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RNA Journal Club 4/30/09

Posted in RNA Journal Club, RNAJC w/ review by YPAA on May 3, 2009

A Role for RNAi in the Selective Correction of DNA Methylation Defects

Felipe Karam Teixeira, Fabiana Heredia, Alexis Sarazin, François Roudier, Martine Boccara, Constance Ciaudo, Corinne Cruaud, Julie Poulain, Maria Berdasco, Mario F. Fraga, Olivier Voinnet, Patrick Wincker, Manel Esteller, Vincent Colot

Science 323 (5921): 1600-1604, March 2009.
doi: 10.1126/science.1165313

This week’s summary and expert analysis by Michael Nodine:

DNA methylation of transposable elements occurs through both RNAi-dependent and RNAi-independent mechanisms in plants. Methylation of transposable elements leads to their silencing and maintains genomic stability. Mutations in methylation components, such as the maintenance methyltransferase MET1 and the chromatin remodeler DDM1, lead to a loss of >70% of genomic methylation. The progeny from met1 x wild-type (WT) and ddm1 x WT crosses have reduced methylation despite these mutations being recessive. Furthermore, when these heterozygous plants are selfed and the MET1 and DDM1 loci are restored to the homozygous WT condition, several loci remain hypomethylated. Based on these findings, it has been proposed that once methylation is severely compromised it cannot be restored and thus is permanently lost. However, comparisons between different Arabidopsis accessions revealed that the methylation patterns of repetitive elements were similar across generations. This suggests that a mechanism exists to prevent permanent loss of DNA methylation. That is, there must be a way to specifically and robustly reestablish methylation.

In this study, Teixeira et al. set out to identify the mechanism that underlies this methylation reestablishment. First, they crossed the methylation defective mutant ddm1 with WT, recovered DDM1 F2 plants and selfed these plants for several generations. They then examined the methylation levels of several loci in the heterochromatic knob region of chromosome 4, and found that methylation was restored for ~50% of the repetitive loci examined (remethylatable sequences (R)), but not for the other ~50% (non-remethylatable sequences (NR)). The patterns of NR and R sequences were consistent between different independent lines. Remethylation did not occur in the F1 generation, but was progressive from the F2 generation onwards and led to silencing of transposable elements. In contrasts to previous models, these findings indicate that a robust and targeted remethylation process takes place.

The authors went on to demonstrate that cytosine remethylation occurred in all three sequence contexts (CG, CHG and CHH where H it A, T or C). They also found that NR sequences had stronger dependence on DDM1 for CHH methylation than did R sequences. Since RNAi components have important roles in CHH methylation, this observation led to the hypothesis that RNAi may be involved in remethylation of R sequences. To test this, they examined small RNA datasets and found that R sequences had a strong association with small RNAs especially 24-nt heterochromatic siRNAs (hc-siRNAs), which are involved in RNA-directed DNA methylation. Moreover, when they combined mutations in the RNAi machinery with ddm1 they observed an enhanced loss of methylation at both R and NR sequences suggesting that RNAi plays a role in the methylation at both types of loci. However, mutations in RNAi components (when not combined with ddm1) resulted in decreased methylation at R, but not NR, sequences. To demonstrate that RNAi plays a direct role in remethylation, they examined whether R sequences were remethylated when RNAi was compromised in the initial generation and found that only sporadic and inconsistent remethylation occurred in several independent progeny lines. Together, these results indicate that RNAi is involved in robust remethylation at specific loci.

Based on these findings, the authors propose that there are three types of methylated loci: those that 1) depend solely on maintenance methylation machinery (NR loci), 2) depend on both maintenance methylation and RNAi components (R loci), and 3) depend solely on RNAi components (unaffected in ddm1 mutants). Furthermore, they speculate that this mechanism may allow for the generation of epialleles with differences in transgenerational stability.

Although, the authors performed a thorough analysis of the 500 kb heterchomatic knob region of chromosome 4, their conclusions could have strengthened if they would have performed a more genome-wide bisulfite sequencing approach to test whether remethylation occurs on a large-scale in both euchromatic and heterochromatic regions. Furthermore, it would have been informative if they would have reported whether small RNA levels increase with each generation. This may have yielded insight into the mechanism behind the progressive nature of remethylation. Several outstanding questions remain. What features distinguish remethylatable vs. non-remethylatable sequences? Why does it take so many generations for remethylation to be re-established? Is there a benefit for the observed slow re-establishment vs. a more rapid one?

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