Kismeth: Analyzer of plant methylation states through bisulfite sequencing

<p>Abstract</p> <p>Background</p> <p>There is great interest in probing the temporal and spatial patterns of cytosine methylation states in genomes of a variety of organisms. It is hoped that this will shed light on the biological roles of DNA methylation in the epigenetic control of gene expression. Bisulfite sequencing refers to the treatment of isolated DNA with sodium bisulfite to convert unmethylated cytosine to uracil, with PCR converting the uracil to thymidine followed by sequencing of the resultant DNA to detect DNA methylation. For the study of DNA methylation, plants provide an excellent model system, since they can tolerate major changes in their DNA methylation patterns and have long been studied for the effects of DNA methylation on transposons and epimutations. However, in contrast to the situation in animals, there aren't many tools that analyze bisulfite data in plants, which can exhibit methylation of cytosines in a variety of sequence contexts (CG, CHG, and CHH).</p> <p>Results</p> <p>Kismeth <url>http://katahdin.mssm.edu/kismeth</url> is a web-based tool for bisulfite sequencing analysis. Kismeth was designed to be used with plants, since it considers potential cytosine methylation in any sequence context (CG, CHG, and CHH). It provides a tool for the design of bisulfite primers as well as several tools for the analysis of the bisulfite sequencing results. Kismeth is not limited to data from plants, as it can be used with data from any species.</p> <p>Conclusion</p> <p>Kismeth simplifies bisulfite sequencing analysis. It is the only publicly available tool for the design of bisulfite primers for plants, and one of the few tools for the analysis of methylation patterns in plants. It facilitates analysis at both global and local scales, demonstrated in the examples cited in the text, allowing dissection of the genetic pathways involved in DNA methylation. Kismeth can also be used to study methylation states in different tissues and disease cells compared to a reference sequence.</p

tRNA-derived small RNAs target transposable element transcripts

tRNA-derived RNA fragments (tRFs) are 18-26 nucleotide small RNAs that are not random degradation products, but are rather specifically cleaved from mature tRNA transcripts. Abundant in stressed or viral-infected cells, the function and potential targets of tRFs are not known. We identified that in the unstressed wild-type male gamete containing pollen of flowering plants, and analogous reproductive structure in non-flowering plant species, tRFs accumulate to high levels. In the reference plant Arabidopsis thaliana, tRFs are processed by Dicer-like 1 and incorporated into Argonaute1 (AGO1), akin to a microRNA. We utilized the fact that many plant small RNAs direct cleavage of their target transcripts to demonstrate that the tRF-AGO1 complex acts to specifically target and cleave endogenous transposable element (TE) mRNAs produced from transcriptionally active TEs. The data presented here demonstrate that tRFs are bona-fide regulatory microRNA-like small RNAs involved in the regulation of genome stability through the targeting of TE transcripts

Erratum: The Non-Coding RNA Journal Club: Highlights on Recent Papers-4. Non-Coding RNA 2016, 2, 9.

Please note that in the published editorial [1], affiliations 1, and 8 contained errors.[...]

The miRNome function transitions from regulating developmental genes to transposable elements during pollen maturation

Animal and plant microRNAs (miRNAs) are essential for the spatio-temporal regulation of development. Together with this role, plant miRNAs have been proposed to target transposable elements (TEs) and stimulate the production of epigenetically active small interfering RNAs. This activity is evident in the plant male gamete containing structure, the male gametophyte or pollen grain. How the dual role of plant miRNAs, regulating both genes and TEs, is integrated during pollen development and which mRNAs are regulated by miRNAs in this cell type at a genome-wide scale are unknown. Here, we provide a detailed analysis of miRNA dynamics and activity during pollen development in Arabidopsis thaliana using small RNA and degradome parallel analysis of RNA end high-throughput sequencing. Furthermore, we uncover miRNAs loaded into the two main active Argonaute (AGO) proteins in the uninuclear and mature pollen grain, AGO1 and AGO5. Our results indicate that the developmental progression from microspore to mature pollen grain is characterized by a transition from miRNAs targeting developmental genes to miRNAs regulating TE activity

The miRNome function transitions from regulating developmental genes to transposable elements during pollen maturation

Animal and plant microRNAs (miRNAs) are essential for the spatio-temporal regulation of development. Together with this role, plant miRNAs have been proposed to target transposable elements (TEs) and stimulate the production of epigenetically active small interfering RNAs. This activity is evident in the plant male gamete containing structure, the male gametophyte or pollen grain. How the dual role of plant miRNAs, regulating both genes and TEs, is integrated during pollen development and which mRNAs are regulated by miRNAs in this cell type at a genome-wide scale are unknown. Here, we provide a detailed analysis of miRNA dynamics and activity during pollen development in Arabidopsis thaliana using small RNA and degradome parallel analysis of RNA end high-throughput sequencing. Furthermore, we uncover miRNAs loaded into the two main active Argonaute (AGO) proteins in the uninuclear and mature pollen grain, AGO1 and AGO5. Our results indicate that the developmental progression from microspore to mature pollen grain is characterized by a transition from miRNAs targeting developmental genes to miRNAs regulating TE activity.sRNA, PARE, and AGO-IP sequencing uncovered the role of miRNAs during pollen development, showing that miRNAs transition from regulating genes involved in development to transposable elements

