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, 151 (5), 964-80

Transcriptional Silencing of Transposons by Piwi and Maelstrom and Its Impact on Chromatin State and Gene Expression

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Transcriptional Silencing of Transposons by Piwi and Maelstrom and Its Impact on Chromatin State and Gene Expression

Grzegorz Sienski et al. Cell.

Abstract

Eukaryotic genomes are colonized by transposons whose uncontrolled activity causes genomic instability. The piRNA pathway silences transposons in animal gonads, yet how this is achieved molecularly remains controversial. Here, we show that the HMG protein Maelstrom is essential for Piwi-mediated silencing in Drosophila. Genome-wide assays revealed highly correlated changes in RNA polymerase II recruitment, nascent RNA output, and steady-state RNA levels of transposons upon loss of Piwi or Maelstrom. Our data demonstrate piRNA-mediated trans-silencing of hundreds of transposon copies at the transcriptional level. We show that Piwi is required to establish heterochromatic H3K9me3 marks on transposons and their genomic surroundings. In contrast, loss of Maelstrom affects transposon H3K9me3 patterns only mildly yet leads to increased heterochromatin spreading, suggesting that Maelstrom acts downstream of or in parallel to H3K9me3. Our work illustrates the widespread influence of transposons and the piRNA pathway on chromatin patterns and gene expression.

Figures

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Figure 1
Figure 1
Maelstrom Is Required for Piwi-Mediated Silencing, but Not for piRNA Biogenesis (A) Shown are β-gal stainings of egg chambers as readout for gypsy silencing. w[1118] flies or indicated VDRC lines for piwi or mael were crossed to tj-GAL4, gypsy-lacZ flies carrying a restrictive flamenco background. (B and C) Displayed are fold changes in steady-state RNA levels of indicated TEs in mael or armi KD ovaries (B, soma; C, germline). Values are averages of three biological replicates (error bars represent SD) and are normalized to control knockdowns. (D) Confocal section of a follicular epithelium stained for Piwi (magenta) and Armi (red) in which armi-RNAi has been clonally activated (marked by GFP; boundary indicated by dashed yellow line). (E) Confocal section of a follicular epithelium stained for Piwi (magenta) and Mael (red) in which mael-RNAi has been clonally activated (marked by GFP; boundary indicated by dashed yellow line). (F) Western blot showing protein levels of Piwi, Armi, Mael, and Tubulin in OSCs transfected with indicated siRNAs. (G) Fold changes in steady-state RNA levels of indicated TEs from OSCs transfected with siRNAs against indicated gene (normalized to siGFP; n = 3; error bars represent SD.) (H) Northern blots showing levels of indicated piRNAs in total RNA from OSCs transfected with siRNAs against indicated genes. To the left, an RNA size marker (nt) is shown. The miR-311 blot serves as loading control. (I) Radiogram of polyacrylamide gel in which Piwi-bound small RNAs (CIP-kinase labeled) isolated from equal amounts of OSCs transfected with indicated siRNAs were separated. RNA size marker (nt) is indicated to the left. (J) Shown are levels of indicated piRNA populations from ovaries of indicated genotypes (normalized to sequenced miRNAs and to respective heterozygotes). Contrasted are piRNA levels from mael−/− ovaries (blue) to piRNA levels from ovaries lacking indicated primary piRNA biogenesis factors (red). See also Figure S1.
Figure 2
Figure 2
Piwi/Mael-Mediated TE Silencing Is a Nuclear Process (A) Cartoon showing protein domains of GFP-tagged Piwi and the residues that have been altered for the mutant analysis. (B) Confocal images of egg chambers expressing the indicated GFP-Piwi constructs in piwi[1]/piwi[2] background. (C) Fold changes in steady-state RNA levels of actin5C and indicated TEs from ovaries expressing indicated GFP-Piwi constructs in piwi[1]/piwi[2] background (normalized to piwi[1]/piwi[2]; GFP-Piwi; n = 3; error bars represent SD). (D) Cartoon showing protein domains of GFP-tagged Mael and the residues that have been altered for the mutant analysis. (E) Confocal images of egg chambers (left) or OSCs (right) expressing the indicated GFP-Mael constructs (GFP) in wild-type background. (F) Summary of the rescue experiments (egg laying and egg hatching) with indicated GFP-Mael constructs in mael[391]/mael[Def] background. (G) Fold changes in steady-state RNA levels of actin5C and indicated TEs from ovaries expressing indicated GFP-Mael constructs in mael[391]/mael[Def] background (n = 3; error bars represent SD).
