SRA- and SET-domain-containing proteins link RNA polymerase V occupancy to DNA methylation.

RNA-directed DNA methylation in Arabidopsis thaliana depends on the upstream synthesis of 24-nucleotide small interfering RNAs (siRNAs) by RNA POLYMERASE IV (Pol IV) and downstream synthesis of non-coding transcripts by Pol V. Pol V transcripts are thought to interact with siRNAs which then recruit DOMAINS REARRANGED METHYLTRANSFERASE 2 (DRM2) to methylate DNA. The SU(VAR)3-9 homologues SUVH2 and SUVH9 act in this downstream step but the mechanism of their action is unknown. Here we show that genome-wide Pol V association with chromatin redundantly requires SUVH2 and SUVH9. Although SUVH2 and SUVH9 resemble histone methyltransferases, a crystal structure reveals that SUVH9 lacks a peptide-substrate binding cleft and lacks a properly formed S-adenosyl methionine (SAM)-binding pocket necessary for normal catalysis, consistent with a lack of methyltransferase activity for these proteins. SUVH2 and SUVH9 both contain SRA (SET- and RING-ASSOCIATED) domains capable of binding methylated DNA, suggesting that they function to recruit Pol V through DNA methylation. Consistent with this model, mutation of DNA METHYLTRANSFERASE 1 (MET1) causes loss of DNA methylation, a nearly complete loss of Pol V at its normal locations, and redistribution of Pol V to sites that become hypermethylated. Furthermore, tethering SUVH9 [corrected] with a zinc finger to an unmethylated site is sufficient to recruit Pol V and establish DNA methylation and gene silencing. These results indicate that Pol V is recruited to DNA methylation through the methyl-DNA binding SUVH2 and SUVH9 proteins, and our mechanistic findings suggest a means for selectively targeting regions of plant genomes for epigenetic silencing.

Sequencing Analysis of NS3/4A, NS5A, and NS5B Genes from Patients Infected with Hepatitis C Virus Genotypes 5 and 6.

Direct-acting antivirals (DAAs) with activity against multiple genotypes of the hepatitis C virus (HCV) were recently developed and approved for standard-of-care treatment. However, sequencing assays to support HCV genotype 5 and 6 analysis are not widely available. Here, we describe the development of a sequencing assay for the NS3/4A, NS5A, and NS5B genes from HCV genotype 5 and 6 patient isolates. Genotype- and subtype-specific primers were designed to target NS3/4A, NS5A, and NS5B for cDNA synthesis and nested PCR amplification. Amplification was successfully performed for a panel of 32 plasma samples from HCV-infected genotype 5 and 6 patients with sequencing data obtained for all attempted samples. LiPA 2.0 (Versant HCV genotype 2.0) is a reverse hybridization line probe assay that is commonly used for genotyping HCV-infected patients enrolled in clinical studies. Using NS3/4A, NS5A, and NS5B consensus sequences, HCV subtypes were determined that were not available for the initial LiPA 2.0 result for genotype 6 samples. Samples amplified here included the following HCV subtypes: 5a, 6a, 6e, 6f, 6j, 6i, 6l, 6n, 6o, and 6p. The sequencing data generated allowed for the determination of the presence of variants at amino acid positions previously characterized as associated with resistance to DAAs. The simple and robust sequencing assay for genotypes 5 and 6 presented here may lead to a better understanding of HCV genetic diversity and prevalence of resistance-associated variants.

Relationship between nucleosome positioning and DNA methylation.

Nucleosomes compact and regulate access to DNA in the nucleus, and are composed of approximately 147 bases of DNA wrapped around a histone octamer. Here we report a genome-wide nucleosome positioning analysis of Arabidopsis thaliana using massively parallel sequencing of mononucleosomes. By combining this data with profiles of DNA methylation at single base resolution, we identified 10-base periodicities in the DNA methylation status of nucleosome-bound DNA and found that nucleosomal DNA was more highly methylated than flanking DNA. These results indicate that nucleosome positioning influences DNA methylation patterning throughout the genome and that DNA methyltransferases preferentially target nucleosome-bound DNA. We also observed similar trends in human nucleosomal DNA, indicating that the relationships between nucleosomes and DNA methyltransferases are conserved. Finally, as has been observed in animals, nucleosomes were highly enriched on exons, and preferentially positioned at intron-exon and exon-intron boundaries. RNA polymerase II (Pol II) was also enriched on exons relative to introns, consistent with the hypothesis that nucleosome positioning regulates Pol II processivity. DNA methylation is also enriched on exons, consistent with the targeting of DNA methylation to nucleosomes, and suggesting a role for DNA methylation in exon definition.

Transcriptome and methylome interactions in rice hybrids.

DNA methylation is a heritable epigenetic mark that controls gene expression, is responsive to environmental stresses, and, in plants, may also play a role in heterosis. To determine the degree to which DNA methylation is inherited in rice, and how it both influences and is affected by transcription, we performed genome-wide measurements of these patterns through an integrative analysis of bisulfite-sequencing, RNA-sequencing, and siRNA-sequencing data in two inbred parents of the Nipponbare (NPB) and indica (93-11) varieties of rice and their hybrid offspring. We show that SNPs occur at a rate of about 1/253 bp between the two parents and that these are faithfully transmitted into the hybrids. We use the presence of these SNPs to reconstruct the two chromosomes in the hybrids according to their parental origin. We found that, unlike genetic inheritance, epigenetic heritability is quite variable. Cytosines were found to be differentially methylated (epimutated) at a rate of 7.48% (1/15 cytosines) between the NPB and 93-11 parental strains. We also observed that 0.79% of cytosines were epimutated between the parent and corresponding hybrid chromosome. We found that these epimutations are often clustered on the chromosomes, with clusters representing 20% of all epimutations between parental ecotypes, and 2-5% in F1 plants. Epimutation clusters are also strongly associated with regions where the production of siRNA differs between parents. Finally, we identified genes with both allele-specific expression patterns that were strongly inherited as well as those differentially expressed between hybrids and the corresponding parental chromosome. We conclude that much of the misinheritance of expression levels is likely caused by epimutations and trans effects.