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. Author manuscript; available in PMC: 2018 Nov 14.
Published in final edited form as: Cell Rep. 2018 Sep 25;24(13):3413–3422.e4. doi: 10.1016/j.celrep.2018.08.081

Co-dependent Assembly of Drosophila piRNA Precursor Complexes and piRNA Cluster Heterochromatin

Gen Zhang 1,3, Shikui Tu 2,3,4, Tianxiong Yu 2, Xiao-Ou Zhang 2, Swapnil S Parhad 1, Zhiping Weng 2,*, William E Theurkauf 1,5,*
PMCID: PMC6235161  NIHMSID: NIHMS1508945  PMID: 30257203

Abstract

In Drosophila, the piRNAs that guide germline transposon silencing are produced from heterochromatic clusters marked by the HP1 homolog Rhino. We show that Rhino promotes cluster transcript association with UAP56 and the THO complex, forming RNA-protein assemblies that are unique to piRNA precursors. UAP56 and THO are ubiquitous RNAprocessing factors, and null alleles of uap56 and the THO subunit gene tho2 are lethal. However, uap56sz15 and mutations in the THO subunit genes thoc5 and thoc7 are viable but sterile and disrupt piRNA biogenesis. The uap56sz15 allele reduces UAP56 binding to THO, and the thoc5 and thoc7 mutations disrupt interactions among the remaining THO subunits and UAP56 binding to the core THO subunit Hpr1. These mutations also reduce Rhino binding to clusters and trigger Rhino binding to ectopic sites across the genome. Rhino thus promotes assembly of piRNA precursor complexes, and these complexes restrict Rhino at cluster heterochromatin.

Graphical Abstract

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In Brief

Zhang et al. show that the piRNAgenerating loci in Drosophila female germline are co-dependently assembled between unique chromatin factor Rhino (with Cutoff and Deadlock) and general RNA-processing factor TREX complex

INTRODUCTION

Transposable elements are ubiquitous genome constituents with the potential to mobilize and trigger catastrophic genome instability (Belancio et al., 2008; Hedges and Deininger, 2007; McClintock, 1950). The PIWI-interacting RNA (piRNA) pathway is an adaptive genome immune system that silences transposons and maintains genome integrity during germline development (Aravin et al., 2007; Brennecke et al., 2007; Ghildiyal and Zamore, 2009; Khurana et al., 2010; Siomi et al., 2010, 2011). The 23–30 nucleotide long piRNAs, loaded into PIWI clade Argonaut proteins, direct sequence-specific transcriptional and post-transcriptional transposon silencing (Brennecke et al., 2007; Girard et al., 2006; Gunawardane et al., 2007; Lau et al., 2006; Malone et al., 2009; Vagin et al., 2006).

In the Drosophila female germline, piRNAs are processed from RNA polymerase II (RNA Pol II) transcripts of discrete genomic domains called piRNA clusters, composed of nested transposon fragments, which provide an archive of invading transposon sequences (Andersen et al., 2017; Bergman et al., 2006; Brennecke et al., 2007; Chen et al., 2016; Mohn et al., 2014; Zhang et al., 2012a, 2014). Transposition of an invading mobile element into a cluster is proposed to trigger adaptation, and the system must therefore have ability to process any inserted sequence into piRNAs. Consistent with this flexibility, cluster transcripts do not have well-defined sequence or secondary structure signatures. This is in striking contrast to the precursors for small interfering RNAs (siRNAs) and microRNAs (miRNAs), which form double-stranded structures that are recognized by proprietary processing machines (Brennecke et al., 2007; Ghildiyal and Zamore, 2009; Iwasaki et al., 2015; Kim et al., 2009; Zhang et al., 2012a, 2014). How transcripts from piRNA clusters are distinguished from bulk RNA Pol II transcripts, including pre-mRNAs and mRNAs, remains an open question.

Germline piRNA clusters in Drosophila ovaries are uniquely marked by the heterochromatin protein 1 (HP1) homolog Rhino (Rhi), which associates with Deadlock (Del) and Cutoff (Cuff) to form the RDC complex (Klattenhoff et al., 2009; Mohn et al., 2014; Zhang et al., 2014). These three factors are co-dependent for localization to cluster heterochromatin, drive non-canonical transcription of piRNA clusters from both genomic strands, suppress cluster transcript splicing and polyadenylation, and promote piRNA biogenesis (Andersen et al., 2017; Chen et al., 2016; Klattenhoff et al., 2009; Mohn et al., 2014; Pane et al., 2011; Parhad et al., 2017; Zhang et al., 2014). Like the founding member of the HP1 family, HP1a, Rhi binds to trimethylated lysine 9 of histone 3 (H3K9me3) through its C-terminal chromo domain (Le Thomas et al., 2014; Mohn et al., 2014; Yu et al., 2015). However, H3K9me3 is broadly distributed over heterochromatin, and Rhi localizes specifically to piRNA clusters (Mohn et al., 2014). It is unclear how Rhi distinguishes between H3K9me3 marks on piRNA clusters and bulk heterochromatin.

UAP56 is a ubiquitously expressed DEAD box protein with conserved functions in RNA processing and export, and null uap56 alleles are lethal (Eberl et al., 1997; Gatfield et al., 2001). However, the uap56sz15 point mutation, when combined with a strong hypomorphic allele (uap5628) that produces low levels of wild-type protein, is viable and supports normal gene expression but leads to sterility and disrupts piRNA biogenesis (Meignin and Davis, 2008; Zhang et al., 2012a). We show that this point mutation reduces UAP56 binding to the heteropentameric THO complex (composed of Hpr1, Tho2, Thoc5, Thoc6, and Thoc7), which interacts with UAP56 in the transcription and export (TREX) complex (Chi et al., 2013; Gatfield et al., 2001; Reed and Cheng, 2005; Rehwinkel et al., 2004; Viphakone et al., 2012). Mutations in the thoc5 gene are sterile and disrupt piRNA production and transposon silencing (Hur et al., 2016). We show that this mutation, and a null allele of thoc7, destabilize the THO complex and block UAP56 co-precipitation with Hpr1. We also show that stable binding to both UAP56 and THO is unique to cluster transcripts and that assembly of these pre-piRNA complexes requires Rhi. Significantly, mutations in thoc7, thoc5, and uap56 that disrupt transposon silencing also reduce Rhi binding to major piRNA clusters and trigger ectopic Rhi binding to heterochromatic and euchromatic H3K9me3 marks across the genome. Rhi thus promotes assembly of pre-piRNA complexes containing UAP56, THO, and cluster transcripts, and these complexes restrict Rhi at piRNA clusters. We propose that this feedforward system drives efficient and specific piRNA biogenesis.

