Epigenetic modifications of the genome are generally stable in somatic cells of multicellular organisms. In germ cells and early embryos, however, epigenetic reprogramming occurs on a genome-wide ...scale, which includes demethylation of DNA and remodeling of histones and their modifications. The mechanisms of genome-wide erasure of DNA methylation, which involve modifications to 5-methylcytosine and DNA repair, are being unraveled. Epigenetic reprogramming has important roles in imprinting, the natural as well as experimental acquisition of totipotency and pluripotency, control of transposons, and epigenetic inheritance across generations. Small RNAs and the inheritance of histone marks may also contribute to epigenetic inheritance and reprogramming. Reprogramming occurs in flowering plants and in mammals, and the similarities and differences illuminate developmental and reproductive strategies.
In flowering plants, heterochromatin is demarcated by the histone variant H2A.W, elevated levels of the linker histone H1, and specific epigenetic modifications, such as high levels of DNA ...methylation at both CG and non-CG sites. How H2A.W regulates heterochromatin organization and interacts with other heterochromatic features is unclear. Here, we create a h2a.w null mutant via CRISPR-Cas9, h2a.w-2, to analyze the in vivo function of H2A.W. We find that H2A.W antagonizes deposition of H1 at heterochromatin and that non-CG methylation and accessibility are moderately decreased in h2a.w-2 heterochromatin. Compared to H1 loss alone, combined loss of H1 and H2A.W greatly increases accessibility and facilitates non-CG DNA methylation in heterochromatin, suggesting co-regulation of heterochromatic features by H2A.W and H1. Our results suggest that H2A.W helps maintain optimal heterochromatin accessibility and DNA methylation by promoting chromatin compaction together with H1, while also inhibiting excessive H1 incorporation.
Deposition of the histone variant H2A.Z by the SWI2/SNF2-Related 1 chromatin remodeling complex (SWR1-C) is important for gene regulation in eukaryotes, but the composition of the Arabidopsis SWR1-C ...has not been thoroughly characterized. Here, we aim to identify interacting partners of a conserved Arabidopsis SWR1 subunit ACTIN-RELATED PROTEIN 6 (ARP6). We isolate nine predicted components and identify additional interactors implicated in histone acetylation and chromatin biology. One of the interacting partners, methyl-CpG-binding domain 9 (MBD9), also strongly interacts with the Imitation SWItch (ISWI) chromatin remodeling complex. MBD9 is required for deposition of H2A.Z at a distinct subset of ARP6-dependent loci. MBD9 is preferentially bound to nucleosome-depleted regions at the 5' ends of genes containing high levels of activating histone marks. These data suggest that MBD9 is a SWR1-C interacting protein required for H2A.Z deposition at a subset of actively transcribing genes.
DNA methylation occurs in 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 preferentially binds histone H3 Lys9 (H3K9) dimethylation and methylates non-CG cytosines that are regulated by H3K9 methylation. We revealed the contributions and redundancies between each non-CG methyltransferase in DNA methylation patterning and in regulating transcription. 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 landscapes of histone modification and small noncoding RNA.
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.
Cytosine methylation is involved in various biological processes such as silencing of transposable elements (TEs) and imprinting. Multiple pathways regulate DNA methylation in different sequence ...contexts, but the factors that regulate DNA methylation at a given site in the genome largely remain unknown. Here we have surveyed the methylomes of a comprehensive list of 86 Arabidopsis gene silencing mutants by generating single-nucleotide resolution maps of DNA methylation. We find that DNA methylation is site specifically regulated by different factors. Furthermore, we have identified additional regulators of DNA methylation. These data and analyses will serve as a comprehensive community resource for further understanding the control of DNA methylation patterning.
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► Genome-wide single-nucleotide resolution methylation maps in 86 silencing mutants ► Complex interplays between different DNA methylation pathways ► Identification of novel regulators of DNA methylation
A genome-wide study of DNA methylation in a comprehensive list of Arabidopsis mutants implicated in gene silencing and histone modification reveals that different pathways regulate DNA methylation site specifically, and the study uncovers new factors that regulate DNA methylation.
