RNA polymerase V targets transcriptional silencing components to promoters of protein‐coding genes

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/96338/1/tpj12034-sup-0010-TableS1.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/96338/2/tpj12034-sup-0006-FigureS4.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/96338/3/tpj12034-sup-0007-FigureS5.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/96338/4/tpj12034-sup-0003-FigureS1.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/96338/5/tpj12034-sup-0008-FigureS6.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/96338/6/tpj12034-sup-0005-FigureS3.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/96338/7/tpj12034-sup-0004-FigureS2.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/96338/8/tpj12034-sup-0009-FigureS7.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/96338/9/tpj12034.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/96338/10/tpj12034-sup-0002-MethodsS1.pd

RNA ‐directed DNA methylation requires stepwise binding of silencing factors to long non‐coding RNA

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/108033/1/tpj12563.pd

Long-range control of gene expression via RNA-directed DNA methylation

<div><p>RNA-mediated transcriptional silencing, in plants known as RNA-directed DNA methylation (RdDM), is a conserved process where small interfering RNA (siRNA) and long non-coding RNA (lncRNA) help establish repressive chromatin modifications. This process represses transposons and affects the expression of protein-coding genes. We found that in <i>Arabidopsis thaliana</i> AGO4 binding sites are often located distant from genes differentially expressed in <i>ago4</i>. Using Hi-C to compare interactions between genotypes, we show that RdDM-targeted loci have the potential to engage in chromosomal interactions, but these interactions are inhibited in wild-type conditions. In mutants defective in RdDM, the frequency of chromosomal interactions at RdDM targets is increased. This includes increased frequency of interactions between Pol V methylated sites and distal genes that are repressed by RdDM. We propose a model, where RdDM prevents the formation of chromosomal interactions between genes and their distant regulatory elements.</p></div

Unorthodox mRNA start site to extend the highly structured leader of retrotransposon Tto1 mRNA increases transposition rate

Retroelement RNAs serve as templates for both translation and reverse transcription into extrachromosomal DNA. DNA copies may be inserted into the host genome to multiply element sequences. This transpositional activity of retroelements is usually restricted to specific conditions, particularly to conditions that impose stress on the host organism. In this work, we examined how the mRNA initiation point, and features of primary and secondary structure, of tobacco retrotransposon Tto1 RNA influence its transpositional activity. We found that the most abundant Tto1 RNA is not a substrate for reverse transcription. It is poorly translated, and its 5′-end does not contain a region of redundancy with the most prominent 3′-end. In contrast, expression of an mRNA with the 5′-end extended by 28 nucleotides allows translation and gives rise to transposition events in the heterologous host, Arabidopsis thaliana. In addition, the presence of extended hairpins and of two short open reading frames in the 5′-leader sequence of Tto1 mRNA suggests that translation does not involve ribosome scanning from the mRNA 5′-end to the translation initiation site

Gene activity corresponds to chromosomal interactions with transcription factor binding sites.

<p>(A) Inactive genes are less likely to engage in chromosomal interactions. The number of chromosomal interactions is plotted as a percentage of total interactions on genes. Inactive and active genes are the 5% of genes with the lowest or highest RNA-seq signals in Col-0 with promoters that are mappable in Hi-C (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006749#sec010" target="_blank">Methods</a>). Expected value from an even distribution of interactions on genes is indicated by a dashed line. Color coding is the same as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006749#pgen.1006749.g001" target="_blank">Fig 1B</a>. Significance score calculated by a t-test of replicates, p≤0.01. (B) Promoter chromosomal interactions correspond to gene expression. Gene expression (calculated from RNA-seq) is plotted for genes with promoters at the lowest 5%, 5% centered at the middle, and the highest 5% for interaction signal in the combined dataset in Col-0. Horizontal and vertical bars indicate median and +/- 1 quartile respectively. Dashed line indicates average gene expression value for all genes. RPKM—reads per kilobase per million. *** p<0.001 Wilcoxon rank-sum test. (C) Genes connected to transcription factors have higher expression levels. RNA-seq based expression counts (RPKM) for genes connected to each transcription factor as detected by DAP-seq compared to the median expression of genes without interactions. The median values derived from each DAP-seq experiment are plotted. Dashed line indicates median expression of total genes. (D) Genes with reduced expression in <i>svp</i> interact with SVP binding sites. ChIP-seq signal of SVP (SVP-GFP / Col -0 RPM) at 250bp regions which loop to genes decreased or increased in <i>svp</i> mutant. Dashed line indicates no ChIP-seq enrichment. ** p<0.01 Wilcoxon rank-sum test.</p

