Assessing the impact of transgenerational epigenetic variation on complex traits.

Loss or gain of DNA methylation can affect gene expression and is sometimes transmitted across generations. Such epigenetic alterations are thus a possible source of heritable phenotypic variation in the absence of DNA sequence change. However, attempts to assess the prevalence of stable epigenetic variation in natural and experimental populations and to quantify its impact on complex traits have been hampered by the confounding effects of DNA sequence polymorphisms. To overcome this problem as much as possible, two parents with little DNA sequence differences, but contrasting DNA methylation profiles, were used to derive a panel of epigenetic Recombinant Inbred Lines (epiRILs) in the reference plant Arabidopsis thaliana. The epiRILs showed variation and high heritability for flowering time and plant height ( approximately 30%), as well as stable inheritance of multiple parental DNA methylation variants (epialleles) over at least eight generations. These findings provide a first rationale to identify epiallelic variants that contribute to heritable variation in complex traits using linkage or association studies. More generally, the demonstration that numerous epialleles across the genome can be stable over many generations in the absence of selection or extensive DNA sequence variation highlights the need to integrate epigenetic information into population genetics studies.

Epigenetic natural variation in Arabidopsis thaliana.

Cytosine methylation of repetitive sequences is widespread in plant genomes, occurring in both symmetric (CpG and CpNpG) as well as asymmetric sequence contexts. We used the methylation-dependent restriction enzyme McrBC to profile methylated DNA using tiling microarrays of Arabidopsis Chromosome 4 in two distinct ecotypes, Columbia and Landsberg erecta. We also used comparative genome hybridization to profile copy number polymorphisms. Repeated sequences and transposable elements (TEs), especially long terminal repeat retrotransposons, are densely methylated, but one third of genes also have low but detectable methylation in their transcribed regions. While TEs are almost always methylated, genic methylation is highly polymorphic, with half of all methylated genes being methylated in only one of the two ecotypes. A survey of loci in 96 Arabidopsis accessions revealed a similar degree of methylation polymorphism. Within-gene methylation is heritable, but is lost at a high frequency in segregating F(2) families. Promoter methylation is rare, and gene expression is not generally affected by differences in DNA methylation. Small interfering RNA are preferentially associated with methylated TEs, but not with methylated genes, indicating that most genic methylation is not guided by small interfering RNA. This may account for the instability of gene methylation, if occasional failure of maintenance methylation cannot be restored by other means.

Analysis of Small RNA Populations Using Hybridization to DNA Tiling Arrays.

Epigenetic response to stress in plants involves changes in DNA methylation, histone modifications, and expression of small noncoding RNAs (sRNA). Here we present the method of analysis of differential expression of sRNA populations using DNA tiling arrays. sRNA extracted from Arabidopsis thaliana plants exposed to pathogen elicitor or control plants were reverse-transcribed into cDNAs, and subsequently hybridized after labeling to a custom-made DNA tiling array covering Arabidopsis chromosome 4. We first designed a control experiment with eight cDNA clones corresponding to sequences located on chromosome 4 and obtained robust and specific hybridization signals. Furthermore, hybridization signals along chromosome 4 were in good agreement with sRNA abundance as previously determined by massive parallel sequence signature (MPSS) in the case of untreated plants, but differed substantially after stress treatment. These results demonstrate the utility of hybridization to DNA tiling arrays to detect major changes in sRNA abundance.

Analysis of small RNA populations using hybridization to DNA tiling arrays.

Small RNA (sRNA) populations extracted from Arabidopsis plants submitted or not to biotic stress, were reverse-transcribed into cDNAs, and these were subsequently hybridized after labelling to a custom-made DNA tiling array covering Arabidopsis chromosome 4. We first designed a control experiment with eight cDNA clones corresponding to sequences located on chromosome 4 and obtained robust and specific hybridization signals. Furthermore, hybridization signals along chromosome 4 were in good agreement with sRNA abundance as previously determined by Massive Parallel Sequence Signature (MPSS) in the case of untreated plants, but differed substantially after stress treatment. These results demonstrate the utility of hybridization to DNA tiling arrays to detect major changes in small RNA populations.

Reinforcement of silencing at transposons and highly repeated sequences requires the concerted action of two distinct RNA polymerases IV in Arabidopsis.

Recent genetic and biochemical studies have revealed the existence in plants of a fourth RNA polymerase, RNAPIV, which mediates siRNA accumulation and DNA methylation-dependent silencing of endogenous repeated sequences. Here, we show that Arabidopsis expresses, in fact, two evolutionarily related forms of RNAPIV, hereafter referred to as RNAPIVa and RNAPIVb. These two forms contain the same second-largest subunit (NRPD2), but differ at least by their largest subunit, termed NRPD1a and NRPD1b. Unlike NRPD1a, NRPD1b possesses a reiterated CTD, a feature that also characterizes the largest subunit of RNAPII. Our data indicate that RNAPIVb is the most abundant form of RNAPIV in Arabidopsis. Selective disruption of either form of RNAPIV indicates that RNAPIVa-dependent siRNA accumulation is not sufficient per se to drive robust silencing at endogenous loci and that high levels of DNA methylation and silencing depend on siRNA that are accumulated through a pathway involving the concerted action of both RNAPIV forms. Taken together, our results imply the existence of a novel two-step mechanism in siRNA synthesis at highly methylated loci, with RNAPIVb being an essential component of a self-reinforcing loop coupling de novo DNA methylation to siRNA production.