RESULTS

The uap56sz15 Allele Reduces Binding to the THO Complex

UAP56 is a conserved DEAD box protein implicated in RNA processing and export, and null mutations in Drosophila uap56 are lethal (Eberl et al., 1997; Gatfield et al., 2001). However, the uap56sz15 point mutation, combined with a strong hypomorphic allele (uap5628) that produces low levels of wild-type protein, is viable but sterile and disrupts germline piRNA biogenesis and transposon silencing (Eberl et al., 1997; Zhang et al., 2012a). We refer to this allelic combination as uap56 mutant in the balance of the text. UAP56 co-localizes with Rhi and binds to cluster transcripts, while the UAP56sz15 protein does not co-localize with Rhi and shows reduced binding to germline cluster transcripts (Zhang et al., 2012a). These findings suggest that uap56sz15 disrupts a protein-protein interaction that is essential to piRNA biogenesis (Zhang et al., 2012a).

To identify proteins that show altered binding to the uap56sz15 gene product, we affinity-purified Venus-tagged UAP56 and UAP56sz15 proteins from wild-type ovaries (Zhang et al., 2012a) and assayed bound proteins by mass spectrometry. The relative abundance of co-precipitating proteins was estimated using iBAQ values (Schwanhäusser et al., 2011), normalized to the Venus tag. We then calculated the average fold difference in protein binding to UAP56sz15venus relative to UAP56venus, from three biological replicates. Figure 1A shows ranked fold differences in protein abundance, with highest fold reduction on the left. All five subunits of the THO complex (labeled red in Figure 1A, inset) rank among the top proteins showing reduced binding to UAP56sz15venus. We independently verified this observation by quantitative western blotting for the THO subunit Hpr1 in UAP56venus and UAP56sz15venus immunoprecipitates (Figures 1B and 1C). For quantification, Hpr1 signal was compared with signal for the Venus tag on the UAP56 fusions. To control for non-linearity in the assay, blots were performed on a series of dilutions of the precipitates. Figure 1C summarizes quantification of four independent biological replicates, each assayed at three different dilutions. These studies revealed a 3-fold reduction in Hpr1 binding to UAP56sz15 relative to wild-type, consistent with our proteomic data. The point mutation in uap56sz15 thus reduces UAP56 binding to the THO complex (Figure 1D).

Figure 1. UAP56-THO Complex Interactions Are Required for piRNA Biogenesis.

Figure 1.

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The uap56sz15 point mutation disrupts piRNA biogenesis. We used IP-mass spectrometry to identify proteins showing altered binding to UAP56sz15 protein relative to wild-type.

(A) Rank-order plot of fold change in protein abundance in UAP56sz15venus precipitates relative to UAP56venus controls. Average fold change was calculated from three biological replicates. The inset shows the top 20 proteins with the highest reduction in binding to UAP56sz15venus.THO subunits are labeled in red.

(B) Western blot for the THO complex protein Hpr1 in UAP56venus and UAP56sz15venus precipitates from wild-type Drosophila ovary lysates. UAP56venus and UAP56sz15venus were detected using rabbit anti-GFP, and Hrp1 was detected with ratanti-Hrp1. The blot was sequentially probed for Hpr1 and GFP, which were detected with distinct fluorescent secondary antibodies. A series of dilutions of each precipitated sample was analyzed.

(C) Plot showing Hpr1 signal normalized to the corresponding Venus tag from four biological replicates. The p values were calculated from t tests.

(D) Schematic representation of TREX complex integrity in wild-type and mutants (uap5628/sz15, thoc5e/1, and thoc7d/Df), on the basis of IP-mass spectrometry.

See also Figure S1 and Tables S1–S3.

THO interacts with UAP56 within the evolutionarily conserved TREX complex and has been previously implicated in germline piRNA biogenesis (Hur et al., 2016; Reed and Cheng, 2005). UAP56 and THO also localize to Rhi nuclear foci (Hur et al., 2016; Zhang et al., 2012a). To determine if THO localization depends on UAP56, we labeled Tho2 in wild-type and uap56 mutant ovaries. In wild-type, Tho2 co-localizes with Rhi nuclear foci throughout oogenesis (Figure S1A, top). In uap56 mutant ovaries, Tho2 shows reduced localization to Rhi foci in early stage egg chambers, and the signal breaks down in later stages egg chambers (Figure S1A, middle). Wild-type interactions between UAP56 and THO are therefore required to maintain THO localization to Rhi nuclear foci.

Intact THO Is Dispensable for Development but Required for piRNA Production

The thoc5e/thoc51 allelic combination (referred to as thoc5 mutant), which disrupts piRNA biogenesis (Hur et al., 2016), appears to be hypomorphic. Low levels of Thoc5 could therefore support zygotic development. By contrast, thoc7d05792 blocks thoc7 splicing and produces no detectable Thoc7 protein, as assayed by western blotting (Figure S1C) (Kim et al., 2011). Furthermore, homozygous thoc7d05792 and hemizygous thoc7d05792/ Df(3L)BSC128 combinations are phenotypically identical (Figure S1C and data not shown). The thoc7d/Df combination thus appears to be null (referred to as thoc7 mutant below). This allelic combination is viable but sterile, disrupts transposon silencing, and reduces germline piRNA production but does not lead to global changes in gene expressions (Figures S1D–S1F). The intact five-member THO complex thus appears to be dispensable for Drosophila development but essential to piRNA production.

To define the role of Thoc7 in assembly of the remaining THO complex components, we used IP-mass spectrometry to characterize proteins that co-precipitate with Hpr1 and Thoc5GFP in wild-type and thoc7 mutants (Tables S1 and S2, respectively). In wild-type ovary extracts, all five THO subunits co-precipitate with both Hpr1 and Thoc5GFP (Tables S1 and S2). In the thoc7 mutant ovaries, in contrast, Tho2 co-precipitates with Hpr1, but UAP56 and other THO subunits are undetectable (thoc7d/Df in Table S1). Similarly, Tho2 was the only subunit that co-precipitated with Hpr1 from thoc5 mutant ovaries (thoc5e/1 in Table S1). In addition, Thoc5 was the only subunit detected when Thoc5GFP was precipitated from thoc7 mutantovaries(thoc7d/DfinTableS2). Finally, no THO subunits co-precipitate with UAP56venus in thoc7 mutant ovaries (thoc7d/Df in Table S3). Consistent with these biochemical observations, Tho2 and UAP56 do not co-localize with Rhi in thoc7 mutant ovaries (Figures S1A and S1B). The results of these proteomic and localization studies are summarized diagrammaticallyinFigure1DandindicatethatlossofThoc7leads to THO complex dissociation into single Thoc5 and Thoc6 subunits and a sub-complex containing Hpr1 and Tho2, which does not stably interact with UAP56.