GMI1, a structural-maintenance-of-chromosomes-hinge domain-containing protein, is involved in somatic homologous recombination in Arabidopsis

DNA double-strand breaks (DSBs) pose one of the most severe threats to genome integrity, potentially leading to cell death. After detection of a DSB, the DNA damage and repair response is initiated and the DSB is repaired by non-homologous end joining and/or homologous recombination. Many components of these processes are still unknown in Arabidopsis thaliana. In this work, we characterized γ-irradiation and mitomycin C induced 1 (GMI1), a member of the SMC-hinge domain-containing protein family. RT-PCR analysis and promoter-GUS fusion studies showed that γ-irradiation, the radio-mimetic drug bleocin, and the DNA cross-linking agent mitomycin C strongly enhance GMI1 expression particularly in meristematic tissues. The induction of GMI1 by γ-irradiation depends on the signalling kinase Ataxia telangiectasia-mutated (ATM) but not on ATM and Rad3-related (ATR). Epistasis analysis of single and double mutants demonstrated that ATM acts upstream of GMI1 while the atr gmi1-2 double mutant was more sensitive than the respective single mutants. Comet assay revealed a reduced rate of DNA double-strand break repair in gmi1 mutants during the early recovery phase after exposure to bleocin. Moreover, the rate of homologous recombination of a reporter construct was strongly reduced in gmi1 mutant plants upon exposure to bleocin or mitomycin C. GMI1 is the first member of its protein family known to be involved in DNA repair

Repression of chromosomal interactions with RdDM targets corresponds to gene repression.

<p>(AB) Genes repressed by AGO4 are inhibited from interacting with RdDM targets. Surface plots of mean interaction signal in Col-0 (A) and <i>ago4</i> (B) for interactions between 1) regions surrounding TSS’s of genes with expression increased in <i>ago4</i> (y-axis) and 2) regions surrounding <i>nrpe1</i> DMRs (x-axis). Height corresponds to average interaction scores (z-axis—see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006749#sec010" target="_blank">Methods</a>). (C) Numbers of genes, with increased expression in <i>ago4</i>, which form chromosomal interactions to <i>nrpe1</i> DMRs. Interactions are plotted in <i>nrpe1</i> or <i>ago4</i> as a ratio to Col-0 for genes with increased expression in <i>ago4</i> and for random genes. Color coding is the same as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006749#pgen.1006749.g001" target="_blank">Fig 1B</a>. (D) Expression changes are greater in <i>ago4</i> than in <i>nrpe1</i>. Venn diagram of differential gene expression calls in <i>ago4</i> (blue) and <i>nrpe1</i> (green). (E) Increased interaction with <i>nrpe1</i> DMRs corresponds to increased gene expression. The positive log2 fold change in gene expression (RNA-seq <i>ago4</i> / Col-0) is plotted for genes which do not show increased interactions to <i>nrpe1</i> DMRs in the <i>ago4</i> mutant and for genes which do show increased interactions to <i>nrpe1</i> DMRs in the <i>ago4</i> mutant. The combined interaction dataset was used. RPKM—reads per kilobase per million. * p<0.05 Wilcoxon rank-sum test. (F) Increased interactions with <i>nrpe1</i> DMRs corresponds to decreased gene expression. The negative log2 fold change in gene expression (RNA-seq <i>ago4</i> / Col-0) is plotted for genes which do not show increased interaction to <i>nrpe1</i> DMRs in the <i>ago4</i> mutant and for genes which do show increased interactions to <i>nrpe1</i> DMRs in the <i>ago4</i> mutant. The combined interaction dataset was used. (G) AGO4 may bind both gene promoters and distant regulatory regions. Plot shows genes with chromosomal interactions in <i>ago4</i>, which have AGO4 binding to the promoter (1kb upstream of transcriptional start sites), to the distant regulatory regions (detected by Hi-C), or to both. Genes increased in <i>ago4</i> (peach) are compared to total genes (green). Color coding for individual replicates is the same as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006749#pgen.1006749.g001" target="_blank">Fig 1B</a>.</p