Intergenic transcription by RNA Polymerase II coordinates Pol IV and Pol V in siRNA-directed transcriptional gene silencing in \u3ci\u3eArabidopsis\u3c/i\u3e

Intergenic transcription by RNA Polymerase II (Pol II) is widespread in plant and animal genomes, but the functions of intergenic transcription or the resulting noncoding transcripts are poorly understood. Here, we show that Arabidopsis Pol II is indispensable for endogenous siRNA-mediated transcriptional gene silencing (TGS) at intergenic low-copy-number loci, despite the presence of two other polymerases—Pol IV and Pol V—that specialize in TGS through siRNAs. We show that Pol II produces noncoding scaffold transcripts that originate outside of heterochromatic, siRNA-generating loci. Through these transcripts and physical interactions with the siRNA effector protein ARGONAUTE4 (AGO4), Pol II recruits AGO4/siRNAs to homologous loci to result in TGS. Meanwhile, Pol II transcription also recruits Pol IV and Pol V to different locations at heterochromatic loci to promote siRNA biogenesis and siRNA-mediated TGS, respectively. This study establishes that intergenic transcription by Pol II is required for siRNA-mediated TGS, and reveals an intricate collaboration and division of labor among the three polymerases in gene silencing

The MBD7 complex promotes expression of methylated transgenes without significantly altering their methylation status.

DNA methylation is associated with gene silencing in eukaryotic organisms. Although pathways controlling the establishment, maintenance and removal of DNA methylation are known, relatively little is understood about how DNA methylation influences gene expression. Here we identified a METHYL-CpG-BINDING DOMAIN 7 (MBD7) complex in Arabidopsis thaliana that suppresses the transcriptional silencing of two LUCIFERASE (LUC) reporters via a mechanism that is largely downstream of DNA methylation. Although mutations in components of the MBD7 complex resulted in modest increases in DNA methylation concomitant with decreased LUC expression, we found that these hyper-methylation and gene expression phenotypes can be genetically uncoupled. This finding, along with genome-wide profiling experiments showing minimal changes in DNA methylation upon disruption of the MBD7 complex, places the MBD7 complex amongst a small number of factors acting downstream of DNA methylation. This complex, however, is unique as it functions to suppress, rather than enforce, DNA methylation-mediated gene silencing

Detection of Pol IV/RDR2-Dependent Transcripts at the Genomic Scale in \u3cem\u3eArabidopsis\u3c/em\u3e Reveals Features and Regulation of siRNA Biogenesis

Twenty-four-nucleotide small interfering (si)RNAs are central players in RNA-directed DNA methylation (RdDM), a process that establishes and maintains DNA methylation at transposable elements to ensure genome stability in plants. The plant-specific RNA polymerase IV (Pol IV) is required for siRNA biogenesis and is believed to transcribe RdDM loci to produce primary transcripts that are converted to double-stranded RNAs (dsRNAs) by RDR2 to serve as siRNA precursors. Yet, no such siRNA precursor transcripts have ever been reported. Here, through genome-wide profiling of RNAs in genotypes that compromise the processing of siRNA precursors, we were able to identify Pol IV/RDR2-dependent transcripts from tens of thousands of loci. We show that Pol IV/RDR2-dependent transcripts correspond to both DNA strands, whereas the RNA polymerase II (Pol II)-dependent transcripts produced upon derepression of the loci are derived primarily from one strand. We also show that Pol IV/RDR2-dependent transcripts have a 5′ monophosphate, lack a poly(A) tail at the 3′ end, and contain no introns; these features distinguish them from Pol II-dependent transcripts. Like Pol II-transcribed genic regions, Pol IV-transcribed regions are flanked by A/T-rich sequences depleted in nucleosomes, which highlights similarities in Pol II- and Pol IV-mediated transcription. Computational analysis of siRNA abundance from various mutants reveals differences in the regulation of siRNA biogenesis at two types of loci that undergo CHH methylation via two different DNA methyltransferases. These findings begin to reveal features of Pol IV/RDR2-mediated transcription at the heart of genome stability in plants

DNA Topoisomerase 1α Promotes Transcriptional Silencing of Transposable Elements through DNA Methylation and Histone Lysine 9 Dimethylation in Arabidopsis

