Title: "Advancing epigenomic profiling using next-generation sequencing technologies"
Speaker: Matt Vaughn, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
Place: Mechanical Engineering (ME) 161; February 26, 2008, Tuesday, 4:30pm


In plants, DNA cytosine methylation plays a central role in transposon silencing, as well as in regulation of transgenes and some endogenous genes. Cytosine methylation of repetitive occurrs in both symmetric (CpG and CpNpG) as well as asymmetric sequence contexts. We have long used the methylation-dependent restriction enzyme McrBc to profile methylated DNA using a genomic tiling microarray covering ~17 Mb of Arabidopsis chromosome 4. Based on this approach, we find that repeated sequences and transposable elements (TEs), especially LTR retrotransposons, are densely methylated, but at least 25% of genes also have detectable methylation in their transcribed regions. This within-gene methylation appears to be coordinated with transcription and, though directly heritable, is highly polymorphic between Arabidopsis ecotypes. The pathways underlying within-gene methylation are unknown, so we have undertaken a candidate gene approach based on comparative phylogenomics and expression profile clustering to identify potenital participants. Based on our criteria, at least 40 additional genes beyond those currently known have a high potential to be involved in DNA methylation. In order to rapidly generate DNA cytosine methylation profiles for these numerous potential methylation pathway mutants, we are developing Illumina sequencing-based methodologies around sodium bisulfite conversion of unmethylated cytosines, immunoprecipitation of methyl-cytosine, and McrBC digestion. Experiences with these varying approaches will be discussed, as well as their effectiveness relative to traditional microarrays.

Associated Reading:
Vaughn MW, Tanurdzic M, Lippman Z, Jiang H, Carrasquillo R, Rabinowicz PD, Dedhia N, McCombie WR, Agier N, Bulski A, Colot V, Doerge RW, Martienssen RA. Epigenetic Natural Variation in Arabidopsis thaliana. PLoS Biol. 2007 Jun 19;5(7):e174

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