Hpr1 and Tho2 are the only THO subunits conserved from yeast to humans (Reed and Cheng, 2005), and Drosophila tho2-null alleles are lethal (Jagut et al., 2013). These findings, with the data presented here, suggest that an Hpr1Tho2 sub-complex is sufficient for viability, but intact THO and wild-type THO binding to UAP56 (forming TREX) are essential for piRNA biogenesis. However, weak interactions between UAP56 and THO subunits are unlikely to be preserved during immuno-precipitation and could support zygotic development.

StableBindingtoUAP56andTHOIsSpecifictoGermline piRNA Cluster Transcripts

Previous studies indicate that germline cluster transcripts coprecipitate with UAP56 and Thoc5, while mature mRNAs fail to associate with either protein (Hur et al., 2016; Zhang et al., 2012a). We have confirmed these observations by RNA immuno-precipitation (RIP) with deep sequencing and found that cluster transcripts also co-precipitate with endogenous Hpr1 (Figure 2). To control specificity in these experiments, we expressed and immuno-precipitated GFP alone, performed immuno-precipitation with a non-specific IgG, and quantified enrichment in precipitates relative to input or control RIP. Nearly identical results were obtained in both cases. To simplify presentation, abundance in RIP relative to the corresponding input is shown (Figure 2).

Figure 2. UAP56 and THO Binding Is Unique to piRNA Cluster Transcripts.

Figure 2.

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(A) Genome browser views of Thoc5GFP, Hpr1,and UAP56venus RIP-seq signal, with IP and input controls, over the 42AB piRNA cluster (class I), a large intron in the Doa gene (class II), and mature spliced RNA from the CG7747 gene (class III). Germline cluster transcripts fall into class I and bind to UAP56 and THO complex subunits. A subset of introns define class II and bind strongly to THO but show weak binding to UAP56. Class III includes mature mRNAs and somatic piRNA cluster transcripts.

(B) Scatterplots showing normalized piRNA clustertranscripts abundance (log10[RPKM + 0.1]) in precipitates of Thoc5GFP, Hpr1, and UAP56venus, relative to input. The last panel on the right shows the rank-ordered fold enrichment (RIP/ input) for each cluster in the experimental precipitates and in GFP and non-specific antibody controls (as indicated in panel).

(C) Scatter and rank-order plot, as described in (B), for exon mapping transcript enrichment. Color-coded contour lines reflecting data point density are overlaid on each scatterplot.

(D) Scatter with contour lines and rank-order plots for intron mapping RNA-seq read RIP enrichment.

See also Figure S2.

Consistent with previous studies, 42AB piRNA cluster transcripts, but not mature mRNAs, bind to Hpr1, Thoc5GFP, and UAP56venus (Figure 2A, 42AB and CG7747) (Hur et al., 2016; Zhang et al., 2012a). However, direct visual inspection of our data also revealed significant binding of intronic transcripts to Hpr1 and Thoc5GFP but not with UAP56venus (Figure 2A, Doa). The scatterplots in Figures 2B–2D confirm that this pattern extends across the transcriptome, with cluster transcripts uniformly enriched with Hpr1, Thoc5GFP, and UAP56, intron transcripts enriched with Hpr1 and Thoc5GFP, but not UAP56, and mature mRNAs showing only background binding to these three proteins.

To quantify intron co-precipitation, we computationally defined introns used in ovaries, as previously described (Zhang et al., 2014), and analyzed introns from protein-coding genes with a minimum of 1 read per kilobase of transcript per million mapped reads (RPKM) in the input. In Thoc5GFP and Hpr1 RIP, 18% and 29% of all introns were enriched by more than 2-fold, respectively, and 14% of all introns were enriched by more than 2-fold with both Thoc5GFP and Hpr1. For this subset of introns, the average fold enrichment (over input) with Hpr1 and Thoc5GFP was 7.5 ± 6.8 and 4.5 ± 3.5, respectively. By contrast, the same set of introns showed an average fold enrichment of only 1.7 ± 1.5 with UAP56venus.

The intron mapping sequences bound by THO could represent unspliced pre-mRNAs or free lariats produced by splicing. To distinguish between these alternatives, we quantified enrichment of RNA sequencing (RNA-seq) reads mapping across 5’ splice sites, 3’ splice sites, and spliced junctions. Consistent with binding to unspliced introns, reads mapping across both 5’ and 3’ splice sites were highly enriched with Thoc5GFP and Hpr1, but not UAP56venus, and splice junction mapping reads were not significantly enriched with UAP56, Hpr1, or Thoc5GFP (Figure S2). The THO complex, but not TREX, thus stably interacts with a significant fraction of unspliced pre-mRNAs.

Our studies define three classes of ovarian RNA Pol II transcripts (Figure 2A). Class I is composed of germline cluster transcripts, which are stably bound by THO and UAP56. Class II includes unspliced pre-mRNAs, which are stably bound by THO (defined by Thoc5GFP and Hpr1) but not UAP56. Class III transcripts include mature mRNAs and uni-strand cluster transcripts, which are not stably bound by THO or UAP56. Signifitercantly, our biochemical studies indicate that piRNA biogenesis, but not mRNA expression, requires wild-type interactions between UAP56 and THO. Stable binding by UAP56 and THO is therefore specific to piRNA precursors, and wildtype interaction between these factors is required for piRNA biogenesis.