RNA-directed DNA methylation (RdDM) and histone H3K9 dimethylation (H3K9me2) are related transcriptional silencing mechanisms that target transposable elements (TEs) and repeats to maintain genome stability in plants. RdDM is mediated by small and long noncoding RNAs produced by the plant-specific RNA polymerases Pol IV and Pol V, respectively. Through a chemical genetics screen with a luciferase-based DNA methylation reporter, LUCL, we found that camptothecin, a compound with anti- cancer properties that targets DNA topoisomerase 1α (TOP1α) was able to de-repress LUCL by reducing its DNA methylation and H3K9me2 levels. Further studies with Arabidopsis top1α mutants showed that TOP1α silences endogenous RdDM loci by facilitating the production of Pol V-dependent long non-coding RNAs, AGONAUTE4 recruitment and H3K9me2 deposition at TEs and repeats. This study assigned a new role in epigenetic silencing to an enzyme that affects DNA topology.Fil: Dinh, Thanh Theresa. University of California Riverside. Center for Plant Cell Biology, Institute of Integrative Genome Biology, Department of Botany and Plant Sciences; Estados Unidos. University of California Riverside. Center for Plant Cell Biology, Institute of Integrative Genome Biology. ChemGen IGERT program; Estados UnidosFil: Gao, Lei. University of California Riverside. Center for Plant Cell Biology, Institute of Integrative Genome Biology, Department of Botany and Plant Sciences; Estados UnidosFil: Liu, Xigang . University of California Riverside. Center for Plant Cell Biology, Institute of Integrative Genome Biology, Department of Botany and Plant Sciences; Estados UnidosFil: Li, Dongming. University of California Riverside. Center for Plant Cell Biology, Institute of Integrative Genome Biology, Department of Botany and Plant Sciences; Estados Unidos. Lanzhou University. School of Life Sciences Plant Biology Laboratory; ChinaFil: Li, Shengben. University of California Riverside. Center for Plant Cell Biology, Institute of Integrative Genome Biology, Department of Botany and Plant Sciences; Estados UnidosFil: Zhao, Yuanyuan. University of California Riverside. Center for Plant Cell Biology, Institute of Integrative Genome Biology, Department of Botany and Plant Sciences; Estados UnidosFil: O'leary, Michael. University of California Riverside. Center for Plant Cell Biology, Institute of Integrative Genome Biology, Department of Botany and Plant Sciences; Estados UnidosFil: Le, Brandon. University of California Riverside. Center for Plant Cell Biology, Institute of Integrative Genome Biology, Department of Botany and Plant Sciences; Estados UnidosFil: Schmitz, Robert J.. The Salk Institute for Biological Studies. Plant Biology Laboratory; Estados UnidosFil: Manavella, Pablo Andrés. Max Planck Institute for Developmental Biology. Department of Molecular Biology; Alemania. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Santa Fe. Instituto de Agrobiotecnologia del Litoral; ArgentinaFil: Li, Shaofang. University of California Riverside. Center for Plant Cell Biology, Institute of Integrative Genome Biology, Department of Botany and Plant Sciences; Estados UnidosFil: Weigel, Detlef. Max Planck Institute for Developmental Biology. Department of Molecular Biology; AlemaniaFil: Pontes, Olga. University of New Mexico. Department of Biology; Estados UnidosFil: Ecker, Joseph R.. The Salk Institute for Biological Studies. Howard Hughes Medical Institute; Estados Unidos. The Salk Institute for Biological Studies. Plant Biology Laboratory; Estados UnidosFil: Chen, Xuemei. University of California Riverside. Center for Plant Cell Biology, Institute of Integrative Genome Biology, Department of Botany and Plant Sciences; Estados Unidos. University of California Riverside. Howard Hughes Medical Institute, ; Estados Unido

siRNAs compete with miRNAs for methylation by HEN1 in Arabidopsis

Plant microRNAs (miRNAs) and small interfering RNAs (siRNAs) bear a 2′-O-methyl group on the 3′-terminal nucleotide. This methyl group is post-synthetically added by the methyltransferase protein HEN1 and protects small RNAs from enzymatic activities that target the 3′-OH. A mutagenesis screen for suppressors of the partial loss-of-function hen1-2 allele in Arabidopsis identified second-site mutations that restore miRNA methylation. These mutations affect two subunits of the DNA-dependent RNA polymerase IV (Pol IV), which is essential for the biogenesis of 24 nt endogenous siRNAs. A mutation in RNA-dependent RNA polymerase 2, another essential gene for the biogenesis of endogenous 24-nt siRNAs, also rescued the defects in miRNA methylation of hen1-2, revealing a previously unsuspected, negative influence of siRNAs on HEN1-mediated miRNA methylation. In addition, our findings imply the existence of a negative modifier of HEN1 activity in the Columbia genetic background

