The plant FYVE domain-containing protein FREE1 associates with microprocessor components to repress miRNA biogenesis.

FYVE domain protein required for endosomal sorting 1 (FREE1), originally identified as a plant-specific component of the endosomal sorting complex required for transport (ESCRT) machinery, plays diverse roles either in endosomal sorting in the cytoplasm or in transcriptional regulation of abscisic acid signaling in the nucleus. However, to date, a role for FREE1 or other ESCRT components in the regulation of plant miRNA biology has not been discovered. Here, we demonstrate a nuclear function of FREE1 as a cofactor in miRNA biogenesis in plants. FREE1 directly interacts with the plant core microprocessor component CPL1 in nuclear bodies and disturbs the association between HYL1, SE and CPL1. Inactivation of FREE1 in the nucleus increases the binding affinity between HYL1, SE, and CPL1 and causes a transition of HYL1 from the inactive hyperphosphorylated version to the active hypophosphorylated form, thereby promoting miRNA biogenesis. Our results suggest that FREE1 has evolved as a negative regulator of miRNA biogenesis and provides evidence for a link between FYVE domain-containing proteins and miRNA biogenesis in plants.

SICKLE modulates lateral root development by promoting degradation of lariat intronic RNA.

Plant lateral roots (LRs) play vital roles in anchorage and uptake of water and nutrients. Here, we reveal that degradation of lariat intronic RNAs (lariRNAs) modulated by SICKLE (SIC) is required for LR development in Arabidopsis (Arabidopsis thaliana). Loss of SIC results in hyper-accumulation of lariRNAs and restricts the outgrowth of LR primordia, thereby reducing the number of emerged LRs. Decreasing accumulation of lariRNAs by over-expressing RNA debranching enzyme 1 (DBR1), a rate-limiting enzyme of lariRNA decay, restored LR defects in SIC-deficient plants. Mechanistically, SIC interacts with DBR1 and facilitates its nuclear accumulation, which is achieved through two functionally redundant regions (SIC1-244 and SIC252-319) for nuclear localization. Of the remaining amino acids in this region, six (SIC245-251) comprise a DBR1-interacting region while two (SICM246 and SICW251) are essential for DBR1-SIC interaction. Reducing lariRNAs restored microRNA (miRNA) levels and LR development in lariRNA hyper-accumulating plants, suggesting that these well-known regulators of LR development mainly function downstream of lariRNAs. Taken together, we propose that SIC acts as an enhancer of DBR1 nuclear accumulation by driving nuclear localization through direct interaction, thereby promoting lariRNA decay to fine-tune miRNA biogenesis and modulating LR development.

H3K9 demethylases IBM1 and JMJ27 are required for male meiosis in Arabidopsis thaliana.

Dimethylation of histone H3 lysine 9 (H3K9me2), a crucial modification for heterochromatin formation and transcriptional silencing, is essential for proper meiotic prophase progression in mammals. We analyzed meiotic defects and generated genome-wide profiles of H3K9me2 and transcriptomes for the mutants of H3K9 demethylases. Moreover, we also identified proteins interacting with H3K9 demethylases. H3K9me2 is usually found at transposable elements and repetitive sequences but is absent from the bodies of protein-coding genes. In this study, we show that the Arabidopsis thaliana H3K9 demethylases IBM1 and JMJ27 cooperatively regulate crossover formation and chromosome segregation. They protect thousands of protein-coding genes from ectopic H3K9me2, including genes essential for meiotic prophase progression. In addition to removing H3K9me2, IBM1 and JMJ27 interact with the Precocious Dissociation of Sisters 5 (PDS5) cohesin complex cofactors. The pds5 mutant shared similar transcriptional alterations with ibm1 jmj27, including meiosis-essential genes, yet without affecting H3K9me2 levels. Hence, PDS5s, together with IBM1 and JMJ27, regulate male meiosis and gene expression independently of H3K9 demethylation. These findings uncover a novel role of H3K9me2 removal in meiosis and a new function of H3K9 demethylases and cohesin cofactors in meiotic transcriptional regulation.

