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. Author manuscript; available in PMC: 2012 Feb 23.
Published in final edited form as: Curr Biol. 2011 Aug 4;21(16):1373–1379. doi: 10.1016/j.cub.2011.06.057

piRNA production requires heterochromatin formation in Drosophila

Prashanth Rangan 1, Colin D Malone 1,3, Caryn Navarro 1,4, Sam P Newbold 1, Patrick S Hayes 1, Ravi Sachidanandam 2, Gregory J Hannon 3, Ruth Lehmann 1,5
PMCID: PMC3205116  NIHMSID: NIHMS321560  PMID: 21820311

Summary

Protecting the genome from transposable element (TE) mobilization is critical for germline development. In Drosophila, Piwi proteins and their bound small RNAs (piRNAs) provide a potent defense against TE activity. TE targeting piRNAs are processed from TE-dense heterochromatic loci termed ‘piRNA clusters’. While piRNA biogenesis from cluster precursors is beginning to be understood, little is known about piRNA cluster transcriptional regulation. Here we show that deposition of histone 3 lysine 9 by the methyltransferase dSETDB1 (egg) is required for piRNA cluster transcription. In the absence of dSETDB1, cluster precursor transcription collapses in germline and somatic gonadal cells and TEs are activated, resulting in germline loss and a block in germline stem cell differentiation. We propose that heterochromatin protects the germline by activating the piRNA pathway.

Results and Discussion

Germline stem cells (GSCs) are unique in that they self-renew but also generate progeny that differentiate into gametes, which give rise to the next generation. During GSC differentiation the genome is acutely vulnerable to chromosomal breaks, either due to meiotic recombination or TE mobilization [1, 2]. To maintain germline genome integrity, organisms have evolved two distinct pathways: checkpoint monitoring of double stranded DNA breaks and transposon suppression via a specialized RNA interference (RNAi) system involving Piwi proteins and their bound small RNAs [3, 4]. There are two RNAi based systems that utilize small-RNAs to target TEs for degradation: the Piwi-interacting small RNA (piRNA) pathway, and the endogenous-small interfering RNA (endo-siRNA) pathway. piRNAs act as the primary defense in the germline [5], while siRNAs act throughout the organism [6],[7]. In germline cells of the Drosophila gonad, piRNAs are bound by three Piwi-clade Argonaute proteins: Piwi, Aubergine (Aub) and Argonaute 3 (AGO3). Piwi and Aub bind piRNAs complementary to active TEs and target them for degradation. This cleavage leads to the production of new piRNAs, which are loaded into AGO3 and target cleavage of antisense transposon transcripts, which produces more small RNAs for Aub and Piwi to bind. These antisense transcripts are derived from piRNA clusters, which typically reside in subtelomeric and pericentric heterochromatin regions, and contain a high-density of fragmented, ancient transposon copies [8, 9]. TEs are also silenced by piRNAs in somatic tissues of the gonad where only Piwi is expressed [10, 11]. While piRNA clusters predominantly produce piRNAs, many also produce significant levels of endo-siRNAs [12, 13], which are processed via a genetically independent machinery. How these transcripts are generated from piRNA clusters is unclear.

To determine whether heterochromatin formation and integrity may play a role in TE regulation during oogenesis, we examined the distribution of trimethylated H3K9 (H3K9me3). This prototypic repressive mark initiates heterochromatin formation by recruiting heterochromatin protein 1 (HP1) (Fig. 1A)[14]. We focused on the GSCs and their niche located at the anterior tip of the ovary in a region called the germarium (Fig. 1B) [15, 16]. During GSC division, one daughter cell maintains contact with the niche and continues to divide as a stem cell, while the other differentiates. As the differentiating daughter cell (pre-cystoblast) moves away from the niche it initiates expression of the differentiation factor Bag of Marbles (Bam) (Fig. 1B) [17, 18]. Subsequently, this daughter, now called the cystoblast, undergoes four mitotic divisions to form an interconnected sixteen-cell germline cyst, producing one oocyte and 15 supporting nurse cells (Fig. 1B)[15, 16]. We observed that while H3K9me3 signal was low in GSCs, as soon as the differentiating daughter left the niche we detected prominent, discrete nuclear H3K9me3 positive foci (Fig. 1C). Once established, these H3K9me3 foci, which typically associated with DAPI-dense regions, persisted throughout oogenesis (Fig. S1A-B2). We termed these foci “Repressive Chromatin Centers” (RCCs). RCCs possess heterochromatic character as they not only contain H3K9me3, but also other heterochromatin markers such as HP1 and tri-methylated histone 4 lysine 20 (H4K20me3) (Fig. 1D)[14, 19, 20].

