Genome-wide, high-resolution DNA methylation profiling using bisulfite-mediated cytosine conversion

Abstract

Methylation of cytosines ((m)C) is essential for epigenetic gene regulation in plants and mammals. Aberrant (m)C patterns are associated with heritable developmental abnormalities in plants and with cancer in mammals. We have developed a genome-wide DNA methylation profiling technology employing a novel amplification step for DNA subjected to bisulfite-mediated cytosine conversion. The methylation patterns detected are not only consistent with previous results obtained with (m)C immunoprecipitation (mCIP) techniques, but also demonstrated improved resolution and sensitivity. The technology, named BiMP (for Bisulfite Methylation Profiling), is more cost-effective than mCIP and requires as little as 100 ng of Arabidopsis DNA.

Figure 1.

Post-amplification quality assessment following random amplification methods used for Bisulfite Methylation Profiling (BiMP). (A) Assessment for amplification bias using locus-specific PCR amplification at the AT3G08650 locus. For each DNA sample, two random amplification methods were performed, “sixN” and “fourN” (as indicated on the left) and assayed for the presence of the target template (see Supplemental Fig. 1A). The second- (2^nd) and fourth-generation (4^th) homozygotes of the met1-3 mutant are indicated. Col is indicated as “na” (not applicable). DNA sample treatment is indicated as either bisulfite-converted (+) or non-treated (−). Each lane corresponds to an equal volume of PCR reaction (10 μL). DNA was separated in a 1% agarose gel and stained with ethidium bromide. The PCR-positive control (pos control) was amplified from genomic Col DNA. The template-free controls (no-template control) correspond to the Sequenase elongation (1^st), random amplification (2^nd), and PCR (PCR) steps (see Methods). The gel images have been manipulated to create a contiguous lane order. (B) Evaluation of repeat representation in randomly amplified reaction products using dot blot hybridizations. Non-treated and bisulfite-converted Columbia DNA samples were amplified by the methods indicated (left) and hybridized to a macroarray (see Methods). The macroarray was spotted with 200 ng of PCR amplicons derived from Col DNA with (+) or without (−) bisulfite-conversion from different genomic repeat sequences (indicated above and described in the Methods). Each column of the macroarray was spotted with two technical replicates, observed as rows on the macroarray. Rectangular borders were added to the image to separate the bisulfite-converted (+) from the nonconverted (−) probes.

Figure 2.

Assessment of the technical reproducibility observed for the BiMP technology. A scatter plot comparison of two technically replicated hybridization datasets (one hybridization per axis) with all signal intensities presented (>3.2 × 10⁶ probe pairs, log₂ scale). From one bisulfite-converted sample, two DNA aliquots (100 ng) were independently amplified, fragmented, labeled, and hybridized (see Methods). The correlation coefficient (r) was calculated using TAS (Affymetrix), and the plot was drawn in the R statistical environment (http://www.r-project.org). (A) Col bisulfite-converted DNA, (B) met1-3 bisulfite-converted DNA.

Figure 3.

Bisulfite methylation profiling results visualized at the chromosomal level: chromosome 4 (A) and a pericentromeric region of chromosome 1 (B). (A) Graphs represent the average signal intensity per 100 kb (see Methods). (Green) Col^BS+ hybridization, (yellow) met1-3^BS+ hybridization, (purple) nontreated DNA hybridization. (X-axis) Physical length of chromosome 4 (NCBI Arabidopsis genome assembly version 5), (arrow) heterochromatic “knob” region. (B) Average hybridization signal intensity profiles across the pericentromeric region of chromosome 1 (chr1:15,520,000–15,600,000). Each tier represents a graph of the hybridization profile corresponding to each dataset (labeled at the right). The hybridization graphs were displayed in the range 0–11 (log₂ scale). The difference graphs, BiMP (Col^BS+) − (met1-3^BS+) and BiMP (met1-3^BS+) − (Col^BS+), compare the BiMP profiles. The signal intensity differences above the applied cut-off (>4.0, horizontal line) are indicated for significantly hypomethylated intervals (green boxes) and hypermethylated intervals (yellow boxes) in met1-3^BS+ relative to Col^BS+.

Figure 4.

Analysis of BiMP results at known DNA methylation targets in Arabidopsis. The graphs were designed as in Fig. 3B. (A) AT4G25530 (FWA) locus. (Red box) Region encoding the methylated repeat sequences in the wild type (Soppe et al. 2000). (B) AT3G23130 (SUP) locus. (Red box) Region that was bisulfite sequenced (see Supplemental Fig. 5).

Figure 5.

Validation of novel methylation polymorphisms detected using BiMP. Graphs were prepared as in Fig. 3B. (A) AT2G36490 (ROS1), (B) AT3G54340 (AP3). (Red boxes) Regions that were bisulfite sequenced. (C,D) Bisulfite sequencing at the AT2G36490 (ROS1) (C) and AT3G54340 (AP3) (D) loci. The DNA methylation level (% methylated, Y-axis) was determined by bisulfite sequencing and presented for each different sequence motif: CG, CNG, and CHH (H = A, T, C) (see Methods). The total number of cytosines analyzed per sequence motif is provided within the parentheses.