IC4R005-Epigenomic-2016-20487381

Project Title

• Genome-wide mapping of cytosine methylation revealed dynamic DNA methylation patterns associated with genes and centromeres in rice

The Background of This Project

Figure 1. Methylation analysis of selected rice DNA sequences based on restriction digestion.

• Cytosine DNA methylation is a conserved epigeneti silencing mechanism in higher eukaryotes. Cytosine methylation plays an important role in many biological processes, including defense against transposon proliferation (Tsukahara et al., 2009), control of genomic imprinting (Morison et al., 2005) and regulation of gene expression (Bird, 2002). In mammals, cytosine methylation is controlled by the de novo methyltransferases DNMT3a/b, and is maintained by the methyltransferase DNMT1 (Goll and Bestor, 2005). Methylated cytosines occur almost exclu- sively at CG dinucleotides in mammalian genomes. However, Lister et al. (2009) recently showed that approximately 15% of methylated cytosines are associated with neighbor.
• Despite significant interest in mapping cytosine methylation in various model eukaryotes, genome-wide mapping has only been accomplished in a few species. Mapping cytosine methylation at a single-base resolution has recently been accomplished in Arabidopsis thaliana (Cokus et al., 2008; Lister et al., 2008) and humans (Lister et al., 2009). The researchers are interested in the high-resolution mapping of DNA methylation associated with plant centromeres. Four of the 12 rice centromeres have been fully or nearly fully sequenced (Yan et al., 2008), providing an unprecedented opportunity to study the methylation associated with centromeric DNA in multicellular eukaryotes. The researchers conducted a methylcytosine immunoprecipitation (mCIP) combined with Illumina sequencing (mCIP-seq) assay in rice. We provide a genome-wide cytosine methylation map of rice and report on the dynamic methylation patterns associated with rice genes and centromeres.

Plant Materials & Treatment

• The fully sequenced rice cultivar ‘Nipponbare’ was used in all experiments. Nipponbare plants were grown in the Biotron glass-house for 2 weeks (30–32℃ and 16 h of light). Genomic DNA was extracted from seedlings and fragmented to 200–1400 bp by sonication. We conducted mCIP following published protocols (Zhang et al., 2006). The mCIP DNA was amplified using a random amplification procedure (Lippman et al., 2005) before sequencing. Three replicates of the mCIP procedure were performed starting with different input DNA, which yielded consistent results based on dot-blot hybridizations.

• The mCIP DNA was subjected to a modified sequencing pipeline on Illumina’s GAiix Genome Analyzer (Illumina, http://www.illumina.com). The mCIP DNA was fragmented by nebulization. The condition of nebulization was optimized to generate fragments with dominant sizes ranging from 200 to 250 bp. After the fragments were purified using a QiaQuick PCR purification kit (Qiagen, http://www.qiagen.com), the ends of fragments were repaired andphosphorylated. A single adenosine triphosphate was added to the 3¢ end of the blunt-ended and phosphorylated DNA fragments. An Illumina proprietary adapter was then ligated to DNA fragments by T4 DNA ligase. A gel purification procedure was carried out to select the fragments sized from 250 to 300 bp. Twelve cycles of PCR with Illumina proprietary PCR primers were used to generate and amplify the library used for Illumina cluster formation. Cluster formation and sequencing on the GAiix platform were performed following the manufacturer’s standard cluster generation and sequencing protocols.

Research Findings

• We performed an mCIP in rice using an antibody that specifically recognizes 5-methylcytosine (5mC). The mCIP DNA was amplified using a random amplification procedure (Lippman et al., 2005). The amplified DNA product was tested for enrichment of methylated sequences by both dot- blot and quantitative PCR analyses. Dot-blot hybridizations using amplified mCIP and input DNA revealed a significant enrichment of the rice retrotransposon noaCRR1, which was previously shown to be significantly methylated (Takata et al., 2007). We identified two DNA fragments, mtDNA1 (3128 bp) and mtDNA2 (2983 bp), from the rice mitochondrial genome. These two fragments were not found in the sequenced rice nuclear genome and showed no methylation, based on analysis of restriction digestions using methylation-sensitive enzymes (Figure 1). Figure 1-a shows an agarose gel with equal quantities of rice genomic DNA digested with HpaII and MspI. Figure 1-b shows the gel was blotted to a membrane and hybridized with three different probes. The differences between the HpaII and MspI lanes hybridized with a noaCRR1 probe indicate that the sequences associated with this retrotransposon are heavily methylated. The two mtDNA segments showed no difference between HpaII and MspI digestion.
  
  

Figure 2. Mapping of DNA methylation along the 12 rice chromosomes.

• We then conducted Illumina sequencing of the amplified mCIP DNA sample and generated a total of 17 310 521 mCIP-seq reads, each with a length of 39 bp. We mapped approximately 43% of the reads to distinct locations in the rice reference genome (Figure S3). The mapped reads were plotted using 100-kb sliding windows along the entire chromosomes, which revealed a clear trend of increased DNA methylation in the pericentromeric regions in all 12 chromosomes (Figure 2).
• This distribution pattern directly resembles the density distribution of repetitive DNA, but is in reverse with that of transcribed genes (Figure 2) (Matsumoto et al., 2005). We conducted an immunofluores- cence assay on meiotic pachytene chromosomes prepared from the sequenced rice variety Nipponbare using the anti-5mC antibody. The pericentromeric region of every rice chromosome showed much brighter fluorescence signals than the distal regions (Figure 3). This immunofluorescence pattern is similar to the distribution pattern of mCIP-seq reads along the chromosomes.
  
  

Figure 3. Immunodetection of 5-methylcytosine (5mC) on meiotic pachytene chromosomes of rice.

Labs working on this Project

• Department of Horticulture, University of Wisconsin-Madison, Madison, WI 53706, USA, and
• U.S. Department of Energy Joint Genome Institute, Walnut Creek, CA 94598, USA

Corresponding Author

• Jiming Jiang (jjiang1@wisc.edu)