Brassica and other plant taxa. The target regions amplified by four primer pairs were found to be consistently free from DNA methylation. Primer pairs for IND.a and NAD were effective within Brassica species, whereas two primer pairs for ATP1 provided reliable controls across a representative range of dicot and monocot angiosperm species. These primer sets may therefore be adopted as controls in plant methylation analysis for a wide range of studies.
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Project description:Cytosine methylation is crucial for gene regulation and silencing of transposable elements in mammals and plants. While this epigenetic mark is extensively reprogrammed in the germline and early embryos of mammals, the extent to which DNA methylation is reset between generations in plants remains largely unknown.Using Arabidopsis as a model, we uncovered distinct DNA methylation dynamics over transposable element sequences during the early stages of plant development. Specifically, transposable elements and their relics show invariably high methylation at CG sites but increasing methylation at CHG and CHH sites. This non-CG methylation culminates in mature embryos, where it reaches saturation for a large fraction of methylated CHH sites, compared to the typical 10-20% methylation level observed in seedlings or adult plants. Moreover, the increase in CHH methylation during embryogenesis matches the hypomethylated state in the early endosperm. Finally, we show that interfering with the embryo-to-seedling transition results in the persistence of high CHH methylation levels after germination, specifically over sequences that are targeted by the RNA-directed DNA methylation (RdDM) machinery.Our findings indicate the absence of extensive resetting of DNA methylation patterns during early plant life and point instead to an important role of RdDM in reinforcing DNA methylation of transposable element sequences in every cell of the mature embryo. Furthermore, we provide evidence that this elevated RdDM activity is a specific property of embryogenesis.
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Project description:We uncover distinct DNA methylation dynamics over genes and repeat sequences during early plant life in Arabidopsis. While gene body methylation, which is restricted to CG sites and concerns 20-30% of all genes is detected at all stages examined, it typically fluctuates over a few CGs, with no coherent pattern. In contrast, transposable elements and their relics as well as other repeat sequences, have consistently high methylation levels at CGs and show increasing CHG and especially CHH methylation during embryogenesis. Remarkably, methylation reaches 100% at many individual CHH sites in the mature embryo, compared to the 10-20% methylation usually observed at CHH sites in seedlings or adult plants. Moreover, the progressive increase in CHG and CHH methylation in embryos mirrors the loss of DNA methylation at CG and CHG sites in the endosperm, suggesting transfer of information from the endosperm to the embryo, presumably in the form of small RNAs. Finally, impairing the embryo to seedling transition through loss of PRC2 activity results in the persistence of high CHH methylation levels after germination and specifically over sequences that are targeted by the RNA-directed DNA methylation (RdDM) machinery. Collectively, our findings indicate a lack of extensive resetting of DNA methylation patterns during early plant life and point instead to an important role of RdDM in targeting DNA methylation to transposable element and other repeat sequences in all cells of the mature embryo.
2017-08-23 | GSE85975 | GEO
Project description:This SuperSeries is composed of the SubSeries listed below.
2019-10-11 | GSE134996 | GEO
Project description:BackgroundTargeted gene silencing is an important approach in both drug development and basic research. However, the selection of a potent suppressor has become a significant hurdle to implementing maximal gene inhibition for this approach. We attempted to construct a 'super suppressor' by combining the activities of two suppressors that function through distinct epigenetic mechanisms.ResultsGene targeting vectors were constructed by fusing a GAL4 DNA-binding domain with a epigenetic suppressor, including CpG DNA methylase Sss1, histone H3 lysine 27 methylase vSET domain, and Kruppel-associated suppression box (KRAB). We found that both Sss1 and KRAB suppressors significantly inhibited the expression of luciferase and copGFP reporter genes. However, the histone H3 lysine 27 methylase vSET did not show significant suppression in this system. Constructs containing both Sss1 and KRAB showed better inhibition than either one alone. In addition, we show that KRAB suppressed gene expression by altering the histone code, but not DNA methylation in the gene promoter. Sss1, on the other hand, not only induced de novo DNA methylation and recruited Heterochromatin Protein 1 (HP1a), but also increased H3K27 and H3K9 methylation in the promoter.ConclusionsEpigenetic studies can provide useful data for the selection of suppressors in constructing therapeutic vectors for targeted gene silencing.
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Project description:Rett syndrome is a severe neurodevelopmental disorder caused by mutations in the methyl-CpG binding protein gene (MECP2). MeCP2 is a methyl-cytosine binding protein that is proposed to function as a transcriptional repressor. However, multiple gene expression studies comparing wild-type and MeCP2-deficient neurons have failed to identify gene expression changes consistent with loss of a classical transcriptional repressor. Recent work suggests that one function of MeCP2 in neurons is to temper the expression of the longest genes in the genome by binding to methylated CA dinucleotides (mCA) within transcribed regions of these genes. Here we explore the mechanism of mCA and MeCP2 in fine tuning the expression of long genes. We find that mCA is not only highly enriched within the body of genes normally repressed by MeCP2, but also enriched within extended megabase-scale regions surrounding MeCP2-repressed genes. Whereas enrichment of mCA exists in a broad region around these genes, mCA together with mCG within gene bodies appears to be the primary driver of gene repression by MeCP2. Disruption of methylation at CA sites within the brain results in depletion of MeCP2 across genes that normally contain a high density of gene-body mCA. We further find that the degree of gene repression by MeCP2 is proportional to the total number of methylated cytosine MeCP2 binding sites across the body of a gene. These findings suggest a model in which MeCP2 tunes gene expression in neurons by binding within the transcribed regions of genes to impede the elongation of RNA polymerase.
