Dynamic DNA cytosine methylation in the Populus trichocarpa genome: tissue-level variation and relationship to gene expression

Kelly J Vining,Todd C Mockler,Kyle R Pomraning,Steven H Strauss,Matteo Pellegrini,Michael Freitag,Larry J Wilhelm,Henry D Priest

doi:10.1186/1471-2164-13-27

Abstract

BackgroundDNA cytosine methylation is an epigenetic modification that has been implicated in many biological processes. However, large-scale epigenomic studies have been applied to very few plant species, and variability in methylation among specialized tissues and its relationship to gene expression is poorly understood.ResultsWe surveyed DNA methylation from seven distinct tissue types (vegetative bud, male inflorescence [catkin], female catkin, leaf, root, xylem, phloem) in the reference tree species black cottonwood (Populus trichocarpa). Using 5-methyl-cytosine DNA immunoprecipitation followed by Illumina sequencing (MeDIP-seq), we mapped a total of 129,360,151 36- or 32-mer reads to the P. trichocarpa reference genome. We validated MeDIP-seq results by bisulfite sequencing, and compared methylation and gene expression using published microarray data. Qualitative DNA methylation differences among tissues were obvious on a chromosome scale. Methylated genes had lower expression than unmethylated genes, but genes with methylation in transcribed regions ("gene body methylation") had even lower expression than genes with promoter methylation. Promoter methylation was more frequent than gene body methylation in all tissues except male catkins. Male catkins differed in demethylation of particular transposable element categories, in level of gene body methylation, and in expression range of genes with methylated transcribed regions. Tissue-specific gene expression patterns were correlated with both gene body and promoter methylation.ConclusionsWe found striking differences among tissues in methylation, which were apparent at the chromosomal scale and when genes and transposable elements were examined. In contrast to other studies in plants, gene body methylation had a more repressive effect on transcription than promoter methylation.

Highlights

DNA cytosine methylation is an epigenetic modification that has been implicated in many biological processes
We found overall patterns of cytosine methylation that are consistent with those seen in Arabidopsis, but observed differences in methylation patterns among tissue types not previously studied
Many of these areas of methylation heterogeneity had low gene density and contained clusters of transposable elements, but one region we examined more closely was a cluster of leucine-rich repeat (LRR) genes

Summary

Methods

Plant material Genomic DNA for most tissues was obtained from P. trichocarpa clone Nisqually-1, the genotype that was used for the published genome sequence [47]. Mature leaves were collected in September 2008, and buds were collected in August-September 2008, December 2008 and March 2009 from two-year-old trees at a field site in Corvallis, Oregon, USA. Fine roots and xylem and phloem ~15 cm below the apical bud were collected in August 2009 from two-year-old Nisqually trees maintained in a lath house at Oregon State University, Corvallis, Oregon. Male and female catkins were collected at anthesis in March, 2009 from mature wild P. trichocarpa in Corvallis, Oregon. The homogenate was incubated at 6065°C for 60 min in sterile 1.5 ml microcentrifuge tubes, followed by extraction with ~500 μl of 24:1 phenol: chloroform. DNA was precipitated with two volumes of ice-cold 95% ethanol at 4°C for 2-24 hours and subsequently pelleted by centrifugation at 13,000 × g for 5 min. DNA concentration was determined using an ND-1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA)

Results

Discussion

Conclusion