Rhi Promotes Stable Association of UAP56 with Cluster Transcripts

The Drosophila HP1 homolog Rhi anchors a chromatin complex that promotes cluster transcription (Andersen et al., 2017;Mohn et al., 2014), suppresses clustertranscript splicing (Zhang et al., 2014) and cluster transcript polyadenylation (Chen et al., 2016), and is essential to germline piRNA biogenesis (Klattenhoff et al., 2009; Mohn et al., 2014; Zhang et al., 2014). To determine if Rhi is required for cluster transcript binding to UAP56 and THO, we performed RIP sequencing (RIP-seq) with Thoc5GFP and UAP56venus in rhi2/KG mutant ovaries. This null combination is referred to as rhi mutant in the balance of the text. This combination reduces overall cluster transcription, but clusters continue to be transcribed (Mohn et al., 2014; Zhang et al., 2014). For example, low-level production of unspliced transcripts continues over much of the left side of the 42AB cluster, and high-level transcription of spliced transcripts from an adjacent partial gypsy12 transposon is induced (Figures 3A and 3B, input) (Zhang et al., 2014). In wild-type controls, transcripts over this entire region bind to UAP56venus and Thoc5GFP (Figure 3A). In rhi mutants, the unspliced transcripts stably associate with Thoc5GFP, but not with UAP56venus (Figures 3B and S3A), and spliced gyspy12 transcripts do not precipitate with either Thoc5GFP or UAP56venus (Figure 3B, highlighted on the right). In the absence of Rhi, unspliced cluster transcripts thus mirror unspliced pre-mRNAs, spliced gypsy12 transcripts mimic mature mRNAs, and neither RNA is processed into piRNAs. As shown in the scatterplots in Figure 3C, the rhi mutation essentially eliminates UAP56venus binding to all cluster transcripts but has a relatively modest effect on cluster transcript binding to Thoc5GFP (Figure 3C, rhi2/KG). In contrast, the rhi mutation does not affect Thoc5GFP binding to pre-mRNAs (Figures 3D and S3B, rhi2/KG). Rhi is therefore required for assembly of piRNA precursor complexes containing cluster transcripts, UAP56, and THO.

Figure 3. Rhi Promotes UAP56 Binding to Cluster Transcripts.

Figure 3.

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(A and B) Genome browser view of the left end of the 42AB piRNA cluster.

(A) RIP-seq signal for UAP56venus and Thoc5GFP from wild-type ovaries.

(B) RIP-seq signal for UAP56venus and Thoc5GFP from rhi2/KG ovaries.

The dashed boxes in (A) and (B) indicate a partial gypsy12 element in 42AB that is spliced in rhi2/KG. This region is expanded on the right side of each panel. In wild-type, the unspliced transcripts from this region bind to UAP56venus and Thoc5GFP. In rhi2/KG, unspliced transcripts bind to Thoc5GFP but not to UAP56venus, while the spliced transcripts do not bind either UAP56venus or Thoc5GFP.

(C) Scatterplots comparing cluster transcript abundance (log10[RPKM + 0.1]) in Thoc5GFP RIP (top row) and UAP56venus RIP (bottom row) relative to the corresponding inputs, from rhi2/KG, thoc7d/Df, and uap5628/sz15 ovaries. The box plot summarizes cluster transcript fold enrichment (RIP/input) in wild-type and mutants. The p values were calculated using Wilcoxon rank-sum tests. GFP RIP served as non-specific control.

(D) Scatterplots of intronic transcript abundance(log10[RPKM + 0.1]) in Thoc5GFP RIP relative to input, from rhi2/KG, thoc7d/Df, and uap5628/sz15 ovaries. The box plot summarizes the fold enrichment (RIP/input) in the mutants for the 14% of introns that are enriched by more than 2-fold in both Hpr1 and Thoc5GFP in wild-type (Figure 3). The p values were calculated using Wilcoxon rank-sum tests. GFP RIP served as non-specific control.

See also Figure S3.

To determine if UAP56 and THO subunits are interdependent for binding to cluster transcripts, we performed Thoc5GFP RIP-seq from uap56 and thoc7 mutant ovaries and UAP56venus RIP-seq from thoc7 mutant ovaries (Figure 3C). The thoc7 mutation completely abrogates cluster transcript binding to Thoc5GFP and significantlyreduces cluster transcript binding to UAP56venus (Figures 3C and S3A, thoc7d/Df). The thoc7 mutation also blocks Tho5GFP binding to unspliced introns (Figures 3D and S3B, thoc7d/Df). In uap56 mutant ovaries, in contrast, cluster transcript and intron binding to Thoc5GFP show only modest reductions (Figures 3C, 3D, and S3, uap5628/sz15). Stable THO subunit binding to RNA thus requires an intact THO complex but does not require wild-type interactions with UAP56.

UAP56andTHORestrictRhiatpiRNAClusterChromatin

The studies presented here, with extensive works from a number of laboratories, indicate that Rhi has a central role in producing piRNA precursors (Andersen et al., 2017; Hur et al., 2016; Klattenhoff et al., 2009; Mohn et al., 2014; Zhang et al., 2012a, 2014). The chromo domain of Rhi binds to H3K9me3, and this mark is present at clusters but is also broadly distributed over heterochromatin. Rhi localization to H3K9me3 marks on clusters is therefore critical to the specificity of piRNA biogenesis, but how this is achieved is not understood.

Rhi forms distinct nuclear foci in thoc5, thoc7, and uap56 mutant ovaries (Figure S1) (Hur et al., 2016; Zhang et al., 2012a), but cytological localization is not sufficient to determine if these Rhi foci correspond to clusters. We therefore used chromatin immuno-precipitation sequencing (ChIP-seq) to directly determine Rhi localization in thoc7, thoc5, and uap56 mutants and in w1 controls. Because Rhi binds to H3K9me3, we also profiled this histone modification in all four genotypes. These studies revealed a striking genome-wide redistribution of Rhi in all three mutants (Figure 4). Figure 4A shows Rhi ChIPseq signal over the right arm of Drosophila chromosome 2. In wild-type ovaries, Rhi is highly enriched at the major germline piRNA cluster 42AB (Figure 4A, gray dashed box). Note that H3K9me3 is present at this cluster but is also widely distributed over pericentromeric regions that do not have Rhi signal (Figure 4A, compare w1 red track with blue track). In the three TREX component mutants, in contrast, Rhi shows reduced binding to 42AB and ectopic localization to both pericentromeric heterochromatin and euchromatin (Figure 4A, compare the red tracks in the mutants with the w1 control). Figure 4B shows an enlarged view of the ChIP-seq profiles at 42AB. Rhi is shown for w1 and all three mutants, with H3K9me3 distribution in wild-type. As shown in Figure S4B, the TREX mutants do not significantly alter H3K9me3 at this cluster. We independently confirmed the reduction in Rhi binding to 42AB by ChIP-qPCR (Figure 4D). THO and UAP56 thus restrict Rhi to germline piRNA clusters.

