SUVH1, a Su(var)3-9 family member, promotes the expression of genes targeted by DNA methylation.

Transposable elements are found throughout the genomes of all organisms. Repressive marks such as DNA methylation and histone H3 lysine 9 (H3K9) methylation silence these elements and maintain genome integrity. However, how silencing mechanisms are themselves regulated to avoid the silencing of genes remains unclear. Here, an anti-silencing factor was identified using a forward genetic screen on a reporter line that harbors a LUCIFERASE (LUC) gene driven by a promoter that undergoes DNA methylation. SUVH1, a Su(var)3-9 homolog, was identified as a factor promoting the expression of the LUC gene. Treatment with a cytosine methylation inhibitor completely suppressed the LUC expression defects of suvh1, indicating that SUVH1 is dispensable for LUC expression in the absence of DNA methylation. SUVH1 also promotes the expression of several endogenous genes with promoter DNA methylation. However, the suvh1 mutation did not alter DNA methylation levels at the LUC transgene or on a genome-wide scale; thus, SUVH1 functions downstream of DNA methylation. Histone H3 lysine 4 (H3K4) trimethylation was reduced in suvh1; in contrast, H3K9 methylation levels remained unchanged. This work has uncovered a novel, anti-silencing function for a member of the Su(var)3-9 family that has previously been associated with silencing through H3K9 methylation

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

RNA polymerase V-dependent small RNAs in Arabidopsis originate from small, intergenic loci including most SINE repeats.

In plants, heterochromatin is maintained by a small RNA-based gene silencing mechanism known as RNA-directed DNA methylation (RdDM). RdDM requires the non-redundant functions of two plant-specific DNA-dependent RNA polymerases (RNAP), RNAP IV and RNAP V. RNAP IV plays a major role in siRNA biogenesis, while RNAP V may recruit DNA methylation machinery to target endogenous loci for silencing. Although small RNA-generating regions that are dependent on both RNAP IV and RNAP V have been identified previously, the genomic loci targeted by RNAP V for siRNA accumulation and silencing have not been described extensively. To characterize the RNAP V-dependent, heterochromatic siRNA-generating regions in the Arabidopsis genome, we deeply sequenced the small RNA populations of wild-type and RNAP V null mutant (nrpe1) plants. Our results showed that RNAP V-dependent siRNA-generating loci are associated predominately with short repetitive sequences in intergenic regions. Suppression of small RNA production from short repetitive sequences was also prominent in RdDM mutants including dms4, drd1, dms3 and rdm1, reflecting the known association of these RdDM effectors with RNAP V. The genomic regions targeted by RNAP V were small, with an estimated average length of 238 bp. Our results suggest that RNAP V affects siRNA production from genomic loci with features dissimilar to known RNAP IV-dependent loci. RNAP V, along with RNAP IV and DRM1/2, may target and silence a set of small, intergenic transposable elements located in dispersed genomic regions for silencing. Silencing at these loci may be actively reinforced by RdDM

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

Reabilitação protética da disfunção velofaríngea: prótese de palato e obturador faríngeo

The seed maturation program only occurs during late embryogenesis, and repression of the program is pivotal for seedling development. However, the mechanism through which this repression is achieved in vegetative tissues is poorly understood. Here we report a microRNA (miRNA)-mediated repression mechanism operating in leaves. To understand the repression of the embryonic program in seedlings, we have conducted a genetic screen using a seed maturation gene reporter transgenic line in Arabidopsis (Arabidopsis thaliana) for the isolation of mutants that ectopically express seed maturation genes in leaves. One of the mutants identified from the screen is a weak allele of ARGONAUTE1 (AGO1) that encodes an effector protein for small RNAs. We first show that it is the defect in the accumulation of miRNAs rather than other small RNAs that causes the ectopic seed gene expression in ago1. We then demonstrate that overexpression of miR166 suppresses the derepression of the seed gene reporter in ago1 and that, conversely, the specific loss of miR166 causes ectopic expression of seed maturation genes. Further, we show that ectopic expression of miR166 targets, type III homeodomain-leucine zipper (HD-ZIPIII) genes PHABULOSA (PHB) and PHAVOLUTA (PHV), is sufficient to activate seed maturation genes in vegetative tissues. Lastly, we show that PHB binds the promoter of LEAFY COTYLEDON2 (LEC2), which encodes a master regulator of seed maturation. Therefore, this study establishes a core module composed of a miRNA, its target genes (PHB and PHV), and the direct target of PHB (LEC2) as an underlying mechanism that keeps the seed maturation program off during vegetative development

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