Figure 1. dSETDB1 mediates formation of heterochromatic RCCs in the pre-cystoblast and is required for GSC differentiation.

Figure 1

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(A) Schematic of dSETDB1 function. (B) Schematic of the GSC niche. (C–D) Germaria of wild-type marked by Vasa (green), DAPI (blue) and (C) anti-H3K9me3 (red) (D) anti-H4K20me3 (red) showing discrete foci in the germline (white arrow). White asterisks mark GSCs and white stippled line mark the pre-cystoblasts. Wild-type pre-cystoblasts contain 3.6 ± 0.4 nuclear foci with one large focus (n=15). RCCs associate with (E) centromere marker CID (green) (n = 20), (F) telomeric cap marker HOAP (n = 80) showing the proximity of these regions to RCCs. (G) Schematic of a RCC (red) showing large heterochromatic domain associated with centromeric (green) and telomeric regions (green). Germaria of (H) bam and (I) zpg mutants stained for anti-Vasa (green), anti-H3K9me3 (red) and DAPI (blue) showing GSCs lack prominent RCCs. egg1473/ Df(2R)Dll-Mp mutant germarium stained for anti-Vasa (green), DAPI (blue) lack RCCs stained for by both (J) anti-H3K9me3 and (K) anti-H4K20me3. Germaria of (L) egg1473/CyO (M) egg1473/ Df(2R)Dll-Mp stained for with anti-Vasa (blue) and anti-1B1 (red), which marks the spectrosomes, shows that dSETDB1 mutants accumulate undifferentiated cells (2.7 ± 0.8, n = 20 respectively and 7.9 ± 3.5, n = 46). (N-O) Germaria of (N) egg1473/CyO and (O) egg1473/Df(2R)Dll that also carry Bam:GFP transgene stained for anti-1B1 (red), anti-Vasa (blue), anti-GFP (green) showing that dSETDB1 mutant germaria do not express Bam GFP. Bottom schematics summarizes confocal images above. See also Figure S1

To correlate H3K9me3 with heterochromatic regions in vivo we asked whether RCCs are located in proximity of centromeric and telomeric regions. Centromeres can be visualized by immunohistology of CENP-A H3-like proteins (CID)[21], and the telomeric caps by HP1 origin recognition complex subunit 2 (HOAP)[22]. When we stained for RCCs, by H3K9me3 or H4K20me3, we found a partial overlap with HOAP, and a close association with CID in germline cells throughout oogenesis (Fig. 1E-F). Repressive foci similar to RCCs are also present in the somatic cells that surround and associate with germline cells throughout oogenesis (Fig. S1B-B2). This suggests that telomeres, centromeres and their adjacent regions are organized into large heterochromatic structures in the gonads (Fig. 1G).

To more precisely determine the developmental sequence of RCC formation, we analyzed two genetic mutants: a GSC differentiation defective mutant, Bag of marbles (bam) and a mutant that lacks differentiated progeny, zero population growth (zpg) [17, 18, 23]. In the wild type, GSCs are maintained by decapentaplegic (DPP), a Drosophila BMP2/4 ortholog secreted from the niche [24]. Activation of the Dpp receptor, Thickveins (Tkv), directly represses transcription of bam [25]. In bam mutants, single undifferentiated germ cells accumulate into a tumorous mass. Based on the expression of Dpp reporters, only those germ cells closest to the niche, the GSCs, respond to the Dpp signal, while the pre-cystoblasts do not [26, 27]. Consistent with our observations in the wild-type ovary, in bam mutant ovaries, we detected low H3K9me3 signal in a few cells which corresponded to GSCs as judged by their proximity to the niche, while the tumor composed of pre-cystoblasts displayed prominent RCCs (Fig. 1H). In zpg mutant germaria, pre-cystoblasts die upon completing cell division, leaving only GSCs [17, 23]. Again, we observed that GSCs in zpg mutant germ cells showed little H3K9me3 staining (Fig. 1I). We conclude that prominent heterochromatin marks are established in the germline as GSCs differentiate.

