Strand-biased DNA methylation associated with centromeric regions in Arabidopsis

Abstract

The Arabidopsis genome project assembled 15 megabases of heterochromatic sequence, facilitating investigations of heterochromatin assembly, maintenance, and structure. In many species, large quantities of methylcytosine decorate heterochromatin; these modifications are typically maintained by methyltransferases that recognize newly replicated hemimethylated DNA. We assessed the extent and patterns of Arabidopsis heterochromatin methylation by amplifying and sequencing genomic DNA treated with bisulfite, which converts cytosine, but not methylcytosine, to uracil. This survey revealed unexpected asymmetries in methylation patterns, with one helix strand often exhibiting higher levels of methylation. We confirmed these observations both by immunoprecipitating methylated DNA strands and by restriction enzyme digestion of amplified, bisulfite-treated DNA. We also developed a primer-extension assay that can monitor the methylation status of an entire chromosome, demonstrating that strand-specific methylation occurs predominantly in the centromeric regions. Conventional models for methylation maintenance do not explain these unusual patterns; instead, new models that allow for strand specificity are required. The abundance of Arabidopsis strand-specific modifications points to their importance, perhaps as epigenetic signals that mark the heterochromatic regions that confer centromere activity.

Fig. 1.

Strand-specific methylation of centromeric DNA sequences. (A) Sequence chromatogram from the CEN5 BAC T3P1, nucleotides 3690–3721 (25). The unmodified sequence (middle) is compared with sequences generated by bisulfite treatment (top and bottom); unmethylated cytosines (arrows) and cytosines protected by methylation (purple shading) are indicated. (B) Independent sequences (upper and lower strands) of a 240-bp CEN2 fragment amplified from bisulfite-treated DNA (26). Black boxes indicate the position of cytosines in the original sequence.

Fig. 2.

Variation in methylation content. Average percent methylcytosine for the sequences reported in Tables 1 and 2. Sequences showing either significant (A) or insignificant (B) differences between upper and lower strands are indicated; error bars are SD; sequence numbers correspond to those of Tables 1 and 2.

Fig. 3.

Strand-biased methylation across chromosomes 2 and 4. Shown are scale drawings of chromosome 2 and a portion of chromosome 4, depicting rDNA (NOR2,4), the genetically defined centromeres (CEN2,4), pericentromeric regions including the chromosome 4 knob (yellow), a mitochondrial insertion in CEN2 (orange), and the 180-bp repeat array (red) (25). (A) Circles, regions with significantly different methylation levels between the two complementary strands. (B) Abundance of hemimethylated Sau3A sites along 255 BAC and P1 clones spanning chromosome 2 and 43 BAC clones surrounding CEN4; gaps correspond to unsequenced portions of the chromosome (25, 33). Relative signals from nick translation products after a Sau3A and MboI digests are plotted on the vertical axis.

Fig. 4.

Restriction digestion assay for strand-specific methylation. PCR products amplified from selected regions (Tables 1 and 2) of bisulfite-treated DNA are numbered, and the extent of digestion with the indicated enzyme was measured with NIH image software. Primers survey the same restriction site on the upper and lower strands; differences in primer location sometimes resulted in different product lengths.

Fig. 5.

Immunoprecipitation assay for strand-specific methylation. Genomic DNA was melted and immunoprecipitated with α-5-methylcytosine antibodies; strand-specific PCR-amplified selected regions (Table 1 and 2) from independent batches of DNA as indicated; amplification of input DNA verified primer quality. U, upper strand; L, lower strand; boxes indicate strands with at least a 5-fold difference in methylation levels. In B, three independent immunoprecipitations (IP1-IP3) were performed, demonstrating reproducibility within a given sample.

Fig. 6.

Models for generating strand-biased DNA methylation. Strand-specific biases in methylation could be generated by DNA methyltransferase binding at specific sites (A), interference with DNA methyltransferase activity by centromere binding proteins (Cenp) (B), or closely coordinating DNA methyltransferase activity with leading or lagging strand synthesis (C).