Maternally inherited piRNAs direct transient heterochromatin formation at active transposons during early Drosophila embryogenesis
- CRUK Cambridge Institute, University of Cambridge, Li Ka Shing Centre, United Kingdom
Figures
Figure 1 with 2 supplements
A transient burst of transposon expression during Drosophila embryogenesis.
(A) Schematic of Drosophila embryogenesis indicating Bownes stages and collected time points. (B) Bar graphs showing contribution of transposon derived reads to the transcriptome of control w1118 …
Figure 1—figure supplement 1
Correlation of embryo collection time points between datasets.
(A) Heatmaps showing expression changes in RNA-seq (in log2 reads per million; n = 2) of control w1118 embryos for the indicated time points and selected genes with dynamic expression during …
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Figure 1—figure supplement 1—source data 1
Gene- and transposable element-mapping reads per million across embryogenesis RNA-seq time points for plots shown in Figure 1B, C, Figure 1—figure supplement 1A, B, Figure 1—figure supplement 2A, F, Figure 2A, and Figure 2—figure supplement 1H, J.
- https://cdn.elifesciences.org/articles/68573/elife-68573-fig1-figsupp1-data1-v2.txt
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Figure 1—figure supplement 1—source data 2
Signal intensity of genes and transposon open reading frames in TMT-MS time-course experiment for plots shown in Figure 1—figure supplement 2A, C, D, F, Figure 2B, and Figure 2—figure supplement 1I, K.
- https://cdn.elifesciences.org/articles/68573/elife-68573-fig1-figsupp1-data2-v2.txt
Figure 1—figure supplement 2
Transposon protein and the known cohort of piRNA co-transcriptional gene silencing (coTGS) factors are present during embryogenesis.
(A) Heatmaps showing expression changes in RNA-seq (left; in log2 reads per million; n = 2) and protein abundance in TMT-MS (right; log2 signal intensity; n = 3) of control w1118 embryos for …
Figure 2 with 1 supplement
piRNA co-transcriptional gene silencing (coTGS) factors are maternally inherited and localize to somatic cells of the Drosophila embryo.
(A) Bar graphs showing Piwi RNA expression (in rpm) at the indicated time points in control w1118 embryos. Error bars show standard deviation (n = 2). (B) Bar graphs showing Piwi protein signal …
Figure 2—figure supplement 1
Maternally inherited co-transcriptional gene silencing (coTGS) factors localize to nuclei of pole and somatic cells during embryogenesis.
(A) Stand-still images from Video 2 obtained by light-sheet fluorescent live microscopy of embryos derived from parents expressing GFP-Panx (green) and H2Av-RFP (red) for the indicated time points. …
Figure 3 with 1 supplement
Transposon insertions targeted by piRNAs in embryos show epigenetic changes characteristic of co-transcriptional gene silencing.
(A) Heatmaps (top) and metaplots (bottom) showing H3K9me3 ChIP-seq signal (in rpm) for the indicated embryonic stages and adult tissues at 117 euchromatic, w1118-specific roo insertions (n = 2). …
Figure 3—figure supplement 1
Transposons targeted by maternally inherited piRNAs show hallmarks of co-transcriptional gene silencing (coTGS).
(A) Bar graphs showing H3K9me3 signal intensity for the indicated time points at roo insertions. Error bars show standard deviation (n = 2). (B) Heatmaps (top) and metaplots (bottom) showing H3K9me3 …
Figure 4 with 1 supplement
Degradation of Piwi protein in ovaries resembles mutant phenotypes.
(A) Cartoon illustrating the Piwi protein degradation strategy using the auxin-inducible AID-TIR1 system. (B) Confocal fluorescent microscopy images showing ovary egg chambers of GFP-AID-Piwi; …
Figure 4—figure supplement 1
Piwi degradation in ovaries resembles knockdown and mutant phenotypes.
(A) Confocal fluorescent microscopy images showing ovary egg chambers of GFP-AID-Piwi; OsTIR1 flies fed with yeast paste containing 5 mM auxin for the indicated time. Blue = DAPI, green = GFP-AID-Piw…
Figure 5 with 1 supplement
Degradation of maternally deposited Piwi in embryos leads to transposon deregulation.
(A) Schematic of embryo auxin treatments and sample collection for RNA-seq and ChIP-seq experiments. (B) Western blot showing abundance of GFP-AID-Piwi fusion protein in embryos treated with 5 mM …
Figure 5—figure supplement 1
Piwi depletion in embryos leads to epigenetic changes at transposable element (TE) insertions targeted by maternally inherited piRNAs.
(A) Heatmap comparing expression profiles of selected genes (same as shown in Figure 1—figure supplement 1) in RNA-seq (in log2 reads per million) of 2.5–3 hr PBS or auxin-treated embryos of …
Author response image 1
Gene expression and H3K9me3 occupancy of genes in proximity of euchromatic TE insertions.
IGV genome browser screenshot showing H3K9me3 ChIP-seq signal for the indicated genes on chromosome 2R carrying a w1118-specific roo insertion. Blue tracks show gene expression for indicated embryo …
Author response image 2
Transposons sensitive to Piwi reduction in larvae showed no change in our study.
(A) MA plot showing base mean expression (log10 scale) of transposon RNAs relative to their fold-change (log2 scale) in GFP-AID-Piwi; OsTIR1 embryos treated with 5mM auxin versus control (n=3). …
Videos
Video 1
GFP-Piwi live imaging.
Light-sheet fluorescent live microscopy of embryos derived from parents expressing GFP-Piwi (green) and H2Av-RFP (red) in developing embryos for indicated time after egg laying.