We next asked whether mutants in Drosophila H3K9 methyltransferase affected RCC formation and GSC differentiation. We chose to study dSETDB1 (egg), one of two Drosophila H3K9 methyltransferases which had previously been shown to be required for deposition of H3K9me3 in the germarium and for female fertility [14, 28]. We found that H3K9me3 and H4K20me3 were absent in dSETDB1 mutants (Fig. 1J-K)[14]. To determine if dSETDB1 is required for the GSC to cystoblast transition we stained wild-type and dSETDB1 mutant ovaries with antibodies against the germ cell marker Vasa, and the spectrosome marker 1B1. Spectrosomes are present in GSCs, the intermediate pre-cystoblast, and the differentiating cystoblast (Fig. 1B)[29]. 1B1 also stains the fusome, a branched organelle found in differentiating multicellular cysts (Fig. 1B)[29]. dSETDB1 mutant ovaries contained an increased number of spectrosome-positive cells compared to wild-type (Fig. 1L-M) and failed to develop fusome-containing cysts (Fig. 1M). The undifferentiated cells in dSETDB1 mutants did not express the differentiation marker Bam but did express phosphorylated Mad (pMad), the mediator of the Dpp signal and a marker of GSC in the wild type (Fig. 1N-O, Fig. S1C-F), suggesting an accumulation of “GSC-like” cells [26, 27]. To determine whether these “GSC-like” cells were capable of differentiating, we forced expression of Bam under the control of a heat shock promoter and observed the development of multi-cellular cysts, as revealed by the formation of fusomes (Fig. S1G-H). This accumulation of “GSC-like” cells is not due to change in niche size or increased GSC division (Fig. S1I-J)[28]. We conclude that dSETDB1 is required for Bam-dependent differentiation of GSCs.

Because we observed repressive marks in both the soma and germline, we wanted to determine where dSETDB1 was required for GSC differentiation. We performed tissue-specific knockdown of dSETDB1 using an inducible short hairpin RNA (shRNA) [30]. We crossed flies carrying shRNA-dSETDB1 under the GAL4 responsive UAS promoter with flies carrying the germline specific nos-Gal4::VP16 (Fig. 2A)[31] or C587-Gal4, which drives GAL4 in the somatic inner sheath cells that intermingle with germ cells (Fig. 2B,) [32]. We found that reducing dSETDB1 levels in either tissue resulted in females that laid few eggs (Fig. S2A) (Table S1). To determine the cause of this ovarian defect we stained for Vasa and 1B1. For the germline-knockdown we observed an age-dependent increase in the phenotype, as has been observed with other shRNAs using germline specific drivers [30]. In 2–3 day-old flies, GSCs were present and we observed a few differentiating cysts, most of which degenerated leaving only a few mature egg chambers (Fig. 2A1-C), which frequently developed into eggs with dorso-ventral patterning defects (spindle phenotype) (Table S1). In 7–10 day-old flies, undifferentiated germ cells accumulated as seen in the genetic mutant (Fig. 2A2-C). For the somatic knockdown, we observed an age-independent accumulation of undifferentiated cells resembling the dSETDB1 mutant phenotype. (Figure 2B1-C). Based on these results, we conclude that dSETDB1 expression in both germline and germarial somatic cells, is required for oogenesis and differentiation.

Figure 2. dSETDB1 is required in both soma and germline and piRNA clusters carry H3K9me3 marks.

