Daofeng Li

PMRD: plant microRNA database

MicroRNAs (miRNA) are ∼21 nucleotide-long non-coding small RNAs, which function as post-transcriptional regulators in eukaryotes. miRNAs play essential roles in regulating plant growth and development. In recent years, research into the mechanism and consequences of miRNA action has made great progress. With whole genome sequence available in such plants as Arabidopsis thaliana, Oryza sativa, Populus trichocarpa, Glycine max, etc., it is desirable to develop a plant miRNA database through the integration of large amounts of information about publicly deposited miRNA data. The plant miRNA database (PMRD) integrates available plant miRNA data deposited in public databases, gleaned from the recent literature, and data generated in-house. This database contains sequence information, secondary structure, target genes, expression profiles and a genome browser. In total, there are 8433 miRNAs collected from 121 plant species in PMRD, including model plants and major crops such as Arabidopsis, rice, wheat, soybean, maize, sorghum, barley, etc. For Arabidopsis, rice, poplar, soybean, cotton, medicago and maize, we included the possible target genes for each miRNA with a predicted interaction site in the database. Furthermore, we provided miRNA expression profiles in the PMRD, including our local rice oxidative stress related microarray data (LC Sciences miRPlants_10.1) and the recently published microarray data for poplar, Arabidopsis, tomato, maize and rice. The PMRD database was constructed by open source technology utilizing a user-friendly web interface, and multiple search tools. The PMRD is freely available at http://bioinformatics.cau.edu.cn/PMRD. We expect PMRD to be a useful tool for scientists in the miRNA field in order to study the function of miRNAs and their target genes, especially in model plants and major crops.

The human epigenome browser at Washington University

To the Editor: Advances in next-generation sequencing have reshaped the landscape of genomic and epigenomic research. Large consortia such as the Encyclopedia of DNA Elements, the Roadmap Epigenomics Mapping Consortium and The Cancer Genome Atlas have generated tens of thousands of sequencingbased genome-wide datasets, creating a reference and resource for the scientific community. Small groups of researchers now can rapidly obtain huge volumes of genomic data, which need to be placed in the context of the consortium data for comparison. These data are often accompanied by rich metadata describing the sample and experiment, which is critical for their interpretation. Visualizing, navigating and interpreting such data in a meaningful way is a daunting challenge1. We developed the Human Epigenome Browser to host Human Epigenome Atlas data produced by the Roadmap Epigenomics Project2 and to support navigation of the Atlas and its interactive visualization, integration, comparison and analysis (http://epigenomegateway.wustl.edu/; see Supplementary Note and Supplementary Protocol for main components and use). The Browser is web-based, and it extends the seminal concept introduced by the University of California Santa Cruz Cancer Genomics Browser3 to support large, sequencing-based datasets. Epigenome measurements are displayed as genome heatmaps in which color gradients reflect signal strength (Fig. 1 and Supplementary Fig. 1). Metadata such as cell type, assay type, epigenetic mark and phenotype of the sample are encoded numerically and displayed in different colors by a metadata heatmap next to the genome heatmap (Fig. 1 and Supplementary Figs. 2–4). Investigators can zoom and pan in a ‘Google Maps’–like style to examine dozens to

DNA hypomethylation within specific transposable element families associates with tissue-specific enhancer landscape

Transposable element (TE)-derived sequences comprise half of the human genome and DNA methylome and are presumed to be densely methylated and inactive. Examination of genome-wide DNA methylation status within 928 TE subfamilies in human embryonic and adult tissues identified unexpected tissue-specific and subfamily-specific hypomethylation signatures. Genes proximal to tissue-specific hypomethylated TE sequences were enriched for functions important for the relevant tissue type, and their expression correlated strongly with hypomethylation within the TEs. When hypomethylated, these TE sequences gained tissue-specific enhancer marks, including monomethylation of histone H3 at lysine 4 (H3K4me1) and occupancy by p300, and a majority exhibited enhancer activity in reporter gene assays. Many such TEs also harbored binding sites for transcription factors that are important for tissue-specific functions and showed evidence of evolutionary selection. These data suggest that sequences derived from TEs may be responsible for wiring tissue type–specific regulatory networks and may have acquired tissue-specific epigenetic regulation.