The RNA-directed DNA methylation (RdDM) pathway in plants controls gene expression via cytosine DNA methylation. The ability to manipulate RdDM would shed light on the mechanisms and applications of ...DNA methylation to control gene expression. Here, we identified diverse RdDM proteins that are capable of targeting methylation and silencing in Arabidopsis when tethered to an artificial zinc finger (ZF-RdDM). We studied their order of action within the RdDM pathway by testing their ability to target methylation in different mutants. We also evaluated ectopic siRNA biogenesis, RNA polymerase V (Pol V) recruitment, targeted DNA methylation, and gene-expression changes at thousands of ZF-RdDM targets. We found that co-targeting both arms of the RdDM pathway, siRNA biogenesis and Pol V recruitment, dramatically enhanced targeted methylation. This work defines how RdDM components establish DNA methylation and enables new strategies for epigenetic gene regulation via targeted DNA methylation.
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•A panel of RdDM factors can target DNA methylation when fused to an artificial ZF•ZF-RdDM fusions in different mutant backgrounds reveal RdDM hierarchy of action•MORC6 can target DNA methylation•Co-targeting of Pol IV and Pol V synergistically enhances DNA methylation targeting
Comprehensive investigation into the genetic pathway of RNA-directed DNA methylation in Arabidopsis defines epistatic relationships and allows for efficient manipulation of DNA methylation to specifically modify developmental phenotypes.
Most transgenic crops are produced through tissue culture. The impact of utilizing such methods on the plant epigenome is poorly understood. Here we generated whole-genome, single-nucleotide ...resolution maps of DNA methylation in several regenerated rice lines. We found that all tested regenerated plants had significant losses of methylation compared to non-regenerated plants. Loss of methylation was largely stable across generations, and certain sites in the genome were particularly susceptible to loss of methylation. Loss of methylation at promoters was associated with deregulated expression of protein-coding genes. Analyses of callus and untransformed plants regenerated from callus indicated that loss of methylation is stochastically induced at the tissue culture step. These changes in methylation may explain a component of somaclonal variation, a phenomenon in which plants derived from tissue culture manifest phenotypic variability. DOI:http://dx.doi.org/10.7554/eLife.00354.001.
In honey bees (Apis mellifera), the development of a larva into either a queen or worker depends on differential feeding with royal jelly and involves epigenomic modifications by DNA ...methyltransferases. To understand the role of DNA methylation in this process we sequenced the larval methylomes in both queens and workers. We show that the number of differentially methylated genes (DMGs) in larval head is significantly increased relative to adult brain (2,399 vs. 560) with more than 80% of DMGs up-methylated in worker larvae. Several highly conserved metabolic and signaling pathways are enriched in methylated genes, underscoring the connection between dietary intake and metabolic flux. This includes genes related to juvenile hormone and insulin, two hormones shown previously to regulate caste determination. We also tie methylation data to expressional profiling and describe a distinct role for one of the DMGs encoding anaplastic lymphoma kinase (ALK), an important regulator of metabolism. We show that alk is not only differentially methylated and alternatively spliced in Apis, but also seems to be regulated by a cis-acting, anti-sense nonprotein-coding transcript. The unusually complex regulation of ALK in Apis suggests that this protein could represent a previously unknown node in a process that activates downstream signaling according to a nutritional context. The correlation between methylation and alternative splicing of alk is consistent with the recently described mechanism involving RNA polymerase II pausing. Our study offers insights into diet-controlled development in Apis.
In eukaryotes, DNA wraps around histones to form nucleosomes, which are compacted into chromatin. DNA-templated processes, including transcription, require chromatin disassembly and reassembly ...mediated by histone chaperones. Additionally, distinct histone variants can replace core histones to regulate chromatin structure and function. Although replacement of H2A with the evolutionarily conserved H2A.Z via the SWR1 histone chaperone complex has been extensively studied, in plants little is known about how a reduction of H2A.Z levels can be achieved. Here, we show that NRP proteins cause a decrease of H2A.Z-containing nucleosomes in Arabidopsis under standard growing conditions. nrp1-1 nrp2-2 double mutants show an over-accumulation of H2A.Z genome-wide, especially at heterochromatic regions normally H2A.Z-depleted in wild-type plants. Our work suggests that NRP proteins regulate gene expression by counteracting SWR1, thereby preventing excessive accumulation of H2A.Z.
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