Figure 3
Figure 3
The piRNA Pathway Silences TEs at the Transcriptional Level (A) Experimental scheme of genome-wide profiling experiments performed for this study. (B) Scatter plot of RPKM values (log2) for all TEs (n = 125) in GFP (control) or piwi knockdown samples based on RNA-seq. Four TE groups are color coded. (C) Scatter plot of RPKM values (log2) for all TEs (n = 125) in GFP (control) or mael knockdown samples based on RNA-seq. Four TE groups are color coded. (D) Displayed are fold changes of TE expression (groups I–III; colors as in B) in OSCs transfected with indicated siRNAs (normalized to control cells) at the level of steady-state sense RNA (RNA-seq; heatmap), Pol II occupancy (ChIP-seq), or nascent sense RNA (GRO-seq). The piRNA-seq diagram indicates Piwi-bound piRNA levels mapping antisense to indicated TEs. (E) Density profiles of normalized reads from RNA-seq (top), Pol II ChIP-seq (middle), and GRO-seq (bottom) experiments on mdg1 (group I) and F-element (group III). Orange line indicates levels in control cells, and solid signal indicates levels in piwi KD cells. (F–H) Box plots showing fold changes (log2) in the expression of group I, group II, and group III TEs based on RNA-seq (F), Pol II ChIP-seq (G), or GRO-seq (H) upon piwi KD (compared to control; p values based on Wilcoxon rank-sum test). Box plots show median (line), 25th–75th percentile (box) ± 1.5 interquartile range; circles represent outliers. Contrasted are sense and antisense reads (RNA-seq and GRO-seq) and IP versus input (Pol II ChIP-seq). See also Figure S2.
Figure 4
Figure 4
Piwi-RISC Mediates TGS of TEs in Ovarian Somatic Cells (A) Scheme of a Drosophila ovariole and an individual egg chamber (somatic cells in green, germline cells in beige). Indicated is the classification of TEs according to Malone et al. (2009). (B) Scatter plot of Pol II ChIP-seq RPKM values (log2) for all TEs (n = 125; color code as in A) from control KD ovaries (tj-GAL4 > RNAi tej) versus armi KD ovaries (tj-GAL4 > RNAi armi). (C) Density profiles of normalized Pol II ChIP-seq reads on ZAM and gypsy (soma dominant) and Burdock and HeT-A (germline dominant). Orange line indicates levels in control, and solid signal indicates levels in armi KD ovaries. (D) Box plots indicating fold enrichments (log2) of Pol II ChIP-seq reads on TEs belonging to the indicated classes. Contrasted are IP (Pol II) versus input (p values based on Wilcoxon rank-sum test). Box plots are as in Figure 3. (E) Normalized Pol II ChIP-seq read density on the gypsy-lacZ reporter in control ovaries (black line) versus armi KD ovaries (red line). Small inset displays the fold change (armi KD versus control) of Pol II occupancy on the reporter. (F) Shown to the left are β-gal stainings of egg chambers from gypsy-restrictive ovaries (top) and gypsy-permissive ovaries (bottom) harboring the gypsy-lacZ reporter (Sarot et al., 2004). In the center, piRNA levels (black, restrictive strain; red, permissive strain) mapping to the indicated TEs (sense up, antisense down; normalized to 1Mio miRNAs) are displayed, and the portion of gypsy present in the gypsy-lacZ reporter (cartoon at top) is indicated. (G) Shown is the Pol II ChIP-qPCR analysis on the gypsy-reporter (primers 1 and 2 indicated in F) in ovaries from restrictive versus permissive strains (enrichments calculated over intergenic region; n = 3; error bars represent SD.). See also Figure S3.