Widespread contribution of transposable elements to the innovation of gene regulatory networks

Transposable elements (TEs) have been shown to contain functional binding sites for certain transcription factors (TFs). However, the extent to which TEs contribute to the evolution of TF binding sites is not well known. We comprehensively mapped binding sites for 26 pairs of orthologous TFs in two pairs of human and mouse cell lines (representing two cell lineages), along with epigenomic profiles, including DNA methylation and six histone modifications. Overall, we found that 20% of binding sites were embedded within TEs. This number varied across different TFs, ranging from 2% to 40%. We further identified 710 TF-TE relationships in which genomic copies of a TE subfamily contributed a significant number of binding peaks for a TF, and we found that LTR elements dominated these relationships in human. Importantly, TE-derived binding peaks were strongly associated with open and active chromatin signatures, including reduced DNA methylation and increased enhancer-associated histone marks. On average, 66% of TE-derived binding events were cell type-specific with a cell type-specific epigenetic landscape. Most of the binding sites contributed by TEs were species-specific, but we also identified binding sites conserved between human and mouse, the functional relevance of which was supported by a signature of purifying selection on DNA sequences of these TEs. Interestingly, several TFs had significantly expanded binding site landscapes only in one species, which were linked to species-specific gene functions, suggesting that TEs are an important driving force for regulatory innovation. Taken together, our data suggest that TEs have significantly and continuously shaped gene regulatory networks during mammalian evolution.

Exploring long-range genome interactions using the WashU Epigenome Browser

To the Editor : Eukar yotic chromosomes are a highly organized three-dimensional entity folded through a tightly regulated process1,2 with important functions that include bringing distal regulatory elements into the vicinity of their target gene promoters and arranging the chromosomes into distinct compartments3–6. Recent technological innovations, including chromosome conformation capture carbon copy (5C), Hi-C and chromatin interaction analysis by paired-end tag sequencing (ChIA-PET), have facilitated the discovery of chromosomal organization principles and folding architectures at unprecedented scales and resolution. Each technology also comes with corresponding computational tools7,8 to process and visualize its specific data type (Supplementary Note 1). However, visualizing and navigating long-range interaction data, as well as integrating these interactions with other epigenomics data, remains a much-desired capability and a daunting challenge9. We have extended the WashU Epigenome Browser10 (http:// epigenomegateway.wustl.edu/), which currently hosts thousands of epigenome and transcriptome data sets for multiple cell types, tissues, individuals and species, to support multiple types of long-range genome interaction data. This enables investigators to explore epigenomic data in the context of higher-order chromosomal domains and to generate multiple types of intuitive, publication-quality figures of interactions (see tutorial in Supplementary Note 2). In Figure 1 we display the histonemodification profile and long-range interaction data of two human cell lines (IMR90 and K562) side by side and note that regions can exhibit similar interaction patterns while showing different histone modifications (such as the boundary region between domains 1 and 2) (Supplementary Methods). These observations are consistent with the hypothesis that chromatin domains are stable across cell types but can have different epigenetic profiles in different cells. Genes within each domain are regulated epigenetically in a cell type– specific manner5. Integrating higherorder chromatin interaction data with other genomic data could potentially reveal novel insights about mechanisms underlying gene and genome regulation. Pairs of interacting regions can be joined by arcs (Fig. 1b)—a suitable representation for sparse interactions—as is common with ChIA-PET and sometimes found in 5C data sets, or they can be indicated by filled rectangles in a heat map (Fig. 1c) for dense interactions (as is typical for Hi-C and some regions in 5C data sets). Investigators can click on the arcs or heat-map cells to invoke a companion Browser panel (Supplementary Fig. 1), which displays epigenomic data over the distal interacting locus. This companion panel can be navigated independently, enabling comparison of data patterns of interacting loci in the same view. Thus, investigators can observe several loci that are distant in their genomic coordinates but are inferred to be spatially close to each other in the nucleus. Both the “arc” and “heat-map” modes display only interactions that are contained within the current browsing range while omitting interactions beyond the range. To visualize the complete set of interactions, investigators can invoke the “Circlet View” (Supplementary Fig. 2), in which the chromosomal axis curls to form a circle and interactions are displayed as arcs inside the circle. Investigators can choose to display a single chromosome or to include interacting chromosomes to achieve a wholegenome perspective of the interactions. Investigators can also toggle between “thin,” “full” or “density” mode for any data track (Supplementary Figs. 3–5). “Gene Set View” and genomic juxtaposition can be combined with the long-range interaction function to focus on the interaction events in a subregion of the genome (Fig. 1). An investigator’s own long-range interaction data can be displayed on the Browser via the custom track or Data Hub function. The Browser currently hosts over 100 genomewide chromatin interaction data sets for human, mouse and fly. We expect such data to become increasingly available, and the browser will be a helpful tool for exploring how eukaryotic genomes function as nonlinear systems.