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Project description:DNA methyltransferases (DNMTs) are thought to be involved in the cellular response to DNA damage, thus linking DNA repair mechanisms with DNA methylation. This study presents a novel method of targeted DNA methylation that utilizes endogenous DNA double strand break repair pathways and applies it to the neurodegenerative disease gene C9orf72. A double strand break induced by CRISPR/cas9 in the promoter of C9orf72 is sufficient to induce DNA methylation, and methylation can be precisely targeted through the process of homology directed repair (HDR) via delivery of an in vitro methylated exogenous repair template. Long methylated double stranded DNA templates induce more methylation than shorter templates and with higher efficiency than a dCas9-DNMT3a fusion protein construct. Genome-wide methylation analysis reveals no significant off-target methylation changes when inducing methylation via HDR, whereas the dCas9-DNMT3a fusion construct causes significant off-target methylation at over 67,000 sites. This method is applied to generate a patient derived iPSC model of amyotrophic lateral sclerosis and frontotemporal dementia (ALS/FTD) that exhibits stable DNA methylation patterns similar to those seen in patients. Using this model, it’s shown for the first time that DNA methylation of the 5’ regulatory region directly reduces C9orf72 expression and increases histone H3K9 tri-methylation levels.
2019-10-11 | GSE134995 | GEO
Project description:DNA methyltransferases (DNMTs) are thought to be involved in the cellular response to DNA damage, thus linking DNA repair mechanisms with DNA methylation. This study presents a novel method of targeted DNA methylation that utilizes endogenous DNA double strand break repair pathways and applies it to the neurodegenerative disease gene C9orf72. A double strand break induced by CRISPR/cas9 in the promoter of C9orf72 is sufficient to induce DNA methylation, and methylation can be precisely targeted through the process of homology directed repair (HDR) via delivery of an in vitro methylated exogenous repair template. Long methylated double stranded DNA templates induce more methylation than shorter templates and with higher efficiency than a dCas9-DNMT3a fusion protein construct. Genome-wide methylation analysis reveals no significant off-target methylation changes when inducing methylation via HDR, whereas the dCas9-DNMT3a fusion construct causes significant off-target methylation at over 67,000 sites. This method is applied to generate a patient derived iPSC model of amyotrophic lateral sclerosis and frontotemporal dementia (ALS/FTD) that exhibits stable DNA methylation patterns similar to those seen in patients. Using this model, it’s shown for the first time that DNA methylation of the 5’ regulatory region directly reduces C9orf72 expression and increases histone H3K9 tri-methylation levels.
2019-10-11 | GSE134994 | GEO
Project description:Changes in DNA methylation are required for the formation of germinal centers (GCs), but the mechanisms of such changes are poorly understood. Activation-induced cytidine deaminase (AID) has been recently implicated in DNA demethylation through its deaminase activity coupled with DNA repair. We investigated the epigenetic function of AID in vivo in germinal center B cells (GCBs) isolated from wild-type (WT) and AID-deficient (Aicda(-/-)) mice. We determined that the transit of B cells through the GC is associated with marked locus-specific loss of methylation and increased methylation diversity, both of which are lost in Aicda(-/-) animals. Differentially methylated cytosines (DMCs) between GCBs and naive B cells (NBs) are enriched in genes that are targeted for somatic hypermutation (SHM) by AID, and these genes form networks required for B cell development and proliferation. Finally, we observed significant conservation of AID-dependent epigenetic reprogramming between mouse and human B cells.
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Project description:Apple latent spherical virus (ALSV) is an efficient virus-induced gene silencing vector in functional genomics analyses of a broad range of plant species. Here, an Agrobacterium-mediated inoculation (agroinoculation) system was developed for the ALSV vector, and virus-induced transcriptional gene silencing (VITGS) is described in plants infected with the ALSV vector. The cDNAs of ALSV RNA1 and RNA2 were inserted between the cauliflower mosaic virus 35S promoter and the NOS-T sequences in a binary vector pCAMBIA1300 to produce pCALSR1 and pCALSR2-XSB or pCALSR2-XSB/MN. When these vector constructs were agroinoculated into Nicotiana benthamiana plants with a construct expressing a viral silencing suppressor, the infection efficiency of the vectors was 100%. A recombinant ALSV vector carrying part of the 35S promoter sequence induced transcriptional gene silencing of the green fluorescent protein gene in a line of N. benthamiana plants, resulting in the disappearance of green fluorescence of infected plants. Bisulfite sequencing showed that cytosine residues at CG and CHG sites of the 35S promoter sequence were highly methylated in the silenced generation zero plants infected with the ALSV carrying the promoter sequence as well as in progeny. The ALSV-mediated VITGS state was inherited by progeny for multiple generations. In addition, induction of VITGS of an endogenous gene (chalcone synthase-A) was demonstrated in petunia plants infected with an ALSV vector carrying the native promoter sequence. These results suggest that ALSV-based vectors can be applied to study DNA methylation in plant genomes, and provide a useful tool for plant breeding via epigenetic modification.
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