Figure 4. The TREX Complex Restricts Rhi to H3K9me3 Marks on piRNA Clusters.

Figure 4.

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(A–C) Genome browser view of chromosome 2R (A), the piRNA cluster at 42AB (B), and ectopic Rhi peaks at CG6470 (C). Red tracks are Rhi ChIPseq signal from w1, thoc7d/Df, thoc5e/1, and uap5628/sz15. The blue track is H3K9me3 ChIP-seq signal from w1. The annotated piRNA clusters are highlighted in orange. The genomic positions of computationally defined Rhi ChIP-seq peaks for each genotype are color highlighted in the Rhi domain track. In wild-type, Rhi is largely confined to piRNA clusters. In thoc7d/Df, thoc5e/1, and uap5628/sz15, cluster binding is reduced, and new peaks are present across the chromosome arm.

(D) Rhi ChIP-qPCR for 42AB and CG6470, with rp49, cluster2, and flam as controls. Arrowheads in the gene models in (B) and (C) indicate the location of qPCR primers. Significance of ChIP signal in the mutants relative to w1 was determined using t test from four biological replicates. *p < 0.05, **p < 0.001.

(E) Pie charts showing the number of Rhi domains that overlap with annotated piRNA clusters (orange charts) and Rhi domains that overlap with H3K9me3 domains in each genotype (blue charts).

(F) A speculative feedforward mechanism for piRNA cluster heterochromatin assembly. We propose that the RDC, through the Rhi chromo domain, samples H3K9me3 marks throughout the genome, but binding at transcriptional silent chromatin is unstable (1). In contrast, RDC binding to H3K9me3 marks at transcribed piRNA clusters is followed by Cuff association with capped cluster transcripts, which blocks cap binding by the cap binding complex, stalling splicing and stabilizing UAP56 and THO binding (pre-piRNP) (3). Within this chromatin-bound protein-RNA complex, the RDC does not exchange with the soluble pool, driving the complex to H3K9me3 marks on clusters (4). Deadlock then recruits transcription factors (TFIIA-S, Moonshiner, and TRF2) (Andersen et al., 2017) to trigger capped non-canonical transcription on both strands (5), which enhances prepiRNP assembly and RDC localization (6).

See also Figure S4 and Table S4.

In wild-type ovaries, Rhi overlaps with H3K9me3 marks at piRNA clusters (Figures 4A and 4B, Figure S4B for H3K9me3 signal in the mutants) (Klattenhoff et al., 2009; Mohn et al., 2014; Parhad et al., 2017). In thoc7, thoc5, and uap56 mutants, ectopic Rhi peaks also correspond to H3K9me3 domains. In wild-type, these domains are frequently marked by H3K9me3 but do not bind Rhi. Figure 4C shows an example of this class of ectopic locus, which is marked by H3K9me3 in all genotypes (Figure S4B for H3K9me3 signal in the mutants). We independently confirmed Rhi binding at this site by ChIP-qPCR (Figure 4D). To extend this analysis genome wide, we used MACS2 (Zhang et al., 2008) to computationally define high-confidence Rhi domains from two independent biological replicates from each genotype (STAR Methods). The genomic positions of the high-confidence Rhi domains are shown in the “Rhino domain” track in the genome browser views in Figures 4 and S4A. In wild-type ovaries, approximately two-thirds of Rhi peaks map to piRNA clusters (Figure 4E, orange pie chart). Each of the mutants leads to an increase in Rhi peaks mapping outside piRNA clusters (Figure 4E, orange pie charts). Intriguingly, these mutants also increased the number of Rhi peaks mapping to clusters, which reflects increased Rhi binding to a number of minor clusters that normally show only weak Rhi accumulation (Figure S4A), coupled with a general decline in peak intensity at major clusters.

We then used MACS2 (Zhang et al., 2008) to call H3K9me3 domains in each genotype and determined the overlap between Rhi and H3K9me3 domains (Figure 4E, blue pie charts). Strikingly, 92%–94% of Rhi domains overlap with H3K9me3 marks in wild-type and the TREX component mutants. The thoc7, thoc5, and uap56 mutations thus trigger Rhi binding to H3K9me3 domains outside piRNA clusters. In the example shown in Figure 4C, these mutations lead to Rhi binding to a site that is marked by H3K9me3 in all genotypes (H3K9me3 profiles in mutants shown in Figure S4B). However, each of the TREX mutations produces a significant number of new H3K9me3 domains, and Rhi binds to some of these genotypespecific H3K9me3 sites (Table S4). However, 68%–80% of Rhi peaks in the mutants correspond to H3K9me3 domains that are present in control ovaries. Stable TREX is therefore required to restrict Rhi to cluster chromatin and suppresses H3K9me3 modification of other chromatin domains.

Rhi promotes cluster transcription and piRNA biogenesis (Andersen et al., 2017; Klattenhoff et al., 2009; Mohn et al., 2014; Zhang et al., 2014), raising the possibility that ectopic binding may enhance transcription and trigger piRNA production from ectopic loci. We observed a modest increase in steady-state transcript accumulation at ectopic Rhi loci in the TREX mutants (Figure S4C). However, this is not linked to enhanced piRNA production (Figure S4D). These findings suggest that Rhi, presumably acting through the RDC, triggers transcription at ectopic sites (Andersen et al., 2017). However, in the absence of the TREX, Rhi binding cannot induce piRNA production.

DISCUSSION

In Drosophila ovaries, primary piRNAs are derived from heterochromatic clusters composed of nested transposon fragments (Brennecke et al., 2007). These loci serve as an archive of invading transposon sequences, and transposition of an invading element into a cluster is proposed to trigger adaptation, as the inserted sequences are incorporated into cluster transcripts and processed into mature piRNAs (Bergman et al., 2006; Brennecke et al., 2007; Muerdter et al., 2012). Clusters are therefore proposed to determine piRNA pathway specificity and adaptability. However, the mechanisms that specify cluster location and differentiate cluster transcripts from gene transcripts are not understood. We present evidence that cluster chromatin promotes assembly of pre-piRNA complexes defined by stable UAP56 and THO binding and that assembly of these complexes restricts Rhi to clusters chromatin.

