Epigenomic Diversity in a Global Collection of Arabidopsis thaliana Accessions.

The epigenome orchestrates genome accessibility, functionality, and three-dimensional structure. Because epigenetic variation can impact transcription and thus phenotypes, it may contribute to adaptation. Here, we report 1,107 high-quality single-base resolution methylomes and 1,203 transcriptomes from the 1001 Genomes collection of Arabidopsis thaliana. Although the genetic basis of methylation variation is highly complex, geographic origin is a major predictor of genome-wide DNA methylation levels and of altered gene expression caused by epialleles. Comparison to cistrome and epicistrome datasets identifies associations between transcription factor binding sites, methylation, nucleotide variation, and co-expression modules. Physical maps for nine of the most diverse genomes reveal how transposons and other structural variants shape the epigenome, with dramatic effects on immunity genes. The 1001 Epigenomes Project provides a comprehensive resource for understanding how variation in DNA methylation contributes to molecular and non-molecular phenotypes in natural populations of the most studied model plant.

Limited Contribution of DNA Methylation Variation to Expression Regulation in Arabidopsis thaliana.

The extent to which epigenetic variation affects complex traits in natural populations is not known. We addressed this question using transcriptome and DNA methylation data from a sample of 135 sequenced A. thaliana accessions. Across individuals, expression was significantly associated with cis-methylation for hundreds of genes, and many of these associations remained significant after taking SNP effects into account. The pattern of correlations differed markedly between gene body methylation and transposable element methylation. The former was usually positively correlated with expression, and the latter usually negatively correlated, although exceptions were found in both cases. Finally, we developed graphical models of causality that adapt to a sample with heavy population structure, and used them to show that while methylation appears to affect gene expression more often than expression affects methylation, there is also strong support for both being independently controlled. In conclusion, although we find clear evidence for epigenetic regulation, both the number of loci affected and the magnitude of the effects appear to be small compared to the effect of SNPs.

The LORE1 insertion mutant resource.

Long terminal repeat (LTR) retrotransposons are closely related to retroviruses, and their activities shape eukaryotic genomes. Here, we present a complete Lotus japonicus insertion mutant collection generated by identification of 640 653 new insertion events following de novo activation of the LTR element Lotus retrotransposon 1 (LORE1) (http://lotus.au.dk). Insertion preferences are critical for effective gene targeting, and we exploit our large dataset to analyse LTR element characteristics in this context. We infer the mechanism that generates the consensus palindromes typical of retroviral and LTR retrotransposon insertion sites, identify a short relaxed insertion site motif, and demonstrate selective integration into CHG-hypomethylated genes. These characteristics result in a steep increase in deleterious mutation rate following activation, and allow LORE1 active gene targeting to approach saturation within a population of 134 682 L. japonicus lines. We suggest that saturation mutagenesis using endogenous LTR retrotransposons with germinal activity can be used as a general and cost-efficient strategy for generation of non-transgenic mutant collections for unrestricted use in plant research.

Characterization of the Dictyostelium homolog of chromatin binding protein DET1 suggests a conserved pathway regulating cell type specification and developmental plasticity.

DET1 (De-etiolated 1) is a chromatin binding protein involved in developmental regulation in both plants and animals. DET1 is largely restricted to multicellular eukaryotes, and here we report the characterization of a DET1 homolog from the social amoeba Dictyostelium discoideum. As in other species, Dictyostelium DET1 is nuclear localized. In contrast to other species, where it is an essential protein, loss of DET1 is nonlethal in Dictyostelium, although viability is significantly reduced. The phenotype of the det1(-) mutant is highly pleiotropic and results in a large degree of heterogeneity in developmental parameters. Loss of DET1 results in delayed and abnormal development with enlarged aggregation territories. Mutant slugs displayed cell type patterning with a bias toward the prestalk pathway. A number of DET1-interacting proteins are conserved in Dictyostelium, and the apparently conserved role of DET1 in regulatory pathways involving the bZIP transcription factors DimB, c-Jun, and HY5 suggests a highly conserved mechanism regulating development in multicellular eukaryotes. While the mechanism by which DET1 functions is unclear, it appears that it has a key role in regulation of developmental plasticity and integration of information on environmental conditions into the developmental program of an organism.