Meiocyte-Specific and AtSPO11-1-Dependent Small RNAs and Their Association with Meiotic Gene Expression and Recombination.

Meiotic recombination ensures accurate chromosome segregation and results in genetic diversity in sexually reproducing eukaryotes. Over the last few decades, the genetic regulation of meiotic recombination has been extensively studied in many organisms. However, the role of endogenous meiocyte-specific small RNAs (ms-sRNAs; 21-24 nucleotide [nt]) and their involvement in meiotic recombination are unclear. Here, we sequenced the total small RNA (sRNA) and messenger RNA populations from meiocytes and leaves of wild type Arabidopsis (Arabidopsis thaliana) and meiocytes of spo11-1, a mutant defective in double-strand break formation, and we discovered 2,409 ms-sRNA clusters, 1,660 of which areSPORULATION 11-1 (AtSPO11-1)-dependent. Unlike mitotic small interfering RNAs that are enriched in intergenic regions and associated with gene silencing, ms-sRNAs are significantly enriched in genic regions and exhibit a positive correlation with genes that are preferentially expressed in meiocytes (i.e. Arabidopsis SKP1-LIKE1 and RAD51), in a fashion unrelated to DNA methylation. We also found that AtSPO11-1-dependent sRNAs have distinct characteristics compared with ms-sRNAs and tend to be associated with two known types of meiotic recombination hotspot motifs (i.e. CTT-repeat and A-rich motifs). These results reveal different meiotic and mitotic sRNA landscapes and provide new insights into how sRNAs relate to gene expression in meiocytes and meiotic recombination.

A Comprehensive Map of Intron Branchpoints and Lariat RNAs in Plants.

Lariats are formed by excised introns, when the 5' splice site joins with the branchpoint (BP) during splicing. Although lariat RNAs are usually degraded by RNA debranching enzyme 1, recent findings in animals detected many lariat RNAs under physiological conditions. By contrast, the features of BPs and to what extent lariat RNAs accumulate naturally are largely unexplored in plants. Here, we analyzed 948 RNA sequencing data sets to document plant BPs and lariat RNAs on a genome-wide scale. In total, we identified 13,872, 5199, 29,582, and 13,478 BPs in Arabidopsis (Arabidopsis thaliana), tomato (Solanum lycopersicum), rice (Oryza sativa), and maize (Zea mays), respectively. Features of plant BPs are highly similar to those in yeast and human, in that BPs are adenine-preferred and flanked by uracil-enriched sequences. Intriguingly, ∼20% of introns harbor multiple BPs, and BP usage is tissue-specific. Furthermore, 10,580 lariat RNAs accumulate in wild-type Arabidopsis plants, and most of these lariat RNAs originate from longer or retroelement-depleted introns. Moreover, the expression of these lariat RNAs is accompanied by the incidence of back-splicing of parent exons. Collectively, our results provide a comprehensive map of intron BPs and lariat RNAs in four plant species and uncover a link between lariat turnover and splicing.

Anaphase-promoting complex/cyclosome regulates RdDM activity by degrading DMS3 in Arabidopsis.

During RNA-directed DNA methylation (RdDM), the DDR complex, composed of DRD1, DMS3, and RDM1, is responsible for recruiting DNA polymerase V (Pol V) to silence transposable elements (TEs) in plants. However, how the DDR complex is regulated remains unexplored. Here, we show that the anaphase-promoting complex/cyclosome (APC/C) regulates the assembly of the DDR complex by targeting DMS3 for degradation. We found that a substantial set of RdDM loci was commonly de-repressed in apc/c and pol v mutants, and that the defects in RdDM activity resulted from up-regulated DMS3 protein levels, which finally caused reduced Pol V recruitment. DMS3 was ubiquitinated by APC/C for degradation in a D box-dependent manner. Competitive binding assays and gel filtration analyses showed that a proper level of DMS3 is critical for the assembly of the DDR complex. Consistent with the importance of the level of DMS3, overaccumulation of DMS3 caused defective RdDM activity, phenocopying the apc/c and dms3 mutants. Moreover, DMS3 is expressed in a cell cycle-dependent manner. Collectively, these findings provide direct evidence as to how the assembly of the DDR complex is regulated and uncover a safeguarding role of APC/C in the regulation of RdDM activity.