UAP56-THO Interactions Are Critical to piRNA Biogenesis

UAP56 and the THO complex are conserved RNA splicing and export factors (Reed and Cheng, 2005), and null alleles of uap56 and the core THO subunit tho2 are lethal (Eberl et al., 1997; Gatfield et al., 2001; Jagut et al., 2013). However, the uap56sz15 allele and mutations in thoc5 and thoc7 are viable but sterile, and disrupt piRNA biogenesis (Hur et al., 2016; Zhang et al., 2012a). We show that the uap56sz15 mutation reduces UAP56 binding to THO and that the thoc5 and thoc7 mutations lead to dissociation of the remaining THO subunits and block interactions between UAP56 and the remaining subunits. High-affinity interactions between UAP56 and the THO complex are therefore required for piRNA biogenesis but dispensable for viability. Intriguingly, Hpr1 binding to Tho2 is retained in viable thoc5 and thoc7 mutants, and Hpr1 and Tho2 are the only THO subunits conserved from yeast to humans (Reed and Cheng, 2005). These observations suggest that an Hpr1-Tho2 heterodimer is sufficient to support basic cellular functions and zygotic development, but this remains to be rigorously tested.

Previous studies indicated that germline piRNA cluster transcripts co-precipitate with UAP56 and the THO subunit Thoc5 (Hur et al., 2016; Zhang et al., 2012a). Here we show that germline cluster transcripts are stably bound by UAP56 and THO, a significant fraction of unspliced pre-mRNAs are stably bound by THO but show only weak binding to UAP56, and mature mRNAs and somatic piRNA cluster transcripts are not enriched with UAP56venus, Hpr1, or Thoc5GFP. Significantly, rhi mutations block stable UAP56 binding to germline cluster transcripts but do not prevent THO binding to cluster transcripts or premRNAs. Rhi is therefore required for stable binding of UAP56 to cluster transcripts, generating complexes that are specific to germline piRNA precursors.

How does Rhi promote assembly of these RNA-protein complexes? Rhi interacts with the linker protein Del and the DXO homolog Cuff, forming the RDC complex, which promotes cluster transcription and suppresses cluster transcript splicing and polyadenylation (Chen et al., 2016; Mohn et al., 2014; Parhad et al., 2017; Zhang et al., 2014). A number of observations suggest that Rhi, functioning through Cuff, may indirectly promote UAP56 binding to cluster transcripts. Recent studies indicate that Rhi recruits transcription initiation factors that drive RNA Pol II transcription from both genomic strands (Andersen et al., 2017). The resulting “non-canonical” transcripts are capped, and cap binding by the nuclear cap binding complex (CBC) promotes efficient splicing and polyadenylation (Proudfoot et al., 2002). However, cluster transcripts are not spliced or polyadenylated (Chen et al., 2016; Zhang et al., 2014). Cuff is a homolog of the decapping enzyme DXO, but the catalytic pocket is not conserved, and residues that interact with the RNA backbone are retained (Zhang et al., 2014). These observations suggest that Rhi localizes Cuff to clusters, where it binds nascent capped transcripts, blocking access to the CBC. This may indirectly suppress splicing and polyadenylation, generating complexes with stably bound THO and UAP56. By contrast, UAP56 appears to only transiently associate with pre-mRNAs, reflected in the weak enrichment observed in our native RIP-seq assays. Supporting this model, cuff mutations that block piRNA production also lead to a pronounced increase in cluster transcript splicing (Zhang et al., 2014).

Rhi binds to H3K9me3 in vitro and co-localizes with H3K9me3 at piRNA clusters (Le Thomas et al., 2014; Mohn et al., 2014; Yu et al., 2015). However, clusters represent only a small fraction of the chromatin marked by H3K9me3 (Mohn et al., 2014), and it is unclear how Rhi is restricted to these specialized domains. Here we show that mutations that disrupt the TREX significantly reduce Rhi association with major piRNA clusters and trigger ectopic Rhi localization to euchromatic and heterochromatic sites that are marked by H3K9me3. Stable interactions between UAP56 and an intact THO are therefore required to restrict Rhi to H3K9me3 marks on piRNA clusters.

A Feedforward Model for Cluster Chromatin Assembly

Knock down of cuff or del essentially eliminates nuclear Rhi foci, and ChIP-seq indicates that Rhi does not bind to clusters or ectopic sites (Mohn et al., 2014). In contrast, Cuff and Rhi still form foci in TREX mutants (Hur et al., 2016; Zhang et al., 2012a), and our Rhi ChIP-seq studies indicate that many of these foci do not correspond to clusters. On the basis of these observations, and the data presented here, we propose that Rhi, within the RDC, samples H3K9me3 marks throughout the genome. However, most of these domains are transcriptionally silent, and RDC binding is unstable (Figure 4F, step 1). piRNA clusters, in contrast, are transcribed and marked by H3K9me3. At these sites, the RDC binds to H3K9me3 through Rhi, and we speculate that capped nascent transcripts are bound through Cuff, which blocks CBC binding, suppressing splicing and polyadenylation (Chen et al., 2016; Zhang et al., 2014) and triggering stable UAP56 binding (Figure 4F, steps 2 and 3). Assembly into these higher order pre-piRNA complexes prevents RDC exchange with the soluble pool (Figure 4F, step 4). Through Del and Moonshiner, the RDC also triggers non-canonical transcription from both genomic strands (Figure 4F, step 5) (Andersen et al., 2017), enhancing RDC binding (Figure 4F, step 6). We note that some cluster transcription persists in rhi mutants, often from one genomic strand, and several major piRNA clusters have flanking canonical RNA Pol II promoters. We speculate that RDC binding to these transcripts initiates this feedforward system, which drives Rhi binding to cluster heterochromatin and promotes piRNA precursor complex assembly.