Dynamics of a novel centromeric histone variant CenH3 reveals the evolutionary ancestral timing of centromere biogenesis.

The centromeric histone H3 variant (CenH3) serves to target the kinetochore to the centromeres and thus ensures correct chromosome segregation during mitosis and meiosis. The Dictyostelium H3-like variant H3v1 was identified as the CenH3 ortholog. Dictyostelium CenH3 has an extended N-terminal domain with no similarity to any other known proteins and a histone fold domain at its C-terminus. Within the histone fold, α-helix 2 (α2) and an extended loop 1 (L1) have been shown to be required for targeting CenH3 to centromeres. Compared to other known and putative CenH3 histones, Dictyostelium CenH3 has a shorter L1, suggesting that the extension is not an obligatory feature. Through ChIP analysis and fluorescence microscopy of live and fixed cells, we provide here the first survey of centromere structure in amoebozoa. The six telocentric centromeres were found to mostly consist of all the DIRS-1 elements and to associate with H3K9me3. During interphase, the centromeres remain attached to the centrosome forming a single CenH3-containing cluster. Loading of Dictyostelium CenH3 onto centromeres occurs at the G2/prophase transition, in contrast to the anaphase/telophase loading of CenH3 observed in metazoans. This suggests that loading during G2/prophase is the ancestral eukaryotic mechanism and that anaphase/telophase loading of CenH3 has evolved more recently after the amoebozoa diverged from the animal linage.

Evolution of self-incompatibility in the Brassicaceae: Lessons from a textbook example of natural selection.

Self-incompatibility (SI) is a self-recognition genetic system enforcing outcrossing in hermaphroditic flowering plants and results in one of the arguably best understood forms of natural (balancing) selection maintaining genetic variation over long evolutionary times. A rich theoretical and empirical population genetics literature has considerably clarified how the distribution of SI phenotypes translates into fitness differences among individuals by a combination of inbreeding avoidance and rare-allele advantage. At the same time, the molecular mechanisms by which self-pollen is specifically recognized and rejected have been described in exquisite details in several model organisms, such that the genotype-to-phenotype map is also pretty well understood, notably in the Brassicaceae. Here, we review recent advances in these two fronts and illustrate how the joint availability of detailed characterization of genotype-to-phenotype and phenotype-to-fitness maps on a single genetic system (plant self-incompatibility) provides the opportunity to understand the evolutionary process in a unique perspective, bringing novel insight on general questions about the emergence, maintenance, and diversification of a complex genetic system.

Transposons: a blessing curse.

The genomes of most plant species are dominated by transposable elements (TEs). Once considered as 'junk DNA', TEs are now known to have a major role in driving genome evolution. Over the last decade, it has become apparent that some stress conditions and other environmental stimuli can drive bursts of activity of certain TE families and consequently new TE insertions. These can give rise to altered gene expression patterns and phenotypes, with new TE insertions sometimes causing flanking genes to become transcriptionally responsive to the same stress conditions that activated the TE in the first place. Such connections between TE-mediated increases in diversity and an accelerated rate of genome evolution provide powerful mechanisms for plants to adapt more rapidly to new environmental conditions. This review will focus on environmentally induced transposition, the mechanisms by which it alters gene expression, and the consequences for plant genome evolution and breeding.

DNA methylation in Arabidopsis has a genetic basis and shows evidence of local adaptation.

