Large-scale survey of cytosine methylation of retrotransposons and the impact of readout transcription from long terminal repeats on expression of adjacent rice genes

Abstract

Transposable elements (TEs) represent approximately 45% of the human genome and 50-90% of some grass genomes. While most elements contain inactivating mutations, others are reversibly inactivated (silenced) by epigenetic mechanisms, including cytosine methylation. Previous studies have shown that retrotransposons can influence the expression of adjacent host genes. In this study, the methylation patterns of TEs and their flanking sequences in different tissues were undertaken using a novel technique called transposon methylation display (TMD). TMD was successfully applied on a highly copied (approximately 1000 copies), newly amplified LTR retrotransposon family in rice called Dasheng. We determined that the methylation status of a subset of LTRs varies in leaves vs. roots. In addition, we determined that tissue-specific LTR methylation correlated with tissue-specific expression of the flanking rice gene. Genes showing tissue-specific expression were in opposite orientation relative to the LTR. Antisense transcripts were detected in the tissue where the sense transcripts from that gene were not detected. Comparative analysis of Dasheng LTR methylation in the two subspecies, japonica vs. indica revealed LTR-mediated differences in subspecies gene expression. Subspecies-specific expression was due either to polymorphic Dasheng insertion sites between the two subspecies or to subspecies-specific methylation of LTRs at the same locus accounted for observed differences in the expression of adjacent genes.

Figure 1.—

Transposon methylation display (TMD) in Oryza species. (A) LTR and flanking sequences are shown together with the cleavage sites of HpaII (H) and MspI (M) and the position of primers used in TMD reactions (see materials and methods). (B) Autoradiograph of transposon methylation display using P and the adapter primer +(AT).

Figure 2.—

Methylation of CCGG sites in the LTR. (A) LTR and flanking sequences are shown together with the cleavage sites of the restriction enzymes (H, HpaII, M, MspI) in the LTR and the position of primers used in PCR analysis (see materials and methods). (B) PCR analysis (using primers P2 and P1) of LTR methylation in leaves using undigested genomic DNA or DNA digested with either HpaII or MspI as template. Dash8 pattern was used as a control for the digestion quality because the same DNA templates (Oryza sativa, see supplemental Table 1 at http://www.genetics.org/supplemental/) were used for Dash5, -6, -7, and -8. For a negative control, H₂O was used as a template in PCR reaction using primers P1 and P2.

Figure 3.—

Transposon methylation display reveals tissue-specific methylation in DNA flanking LTRs in leaves (L) and roots (R). Autoradiograph of transposon methylation display using P and the adapter primer +(TT) (see Figure 1A). The arrows indicate sites that show tissue-specific methylation. A standard AFLP [primers reaction (using MseI and EcoRI restriction enzymes] was used for DNA quality control in leaves and roots (Vos et al. 1995).

Figure 4.—

LTR methylation at loci showed tissue-specific chimeric transcripts. (A) LTR and flanking sequences are shown together with the cleavage sites of the restriction enzymes (H, HpaII; M, MspI) in the LTR and the position of primers used in PCR analysis (see materials and methods). (B) PCR analysis (using primers P2 and P1) of LTR methylation in leaves (L) and roots (R) using undigested genomic DNA or DNA digested with either HpaII or MspI as template. The presence of a band in both the HpaII and MspI lanes indicates that the CCGG sites in the LTR sequence are methylated (because the fragment was not digested, see materials and methods). (C) RT–PCR analysis of chimeric transcripts in leaves and roots of japonica using a primer from the LTR (P3) and a second primer derived from the flanking sequence (P1). LR* is a negative control (RNA templates treated with RNase) and actin was used as a control for RNA quality.

Figure 5.—

Expression analysis of putative genes adjacent to Dasheng LTRs in leaves (L) and roots (R) of japonica and indica rice tissue. The relative position of Dasheng and flanking genes (including exons and introns) in japonica and/or indica is shown schematically. The LTR methylation status and the readout transcription (in the tissue where the LTR is unmethylated) are indicated (see key at the bottom). (Right) RT–PCR products (gene sense transcripts) after using gene specific primers are shown. LR* and actin are controls (see Figure 4 legend).

Figure 6.—

Detection of antisense transcripts. RT–PCR analysis of antisense transcripts in leaves (L) and roots (R) of japonica and indica using gene specific primers. LR* is a negative control (RNA templates treated with RNase).