A Dominant Mutation in mediator of paramutation2, One of Three Second-Largest Subunits of a Plant-Specific RNA Polymerase, Disrupts Multiple siRNA Silencing Processes

Paramutation involves homologous sequence communication that leads to meiotically heritable transcriptional silencing. We demonstrate that mop2 (mediator of paramutation2), which alters paramutation at multiple loci, encodes a gene similar to Arabidopsis NRPD2/E2, the second-largest subunit of plant-specific RNA polymerases IV and V. In Arabidopsis, Pol-IV and Pol-V play major roles in RNA–mediated silencing and a single second-largest subunit is shared between Pol-IV and Pol-V. Maize encodes three second-largest subunit genes: all three genes potentially encode full length proteins with highly conserved polymerase domains, and each are expressed in multiple overlapping tissues. The isolation of a recessive paramutation mutation in mop2 from a forward genetic screen suggests limited or no functional redundancy of these three genes. Potential alternative Pol-IV/Pol-V–like complexes could provide maize with a greater diversification of RNA–mediated transcriptional silencing machinery relative to Arabidopsis. Mop2-1 disrupts paramutation at multiple loci when heterozygous, whereas previously silenced alleles are only up-regulated when Mop2-1 is homozygous. The dramatic reduction in b1 tandem repeat siRNAs, but no disruption of silencing in Mop2-1 heterozygotes, suggests the major role for tandem repeat siRNAs is not to maintain silencing. Instead, we hypothesize the tandem repeat siRNAs mediate the establishment of the heritable silent state—a process fully disrupted in Mop2-1 heterozygotes. The dominant Mop2-1 mutation, which has a single nucleotide change in a domain highly conserved among all polymerases (E. coli to eukaryotes), disrupts both siRNA biogenesis (Pol-IV–like) and potentially processes downstream (Pol-V–like). These results suggest either the wild-type protein is a subunit in both complexes or the dominant mutant protein disrupts both complexes. Dominant mutations in the same domain in E. coli RNA polymerase suggest a model for Mop2-1 dominance: complexes containing Mop2-1 subunits are non-functional and compete with wild-type complexes

Maize <em>Unstable factor for orange1</em> Is Required for Maintaining Silencing Associated with Paramutation at the <em>pericarp color1</em> and <em>booster1</em> Loci

<div><p>To understand the molecular mechanisms underlying paramutation, we examined the role of <em>Unstable factor for orange1</em> (<em>Ufo1</em>) in maintaining paramutation at the maize <em>pericarp color1</em> (<em>p1</em>) and <em>booster1</em> (<em>b1</em>) loci. Genetic tests revealed that the <em>Ufo1-1</em> mutation disrupted silencing associated with paramutation at both <em>p1</em> and <em>b1</em>. The level of up regulation achieved at <em>b1</em> was lower than that at <em>p1</em>, suggesting differences in the role <em>Ufo1-1</em> plays at these loci. We characterized the interaction of <em>Ufo1-1</em> with two silenced <em>p1</em> epialleles, <em>P1-rr</em>′ and <em>P1-pr^TP</em>, that were derived from a common <em>P1-rr</em> ancestor. Both alleles are phenotypically indistinguishable, but differ in their paramutagenic activity; <em>P1-rr′</em> is paramutagenic to <em>P1-rr</em>, while <em>P1-pr^TP</em> is non-paramutagenic. Analysis of cytosine methylation revealed striking differences within an enhancer fragment that is required for paramutation; <em>P1-rr</em>′ exhibited increased methylation at symmetric (CG and CHG) and asymmetric (CHH) sites, while <em>P1-pr^TP</em> was methylated only at symmetric sites. Both silenced alleles had higher levels of dimethylation of lysine 9 on histone 3 (H3K9me2), an epigenetic mark of silent chromatin, in the enhancer region. Both epialleles were reactivated in the <em>Ufo1-1</em> background; however, reactivation of <em>P1-rr′</em> was associated with dramatic loss of symmetric and asymmetric cytosine methylation in the enhancer, while methylation of up-regulated <em>P1-pr^TP</em> was not affected. Interestingly, <em>Ufo1-1</em>–mediated reactivation of both alleles was accompanied with loss of H3K9me2 mark from the enhancer region. Therefore, while earlier studies have shown correlation between H3K9me2 and DNA methylation, our study shows that these two epigenetic marks are uncoupled in the <em>Ufo1-1</em>–reactivated <em>p1</em> alleles. Furthermore, while CHH methylation at the enhancer region appears to be the major distinguishing mark between paramutagenic and non-paramutagenic <em>p1</em> alleles, H3K9me2 mark appears to be important for maintaining epigenetic silencing.</p> </div