Figure 2

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(A) Schematic of expression pattern of nos Gal4 in the germarium. (A1) Germaria after germline specific dSETDB1 knockdown shows a block in differentiation at the cyst stage after 2–3 days (white arrow). (A2) After 7–10 days germaria show an accumulation of undifferentiated cells monitored by spectrosomes (red). (B) Schematic of expression pattern of c587 Gal4 in the germarium. (B1) Germarium after soma-specific dSETDB1 knockdown shows an accumulation of undifferentiated cells as soon as 2–3 days after eclosion. (C) Quantification of the phenotypes 2–3 days and 7–10 days in soma- and germline-specific knockdown of dSETDB1. Germaria containing tumors (black), degenerating after 16 cell-cyst stage (dark gray), before stage 6 (light gray), ovarioles that had lost stem cells (white). (D) Distribution of repressive marks (red) on various repeat elements expressed as a percentage of total H3K9me3 marks (multiple mappers). (E) Representative examples of the distribution of repressive marks showing enrichment of repressive marks on the 4th chromosome telomeric piRNA cluster (bottom panel) compared to nanos (top panel) (unique mappers). Repeat elements as annotated by repeat master are shown as black lines. See also Figure S2

To gain more insight into the germline and somatic function of dSETDB1, we followed RCC formation using the heterochromatin marker H4K20me3 [19, 33]. In the few late stage egg chambers that formed in 2–3 day old flies from the germline dSETDB1 knockdown, we observed a loss of H4K20me3 staining specifically in the germline but no change in the soma, indicating that loss of germline dSETDB1 does not affect H4K20me3 foci in somatic cells (Fig. S2B-D2). However, in the somatic knock down (Fig. S2E-E2), we observed a loss of repressive marks in both the germline and soma indicating that the function of dSETDB1 in the soma affects germline RCC formation, possibly by blocking GSC differentiation. Thus dSETDB1 acts to regulate heterochromatin formation in the germline directly and indirectly through its somatic function.

To identify genomic targets of dSETDB1 in oogenesis we profiled genome-wide H3K9me3 occupancy by ChIP-seq in the wild-type ovaries. We used the entire ovary because RCCs once formed are stably maintained throughout oogenesis. After chromatin-immunoprecipitation with H3K9me3 antibody, the recovered DNA was cloned, sequenced and mapped to the Drosophila genome. H3K9me3 was highly enriched at transposons and other repetitive loci (~90%) (Fig. 2D). Because of the repetitive nature of transposable elements we analyzed uniquely mapping sequence reads to the genome and compared these to the actively transcribed germline gene nanos, as a negative control. We found that piRNA clusters, which are mostly present in the pericentric or subtelomeric heterochromatin, but not in actively transcribed genes such as nanos, were marked by H3K9me3 (Fig. 2E) (Fig. S3A-C).

To determine how H3K9 methylation affects piRNA production, we cloned and sequenced small RNAs from dSETDB1 mutant ovaries, which lack RCCs, and compared them to bam mutants as a control, which display a morphologically similar phenotype but form prominent RCCs (Fig 1H-K). To account for differences in sequencing quality and depth, small RNAs were normalized to gene-derived, antisense mapping, endogenous small-interfering RNAs (endo-siRNAs), similar to previously published analyses [10]. piRNA (23-29nt) levels were significantly reduced in dSETDB1 mutant ovaries (Fig. 3A-C), along with endo-siRNAs derived from major germline and somatic piRNA clusters (Fig. 3D). This loss of endo-siRNAs is specific to piRNA clusters as production from an euchromatic endo-siRNA producing locus, esi-2 [12, 13, 34], which is also marked by H3K9me3, increases in dSETDB1 mutants (Fig. S3D-E). This increase in esi-2 endo-siRNAs is likely due to the loss of silencing chromatin marks at this locus, allowing for increased transcription and likewise, increased siRNA production. This is in contrast to the heterochromatic piRNA clusters, which show a dramatic loss of both si- and piRNAs from all clusters examined, suggesting a direct defect in cluster transcription rather than an effect on downstream transcript processing by either the si- or piRNA machinery. Therefore, we sought to directly test the effect of dSETDB1 loss on piRNA cluster transcription.

Figure 3. dSETDB1 is required for piRNA production and transposon control.