Heterochromatic silencing is reinforced by ARID1-mediated small RNA movement in Arabidopsis pollen.

In the plant male germline, transposable elements (TEs) are reactivated in the companion vegetative nucleus, resulting in siRNA production and the intercellular movement of these siRNAs to reinforce TE silencing in sperm. However, the mechanism by which siRNA movement is regulated remains unexplored. Here we show that ARID1, a transcription factor which is constitutively expressed in the vegetative nucleus but dynamically accumulates in the generative cell (the progenitor of sperm) to promote the second pollen mitosis, mediates siRNA movement to reinforce heterochromatic silencing in the male germline. We looked for regulators involved in the accumulation of ARID1 in the generative cell, and found that AGO9, a germline-specific AGO in Arabidopsis, is required for the accumulation of ARID1 in the generative cell. Mutations in either ARID1 or AGO9 lead to the interruption of not only the second pollen mitosis but also the movement of siRNA from the vegetative nucleus to the male germline, resulting in the release of heterochromatic silencing in the male germline. Moreover, conditional knockdown of ARID1 in the generative cell causes reduced heterochromatic silencing in both bicellular and mature pollen. This study provides insights into how a spatiotemporal transcription factor coordinates heterochromatic silencing and male germline maturation.

Spliceosome disassembly factors ILP1 and NTR1 promote miRNA biogenesis in Arabidopsis thaliana.

The intron-lariat spliceosome (ILS) complex is highly conserved among eukaryotes, and its disassembly marks the end of a canonical splicing cycle. In this study, we show that two conserved disassembly factors of the ILS complex, Increased Level of Polyploidy1-1D (ILP1) and NTC-Related protein 1 (NTR1), positively regulate microRNA (miRNA) biogenesis by facilitating transcriptional elongation of MIRNA (MIR) genes in Arabidopsis thaliana. ILP1 and NTR1 formed a stable complex and co-regulated alternative splicing of more than a hundred genes across the Arabidopsis genome, including some primary transcripts of miRNAs (pri-miRNAs). Intriguingly, pri-miRNAs, regardless of having introns or not, were globally down-regulated when the ILP1 or NTR1 function was compromised. ILP1 and NTR1 interacted with core miRNA processing proteins Dicer-like 1 and Serrate, and were required for proper RNA polymerase II occupancy at elongated regions of MIR chromatin, without affecting either MIR promoter activity or pri-miRNA decay. Our results provide further insights into the regulatory role of spliceosomal machineries in the biogenesis of miRNAs.

Degradation of unmethylated miRNA/miRNA*s by a DEDDy-type 3' to 5' exoribonuclease Atrimmer 2 in Arabidopsis.

The 3' end methylation catalyzed by HUA Enhancer 1 (HEN1) is a crucial step of small RNA stabilization in plants, yet how unmethylated small RNAs undergo degradation remains largely unknown. Using a reverse genetic approach, we here show that Atrimmer 2 (ATRM2), a DEDDy-type 3' to 5' exoribonuclease, acts in the degradation of unmethylated miRNAs and miRNA*s in Arabidopsis Loss-of-function mutations in ATRM2 partially suppress the morphological defects caused by HEN1 malfunction, with restored levels of a subset of miRNAs and receded expression of corresponding miRNA targets. Dysfunction of ATRM2 has negligible effect on miRNA trimming, and further increase the fertility of hen1 heso1 urt1, a mutant with an almost complete abolishment of miRNA uridylation, indicating that ATRM2 may neither be involved in 3' to 5' trimming nor be the enzyme that specifically degrades uridylated miRNAs. Notably, the fold changes of miRNAs and their corresponding miRNA*s were significantly correlated in hen1 atrm2 versus hen1 Unexpectedly, we observed a marked increase of 3' to 5' trimming of several miRNA*s but not miRNAs in ATRM2 compromised backgrounds. These data suggest an action of ATRM2 on miRNA/miRNA* duplexes, and the existence of an unknown exoribonuclease for specific trimming of miRNA*. This asymmetric effect on miRNA/miRNA* is likely related to Argonaute (AGO) proteins, which can distinguish miRNAs from miRNA*s. Finally, we show that ATRM2 colocalizes and physically interacts with Argonaute 1 (AGO1). Taken together, our results suggest that ATRM2 may be involved in the surveillance of unmethylated miRNA/miRNA* duplexes during the initiation step of RNA-induced silencing complex assembly.