STAR★METHODS

KEY RESOURCES TABLE

REAGENT or RESOURCE SOURCE IDENTIFIER
Antibodies
Rat anti-Tho2 Rehwinkel et al., 2004 N/A
Rat anti-Hpr1 Rehwinkel et al., 2004 N/A
Rabbit anti-Thoc7 Kim et al., 2011 N/A
Rabbit anti-UAP56 Eberl et al., 1997 RRID: AB_2567529
Guinea pig anti-Rhi Klattenhoff et al., 2009 RRID: AB_2568331
Rabbit anti H3K9me3 Abcam Cat# ab8898
Monoclonal Mouse Anti-α-Tubulin Sigma Aldrich Cat# T5168
Rabbit anti-GFP ThermoFisher Scientific Cat# A11122
Alexa Fluor 563 Goat anti Guinea pig IgG ThermoFisher Scientific Cat# A11075
Alexa Fluor 633 Goat anti Guinea pig IgG ThermoFisher Scientific Cat# A21105
Alexa Fluor 488 Goat anti Rat IgG ThermoFisher Scientific Cat# A11006
Alexa Fluor 488 Goat anti Rabbit IgG ThermoFisher Scientific Cat# R37116
IRDye 800CW Goat anti-Rabbit IgG LI-COR Cat# 926–32211
IRDye 680 Goat anti-Rabbit IgG LI-COR Cat# 926–32221
IRDye 800CW Goat anti-Rat IgG LI-COR Cat# 926–32219
IRDye 800CW Goat anti-Mouse IgG LI-COR Cat# 926–32210
Chemicals, Peptides, and Recombinant Proteins
Superscript III ThermoFisher Scientific Cat# 18080–085
RNase OUT ThermoFisher Scientific Cat# 10777–019
TURBO DNase ThermoFisher Scientific Cat# AM2238
RNaseH ThermoFisher Scientific Cat# 18021–071
T4 RNA Ligase ThermoFisher Scientific AM2141
AccuPrime Pfx DNA Polymerase ThermoFisher Scientific Cat# 12344024
UltraPure Phenol:Chloroform:Isoamyl Alcohol (25:24:1, v/v) ThermoFisher Scientific Cat# 15593031
dNTP Set (100 mM) ThermoFisher Scientific Cat# 10297018
dUTP Solution (100 mM) ThermoFisher Scientific Cat# R0133
Hybridase Lucigen Cat# H39500
dNTP mix NEB Cat# N0447L
DNA polymerase I NEB Cat# M0209S
T4 DNA polymerase NEB Cat# M0203L
Klenow DNA polymerase NEB Cat# M0210S
T4 PNK NEB Cat# M0201L
Klenow 3’ to 5’ exo NEB Cat# M0212L
UDG NEB Cat# M0280S
Phusion Polymerase NEB Cat# M0530S
T4 RNA Ligase 2, truncated NEB Cat# M0242L
50% PEG8000 NEB Cat# B1004S
T4 DNA ligase Enzymatics Cat# L6030-HC-L
16% formaldehyde Ted Pella Cat# 18505
Critical Commercial Assays
mirVANA miRNA isolation kit ThermoFisher Scientific Cat# AM1560
RNeasy Mini Kit QIAGEN Cat# 74104
QuantiTect SYBR Green PCR Kits QIAGEN Cat# 204145
Dynabeads Protein G ThermoFisher Scientific Cat# 10004D
Dynabeads Protein A ThermoFisher Scientific Cat# 10001D
GFP-Trap_A beads Chromotek Cat# gta-100
ANTI-FLAG M2 Agarose Affinity Gel Sigma Aldrich Cat# A2220
RNA Clean & Concentrator-5 Zymo Research Cat# R1015
Agencourt AMPure XP Beckman Coulter Cat# A63880
Deposited Data
Scaffold files for Mass Spec results This study https://doi.org/10.17632/j4yktssk9g.1
High throughput Sequencing This study SRP151054
Experimental Models: Organisms/Strains
D. melanogaster/uap5628 Zhang et al., 2012a N/A
D. melanogaster/uap56sz15 Zhang et al., 2012a N/A
D. melanogaster/thoc7d05792 Harvard Exelixis stock collection Stock# d05792
D. melanogaster/Df(3L)BSC128 Bloomington Drosophila Stock Center Stock# 9293
D. melanogaster/thoc5e00906 Harvard Exelixis stock collection Stock# e00906
D. melanogaster/thoc51 Moon et al., 2011 N/A
D. melanogaster/rhino2 Klattenhoff et al., 2009 N/A
D. melanogaster/rhinoKG Klattenhoff et al., 2009 N/A
D. melanogaster/uap56 promoter > UAP56Venus Zhang et al., 2012a N/A
D. melanogaster/uap56 promoter > UAP56sz15Venus Zhang et al., 2012a N/A
D. melanogaster/thoc5 promoter > Thoc5-GFP Moon et al., 2011 N/A
D. melanogaster/vasa promoter > GFP-nls Zhang et al., 2014 N/A
D. melanogaster/nanos promoter > Gal4 William Theurkauf lab N/A
D. melanogaster/w1 William Theurkauf lab N/A
Oligonucleotides
Random primers ThermoFisher Scientific Cat# 48190011
Primers for ChIP-qPCR, see Table S5 Klattenhoff et al. and This study N/A
Software and Algorithms
Prism 7 GraphPad Prism https://www.graphpad.com/
Image Studio Lite LI-COR https://www.licor.com/bio/products/software/image_studio_lite/
RStudio N/A https://www.rstudio.com/
ImageJ N/A https://imagej.nih.gov/ij/
Scaffold Proteome Software http://www.proteomesoftware.com/products/scaffold/
UCSC Genome Browser Kent et al., 2002 https://genome.ucsc.edu/cgi-bin/hgGateway
Bowtie Langmead et al., 2009 N/A
BEDTools Quinlan and Hall, 2010 N/A
TopHat Trapnell et al., 2009 N/A
BWA Li and Durbin, 2009 N/A
MACS2 Zhang et al., 2008 N/A
Picard Tools N/A http://broadinstitute.github.io/picard/
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CONTACT FOR REAGENT AND RESOURCE SHARING

Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact William Theurkauf (william.theurkauf@umassmed.edu).

EXPERIMENTAL MODEL AND SUBJECT DETAILS

All flies were raised at 25°C on cornmeal medium. All experiments were performed on ovaries from 2–4 days old female Drosophila melanogaster raised in the presence of yeast paste. Ovaries were dissected at room temperature in Robb’s buffer (100 mM HEPES ph 7.4, 100 mM sucrose, 55 mM Potassium Acetate, 40 mM Sodium Acetate, 10 mM glucose, 1.2 mM MgCl2, and 1 mM CaCl2).

METHOD DETAILS

Immunofluorescent staining and image analysis

Fixation and immuno-staining of Drosophila ovaries was performed with Buffer A protocol as described previously (Theurkauf, 1994). The images were acquired with Leica TCS SP8 confocal microscope. The line scan analyses were done in ImageJ (Schneider et al., 2012).