Figure 3

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(A) Size profiles of small RNA populations in bam (black) and dSETDB1 (red) mutant ovaries. Sequence reads were normalized to number of gene-derived, antisense mapping, endo-siRNAs (B) Quantification, via bar graph, showing the Log2-fold changes in total normalized, uniquely-mapping cluster-derived small RNA (piRNAs + siRNAs) levels, comparing dSETDB1 (red) to bam mutant levels. (C) Genome-uniquely mapping piRNAs (23-29 nt), normalized to antisense, genic endo-siRNAs, from bam (black) and dSETDB1 (red) mutant ovaries are plotted across 42AB and flamenco piRNA clusters. Reads above the x-axis indicate mapping ‘sense’ to the genomic sequence, while reads below indicated ‘antisense’ mapping. (D) Genome-uniquely mapping endo-siRNAs normalized to antisense, genic endo-siRNAs, from bam (black) and dSETDB1 (red) mutant ovaries are plotted across 42AB and flamenco piRNA clusters (E) qPCR measuring transcripts from genomic sense (grey) and genomic antisense (black) in dSETDB1 mutants and bam mutants showing reduction of antisense transcripts from 42AB cluster and sense from Cluster 2 and flamenco in dSETDB1 mutants. (F) Germline transposons (HeT-A, TART) and somatic transposons (gypsy, Zam) measured by qPCR are specifically up-regulated in dSETDB1 mutants relative to bam mutant. (G) Germline transposons (HeT-A, TART) and somatic transposons (gypsy, Zam) measured by qPCR are preferentially up-regulated in the germline and somatic knockdowns respectively. See also Figure S3

To determine if the loss of piRNAs in dSETDB1 mutants is due to a loss of precursor transcription, we carried out strand-specific qPCR to measure transcript levels from the germline specific 42AB cluster, germline and somatic expressed Cluster 2, and the soma specific flamenco cluster [35]. The 42AB cluster contains a mixture of sense and antisense oriented transposon fragments that requires bidirectional transcription to ensure antisense transposon transcript production for the piRNA pathway[8] We found that production of at least one strand of 42AB cluster is affected in dSETDB1 mutants compared to bam mutants. In Cluster 2 and flamenco, which contain transposon fragments oriented in the same direction, unidirectional transcription is sufficient to generate antisense transposon precursors [8]. We also observed diminished transcription in dSETDB1 mutants compared to bam mutants (Fig. 3E). qPCRs measuring native transcript levels in Bam mutants probably under-represent wild-type levels of cluster transcripts because they are processed by the active piRNA and siRNA pathways. Thus, dSETDB1 is required for transcription of both bi- and uni-directionally transcribed clusters, in both germline and somatic tissues of the gonad. This is in stark contrast to what is seen for the HP1 homolog, Rhino, which is selectively required for the transcription of only bidrectional clusters and specifically so in germline cells [35]. Therefore, we have identified a unifying mark dictating piRNA cluster transcription prior to piRNA production in germline and somatic cells. Our results are consistent with Rhino recognizing H3K9me3 at bi-directional germline clusters, but there must then be an alternative Rhino-independent mechanism directing somatic and uni-directional germline cluster transcription. These results strongly suggest that repressive marks deposited by dSETDB1 are required for transcription from all major piRNA clusters, although how these marks are targeted to piRNA clusters remains unknown.

Finally we wanted to assess whether loss of piRNA function in dSETDB1 mutants also results in increased TE levels, as has been shown for piRNA pathway mutants involved in post-transcriptional piRNA processing. We assessed TE levels in dSETDB1 mutant ovaries, again using bam mutants as a control. In dSETDB1 mutants we found higher levels of both germline (HeT-A and TART) and somatic (gypsy and ZAM) expressed transposons by qPCR of steady-state transcript levels (Fig. 3F). To determine if dSETDB1-dependent H3K9me3 protects germline and somatic cells separately, we knocked down dSETDB1 in either germline or somatic cells of the ovary as before. As expected, germline knockdown led to the preferential de-repression of germline transposons, while somatic knockdown resulted in the selective de-repression of somatic transposons (Fig. 3G). Therefore, dSETDB1 plays a critical role in silencing transposons in both the germline and somatic cells of the ovary.