Temperature-dependent survival of Turnip crinkle virus-infected Arabidopsis plants relies on an RNA silencing-based defense that requires DCL2, AGO2, and HEN1

ABSTRACT While RNA silencing is a potent antiviral defense in plants, well-adapted plant viruses are known to encode suppressors of RNA silencing (VSR) that can neutralize the effectiveness of RNA silencing. As a result, most plant genes involved in antiviral silencing were identified by using debilitated viruses lacking silencing suppression capabilities. Therefore, it remains to be resolved whether RNA silencing plays a significant part in defending plants against wild-type viruses. We report here that, at a higher plant growth temperature (26°C) that permits rigorous replication of Turnip crinkle virus (TCV) in Arabidopsis, plants containing loss-of-function mutations within the Dicer-like 2 (DCL2), Argonaute 2 (AGO2), and HEN1 RNA methyltransferase genes died of TCV infection, whereas the wild-type Col-0 plants survived to produce viable seeds. To account for the critical role of DCL2 in ensuring the survival of wild-type plants, we established that higher temperature upregulates the activity of DCL2 to produce viral 22-nucleotide (nt) small interfering RNAs (vsRNAs). We further demonstrated that DCL2-produced 22-nt vsRNAs were fully capable of silencing target genes, but that this activity was suppressed by the TCV VSR. Finally, we provide additional evidence supporting the notion that TCV VSR suppresses RNA silencing through directly interacting with AGO2. Together, these results have revealed a specialized RNA silencing pathway involving DCL2, AGO2, and HEN1 that provides the host plants with a competitive edge against adapted viruses under environmental conditions that facilitates robust virus reproduction.

Functional DNA methylation differences between tissues, cell types, and across individuals discovered using the M&M algorithm

DNA methylation plays key roles in diverse biological processes such as X chromosome inactivation, transposable element repression, genomic imprinting, and tissue-specific gene expression. Sequencing-based DNA methylation profiling provides an unprecedented opportunity to map and compare complete DNA methylomes. This includes one of the most widely applied technologies for measuring DNA methylation: methylated DNA immunoprecipitation followed by sequencing (MeDIP-seq), coupled with a complementary method, methylation-sensitive restriction enzyme sequencing (MRE-seq). A computational approach that integrates data from these two different but complementary assays and predicts methylation differences between samples has been unavailable. Here, we present a novel integrative statistical framework M&M (for integration of MeDIP-seq and MRE-seq) that dynamically scales, normalizes, and combines MeDIP-seq and MRE-seq data to detect differentially methylated regions. Using sample-matched whole-genome bisulfite sequencing (WGBS) as a gold standard, we demonstrate superior accuracy and reproducibility of M&M compared to existing analytical methods for MeDIP-seq data alone. M&M leverages the complementary nature of MeDIP-seq and MRE-seq data to allow rapid comparative analysis between whole methylomes at a fraction of the cost of WGBS. Comprehensive analysis of nineteen human DNA methylomes with M&M reveals distinct DNA methylation patterns among different tissue types, cell types, and individuals, potentially underscoring divergent epigenetic regulation at different scales of phenotypic diversity. We find that differential DNA methylation at enhancer elements, with concurrent changes in histone modifications and transcription factor binding, is common at the cell, tissue, and individual levels, whereas promoter methylation is more prominent in reinforcing fundamental tissue identities.

