Nucleosomes compact and regulate access to DNA in the nucleus, and are composed of approximately 147 bases of DNA wrapped around a histone octamer1, 2. Here we report a genome-wide nucleosome positioning analysis of Arabidopsis thaliana utilizing massively parallel sequencing of mononucleosomes. By combining this data with profiles of DNA methylation at single base resolution, we identified ten 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 suggest that nucleosome positioning strongly 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 suggesting 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 Pol II was also enriched on exons relative to introns, consistent with the hypothesis that nucleosome positioning regulates Pol II processivity. DNA methylation is enriched on exons, consistent with the targeting of DNA methylation to nucleosomes, and suggesting a role for DNA methylation in exon definition.

RNA-directed DNA methylation (RdDM) in Arabidopsis thaliana depends on the upstream synthesis of 24-nucleotide small interfering RNAs (siRNAs) by RNA POLYMERASE IV (Pol IV)1,2 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 DNA3-7. The SU(VAR)3-9 homologs SUVH2 and SUVH9 act in this downstream step but the mechanism of their action is unknown8,9. 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 proteins8. SUVH2 and SUVH9 both contain SET- and RING-ASSOCIATED (SRA) domains capable of binding methylated DNA8, 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 SUVH2 with a zinc finger to an unmethylated site is sufficient to recruit Pol V and establish DNA methylation and gene silencing. These results suggest 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.

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.bioinformatics | plant biology D NA methylation is an epigenetic mark that can often lead to the repression of gene expression (1, 2). It is enriched in heterochromatin and, when present at regulatory sites, usually acts as a repressor of expression, most notably in transposons (3). However, it is also found over coding regions, where it likely does not directly affect transcription and is associated with moderately expressed genes (2, 4-6). In plants, DNA methylation occurs in three different contexts: CG, CHG, and CHH (where H is any nucleotide but G). In Arabidopsis, each context is maintained by different enzymes: MET1 for CG sites, CMT3 for CHG sites and DRM2 for CHH sites. CG and CHG sites are symmetric across the two DNA strands, which is thought to be important for the maintenance of methylation at these sites following DNA replication. In contrast, CHH sites are not symmetric, and their methylation is mediated by RNA-directed DNA methylation pathways (RdDM), which use siRNAs to initiate de novo methylation (3). Cellular methylation states tend to persist during cell division, and recent studies in Arabidopsis have also shown that DNA methylation is faithfully inherited across generations (7,8). Nonetheless, we are only beginning to understand how different...

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. Globally, 130 to 150 million people have chronic hepatitis C virus (HCV) infection. Without treatment, chronic hepatitis may lead to liver cirrhosis and hepatocellular carcinoma and the possible need for a liver transplantation (1-3). Hepatitis C virus has high genetic diversity and is classified into seven genotypes and at least 67 subtypes among the genotypes (4, 5). At the nucleotide level for the open reading frame, there is a 31 to 33% difference between genotypes and 20 to 25% difference between subtypes (6).HCV genotype 1 is the most prevalent genotype and is widely distributed across the globe. Genotype 1 accounts for the majority of the HCV cases in North America, northern and western Europe, South America, Asia, and Australia (7,8). Similarly, genotypes 2 and 3 have broad geographical distribution. In contrast, genotypes 4, 5, and 6 are common only in specific regions. Genotype 4 and genotype 5 are endemic to Egypt and South Africa, respectively. Genotype 6 has the highest prevalence in Southeast Asia and southern China. Although genotypes 5 and 6 are not widespread, there are an estimated 9.8 million and 1.4 million people infected with genotypes 5 and 6, respectively, worldwide (9-13). Better treatment options are needed for genotype 5-and 6-infected patients.Due to the high prevalence and broad geographical distribution of genotype 1, early HCV research and development efforts were focused on this genotype. With the recent approval of HCV direct-acting antivirals (DAAs) active against multiple genotypes, such as Sovaldi (...

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