Project description:Cytosine methylation is a key epigenetic mark in many organisms, important for both transcriptional control and genome integrity. While relatively stable during somatic growth, DNA methylation is reprogrammed genome-wide during mammalian reproduction. Reprogramming is essential for zygotic totipotency, and to prevent transgenerational inheritance of epimutations. The extent of DNA methylation reprogramming in plants however remains unclear. Here, we developed sensors reporting with single-cell resolution CG and non-CG methylation in Arabidopsis. Live imaging during reproduction revealed distinct and sex-specific dynamics for both contexts. We found that CHH methylation in the egg cell depends on DRM2 and Pol V, two main actors of RNA-directed DNA methylation, but does not depend on Pol IV. Our sensors provide insight into global DNA methylation dynamics at the single cell level with high temporal resolution, and offer a powerful tool to track CG and non-CG methylation both during development and in response to environmental cues in all organisms with methylated DNA.
Project description:DNA methylation is essential for silencing transposable elements and some genes in higher eukaryotes, implying that this modification must be tightly controlled. However, accidental changes in DNA methylation can be transmitted through mitosis, as in cancer, or meiosis, leading to epiallelic variation. Here, we demonstrate the existence of an efficient and faithful mechanism that protects against transgenerational loss of DNA methylation in the plant Arabidopsis. This process is specific to the subset of heavily methylated genomic repeats that are targeted by the RNAi machinery, and does not spread into flanking regions. Remethylation is often progressive over two to four sexual generations. This differential and incremental correction of epigenetic defects may preserve genome stability while increasing adaptive opportunities.
Project description:The centromere is a pivotal chromatin domain that ensures accurate chromosome segregation during cell division. However, the epigenome regulation of the centromere and its impact on centromere function remain largely elusive. Here in the model plant Arabidopsis, we show that CCR4, the catalytic subunit of the RNA deadenylation complex CCR4-NOT, is essential for maintenance of the centromere epigenome and chromosome integrity. We demonstrate that CCR4 is involved in shortening of the poly(A) tails of transcripts originated from centromeric transposons and repeats, thereby promoting the production of small interfering RNAs (siRNAs). The CCR4-dependent siRNAs guide non-CG DNA methylation at centromere repeats, and CCR4 cooperates with canonical DNA methylation pathways to enhance centromeric H3K9 methylation and ensure mitotic chromosome stability. Our study illustrates the crucial role of RNA quality control in RNA interference and reveals the elaborate mechanism that safeguards plant centromeres through epigenomic regulation.
Project description:The centromere is a pivotal chromatin domain that ensures accurate chromosome segregation during cell division. However, the epigenome regulation of the centromere and its impact on centromere function remain largely elusive. Here in the model plant Arabidopsis, we show that CCR4, the catalytic subunit of the RNA deadenylation complex CCR4-NOT, is essential for maintenance of the centromere epigenome and chromosome integrity. We demonstrate that CCR4 is involved in shortening of the poly(A) tails of transcripts originated from centromeric transposons and repeats, thereby promoting the production of small interfering RNAs (siRNAs). The CCR4-dependent siRNAs guide non-CG DNA methylation at centromere repeats, and CCR4 cooperates with canonical DNA methylation pathways to enhance centromeric H3K9 methylation and ensure mitotic chromosome stability. Our study illustrates the crucial role of RNA quality control in RNA interference and reveals the elaborate mechanism that safeguards plant centromeres through epigenomic regulation.