Video 2
GFP-Panx live imaging.
Light-sheet fluorescent live microscopy of embryos derived from parents expressing GFP-Panx (green) and H2Av-RFP (red) in developing embryos for indicated time after egg laying.
Video 3
GFP-Nxf2 live imaging.
Light-sheet fluorescent live microscopy of embryos derived from parents expressing GFP-Nxf2 (green) and H2Av-RFP (red) in developing embryos for indicated time after egg laying.
Video 4
GFP-AID-Piwi live imaging.
Light-sheet fluorescent live microscopy of embryos derived from parents expressing GFP-AID-Piwi (green) and OsTIR1. Time course shows early embryo treated with 5 mM auxin for indicated time points.
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Gene (Drosophila melanogaster) | nxf2 | FlyBase | FBgn0036640 | |
| Gene (Drosophila melanogaster) | panx | FlyBase | FBgn0034617 | |
| Gene (Drosophila melanogaster) | piwi | FlyBase | FBgn0004872 | |
| Gene (Drosophila melanogaster) | roo | FlyBase | FBgn0043856 | |
| Antibody | Anti-GFP (chicken polyclonal) | Abcam | Cat# ab13970 RRID:AB_300798 | WB (1:5000) IF (1:1000) |
| Antibody | Anti-Piwi (rabbit polyclonal) | DOI: 10.1016/j.cell.2007.01.043 | N/A | WB (1:1000) IF (1:500) |
| Antibody | Anti-Tubulin (mouse monoclonal) | Abcam | Cat# ab44928, RRID:AB_2241150 | WB (1:5000) |
| Antibody | Anti-Myc tag (rabbit polyclonal) | Abcam | Cat# ab9106, RRID:AB_307014 | WB (1:1000) |
| Antibody | Anti-H3K4me2 (rabbit polyclonal) | Merck Millipore | Cat# 07-030, RRID:AB_310342 (Lot# 2971019) | ChIP (1:50) |
| Antibody | Anti-H3K9me3 (rabbit polyclonal) | Active Motif | Cat# 39161, RRID:AB_2532132 (Lot# 15617003) | ChIP (1:50) |
| Antibody | Anti-Mouse IgG Alexa Fluor 488 (goat polyclonal) | Thermo Fisher Scientific | Cat# A-11029, RRID:AB_2534088 | IF (1:500) |
| Antibody | Anti-Mouse IgG Alexa Fluor 555 (goat polyclonal) | Thermo Fisher Scientific | Cat# A-21424, RRID:AB_141780 | IF (1:500) |
| Antibody | Anti-Rabbit IgG Alexa Fluor 647 (goat polyclonal) | Thermo Fisher Scientific | Cat# A-21245, RRID:AB_2535813 | IF (1:500) |
| Commercial assay or kit | RIPA Lysis and Extraction Buffer | Thermo Fisher Scientific | Cat# 89901 | |
| Commercial assay or kit | cOmplete, Mini, EDTA-free Protease Inhibitor Cocktail | Sigma-Aldrich | Cat#11836170001 | |
| Commercial assay or kit | RNasin Plus RNase Inhibitor | Promega | Cat# N2615 | |
| Commercial assay or kit | Blood and Cell Culture DNA Mini Kit | Qiagen | Cat# 13323 | |
| Commercial assay or kit | RNeasy Mini Kit | Qiagen | Cat# 74106 | |
| Commercial assay or kit | NEBNext Poly(A) mRNA Magnetic Isolation Module | NEB | Cat# E7490L | |
| Commercial assay or kit | NEBNext Ultra Directional RNA Library Prep Kit for Illumina | NEB | Cat# E7420L | |
| Commercial assay or kit | NEBNext Ultra II DNA Library Prep Kit for Illumina | NEB | Cat# E7645L | |
| Commercial assay or kit | Invitrogen SuperScript IV Reverse Transcriptase | Thermo Fisher Scientific | Cat# 18090050 | |
| Commercial assay or kit | Indole-3-acetic acid sodium salt | Sigma-Aldrich | Cat# I5148-10G | |
| Commercial assay or kit | Pierce 16% formaldehyde (w/v), methanol-free | Thermo Fisher Scientific | Cat# 28908 | |
| Commercial assay or kit | MinElute PCR Purification Kit | Qiagen | Cat# 28004 | |
| Software, algorithm | Fiji | ImageJ | RRID:SCR_002285 | |
| Software, algorithm | Zeiss ZEN Imaging Software | Zeiss | RRID:SCR_018163 | |
| Software, algorithm | Proteome Discoverer 2.1 | Thermo Fisher Scientific | RRID:SCR_014477 | |
| Software, algorithm | R | RCoreTeam | N/A | |
| Software, algorithm | STAR | DOI:10.1093/bioinformatics/bts635 | RRID:SCR_015899 | |
| Software, algorithm | TEMP | https://github.com/JialiUMassWengLab/TEMPZhuang et al., 2014 | RRID:SCR_001788 | |
| Software, algorithm | Prodigal | https://github.com/hyattpd/Prodigal | N/A | |
| Software, algorithm | DEseq2 | DOI:10.1186/s13059-014-0550-8 | RRID:SCR_015687 | |
| Software, algorithm | Image Studio Lite | LI-COR | RRID:SCR_013715 |
Additional files
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Supplementary file 1
List of oligonucleotides for qRT-PCR and RNA-FISH.
- https://cdn.elifesciences.org/articles/68573/elife-68573-supp1-v2.xlsx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/68573/elife-68573-transrepform-v2.pdf