Figure 5
Figure 5
Loss of Piwi, but Not Mael, Leads to Decreased H3K9me3 on TEs (A) Profiles of normalized H3K9me3 signals on indicated TE consensus sequences (group I versus group III) in piwi KD cells (red line), mael KD cells (green line), and GFP KD cells (solid gray). (B) Profiles of transcriptional activity (Pol II ChIP-seq, GRO-seq, RNA-seq) and H3K9me3 density of OSCs after indicated knockdowns (left) in a 100 kb window (chr. 3L; 63E) that contains an mdg1 insertion (only genome unique reads are displayed). (C) Heatmaps presenting Pol II occupancy, GRO-seq signal, and H3K9me3 ChIP-seq signal within 50 kb windows centered on all 24 euchromatic mdg1 insertions (oriented 5′ to 3′) in control, piwi, or mael KD cells. Red arrows mark insertions within H3K27me3 domains; the insertion marked by the blue arrow is the one displayed in (B). (D and E) Metaplots for the genomic regions flanking the average euchromatic mdg1 insertion; displayed are changes in Pol II occupancy (D), normalized H3K9me3 signal (D), normalized GRO-seq signal (E), and normalized RNA-seq signal (E) in control KD (black), piwi KD (red), or mael KD cells (green). See also Figure S4.
Figure 6
Figure 6
Euchromatic H3K9me3 Islands Correlate with TE Insertions and Depend on Piwi (A) Metaplots for the genomic regions flanking the average euchromatic insertion of indicated TEs (copy number indicated) belonging to group I or III; displayed are changes in Pol II occupancy (compared to control KD) and normalized H3K9me3 signal in control KD (black), piwi KD (red), or mael KD cells (green). (B) Heatmaps showing signal of H3K9me3 (left) or Pol II occupancy (right) in 50 kb windows centered on euchromatic H3K9me3 peaks (n = 466) and in a random set of euchromatic 50 kb windows (n = 466) in control KD, piwi KD, or mael KD cells. Windows with euchromatic H3K9me3 peaks were sorted according to the loss of signal in piwi KD cells, and five equally sized bins were defined (I–V). To the right, positions of all TE insertions within the euchromatic H3K9me3 windows are displayed. (C) Metaplots of H3K9me3 signals (individual plots for the five bins defined in B) for the 50 kb window flanking the average euchromatic H3K9me3 peak in control (black), piwi KD (red), or mael KD cells (green). (D) Distribution of group I (red) or III (white) TE insertions in the 50 kb windows centered on all H3K9me3 peaks (n = 466). (E) Percentages of all group I (red) or all group III (white) TE insertions found within 5 kb of euchromatic H3K9me3 summits in comparison to an average and randomly selected set of control regions. Box plots are as in Figure 3. (F) Percentages of insertions of indicated individual TEs found within 5 kb of euchromatic H3K9me3 summits (p values based on binominal test using random control areas).
Figure 7
Figure 7
The Impact of TEs and the piRNA Pathway on Gene Expression (A) Transcriptional activity (Pol II ChIP-seq, GRO-seq, RNA-seq) and H3K9me3 density at the expanded locus. OSC-specific insertion site of gypsy indicated with gray line; ex TSS, with dashed line. (B) Changes in mRNA abundance for the set of expressed genes (RPKM >5 in one library) between piwi KD and control KD cells. The upper plot contrasts genes with no insertion (gray) and genes with an insertion of a group I TE in sense orientation (red). The lower plot contrasts genes with no insertion (gray) and genes with no group I TE but with a group III TE insertion (yellow). (C) Box plot showing the changes (log2) in RNA-seq RKPM levels for the three gene groups defined in (B) upon knockdown of Piwi (left) or Mael (right). (D) Venn diagram displaying the number of upregulated genes upon depletion of the indicated piRNA pathway components (criteria: armi and piwi KDs >4-fold, mael KD >2-fold; compared to GFP KD). (E) Shown is the percentage of all upregulated genes (red) with TE insertions belonging to class I, II, or III in close proximity (± 5 kb) in comparison to random control genes (blue). (F) Box plots displaying fold changes (log2) in Pol II occupancy (left) or GRO-seq signal (right) upon piwi KD versus GFP KD for upregulated genes with TE insertion (n = 28) versus all expressed genes with no TE insertion (n = 5272; RPKM >5). Box plots are as in Figure 3. (G) Cartoon showing the three categories of how TE insertions impact gene loci. For category A (repressive chromatin influence), the TE insertion is close to the gene's TSS and results in TSS repression via H3K9me3 spreading. For category B (promoter addition), the TE insertion serves as an ectopic promoter through transcriptional bleeding. For category C (neutral chromatin influence), the TE insertion does not dampen gene expression but triggers local H3K9me3 spreading. Examples for each category are given below. See also Figure S5.