Project description:In diverse eukaryotes, constitutively silent sequences, such as transposons and repeats, are marked by methylation at histone H3 lysine 9 (H3K9me). Despites the conservation and importance in the genome integrity, mechanisms to exclude H3K9m from active genes remained largely unexplored. Here we show in Arabidopsis that the exclusion depends on a histone demethylase gene, IBM1 (increase in BONSAI methylation); loss-of-function ibm1 mutation caused ectopic H3K9me in thousands of genes, which accompanies genic DNA methylation at non-CG sites. The ibm1-induced genic H3K9me depended on both histone methylase KYP/SUVH4 and DNA methylase CMT3, suggesting interdependence of two epigenetic marks – H3K9me and non-CG methylation. Notably, IBM1 enhanced loss of H3K9m in transcriptionally de-repressed sequences. Furthermore, disruption of transcription in genes induced ectopic non-CG methylation, mimicking the loss of IBM1 function. We propose that active chromatin is stabilized by the autocatalytic loop of transcription and H3K9 demethylation. This process counteracts accumulation of silent epigenetic marks, H3K9me and non-CG methylation, which is also autocatalytic.
Project description:DNA methylation is essential for silencing transposable elements and some genes in higher eukaryotes, implying that this modification must be tightly controlled. However, accidental changes in DNA methylation can be transmitted through mitosis, as in cancer, or meiosis, leading to epiallelic variation. Here, we demonstrate the existence of an efficient and faithful mechanism that protects against transgenerational loss of DNA methylation in the plant Arabidopsis. This process is specific to the subset of heavily methylated genomic repeats that are targeted by the RNAi machinery, and does not spread into flanking regions. Remethylation is often progressive over two to four sexual generations. This differential and incremental correction of epigenetic defects may preserve genome stability while increasing adaptive opportunities. 2 samples examined: wild type, and ddm1 mutant.
Project description:RNA silencing is a mechanism for regulating gene expression at the transcriptional and post-transcriptional levels. Its functions include regulating endogenous gene expression and protecting the cell against viruses and invading transposable elements (TEs). A key component of the mechanism is small RNAs (sRNAs) of 21-24 nucleotides (nt) in length, which direct the silencing machinery in a sequence specific manner to target nucleic acids. sRNAs of 24 nt are involved in methylation of cytosine residues of target loci in three sequence contexts (CG, CHG and CHH), referred to as RNA-directed DNA methylation (RdDM). We previously demonstrated that 24 nt sRNAs are mobile from shoot to root in Arabidopsis thaliana. In this study we demonstrated that methylation of thousands of loci in root tissues is dependent upon mobile sRNAs from the shoot. Furthermore, we found that mobile sRNA-dependent DNA methylation occurs predominantly in non-CG contexts. These findings were made using base-resolution next generation sequencing approaches and genome wide analyses. Specific classes of short TEs are the predominant targets of mobile sRNA-dependent DNA methylation; classes typically found in gene-rich euchromatic regions. Mobile sRNA-regulated genes were also identified. Mechanistically, we demonstrate that mobile sRNA-dependent non-CG methylation is largely independent of the CMT2/3 RdDM pathway but dependent upon the DRM1/DRM2 RdDM pathway. This is in contrast to non-mobile sRNA-dependent DNA methylation, which predominantly depends upon the CMT2/3 RdDM pathway. These data are complementary to the small RNA sequencing data from Arabidopsis root grafts described in Molnar et al (Science, 2010 May 14;328(5980):872-5).
Project description:The centromere is a pivotal chromatin domain that ensures accurate chromosome segregation during cell division. However, the epigenome regulation of the centromere and its impact on centromere function remain largely elusive. Here in the model plant Arabidopsis, we show that CCR4, the catalytic subunit of the RNA deadenylation complex CCR4-NOT, is essential for maintenance of the centromere epigenome and chromosome integrity. We demonstrate that CCR4 is involved in shortening of the poly(A) tails of transcripts originated from centromeric transposons and repeats, thereby promoting the production of small interfering RNAs (siRNAs). The CCR4-dependent siRNAs guide non-CG DNA methylation at centromere repeats, and CCR4 cooperates with canonical DNA methylation pathways to enhance centromeric H3K9 methylation and ensure mitotic chromosome stability. Our study illustrates the crucial role of RNA quality control in RNA interference and reveals the elaborate mechanism that safeguards plant centromeres through epigenomic regulation.