Comprehensive Analysis of Silencing Mutants Reveals Complex Regulation of the Arabidopsis Methylome

Abstract

In our study, we surveyed the methylomes of 86 gene-silencing mutants by whole-genome bisulfite sequencing (BS-seq) and revealed interactions between DNA methylation pathways as well as new regulators of DNA methylation. One of the observations that we made was that mutation in histone deacetylase 6 (HDA6) resulted in loss of methylation at a small subset of sites in the genome. It has recently come to our attention that our hda6 mutant line was contaminated and thus our hda6 mutant data set did not represent data for a null mutation of HDA6. We therefore repeated the BS-seq experiments using two different hda6 mutant alleles, hda6-6 and hda6-7 (seeds generously provided by Craig Pikaard), as well as an additional wild-type control. In contrast to the limited subset of sequences that we had initially reported to be affected, we found global reduction of methylation primarily at cytosines in CHG and CHH (H = A, T, or C) sequence contexts across the genome. We would therefore now conclude, as reflected in a corrected panel of the original Figure 4A below, that HDA6 plays a stronger role in regulating methylation than what we had reported and appears to be an important factor shaping the Arabidopsis methylome, consistent with a recent study (Blevins et al., 2014). We have deposited the new sequencing data to GEO (GEO: GSE39901) and have updated our genome browser (http:// genomes.mcdb.ucla.edu/AthBSseq/). We regret this error and apologize for any inconvenience this may have caused.

Mobile small RNAs and their role in regulating cytosine methylation of DNA

“…It seems likely that this is due to the substantial overlap between CHH E class loci with D and E type loci in CG and CHG contexts that we have previously demonstrated. 46 We hypothesize that loss of methylation in the CHG and CG contexts caused by disruption to the RNA-independent maintenance pathways has a knock-on effect on CHH methylation for these loci, consistent with the findings of Stroud et al 54 Class A & B loci, which represent direct and indirect methylation by the mobile signal behave identically for the examined mutants in the Stroud et al set. For CHG context methylation, Figure 2.…”

“…To investigate further the mechanisms by which the classes of sRNA-methylation interaction are differentiated we compare the methylation profiles of the class A & B loci in 86 mutants of RNA silencing components using the data of Stroud et al 54 to those observed at the E & F class loci and relate these profiles to the model 19 of RdDM initiation, establishment, RNA-dependent maintenance and RNA-independent maintenance, and to the DDM1 chromatin remodelling pathway. 25 Examining the behavior of the identified loci in the Stroud et al knockout mutant dataset (Fig.…”

Dicer-independent RNA-directed DNA methylation in Arabidopsis

“…Functional redundancies among the different DCL proteins have been reported or suggested in many studies [27][28][29][30]. Despite the role of DCL3 in generating 24-nt siRNAs, the dcl3 mutant does not exhibit a severe decrease in DNA methylation levels at RdDM loci [31]. It has been thought that other DCL proteins could also process RDR products, compensating for DCL3 when it is absent, and that the 21/22-nt siRNAs, though less efficient, could also be fed into Argonaute (AGO) proteins [20].…”

Pollen-Specific Activation of Arabidopsis Retrogenes Is Associated with Global Transcriptional Reprogramming

“…Transcribed protein-coding genes are associated with permissive chromatin marks. In contrast, transcriptionally repressed genes and repetitive elements are typically labeled by histone H3 Lysine 27 trimethylation (H3K27me3), histone H3 Lysine 9 dimethylation (H3K9me2), and/or high-density DNA methylation in all cytosine sequence contexts in plants Stroud et al, 2013). While H3K27me3 ensures tissue-specific developmental transcription (Lafos et al, 2011), the role of H3K9me2 and promoter DNA methylation is to minimize the activities of repetitive elements, which frequently include retrotransposons (Mosher et al, 2009;Slotkin et al, 2009;Ibarra et al, 2012).…”