<i>Ufo1-1</i>-induced methylation modifications of individual cytosine residues in the 443-bp fragment of the P1.2 enhancer of <i>P1-rr′</i> and <i>P1-pr^TP</i>.

<p><i>P1-rr′/p1-ww; Ufo1-1/ufo1</i> and <i>P1-pr^TP/p1-ww; Ufo1-1/ufo1</i> refer to plants that showed gain of pigmentation. Bisulfite sequencing was performed on genomic DNA extracted from pericarp tissue. Genomic DNA from two independent plants per genotype was used for bisulfite sequencing. For each genotype, the percent methylation is shown on the <i>y</i>-axis while the position of cytosine residues in CG, CHG, and CHH context is shown at the <i>x</i>-axis of the bottom graph.</p

Silencing of <i>P1-pr^TP</i>, an epiallele of <i>P1-rr</i>, is correlated with DNA methylation.

<p>A. Loss of pericarp and cob glume pigmentation phenotype of <i>P1-pr^TP</i>. The pericarp pigmentation is present only at the silk attachment point while the cob glumes are pink. B. <i>P1-pr^TP</i> is highly methylated as compared to <i>P1-rr</i>. Leaf genomic DNA of <i>P1-rr</i> and <i>P1-pr^TP</i> was digested with <i>Hpa</i>II and sequentially hybridized with <i>p1</i> probe fragments 15 and 6 (See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002980#pgen-1002980-g005" target="_blank">Figure 5C</a> for location of probes). Size and location of hybridizing bands in kilobase pair is indicated by arrows on the left for probe 15 and on the right for probe 6. C. Methylation map of <i>P1-rr ufo1</i> and <i>P1-pr^TP ufo1</i>. Gene structure is shown at the top and the rest of the figure description is same as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002980#pgen-1002980-g003" target="_blank">Figure 3B</a>.</p

Comparison of H3K9-dimethylation (H3K9me2) levels in <i>P1-rr′</i> and <i>P1-pr^TP</i> in the absence and presence of <i>Ufo1-1</i>.

<p>ChIP assay was performed using pericarp tissues. Chromatin complex was immunoprecipitated with antibodies against H3K9me2. Mouse IgG was used as a negative control (NoAb). Quantitative PCR was performed to quantify the DNA enrichments at the P1.2 kb distal enhancer. The data presented here is the mean ± SE from three biological replicates of three independent ChIP experiments.</p

Specific Tandem Repeats Are Sufficient for Paramutation-Induced Trans-Generational Silencing

