HCF-1 inhibits SKN-1 to Modulate Stress Resistance but Not lifespan in Caenorhabditis Elegans AND Determination of Enrichment Regions for H3K27me3 and Other low-signal, High-noise ChIP-seq Data

1) Caenorhabditis elegans host cell factor-1 (HCF-1) is an evolutionarily conserved longevity determinant. HCF-1 modulates both lifespan and stress resistance by inhibiting the C. elegans homolog of the mammalian FOXO transcription factors, DAF-16. However, the involvement of other components in HCF-1 mediated longevity and stress resistance has not yet been characterized. We show that SKN-1, the C. elegans homolog of the mammalian Nrf proteins and a major orchestrator of the phase II detoxification response that defends against oxidative stress, is regulated by HCF-1 to modulate oxidative stress resistance but not lifespan. Our findings imply a novel regulatory relationship between HCF-1 and SKN-1 that is revealed only in the presence of oxidative stress. 2) Chromatin modifications are a major mechanism through which cells regulate gene expression. ChIP-seq allows for genome-wide profiling of any DNA-binding protein, including histones. To analyze these data, researchers have developed statistical tools that can identify genomic regions enriched for the DNA-binding protein of interest. Some histone marks, however, bind across broad regions of the genome and produce diffuse and high-noise data profiles that are often difficult to analyze. In this paper, I investigate the ability of peak-callers to analyze ChIP-seq data from H3K27me3, a histone mark known to produce broad regions of enrichment. Further, I present an alternative method that is both faster and simpler than the than the other peak callers investigated. This alternative method offers promise in identifying enriched genomic features when a histone mark, like H3K27me3, generates very diffuse and noisy ChIP-seq data patterns

Data from: Proximal methylation features associated with nonrandom changes in gene body methylation

Background: Gene body methylation at CG dinucleotides is a widely conserved feature of methylated genomes but remains poorly understood. The Arabidopsis thaliana strain Cvi has depleted gene body methylation relative to the reference strain Col. Here, we leverage this natural epigenetic difference to investigate gene body methylation stability. Results: Recombinant inbred lines derived from Col and Cvi were used to examine the transmission of distinct gene body methylation states. The vast majority of genic CG methylation patterns are faithfully transmitted over nine generations according to parental genotype, with only 1–4% of CGs either losing or gaining methylation relative to the parent. Genic CGs that fail to maintain the parental methylation state are shared among independent lines, suggesting that these are not random occurrences. We use a logistic regression framework to identify features that best predict sites that fail to maintain parental methylation state. Intermediate levels of CG methylation around a dynamic CG site and high methylation variability across many A. thaliana strains at that site are the strongest predictors. These data suggest that the dynamic CGs we identify are not specific to the Col–Cvi recombinant inbred lines but have an epigenetic state that is inherently less stable within the A. thaliana species. Extending this, variably methylated genic CGs in maize and Brachypodium distachyon are also associated with intermediate local CG methylation. Conclusions: These results provide new insights into the features determining the inheritance of gene body methylation and demonstrate that two different methylation equilibria can be maintained within single individuals

RNA-directed DNA Methylation.

RNA-directed DNA methylation (RdDM) is a biological process in which non-coding RNA molecules direct the addition of DNA methylation to specific DNA sequences. The RdDM pathway is unique to plants, although other mechanisms of RNA-directed chromatin modification have also been described in fungi and animals. To date, the RdDM pathway is best characterized within angiosperms (flowering plants), and particularly within the model plant Arabidopsis thaliana. However, conserved RdDM pathway components and associated small RNAs (sRNAs) have also been found in other groups of plants, such as gymnosperms and ferns. The RdDM pathway closely resembles other sRNA pathways, particularly the highly conserved RNAi pathway found in fungi, plants, and animals. Both the RdDM and RNAi pathways produce sRNAs and involve conserved Argonaute, Dicer and RNA-dependent RNA polymerase proteins. RdDM has been implicated in a number of regulatory processes in plants. The DNA methylation added by RdDM is generally associated with transcriptional repression of the genetic sequences targeted by the pathway. Since DNA methylation patterns in plants are heritable, these changes can often be stably transmitted to progeny. As a result, one prominent role of RdDM is the stable, transgenerational suppression of transposable element (TE) activity. RdDM has also been linked to pathogen defense, abiotic stress responses, and the regulation of several key developmental transitions. Although the RdDM pathway has a number of important functions, RdDM-defective mutants in Arabidopsis thaliana are viable and can reproduce, which has enabled detailed genetic studies of the pathway. However, RdDM mutants can have a range of defects in different plant species, including lethality, altered reproductive phenotypes, TE upregulation and genome instability, and increased pathogen sensitivity. Overall, RdDM is an important pathway in plants that regulates a number of processes by establishing and reinforcing specific DNA methylation patterns, which can lead to transgenerational epigenetic effects on gene expression and phenotype

Genome_Biol_2017_datasets

Zip archive containing four .txt files: 3 tab-delimited datasets and a README (readme also uploaded separately). full_dataset.txt contains methylation and other information from A. thaliana generated as part of this study, full_distachyon_Bd1_1_data.txt contains data from B. distachyon generated by Eichten et al. 2016 (Genome Res.), and full_maize_B73_data.txt contains data from Z. mays generated by Li et al. 2015 (Plant Physiol.). Datasets contain all data used to generate Fig. 5 and Fig. S11. Details on each field in each file found in README

Transcriptional and imprinting complexity in Arabidopsis seeds at single-nucleus resolution