Brassinosteroids inhibit miRNA-mediated translational repression by decreasing AGO1 on the endoplasmic reticulum.

Translational repression is a conserved mechanism in microRNA (miRNA)-guided gene silencing. In Arabidopsis, ARGONAUTE1 (AGO1), the major miRNA effector, localizes in the cytoplasm for mRNA cleavage and at the endoplasmic reticulum (ER) for translational repression of target genes. However, the mechanism underlying miRNA-mediated translational repression is poorly understood. In particular, how the subcellular partitioning of AGO1 is regulated is largely unexplored. Here, we show that the plant hormone brassinosteroids (BRs) inhibit miRNA-mediated translational repression by negatively regulating the distribution of AGO1 at the ER in Arabidopsis thaliana. We show that the protein levels rather than the transcript levels of miRNA target genes were reduced in BR-deficient mutants but increased under BR treatments. The localization of AGO1 at the ER was significantly decreased under BR treatments while it was increased in the BR-deficient mutants. Moreover, ROTUNDIFOLIA3 (ROT3), an enzyme involved in BR biosynthesis, co-localizes with AGO1 at the ER and interacts with AGO1 in a GW motif-dependent manner. Complementation analysis showed that the AGO1-ROT3 interaction is necessary for the function of ROT3. Our findings provide new clues to understand how miRNA-mediated gene silencing is regulated by plant endogenous hormones.

The transcriptome variations of Panaxnotoginseng roots treated with different forms of nitrogen fertilizers.

BACKGROUND: The sensitivity of plants to ammonia is a worldwide problem that limits crop production. Excessive use of ammonium as the sole nitrogen source results in morphological and physiological disorders, and retarded plant growth. RESULTS: In this study we found that the root growth of Panax notoginseng was inhibited when only adding ammonium nitrogen fertilizer, but the supplement of nitrate fertilizer recovered the integrity, activity and growth of root. Twelve RNA-seq profiles in four sample groups were produced and analyzed to identify deregulated genes in samples with different treatments. In comparisons to NH[Formula: see text] treated samples, ACLA-3 gene is up-regulated in samples treated with NO[Formula: see text] and with both NH[Formula: see text] and NO[Formula: see text], which is further validated by qRT-PCR in another set of samples. Subsequently, we show that the some key metabolites in the TCA cycle are also significantly enhanced when introducing NO[Formula: see text]. These potentially enhance the integrity and recover the growth of Panax notoginseng roots. CONCLUSION: These results suggest that the activated TCA cycle, as demonstrated by up-regulation of ACLA-3 and several key metabolites in this cycle, contributes to the increased Panax notoginseng root yield when applying both ammonium and nitrate fertilizer.

Intercellular delivery of small RNAs in plant gametes.

Small RNAs are 20-24 nucleotides in length. In plants, small RNAs are classified into microRNAs (miRNAs) and small interfering RNAs (siRNAs), based on their biogenesis and molecular features. In contrast to the extensive knowledge of the roles of small RNAs in sporophytic tissues, the distribution and function of small RNAs in gametophytic cells have been less well studied. However, with the improvement of single-cell sorting and RNA sequencing technologies, the distribution of small RNAs, especially siRNAs, between sperm cells and the vegetative cell, as well as the function of sperm-delivered small RNAs during early seed development have been elucidated. This review summarizes work from the past 5 years regarding small RNAs in male gametes, emphasizing the intercellular communication and biological significance of small RNAs in Arabidopsis.