<div><p>Paramutation is a well-studied epigenetic phenomenon in which <i>trans</i> communication between two different alleles leads to meiotically heritable transcriptional silencing of one of the alleles. Paramutation at the <i>b1</i> locus involves RNA-mediated transcriptional silencing and requires specific tandem repeats that generate siRNAs. This study addressed three important questions: 1) are the tandem repeats sufficient for paramutation, 2) do they need to be in an allelic position to mediate paramutation, and 3) is there an association between the ability to mediate paramutation and repeat DNA methylation levels? Paramutation was achieved using multiple transgenes containing the <i>b1</i> tandem repeats, including events with tandem repeats of only one half of the repeat unit (413 bp), demonstrating that these sequences are sufficient for paramutation and an allelic position is not required for the repeats to communicate. Furthermore, the transgenic tandem repeats increased the expression of a reporter gene in maize, demonstrating the repeats contain transcriptional regulatory sequences. Transgene-mediated paramutation required the <i>mediator of paramutation1</i> gene, which is necessary for endogenous paramutation, suggesting endogenous and transgene-mediated paramutation both require an RNA-mediated transcriptional silencing pathway. While all tested repeat transgenes produced small interfering RNAs (siRNAs), not all transgenes induced paramutation suggesting that, as with endogenous alleles, siRNA production is not sufficient for paramutation. The repeat transgene-induced silencing was less efficiently transmitted than silencing induced by the repeats of endogenous <i>b1</i> alleles, which is always 100% efficient. The variability in the strength of the repeat transgene-induced silencing enabled testing whether the extent of DNA methylation within the repeats correlated with differences in efficiency of paramutation. Transgene-induced paramutation does not require extensive DNA methylation within the transgene. However, increased DNA methylation within the endogenous <i>b1</i> repeats after transgene-induced paramutation was associated with stronger silencing of the endogenous allele.</p></div

Representative data showing <i>Ufo1-1</i>–induced reactivation of <i>P1-pr^TP</i>.

<p>A. Pericarp and cob glume pigmentation phenotypes of F₁ progeny obtained from a cross between <i>P1-pr^TP</i> and <i>p1-ww Ufo1-1</i> plants. B. Heritability of <i>Ufo1-1</i>-induced reactivation of <i>P1-pr^TP</i>. F₁ plants showing gain of pericarp pigmentation (red/variegated pericarps) were crossed with <i>p1-ww</i>[<i>4Co63</i>] and test-cross progenies were examined for pericarp and cob glume pigmentation. Expected segregation frequencies are based on assumption that increased pigmentation of <i>P1-pr^TP</i> allele is not heritable in the absence <i>Ufo1-1</i>.</p

<i>Ufo1-1</i> mediates reactivation of <i>P1-rr′</i>.

<p>A. Pericarp and cob glume phenotypes of F₁ progeny plants obtained from a cross between highly suppressed <i>P1-rr′ ufo1</i> and <i>p1-ww Ufo1-1</i>. B. Heritability of <i>Ufo1-1</i>-induced reactivation of <i>P1-rr′</i>. F₁ plants were crossed with <i>p1-ww</i>[<i>4Co63</i>] and progeny was scored for pericarp pigmentation. Expected segregation frequencies are based on the assumption that <i>P1-rr′</i> reverts back to silenced state after segregation of <i>Ufo1-1</i>.</p

<i>Ufo1-1</i> increases <i>B′</i> pigmentation.

<p>Effect of <i>Ufo1-1</i> was assayed in four consecutive backcrosses as shown in the crossing scheme in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002980#pgen.1002980.s003" target="_blank">Figure S3</a>. A. Photo panels from BC₂ generation showing <i>B′</i> plant pigmentation and corresponding <i>P1-wr</i> pericarp and cob glume phenotypes, which were used as phenotypic indicators of <i>Ufo1-1</i> presence. The <i>P1-wr</i> has colorless pericarp and red cob and <i>P1-pr^TP</i> has silk-scarred pericarp and pink cob glume in the <i>ufo1</i> genetic background. In the mutant <i>Ufo1-1/ufo1</i> background <i>P1-wr</i> and <i>P1-pr^TP</i> exhibit dramatic increase in pigmentation and have very dark red pericarp and cob glume. In the wild type <i>ufo1</i> background, the <i>B′</i> allele has light streaky plant pigmentation while in the <i>Ufo1-1/ufo1</i> background <i>B′</i> plants have increased pigmentation as evidenced by the presence of broad darkly pigmented sectors. This increase of <i>B′</i> pigmentation is significant because <i>B′</i> never displays any increase in pigmentation in wild type genetic backgrounds. B. Summary of the results from F₁ and three generations of introgression in the mutant <i>Ufo1-1/ufo1</i> background are shown.</p