Epigenome modulation potentially provides a mechanism for organisms to adapt, within and between generations. However, neither the extent to which this occurs, nor the mechanisms involved are known. Here we investigate DNA methylation variation in Swedish Arabidopsis thaliana accessions grown at two different temperatures. Environmental effects were limited to transposons, where CHH methylation was found to increase with temperature. Genome-wide association studies (GWAS) revealed that the extensive CHH methylation variation was strongly associated with genetic variants in both cis and trans, including a major trans-association close to the DNA methyltransferase CMT2. Unlike CHH methylation, CpG gene body methylation (GBM) was not affected by growth temperature, but was instead correlated with the latitude of origin. Accessions from colder regions had higher levels of GBM for a significant fraction of the genome, and this was associated with increased transcription for the genes affected. GWAS revealed that this effect was largely due to trans-acting loci, many of which showed evidence of local adaptation.

Advanced methylome analysis after bisulfite deep sequencing: an example in Arabidopsis.

Deep sequencing after bisulfite conversion (BS-Seq) is the method of choice to generate whole genome maps of cytosine methylation at single base-pair resolution. Its application to genomic DNA of Arabidopsis flower bud tissue resulted in the first complete methylome, determining a methylation rate of 6.7% in this tissue. BS-Seq reads were mapped onto an in silico converted reference genome, applying the so-called 3-letter genome method. Here, we present BiSS (Bisufite Sequencing Scorer), a new method applying Smith-Waterman alignment to map bisulfite-converted reads to a reference genome. In addition, we introduce a comprehensive adaptive error estimate that accounts for sequencing errors, erroneous bisulfite conversion and also wrongly mapped reads. The re-analysis of the Arabidopsis methylome data with BiSS mapped substantially more reads to the genome. As a result, it determines the methylation status of an extra 10% of cytosines and estimates the methylation rate to be 7.7%. We validated the results by individual traditional bisulfite sequencing for selected genomic regions. In addition to predicting the methylation status of each cytosine, BiSS also provides an estimate of the methylation degree at each genomic site. Thus, BiSS explores BS-Seq data more extensively and provides more information for downstream analysis.

A versatile set of tagged expression vectors to monitor protein localisation and function in Dictyostelium.

We describe here a series of vectors for ectopic expression of tagged proteins in Dictyostelium discoideum. These vectors allow the addition of N- or C-terminal tags (GFP, mRFP, 3xFLAG, 3xHA, 6xMYC or TAP) with an optimised polylinker sequence and no additional amino acid residues at the N- or C-terminus of the protein. The expression cassettes were introduced into vectors containing Blasticidin or Geneticin resistance markers and into integrating as well as extrachromosomal plasmids. The vectors are designed as high and low copy versions and thus allow for a limited expression level control. They are also convenient with regard to complementation, co- and super-transformation. Finally the vectors share standardised cloning sites, so that a gene of interest can be easily transferred between vectors as experimental requirements evolve. These vectors were used to study the localisation of several putative RNA processing proteins including EriA and DicerB.

Overexpressing tagged proteins in plants using a modified gateway cloning strategy.

In recent years, sequence-specific recombination cloning methods such as the Gateway system have become increasingly popular for (over)expressing tagged proteins in high-throughput investigations in many different organisms, including plants. Because of their versatility and ease of use, these methods have gained favor in low- and medium-throughput investigations as well. However, due to the recombination step, the resulting fusion proteins contain long and often highly charged polylinker sequences that can interfere with their physiological function. Furthermore, in some cases the gene of interest must be cloned twice (once with and once without a stop codon) for N- and C-terminal tagging. Here, we present a hybrid combinatorial cloning strategy that overcomes many of these limitations. In the first step, the gene of interest is cloned into an entry vector containing standardized cloning sites with the desired N- or C-terminal tag and an optimized polylinker sequence. A Gateway recombination reaction is used to transfer the protein-tag fusion from the entry clone to a Gateway destination vector with the desired promoter and selectable marker for the organism of interest. As experimental requirements evolve, constructs for expressing the protein of interest with the desired tag, promoter, and selectable marker or other features can rapidly and easily be created.

A modified Gateway cloning strategy for overexpressing tagged proteins in plants.