Figure S1
Figure S1
Related to Figure 1 (A) Displayed are fold changes in steady state RNA levels of indicated TEs in mael null mutant ovaries (normalized to mael heterozygote siblings; values are averages of 3 biological replicates (error bars: StDev.). (B) Confocal images of mael heterozygous (top) or mael[M391]/mael[Def] (bottom) egg chambers stained for Piwi, Aub or AGO3. (C) Displayed are fold changes in piwi steady state mRNA levels in OSCs after transfection with indicated siRNAs. Values are averages of 3 biological replicates (error bars: StDev.) and normalized to GFP siRNA treated cells. (D) Shown are length profiles of small RNAs (normalized to 1 million microRNAs; small insets) isolated from ovaries of mael[M391] heterozygous or mael[391]/mael[Def] flies. siRNA and piRNA populations are indicated. (E) Shown are length profiles of repeat derived ovarian small RNAs (normalized to 1Mio miRNAs) from mael heterozygous and mael[M391]/mael[Def] flies. (red antisense; blue sense). (F) Normalized piRNA profiles (sense up; antisense down; 200nt windows) from mael het. (black) or mael mut. (red) libraries mapping uniquely to the 42AB piRNA cluster. (G) Scatter plot (log2 scale) showing levels of antisense piRNAs mapping to soma dominant (green), intermediate (yellow) or germline dominant (black) TEs in mael het. or mael mut. libraries.
Figure S2
Figure S2
Related to Figure 3 (A) Metagene profiles of normalized RNA-seq, Pol II ChIP-seq and GRO-seq reads around (±0.5 kb) transcriptional start site (TSS) and polyadenylation site (PAS) from indicated siRNA mediated knockdowns (GFP: blue; piwi: red; mael: green). Only genes meeting the following criteria were used: RNA-seq RPKM ≥ 5, length > 1 kb, no overlaps with flanking gene up to 200 nt upstream of annotated TSS; n = 2628; note the strong TSS bias for Pol II occupancy and GRO-seq; note also that only RNA-seq shows the expected drop at the PAS, while Pol II occupancy and GRO-seq do not as expected as they continue to transcribe downstream of the PAS. (B) Heatmap showing TSS data presented in (A) at single gene-resolution. Profiles were sorted for decreasing signal of Pol II ChIP-seq in control knockdowns. (C) Table listing RNA-seq RPKM values for indicated TEs (upper part) or a set of highly expressed genes (lower part) upon indicated siRNA knockdowns in OSC. (D) Scatter plot showing RNA-seq RPKM values (log2) of group I-IV TEs in armi KD versus piwi KD OSCs. (E) Scatter plot showing RNA-seq RPKM values (log2) of group I-IV TEs in mael KD versus piwi KD OSCs. (F) Displayed are fold changes in steady state RNA levels of indicated TEs and genes upon piwi KD or mael KD or piwi+mael KD in OSCs. Note that the gypsy primer pair spans a splice junction, which explains the higher de-repression values compared to the RNA-seq data. Values are averages of 3 biological replicates (error bars: StDev.) and normalized to GFP siRNA treated cells. (G) Box plot analysis indicating fold changes (log2) in RNA-seq RPKM values (left) or Pol II occupancy values (right) for indicated TE groups in armi KD or mael KD cells; for the RNA-seq analysis, reads mapping sense or antisense to TEs were contrasted; p-values were computed with Wilcoxon rank-sum test. Box plots show median (line), 25th–75th percentile (box) ± 1.5 interquartile range; circles represent outliers.
