Highly Integrated Single-Base Resolution Maps of the Epigenome in Arabidopsis

Abstract

Deciphering the multiple layers of epigenetic regulation that control transcription is critical to understanding how plants develop and respond to their environment. Using sequencing-by-synthesis technology we directly sequenced the cytosine methylome (methylC-seq), transcriptome (mRNA-seq), and small RNA transcriptome (smRNA-seq) to generate highly integrated epigenome maps for wild-type Arabidopsis thaliana and mutants defective in DNA methyltransferase or demethylase activity. At single-base resolution we discovered extensive, previously undetected DNA methylation, identified the context and level of methylation at each site, and observed local sequence effects upon methylation state. Deep sequencing of smRNAs revealed a direct relationship between the location of smRNAs and DNA methylation, perturbation of smRNA biogenesis upon loss of CpG DNA methylation, and a tendency for smRNAs to direct strand-specific DNA methylation in regions of RNA-DNA homology. Finally, strand-specific mRNA-seq revealed altered transcript abundance of hundreds of genes, transposons, and unannotated intergenic transcripts upon modification of the DNA methylation state.

Cistrome and Epicistrome Features Shape the Regulatory DNA Landscape

“…To determine how DNA methylation affected binding, we used base-pair methylation maps from Arabidopsis leaf DNA to quantify DAP-seq and ChIP-seq binding at high-scoring motifs that contained 5-methylcytosine. As plant DNA methylation is equally distributed between two mutually exclusive patterns (Cokus et al, 2008;Lister et al, 2008), we classified these motifs into two categories: (1) motifs in mC-all regions identified by dense methylation in all contexts (CHH, CHG, and CG, where H is A, C, or T) associated with silenced genes and transposons ( Figure 7A, inset), and (2) motifs in mCG-only regions exclusively methylated in the CG context, more sparsely distributed, and enriched in expressed genes ( Figure 7B, inset). As a control, we identified a set of motifs that neighbored a methylated region (within 200 bp), but themselves did not contain methylation.…”

Power sequencing

“…1) to sequence at varying levels of detail the transcriptomes of four organisms -the fission yeast Saccharomyces pombe 1 , the budding yeast Saccharomyces cerevisiae 2 , the plant Arabidopsis thaliana 3 and the mouse 4,5 ; for the sequencing step, four of the groups used the Illumina Genome Analyzer system [1][2][3][4] and one used the ABI SOLiD system 5 . Between 30 and 125 million sequences 25 to 39-base-pairs in length were obtained in each study.…”

“…Together, these advances will provide even greater insight into the transcriptional landscapes, regulation of gene expression and alternative splicing. Most importantly, next-generation sequencing has the potential to turn individual laboratories into small genome centres and to allow an individual scientist to determine the entire transcriptome of any source (any organism, tumour samples, tissues from patients with neurodegenerative disorders and so on) in a matter of days, and for only a few thousand In this technique, which was used for analysis of transcriptomes of five organisms [1][2][3][4][5] , the isolated mRNA is analysed by one of three procedures. a, In the first procedure, mRNAs are randomly sheared, linker molecules are attached to their ends, and they are then converted to DNA.…”

Analysis of DNA modifications in aging research

“…In a natural progression, these techniques were combined into a new generation of methods. Whole-genome bisulfite sequencing (WGBS) was first introduced to quantify total genome methylation levels in the plant Arabidopsis (Cokus et al 2008;Lister et al 2008) but was rapidly adapted for use in mammalian genomes ). For WGBS, genomic DNA libraries are created and subsequently bisulfite converted, sequenced, and mapped back to the reference genome.…”