Estimating absolute methylation levels at single-CpG resolution from methylation enrichment and restriction enzyme sequencing methods

Recent advancements in sequencing-based DNA methylation profiling methods provide an unprecedented opportunity to map complete DNA methylomes. These include whole-genome bisulfite sequencing (WGBS, MethylC-seq, or BS-seq), reduced-representation bisulfite sequencing (RRBS), and enrichment-based methods such as MeDIP-seq, MBD-seq, and MRE-seq. These methods yield largely comparable results but differ significantly in extent of genomic CpG coverage, resolution, quantitative accuracy, and cost, at least while using current algorithms to interrogate the data. None of these existing methods provides single-CpG resolution, comprehensive genome-wide coverage, and cost feasibility for a typical laboratory. We introduce methylCRF, a novel conditional random fields-based algorithm that integrates methylated DNA immunoprecipitation (MeDIP-seq) and methylation-sensitive restriction enzyme (MRE-seq) sequencing data to predict DNA methylation levels at single-CpG resolution. Our method is a combined computational and experimental strategy to produce DNA methylomes of all 28 million CpGs in the human genome for a fraction (<10%) of the cost of whole-genome bisulfite sequencing methods. methylCRF was benchmarked for accuracy against Infinium arrays, RRBS, WGBS sequencing, and locus-specific bisulfite sequencing performed on the same human embryonic stem cell line. methylCRF transformation of MeDIP-seq/MRE-seq was equivalent to a biological replicate of WGBS in quantification, coverage, and resolution. We used conventional bisulfite conversion, PCR, cloning, and sequencing to validate loci where our predictions do not agree with whole-genome bisulfite data, and in 11 out of 12 cases, methylCRF predictions of methylation level agree better with validated results than does whole-genome bisulfite sequencing. Therefore, methylCRF transformation of MeDIP-seq/MRE-seq data provides an accurate, inexpensive, and widely accessible strategy to create full DNA methylomes.

The HC-pro protein of potato virus Y interacts with NtMinD of tobacco

Potato virus Y (PVY) infections often lead to altered numbers of host plant chloroplasts, as well as changes in morphology and inhibited photosynthesis. The multifunctional protein helper component-proteinase, HC-Pro, has been identified in PVY-infected leaf chloroplasts. We used yeast two-hybrid and bimolecular fluorescence complementation assays to demonstrate that HC-Pro can interact with the chloroplast division-related factor NtMinD in yeast and tobacco cells, respectively. In addition, we confirmed that residues 271 to 314 in NtMinD are necessary for its interaction with PVY HC-Pro in a yeast two-hybrid analysis using four NtMinD deletion mutants. These residues are necessary for the dimerization of NtMinD, which plays a vital role in chloroplast division. Thus, PVY HC-Pro may affect NtMinD activity by inhibiting the formation of NtMinD homodimers, and this may interfere with chloroplast division and contribute to changes in the numbers of chloroplast per cell observed in PVY-infected plants.

Transcriptional profiling of Medicago truncatula under salt stress identified a novel CBF transcription factor MtCBF4 that plays an important role in abiotic stress responses.

BackgroundSalt stress hinders the growth of plants and reduces crop production worldwide. However, different plant species might possess different adaptive mechanisms to mitigate salt stress. We conducted a detailed pathway analysis of transcriptional dynamics in the roots of Medicago truncatula seedlings under salt stress and selected a transcription factor gene, MtCBF4, for experimental validation.ResultsA microarray experiment was conducted using root samples collected 6, 24, and 48 h after application of 180 mM NaCl. Analysis of 11 statistically significant expression profiles revealed different behaviors between primary and secondary metabolism pathways in response to external stress. Secondary metabolism that helps to maintain osmotic balance was induced. One of the highly induced transcription factor genes was successfully cloned, and was named MtCBF4. Phylogenetic analysis revealed that MtCBF4, which belongs to the AP2-EREBP transcription factor family, is a novel member of the CBF transcription factor in M. truncatula. MtCBF4 is shown to be a nuclear-localized protein. Expression of MtCBF4 in M. truncatula was induced by most of the abiotic stresses, including salt, drought, cold, and abscisic acid, suggesting crosstalk between these abiotic stresses. Transgenic Arabidopsis over-expressing MtCBF4 enhanced tolerance to drought and salt stress, and activated expression of downstream genes that contain DRE elements. Over-expression of MtCBF4 in M. truncatula also enhanced salt tolerance and induced expression level of corresponding downstream genes.ConclusionComprehensive transcriptomic analysis revealed complex mechanisms exist in plants in response to salt stress. The novel transcription factor gene MtCBF4 identified here played an important role in response to abiotic stresses, indicating that it might be a good candidate gene for genetic improvement to produce stress-tolerant plants.