Figure S3
Figure S3
Related to Figure 4 (A) Box-plots showing the changes of the H3K9me3 ChIPseq levels in 1kb bins for the flanking genomic regions (upstream and downstream 5 kb) of mapped insertions of the indicated TEs in piwi (red) or mael (green) knockdowns compared to GFP control KDs. All TEs except F-element and roo (group III) belong to the piRNA-regulated group I. (B) Heatmap showing distribution of H3K9me3 density in genomic regions (50kb windows) around all euchromatic insertions of F-element. Black arrows indicate insertions located in sense orientation within a transcriptionally active unit. (C) Heatmap showing distribution of H3K9me3 density in genomic regions (50kb windows) around all euchromatic insertions of roo. Black arrows indicate insertions located in sense orientation within a transcriptionally active unit. (D) Heatmaps showing signal of H3K9me3 (left) or Pol II occupancy (right) in 50kb windows centered on heterochromatic H3K9me3 peaks (n = 655) in control KD, piwi KD or mael KD cells. Only genome unique reads are displayed. (E) Shown are average profiles of normalized Pol II ChIP-seq signals in the 50kb regions around all euchromatic peaks of H3K9me3 (blue, n = 466) or randomly chosen peaks (gray, n = 466) in OSCs upon indicated knockdowns. Pol II occupancy is not lower in the vicinity of H3K9me3 peaks.
Figure S4
Figure S4
Related to Figure 5 (A) Depicted are individual RNA-seq reads (including spliced reads) from GFP or piwi KD cells mapping to the expanded locus (reads mapping to the flanking transcription unit in blue); note that the first intron is faithfully spliced despite the inserted gypsy element. (B) Changes in mRNA abundance for the set of expressed genes (RPKM > 5 in one library) between mael KD and control KD cells. The upper plot contrasts genes with no insertion (gray) and genes with an insertion of a group I TE in sense orientation (red). The lower plot contrast genes with no insertion (gray) and genes with no group I TE, but with a group III TE insertion (yellow). (C) List of all upregulated genes (4x in piwi and armi KDs, 2x in mael KDs) with mapped TE insertions either in the gene body or in close proximity (up to 5kb). Given are also the RNA-seq RPKM values in control and piwi KD cells.
Figure S5
Figure S5
Related to Figure 7 (A and B) Shown are normalized density profiles of Pol II ChIP-seq (red), GRO-seq (black), RNA-seq (brown) and H3K9me3 ChIP-seq (green) for the indicated OSC knockdowns (left). (A) Shown is the ∼140kb area with an mdg1 insertion upstream of the typically non-expressed gene CG15278. Upon loss of the piRNA pathway transcriptional bleeding from the TE insertion into the CG15278 locus leads to accumulation of RNA reads. (B) Shown is a ∼120kb area with a 17.6 insertion in sense orientation into an intron of the Btk29A transcription unit. This insertion triggers H3K9me3 spreading, which depends on Piwi but only weakly on Mael. Loss of the piRNA pathway does not lead to upregulation of the host gene, classifying this insertion.
Figure S6
Figure S6
Related to Discussion (A) Shown are normalized density profiles of Pol II ChIP-seq (red), GRO-seq (black), RNA-seq (brown), H3K9me3 ChIP-seq (green) and piRNA-seq (light green) for the indicated OSC knockdowns (left). Shown is the ∼20kb area around the transcriptional start site of the flamenco cluster. Shown are only reads mapping uniquely to the genome but we note that nearly all areas in this window are genome-unique. (B) Western blot showing protein levels of Armi, Piwi, Lamin, Mael, HP1 and Histone 3 (H3) in cytoplasmic, nucleoplasmic, soluble and insoluble chromatin fractions of OSCs. The relative amount of each fraction loaded per lane (based on fraction volume) is given below. The following antibodies were used: α-Lamin (ADL67.10, DSHB), α-HP1 (C1A9, DSHB) and α-H3 (Abcam, ab1791).
Figure S7
Figure S7
Related to Discussion Shown are normalized density profiles of Pol II ChIP-seq (red), GRO-seq (black), RNA-seq (brown), H3K9me3 ChIP-seq (green) and piRNA-seq (light green) for the indicated OSC knockdowns (left). Shown is a ∼60kb area that resides in the peri-centromeric heterochromatin of chromosome 2R (cytological position 42A); the position of the mdg1 insertion (minus strand) is indicated; note the absence of piRNAs mapping to this region and the massive spreading of H3K9me3 in mael KD cells.

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