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

High- resolution map of plastid- encoded RNA polymerase binding patterns demonstrates a major role of transcription in chloroplast gene expression

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/174809/1/tpj15882.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/174809/2/tpj15882-sup-0001-Supinfo.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/174809/3/tpj15882_am.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

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

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

Speculative model of gene regulation by RdDM.

<p>(A) In wild-type plants Pol V and AGO4 bind to chromatin and help establish repressive chromatin marks at regulatory regions. These chromatin modifications inhibit chromosomal interactions between genes and distant regulatory regions. (B) When RdDM is not functional, chromosomal interactions are able to occur between promoters and distant regulatory regions bound by transcription factors (orange), thereby affecting gene expression.</p

Państwo demokratyczne, prawne i socjalne: księga jubileuszowa dedykowana profesorowi Zbigniewowi Antoniemu Maciągowi. T. 4, Studia społeczne, polityczne i ekonomiczne

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