Reactive oxygen species-mediated BIN2 activity revealed by single-molecule analysis.

Much evidence has shown that reactive oxygen species (ROS) regulate several plant hormone signaling cascades, but little is known about the real-time kinetics and the underlying molecular mechanisms of the target proteins in the brassinosteroid (BR) signaling pathway. In this study, we used single-molecule techniques to investigate the true signaling timescales of the major BR signaling components BRI1-EMS-SUPPRESSOR 1 (BES1) and BRASSINOSTEROID INSENSITIVE 2 (BIN2) of Arabidopsis thaliana. The rate constants of BIN2 associating with ATP and phosphorylating BES1 were determined to be 0.7 ± 0.4 mM-1  s-1 and 2.3 ± 1.4 s-1 , respectively. Interestingly, we found that the interaction of BIN2 and BES1 was oxygen-dependent, and oxygen can directly modify BIN2. The activity of BIN2 was switched on via modification of specific cysteine (Cys) residues, including C59, C95, C99 and C162. The mutation of these Cys residues inhibited the BR signaling outputs. These findings demonstrate the power of using single-molecule techniques to study the dynamic interactions of signaling components, which is difficult to be discovered by conventional physiological and biochemical methods.

The Arabidopsis anaphase-promoting complex/cyclosome subunit 8 is required for male meiosis.

Faithful chromosome segregation is required for both mitotic and meiotic cell divisions and is regulated by multiple mechanisms including the anaphase-promoting complex/cyclosome (APC/C), which is the largest known E3 ubiquitin-ligase complex and has been implicated in regulating chromosome segregation in both mitosis and meiosis in animals. However, the role of the APC/C during plant meiosis remains largely unknown. Here, we show that Arabidopsis APC8 is required for male meiosis. We used a combination of genetic analyses, cytology and immunolocalisation to define the function of AtAPC8 in male meiosis. Meiocytes from apc8-1 plants exhibit several meiotic defects including improper alignment of bivalents at metaphase I, unequal chromosome segregation during anaphase II, and subsequent formation of polyads. Immunolocalisation using an antitubulin antibody showed that APC8 is required for normal spindle morphology. We also observed mitotic defects in apc8-1, including abnormal sister chromatid segregation and microtubule morphology. Our results demonstrate that Arabidopsis APC/C is required for meiotic chromosome segregation and that APC/C-mediated regulation of meiotic chromosome segregation is a conserved mechanism among eukaryotes.

jaw-1D: a gain-of-function mutation responsive to paramutation-like induction of epigenetic silencing.

The Arabidopsis thaliana gain-of-function T-DNA insertion mutant jaw-1D produces miR319A, a microRNA that represses genes encoding CIN-like TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL FACTORs (TCPs), a family of transcription factors that play key roles in leaf morphogenesis. In this study, we show that jaw-1D is responsive to paramutation-like epigenetic silencing. A genetic cross of jaw-1D with the polycomb gene mutant curly leaf-29 (clf-29) leads to attenuation of the jaw-1D mutant plant phenotype. This induced mutation, jaw-1D*, was associated with down-regulation of miR319A, was heritable independently from clf-29, and displayed paramutation-like non-Mendelian inheritance. Down-regulation of miR319A in jaw-1D* was linked to elevated levels of histone H3 lysine 9 dimethylation and DNA methylation at the CaMV35S enhancer located within the activation-tagging T-DNA of the jaw-1D locus. Examination of 21 independent T-DNA insertion mutant lines revealed that 11 could attenuate the jaw-1D mutant phenotype in a similar way to the paramutation induced by clf-29. These paramutagenic mutant lines shared the common feature that their T-DNA insertion was present as multi-copy tandem repeats and contained high levels of CG and CHG methylation. Our results provide important insights into paramutation-like epigenetic silencing, and caution against the use of jaw-1D in genetic interaction studies.

Intron Lariat RNA Inhibits MicroRNA Biogenesis by Sequestering the Dicing Complex in Arabidopsis.