The Arabidopsis PHD-finger protein EDM2 has multiple roles in balancing NLR immune receptor gene expression

Plant NLR-type receptors serve as sensitive triggers of host immunity. Their expression has to be well-balanced, due to their interference with various cellular processes and dose-dependency of their defense-inducing activity. A genetic “arms race” with fast-evolving pathogenic microbes requires plants to constantly innovate their NLR repertoires. We previously showed that insertion of the COPIA-R7 retrotransposon into RPP7 co-opted the epigenetic transposon silencing signal H3K9me2 to a new function promoting expression of this Arabidopsis thaliana NLR gene. Recruitment of the histone binding protein EDM2 to COPIA-R7-associated H3K9me2 is required for optimal expression of RPP7. By profiling of genome-wide effects of EDM2, we now uncovered additional examples illustrating effects of transposons on NLR gene expression, strongly suggesting that these mobile elements can play critical roles in the rapid evolution of plant NLR genes by providing the “raw material” for gene expression mechanisms. We further found EDM2 to have a global role in NLR expression control. Besides serving as a positive regulator of RPP7 and a small number of other NLR genes, EDM2 acts as a suppressor of a multitude of additional NLR genes. We speculate that the dual functionality of EDM2 in NLR expression control arose from the need to compensate for fitness penalties caused by high expression of some NLR genes by suppression of others. Moreover, we are providing new insights into functional relationships of EDM2 with its interaction partner, the RNA binding protein EDM3/AIPP1, and its target gene IBM1, encoding an H3K9-demethylase

A new role for histone demethylases in the maintenance of plant genome integrity

Histone modifications deposited by the Polycomb repressive complex 2 (PRC2) play a critical role in the control of growth, development, and adaptation to environmental fluctuations of most multicellular eukaryotes. The catalytic activity of PRC2 is counteracted by Jumonji-type (JMJ) histone demethylases, which shapes the genomic distribution of H3K27me3. Here, we show that two JMJ histone demethylases in Arabidopsis, EARLY FLOWERING 6 (ELF6) and RELATIVE OF EARLY FLOWERING 6 (REF6), play distinct roles in H3K27me3 and H3K27me1 homeostasis. We show that failure to reset these chromatin marks during sexual reproduction results in the transgenerational inheritance of histone marks, which cause a loss of DNA methylation at heterochromatic loci and transposon activation. Thus, Jumonji-type histone demethylases play a dual role in plants by helping to maintain transcriptional states through development and safeguard genome integrity during sexual reproduction

Epigenomic Consequences of Immortalized Plant Cell Suspension Culture

Plant cells grown in culture exhibit genetic and epigenetic instability. Using a combination of chromatin immunoprecipitation and DNA methylation profiling on tiling microarrays, we have mapped the location and abundance of histone and DNA modifications in a continuously proliferating, dedifferentiated cell suspension culture of Arabidopsis. We have found that euchromatin becomes hypermethylated in culture and that a small percentage of the hypermethylated genes become associated with heterochromatic marks. In contrast, the heterochromatin undergoes dramatic and very precise DNA hypomethylation with transcriptional activation of specific transposable elements (TEs) in culture. High throughput sequencing of small interfering RNA (siRNA) revealed that TEs activated in culture have increased levels of 21-nucleotide (nt) siRNA, sometimes at the expense of the 24-nt siRNA class. In contrast, TEs that remain silent, which match the predominant 24-nt siRNA class, do not change significantly in their siRNA profiles. These results implicate RNA interference and chromatin modification in epigenetic restructuring of the genome following the activation of TEs in immortalized cell culture

Arabidopsis bioinformatics resources: the current state, challenges, and priorities for the future

Effective research, education, and outreach efforts by the Arabidopsis thaliana community, as well as other scientific communities that depend on Arabidopsis resources, depend vitally on easily available and publicly-shared resources. These resources include reference genome sequence data and an ever-increasing number of diverse data sets and data types. TAIR (The Arabidopsis Information Resource) and Araport (originally named the Arabidopsis Information Portal) are community informatics resources that provide tools, data, and applications to the more than 30,000 researchers worldwide that use in their work either Arabidopsis as a primary system of study or data derived from Arabidopsis. Four years after Araport’s establishment, the IAIC held another workshop to evaluate the current status of Arabidopsis Informatics and chart a course for future research and development. The workshop focused on several challenges, including the need for reliable and current annotation, community-defined common standards for data and metadata, and accessible and user-friendly repositories / tools / methods for data integration and visualization. Solutions envisioned included (1) a centralized annotation authority to coalesce annotation from new groups, establish a consistent naming scheme, distribute this format regularly and frequently, and encourage and enforce its adoption. (2) Standards for data and metadata formats, which are essential, but challenging when comparing across diverse genotypes and in areas with less-established standards (e.g. phenomics, metabolomics). Community-established guidelines need to be developed. (3) A searchable, central repository for analysis and visualization tools. Improved versioning and user access would make tools more accessible. Workshop participants proposed a “one-stop shop” website, an Arabidopsis “Super-Portal” to link tools, data resources, programmatic standards, and best practice descriptions for each data type. This must have community buy-in and participation in its establishment and development to encourage adoption