Our results indicate that dSETDB1 is required for transposon control in the germline and ovarian soma by positively regulating piRNA cluster transcription through the deposition of H3K9me3. To test directly whether transposon de-repression in either the germline or soma is sufficient to cause a block in GSC differentiation, we utilized two models of hybrid dysgenesis. First, we used the germline specific P-element model of hybrid dysgenesis , which results in de-repression of P-element DNA transposons and progeny sterility when a male carrying a copy of an active P-element transposon (Harwich) is crossed to a female devoid of P-elements (w1118) [36] [37]. This was attributed to the absence of maternally supplied piRNAs capable of silencing this transposon [38]. Second, to test for somatic de-repression, we used flamenco mutant lines, which ablate production from the primary somatic piRNA cluster, flamenco, resulting in de-repression of gypsy-family transposable elements [41, 42]. We found that in both cases germaria accumulate undifferentiated cells similar to those observed in dSETDB1 mutants (Fig. 4A-B,D). As with dSETDB1 mutants, these undifferentiated cells do not express Bam, but do stain positive for pMad (Fig. 4A, D-D2). These results indicate that transposon mobilization alone in either germline or soma is sufficient to cause a block in GSC differentiation. Additionally, we observed that the loss of GSC differentiation during P-element dysgenesis can be relieved by removing the Chk-2 kinase, suggesting that transposon de-repression activates a double stranded DNA break checkpoint (Fig 4B-C) [1]. Interestingly, it is known that through viral-packaging, some gypsy-family elements maintain the ability to infect germline cells from the surrounding soma [41, 43], leaving open the possibility that the GSC differentiation block in flamenco mutants is due to gypsy invasion and mobilization within differentiating germline cells. Alternatively, loss of piRNA production from the flamenco locus, as in piwi mutants, could result in the loss of somatic cells that surround germ cells and provide cues for differentiation. Thus transposon upregulation in the germline and soma in dSETDB1 mutants is sufficient to cause a loss of differentiation phenotype.

Figure 4. Up regulation of transposons is sufficient to cause a block in differentiation.

Figure 4

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(A) Germarium of progeny of Harwich males crossed to w1118 females carrying the Bam-GFP transgene stained for anti-Vasa (blue) anti-1B1 (red) and anti-GFP (green) accumulate cells with spectrosomes (red) that do not express Bam (white bracket). Faint Bam-GFP expression is seen at the edge of these tumors (white arrow). Germarium of (B) progeny of Harwich males crossed to w1118 females stained for anti-Vasa (green) and anti-1B1 (red) accumulate undifferentiated cells (white bracket). (C) Removing a copy of chk-2 from this cross partially rescues the block in differentiation with fusomes (white arrow) connecting differentiating cysts cells. (D) Germaria of flamenco mutants stained for 1B1 (blue), Vasa (green) and pMad (red) showing an accumulation of “GSC-like” undifferentiated cells. D1 and D2 are IB1 and pMad channels respectively. (E) Schematic of dSETDB1 regulating piRNA production from both somatic and germline clusters, which in turn suppress transposon expression. Transposon expression either activate a checkpoint in the germline or leads to loss of some somatic cells (intermingling cells in the gonad) leading to a block in GSC differentiation.

From Drosophila to humans, a large fraction of eukaryotic genomes contain transposable elements. Here, we find a novel role for dSETDB1-mediated heterochromatin formation in activating transcription of piRNA clusters and thus triggering piRNA-based control of transposon regulation (Fig. 4E). Interestingly this transcriptional upregulation of the germline piRNA pathway happens at the time when transcription is generally upregulated in the germline to permit differentiation [44], potentially also leading to increases in transposon transcription. Thus production of piRNAs needed to keep transposon activity in check is timed to occur when they are likely most needed to protect the integrity of the genome of the next generation.

Supplementary Material

01

Acknowledgements

We are particularly grateful to all members of the Lehmann lab for discussion and extensive comments on the manuscript and in particular Dr. Daria Siekhaus, Dr. Allison Blum, Dr. and Dr. Thomas Hurd for comments and discussion. We also thank Dr. Alexander Stark for computational assistance. We thank the TRiP at Harvard Medical School (NIH/NIGMS R01-GM084947) for providing transgenic RNAi fly stocks used in this study. We would like to thank the following for sharing fly stocks and antibodies: Dr. Tulle Hazelrigg (egg) as well as the Drosophila Bloomington stock center and Flybase. P.R. is an HHMI Research Associate. R.L. is an HHMI investigator and a member of the Kimmel Center for Stem Cell Biology at NYULMC. G.H. is an HHMI investigator.

Footnotes

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