Lariat RNAs formed as by-products of splicing are quickly degraded by the RNA debranching enzyme 1 (DBR1), leading to their turnover. Null dbr1 mutants in both animals and plants are embryo lethal, but the mechanism underlying the lethality remains unclear. Here we characterized a weak mutant allele of DBR1 in Arabidopsis, dbr1-2, and showed that a global increase in lariat RNAs was unexpectedly accompanied by a genome-wide reduction in miRNA accumulation. The dbr1-2 mutation had no effects on expression of miRNA biogenesis genes or primary miRNAs (pri-miRNAs), but the association of pri-miRNAs with the DCL1/HYL1 dicing complex was impaired. Lariat RNAs were associated with the DCL1/HYL1 dicing complex in vivo and competitively inhibited the binding of HYL1 with pri-miRNA. Consistent with the impacts of lariat RNAs on miRNA biogenesis, over-expression of lariat RNAs reduced miRNA accumulation. Lariat RNAs localized in nuclear bodies, and partially co-localize with HYL1, and both DCL1 and HYL1 were mis-localized in dbr1-2. Together with our findings that nearly four hundred lariat RNAs exist in wild type plants and that these lariat RNAs also associate with the DCL1/HYL1 dicing complex in vivo, we thus propose that lariat RNAs, as decoys, inhibit miRNA processing, suggesting a hitherto unknown layer of regulation in miRNA biogenesis.

Detection of Pol IV/RDR2-dependent transcripts at the genomic scale in Arabidopsis 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.

APETALA2 antagonizes the transcriptional activity of AGAMOUS in regulating floral stem cells in Arabidopsis thaliana.

APETALA2 (AP2) is best known for its function in the outer two floral whorls, where it specifies the identities of sepals and petals by restricting the expression of AGAMOUS (AG) to the inner two whorls in Arabidopsis thaliana. Here, we describe a role of AP2 in promoting the maintenance of floral stem cell fate, not by repressing AG transcription, but by antagonizing AG activity in the center of the flower. We performed a genetic screen with ag-10 plants, which exhibit a weak floral determinacy defect, and isolated a mutant with a strong floral determinacy defect. This mutant was found to harbor another mutation in AG and was named ag-11. We performed a genetic screen in the ag-11 background to isolate mutations that suppress the floral determinacy defect. Two suppressor mutants were found to harbor mutations in AP2. While AG is known to shut down the expression of the stem cell maintenance gene WUSCHEL (WUS) to terminate floral stem cell fate, AP2 promotes the expression of WUS. AP2 does not repress the transcription of AG in the inner two whorls, but instead counteracts AG activity.

An ARID domain-containing protein within nuclear bodies is required for sperm cell formation in Arabidopsis thaliana.

In plants, each male meiotic product undergoes mitosis, and then one of the resulting cells divides again, yielding a three-celled pollen grain comprised of a vegetative cell and two sperm cells. Several genes have been found to act in this process, and DUO1 (DUO POLLEN 1), a transcription factor, plays a key role in sperm cell formation by activating expression of several germline genes. But how DUO1 itself is activated and how sperm cell formation is initiated remain unknown. To expand our understanding of sperm cell formation, we characterized an ARID (AT-Rich Interacting Domain)-containing protein, ARID1, that is specifically required for sperm cell formation in Arabidopsis. ARID1 localizes within nuclear bodies that are transiently present in the generative cell from which sperm cells arise, coincident with the timing of DUO1 activation. An arid1 mutant and antisense arid1 plants had an increased incidence of pollen with only a single sperm-like cell and exhibited reduced fertility as well as reduced expression of DUO1. In vitro and in vivo evidence showed that ARID1 binds to the DUO1 promoter. Lastly, we found that ARID1 physically associates with histone deacetylase 8 and that histone acetylation, which in wild type is evident only in sperm, expanded to the vegetative cell nucleus in the arid1 mutant. This study identifies a novel component required for sperm cell formation in plants and uncovers a direct positive regulatory role of ARID1 on DUO1 through association with histone acetylation.

Intergenic transcription by RNA polymerase II coordinates Pol IV and Pol V in siRNA-directed transcriptional gene silencing in Arabidopsis.

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.