BACKGROUND: Recent developments, including the sequencing of a number of plant genomes, have greatly increased the amount of data available to scientists and has enabled high throughput investigations where many genes are investigated simultaneously. To perform these studies, recombinational cloning methods such as the Gateway system have been adapted to plant transformation vectors to facilitate the creation of overexpression, tagging and silencing vectors on a large scale. RESULTS: Here we present a hybrid cloning strategy which combines advantages of both recombinational and traditional cloning and which is particularly amenable to low-to-medium throughput investigations of protein function using techniques of molecular biochemistry and cell biology. The system consists of a series of twelve Gateway Entry cassettes into which a gene of interest can be inserted using traditional cloning methods to generate either N- or C-terminal fusions to epitope tags and fluorescent proteins. The resulting gene-tag fusions can then be recombined into Gateway-based Destination vectors, thus providing a wide choice of resistance marker, promoter and expression system. The advantage of this modified Gateway cloning strategy is that the entire open reading frame encoding the tagged protein of interest is contained within the Entry vectors so that after recombination no additional linker sequences are added between the tag and the protein that could interfere with protein function and expression. We demonstrate the utility of this system for both transient and stable Agrobacterium-mediated plant transformations. CONCLUSION: This modified Gateway cloning strategy is complementary to more conventional Gateway-based systems because it expands the choice of tags and higher orders of combinations, and permits more control over the linker sequence lying between a protein of interest and an epitope tag, which can be particularly important for studies of protein function.

CUL4 associates with DDB1 and DET1 and its downregulation affects diverse aspects of development in Arabidopsis thaliana.

Cullins are central scaffolding subunits in eukaryotic E3 ligases that facilitate the ubiquitination of target proteins. Arabidopsis contains at least 11 cullin proteins but only a few of them have been assigned biological roles. In this work Arabidopsis cullin 4 is shown to assemble with DDB1, RBX1, DET1 and DDB2 in vitro and in planta. In addition, by using T-DNA insertion and CUL4 antisense lines we demonstrate that corresponding mutants are severely affected in different aspects of development. Reduced CUL4 expression leads to a reduced number of lateral roots, and to abnormal vascular tissue and stomatal development. Furthermore, cul4 mutants display a weak constitutive photomorphogenic phenotype. These results therefore assign an important function to CUL4 during plant development and provide strong evidence that CUL4 assembles together with RBX1 and DDB1 proteins to form a functional E3 ligase in Arabidopsis.

Dimerization of CtIP, a BRCA1- and CtBP-interacting protein, is mediated by an N-terminal coiled-coil motif.

CtIP is a transcriptional co-regulator that binds a number of proteins involved in cell cycle control and cell development, such as CtBP (C terminus-binding protein), BRCA1 (breast cancer-associated protein-1), and LMO4 (LIM-only protein-4). The only recognizable structural motifs within CtIP are two putative coiled-coil domains located near the N and C termini of the protein. We now show that the N-terminal coiled coil (residues 45-160), but not the C-terminal coiled coil, mediates homodimerization of CtIP in mammalian 293T cells. The N-terminal coiled coil did not facilitate binding to LMO4 and BRCA1 proteins in these cells. A protease-resistant domain (residues 27-168) that minimally encompasses the putative N-terminal coiled coil was identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. This region is predicted to contain two smaller coiled-coil regions. The CtIP-(45-160) dimerization domain is helical and dimeric, indicating that the domain does form a coiled coil. The two smaller domains, CtIP-(45-92) and CtIP-(93-160), also formed dimers of lower binding affinity, but with less helical content than the longer peptide. The hydrodynamic radius of CtIP-(45-160) is the same as those of CtIP-(45-92) and CtIP-(93-160), implying that CtIP-(45-160) does not form a single long coiled coil, but a more compact structure involving homodimerization of the two smaller coiled coils, which fold back as a four-helix bundle or other compact structure. These results suggest a specific model for CtIP homodimerization via its N terminus and contribute to an improved understanding of how this protein might assemble other factors required for its role as a transcriptional corepressor.