Seeds are a key life cycle stage for many plants. Seeds are also the basis of agriculture and the primary source of calories consumed by humans1. Here, we employ single-nucleus RNA-sequencing to generate a transcriptional atlas of developing Arabidopsis thaliana seeds, with a focus on endosperm. Endosperm, the primary site of gene imprinting in flowering plants, mediates the relationship between the maternal parent and the embryo2. We identify transcriptionally uncharacterized nuclei types in the chalazal endosperm, which interfaces with maternal tissue for nutrient unloading3,4. We demonstrate that the extent of parental bias of maternally expressed imprinted genes varies with cell-cycle phase, and that imprinting of paternally expressed imprinted genes is strongest in chalazal endosperm. Thus, imprinting is spatially and temporally heterogeneous. Increased paternal expression in the chalazal region suggests that parental conflict, which is proposed to drive imprinting evolution, is fiercest at the boundary between filial and maternal tissues

CRISPR-based targeting of DNA methylation in Arabidopsis thaliana by a bacterial CG-specific DNA methyltransferase.

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

Data from: Natural epigenetic polymorphisms lead to intraspecific variation in Arabidopsis gene imprinting

Imprinted gene expression occurs during seed development in plants and is associated with differential DNA methylation of parental alleles, particularly at proximal transposable elements (TEs). Imprinting variability could contribute to observed parent-of-origin effects on seed development. We investigated intraspecific variation in imprinting, coupled with analysis of DNA methylation and small RNAs, among three Arabidopsis strains with diverse seed phenotypes. The majority of imprinted genes were parentally biased in the same manner among all strains. However, we identified several examples of allele-specific imprinting correlated with intraspecific epigenetic variation at a TE. We successfully predicted imprinting in additional strains based on methylation variability. We conclude that there is standing variation in imprinting even in recently diverged genotypes due to intraspecific epiallelic variation. Our data demonstrate that epiallelic variation and genomic imprinting intersect to produce novel gene expression patterns in seeds

A viral guide RNA delivery system for CRISPR-based transcriptional activation and heritable targeted DNA demethylation in Arabidopsis thaliana.

Plant RNA viruses are used as delivery vectors for their high level of accumulation and efficient spread during virus multiplication and movement. Utilizing this concept, several viral-based guide RNA delivery platforms for CRISPR-Cas9 genome editing have been developed. The CRISPR-Cas9 system has also been adapted for epigenome editing. While systems have been developed for CRISPR-Cas9 based gene activation or site-specific DNA demethylation, viral delivery of guide RNAs remains to be developed for these purposes. To address this gap we have developed a tobacco rattle virus (TRV)-based single guide RNA delivery system for epigenome editing in Arabidopsis thaliana. Because tRNA-like sequences have been shown to facilitate the cell-to-cell movement of RNAs in plants, we used the tRNA-guide RNA expression system to express guide RNAs from the viral genome to promote heritable epigenome editing. We demonstrate that the tRNA-gRNA system with TRV can be used for both transcriptional activation and targeted DNA demethylation of the FLOWERING WAGENINGEN gene in Arabidopsis. We achieved up to ~8% heritability of the induced demethylation phenotype in the progeny of virus inoculated plants. We did not detect the virus in the next generation, indicating effective clearance of the virus from plant tissues. Thus, TRV delivery, combined with a specific tRNA-gRNA architecture, provides for fast and effective epigenome editing

Single-nucleus RNA-seq reveals that MBD5, MBD6, and SILENZIO maintain silencing in the vegetative cell of developing pollen

Silencing of transposable elements (TEs) drives the evolution of numerous redundant mechanisms of transcriptional regulation. Arabidopsis MBD5, MBD6, and SILENZIO act as TE repressors downstream of DNA methylation. Here, we show, via single-nucleus RNA-seq of developing male gametophytes, that these repressors are critical for TE silencing in the pollen vegetative cell, a companion cell important for fertilization that undergoes chromatin decompaction. Instead, other silencing mutants (met1, ddm1, mom1, morc) show loss of silencing in all pollen nucleus types and somatic cells. We show that TEs repressed by MBD5/6 gain chromatin accessibility in wild-type vegetative nuclei despite remaining silent, suggesting that loss of DNA compaction makes them sensitive to loss of MBD5/6. Consistently, crossing mbd5/6 to histone 1 mutants, which have decondensed chromatin in leaves, reveals derepression of MBD5/6-dependent TEs in leaves. MBD5/6 and SILENZIO thus act as a silencing system particularly important when chromatin compaction is compromised

The role of ATXR6 expression in modulating genome stability and transposable element repression in Arabidopsis.

ARABIDOPSIS TRITHORAX-RELATED PROTEIN 5 (ATXR5) AND ATXR6 are required for the deposition of H3K27me1 and for maintaining genomic stability in Arabidopsis Reduction of ATXR5/6 activity results in activation of DNA damage response genes, along with tissue-specific derepression of transposable elements (TEs), chromocenter decompaction, and genomic instability characterized by accumulation of excess DNA from heterochromatin. How loss of ATXR5/6 and H3K27me1 leads to these phenotypes remains unclear. Here we provide extensive characterization of the atxr5/6 hypomorphic mutant by comprehensively examining gene expression and epigenetic changes in the mutant. We found that the tissue-specific phenotypes of TE derepression and excessive DNA in this atxr5/6 mutant correlated with residual ATXR6 expression from the hypomorphic ATXR6 allele. However, up-regulation of DNA damage genes occurred regardless of ATXR6 levels and thus appears to be a separable process. We also isolated an atxr6-null allele which showed that ATXR5 and ATXR6 are required for female germline development. Finally, we characterize three previously reported suppressors of the hypomorphic atxr5/6 mutant and show that these rescue atxr5/6 via distinct mechanisms, two of which involve increasing H3K27me1 levels