Immuno-precipitation, western blotting, proteomics, and RNA IP

Immuno-precipitation (IP) from ovary lysate was performed as described previously (Parhad et al., 2017). For GFP or Venus tagged proteins IP, GFP-Trap A agarose beads (ChromoTek) and ANTI-FLAG M2 agarose beads (Sigma-Aldrich) were used, respectively. For antibody based IP, the antibody was first conjugated to Magnetic Dynabeads protein A/G (Invitrogen) in Citric phosphate buffer (7.10 g Na2HPO4, 11.5g Citric acid in 1 l water, ph 5.6) for 2 hours at room temperature with rotation. For RNA isolation, beads were resuspended in RTL buffer and the supernatant was processed using the RNeasy mini kit (QIAGEN). The proteomics samples were prepared and processed as described previously (Parhad et al., 2017). The western blots were detected by LI-COR Odyssey Infrared Imaging System. The fluorescent band intensities were quantified using Image Studio Lite (LI-COR).

High-throughput sequencing

Strand specific RNA-seq libraries were constructed as described previously (Zhang et al., 2012b) with modification in the rRNA depletion procedure using enzymatic digestion of rRNA by Hybridase™ Thermostable RNase H (Epicenter) with a comprehensive mixture of antisense rRNA oligos (Fu et al., 2018). The small RNaseq library is constructed as detailed previously (Li et al., 2009) with 2S rRNA depletion as described in (Zhang et al., 2011). The ChIPseq libraries were prepared as described previously (Zhang et al., 2014), with ovaries from 120 females per ChIP. RNaseq and ChIPseq libraries were paired-end sequenced, and small RNaseq libraries were single-end sequenced on the Nextseq 500 platform (Illumina).

ChIP-qPCR

The qPCR was performed using the QuantiTect SYBR Green PCR Kit (QIAGEN) in Step ONE plus real time PCR system (Applied Biosystem). PCR primer sequences were described previously (Klattenhoff et al., 2009; Zhang et al., 2012a) and presented in Table S5. Raw fluorescent amplification intensity from ChIP qPCR was used in DART-qPCR to estimate relative abundance (Peirson et al., 2003). Fold enrichment for precipitates was relative to input, and statistics and graphing was performed using Prism 7 (GraphPad).

Bioinformatics Analysis

The bioinformatics analysis was performed as described previously (Li et al., 2009; Parhad et al., 2017; Zhang et al., 2014). RNA-seq: The raw RNaseq reads were mapped to Drosophila genome (dm3) using TopHat 2.0.8 (Trapnell et al., 2009) with default parameters. The gene annotation was obtained from Flybase r5.50. The piRNA clusters annotation was described previously (Brennecke et al., 2007). The transcript abundance (rpkm: Reads Per Kilobase per Million mapped reads) was counted by BEDTools (Quinlan and Hall, 2010) and normalized to total number of mapped reads, after excluding rRNA mapping reads. The transposon family expression was calculated by proportionally collecting the reads that were aligned to the genomic annotation of transposons. We did differentially expression analysis of transposons and protein-coding genes with edgeR (Robinson et al., 2010) between thoc7d/Df and wild-type from two biological replicates. The replicated RNaseq from wild-type were published previously and deposited in NCBI Sequence Read Archive: SRP111075 (Parhad et al., 2017). The raw small RNaseq reads were mapped to Drosophila genome (dm3) using bowtie (Langmead et al., 2009) by allowing no mismatches after removing the 30 end linker sequence. We counted unique mapped reads for piRNA clusters. The read counts for piRNA clusters were normalized to the number of reads mapping to miRNA.

The raw ChIPseq reads were mapped to Drosophila genome (dm3) using by BWA 0.6.2 (Li and Durbin, 2009). The duplicated reads were discarded as PCR duplication by Picard tools (http://broadinstitute.github.io/picard). We called Rhi domains in each genotype from uniquely mapped ChIPseq reads using MACS2 (Zhang et al., 2008) with following parameters: macs2 callpeak -q 0.01 –ratio 2. For each replicate, Rhi domains within one kilo base pairs were merged. The Rhi domains present in both biological replicates were kept as high confidence Rhi domains for further analysis. Rhi domains mapping to mitochondrial genome were discarded. To quantify the long RNA and small RNA abundance in the Rhi domains across genotypes, the Rhi domain from each genotype are merged and assigned back to each genotype, which is used to quantify the long RNA and small RNA abundance.

QUANTIFICATION AND STATISTICAL ANALYSIS

The statistical method and sample size are reported in the Figures and corresponding legends. The statistical tests for quantitative western and ChIP-qPCR were t test for at least three biological replicates. The statistical tests for deep sequencing data were Wilcoxon Rank-Sum test. The minimal p-value reported was 2.2e-16. We used R and Prism 7 (GraphPad) to do the statistical test.

Supplementary Material

1
2

Highlights.

  • UAP56 and THO complex binding distinguishes cluster transcripts from pre-mRNAs

  • The HP1 homolog Rhino promotes assembly of piRNA precursor complexes

  • Assembly of piRNA precursor complexes restricts Rhino to cluster heterochromatin

ACKNOWLEDGMENTS

We would like to thank the members of the Theurkauf and Weng labs for their insightful discussions and comments throughout the project; Elisa Izaurralde (Max Planck Institute) for anti-Hpr1 and anti-Tho2 antibodies; Yun Doo Chung (University of Seoul) for anti-Thoc7 antibody, thoc51, and Thoc5GFP transgenic stocks; the Bloomington and Harvard Exelixis stock centers for fly strains; and John Leszyk and the Mass Spectrometry Facility of the University of Massachusetts Medical School (UMass Med) for the proteomics service. This work was supported by NIH grants R01 HD049116 and P01 HD078253 to W.E.T.

Footnotes

DECLARATION OF INTERESTS

The authors declare no competing interests.

SUPPLEMENTAL INFORMATION

Supplemental Information includes four figures and five tables and can be found with this article online at https://doi.org/10.1016/j.celrep.2018.08.081.

DATA AND SOFTWARE AVAILABILITY

High-throughput sequencing data was deposited in the NCBI Sequence read archive (SRA: SRP151054). The scaffold files for Mass spectrometry analysis were deposited in Mendeley Data (https://doi.org/10.17632/j4yktssk9g.1)

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Associated Data

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Supplementary Materials

1
NIHMS1508945-supplement-1.pdf (11.9MB, pdf)
2
NIHMS1508945-supplement-2.pdf (14.6MB, pdf)

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