Intergenic transcription by RNA Polymerase II coordinates Pol IV and Pol V in siRNA-directed transcriptional gene silencing in \u3ci\u3eArabidopsis\u3c/i\u3e

Intergenic transcription by RNA Polymerase II (Pol II) is widespread in plant and animal genomes, but the functions of intergenic transcription or the resulting noncoding transcripts are poorly understood. Here, we show that Arabidopsis Pol II is indispensable for endogenous siRNA-mediated transcriptional gene silencing (TGS) at intergenic low-copy-number loci, despite the presence of two other polymerases—Pol IV and Pol V—that specialize in TGS through siRNAs. We show that Pol II produces noncoding scaffold transcripts that originate outside of heterochromatic, siRNA-generating loci. Through these transcripts and physical interactions with the siRNA effector protein ARGONAUTE4 (AGO4), Pol II recruits AGO4/siRNAs to homologous loci to result in TGS. Meanwhile, Pol II transcription also recruits Pol IV and Pol V to different locations at heterochromatic loci to promote siRNA biogenesis and siRNA-mediated TGS, respectively. This study establishes that intergenic transcription by Pol II is required for siRNA-mediated TGS, and reveals an intricate collaboration and division of labor among the three polymerases in gene silencing

siRNAs compete with miRNAs for methylation by HEN1 in \u3ci\u3eArabidopsis\u3c/i\u3e (Supplementary Material)

Legends of Supplemental Figure

siRNAs compete with miRNAs for methylation by HEN1 in \u3ci\u3eArabidopsis\u3c/i\u3e

Plant microRNAs (miRNAs) and small interfering RNAs (siRNAs) bear a 2\u27-O-methyl group on the 3\u27-terminal nucleotide. This methyl group is post-synthetically added by the methyltransferase protein HEN1 and protects small RNAs from enzymatic activities that target the 3\u27-OH. A mutagenesis screen for suppressors of the partial loss-offunction hen1-2 allele in Arabidopsis identified second-site mutations that restore miRNA methylation. These mutations affect two subunits of the DNA-dependent RNA polymerase IV (Pol IV), which is essential for the biogenesis of 24 nt endogenous siRNAs. A mutation in RNA-dependent RNA polymerase 2, another essential gene for the biogenesis of endogenous 24-nt siRNAs, also rescued the defects in miRNA methylation of hen1-2, revealing a previously unsuspected, negative influence of siRNAs on HEN1-mediated miRNA methylation. In addition, our findings imply the existence of a negative modifier of HEN1 activity in the Columbia genetic background

Development of a luciferase-based reporter of transcriptional gene silencing that enables bidirectional mutant screening in Arabidopsis thaliana

BACKGROUND: Cytosine methylation is an important chromatin modification that maintains genome integrity and regulates gene expression through transcriptional gene silencing. Major players in de novo methylation guided by siRNAs (known as RNA-directed DNA methylation, or RdDM), maintenance methylation, and active demethylation have been identified in Arabidopsis. However, active demethylation only occurs at a subset of RdDM loci, raising the question of how the homeostasis of DNA methylation is achieved at most RdDM loci. To identify factors that regulate the levels of cytosine methylation, we aimed to establish a transgenic reporter system that allows for forward genetic screens in Arabidopsis. RESULTS: We introduced a dual 35 S promoter (d35S) driven luciferase reporter, LUCH, into Arabidopsis and isolated a line with a moderate level of luciferase activity. LUCH produced transgene-specific 24 nucleotide siRNAs and its d35S contained methylated cytosine in CG, CHG and CHH contexts. Treatment of the transgenic line with an inhibitor of cytosine methylation de-repressed luciferase activity. Mutations in several components of the RdDM pathway but not the maintenance methylation genes resulted in reduced d35S methylation, especially CHH methylation, and de-repression of luciferase activity. A mutation in MOM1, which is known to cooperate with RdDM to silence transposons, reduced d35S DNA methylation and de-repressed LUCH expression. A mutation in ROS1, a cytosine demethylation enzyme, increased d35S methylation and reduced LUCH expression. CONCLUSION: We developed a luciferase-based reporter, LUCH, which reports both DNA methylation directed by small RNAs and active demethylation by ROS1 in Arabidopsis. The moderate basal level of LUCH expression allows for bi-directional genetic screens that dissect the mechanisms of DNA methylation as well as demethylation