Dimerisation of the PICTS complex via LC8/Cut-up drives co-transcriptional transposon silencing in Drosophila
- Cancer Research UK Cambridge Institute, University of Cambridge, Li Ka Shing Centre, United Kingdom
- Department of Biochemistry and Biophysics, Oregon State University, United States
Figures
Figure 1 with 1 supplement
Ctp associates with the PICTS complex and is required for transposon silencing in the fly germline.
(A) Enrichment plot showing the fold-change of PICTS complex proteins and Piwi in the GFP-Panx and GFP-Nxf2 IPs vs the control (n = 4). (B) Heatmap showing expression levels of ctp, cdlc2, panx, nxf2…
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Figure 1—source data 1
Differential expression analysis of ovary RNA-seq data used for volcano plots shown in Figure 1D and Figure 1E.
- https://cdn.elifesciences.org/articles/65557/elife-65557-fig1-data1-v1.xlsx
Figure 1—figure supplement 1
Ctp is highly conserved and essential for germline transposon repression.
(A) Protein sequence alignment of Ctp orthologs across the indicated species. Known secondary structural elements are shown below and asterisks indicate residues that form the main contacts with the …
Figure 2 with 1 supplement
Ctp is required for transposon silencing and H3K9me3 deposition at transposon loci in ovarian somatic cells.
(A) MA plot showing expression level against fold-change (FC) for genes and transposons in siCtp vs siGFP. Red dotted lines indicate log2FC = 2. TE, transposable element. (B) As in A but comparing si…
Figure 2—figure supplement 1
Transposon de-repression upon loss of Ctp is associated with changes at the chromatin level.
(A) MA plots showing expression level against fold-change (FC) for genes in siCtp (left) or siPanx (right) vs siGFP. Red dotted lines indicate log2FC = 2 and the black dotted box indicates …
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Figure 2—figure supplement 1—source data 1
Differential expression analysis of OSC RNA-seq data used for plots in Figure 2 and Figure 2—figure supplement 1.
- https://cdn.elifesciences.org/articles/65557/elife-65557-fig2-figsupp1-data1-v1.xlsx
Figure 3 with 1 supplement
Ctp is involved in co-transcriptional gene silencing of transposons.
(A) Immunofluorescence images showing the subcellular localisation of 3xFLAG-Ctp transiently transfected into OSCs upon the indicated knockdown. Scale bar = 5 µm. (B) Western blot for cytosolic …
Figure 3—figure supplement 1
Ctp is a TGS factor.
(A) Immunofluorescence images showing the localisation of 3xFLAG-Ctp in OSCs with the indicated knockdown. Scale bar = 5 µm. (B) Western blot analyses from OSC lysates showing the expression of the …
Figure 4 with 1 supplement
Ctp associates with PICTS via two highly conserved motifs in the carboxy-terminal region of Panx.
(A) Volcano plot showing enrichment against significance for proteins identified by mass spectrometry that co-purify with 3xFLAG-Ctp from OSC lysates compared to the 3xFLAG-mCherry control (n = 3). …
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Figure 4—source data 1
Source data for volcano plot shown in Figure 4A.
- https://cdn.elifesciences.org/articles/65557/elife-65557-fig4-data1-v1.csv
Figure 4—figure supplement 1
Two recognition motifs in the Panx C-terminus interact with Ctp.
(A) Western blot analyses for the IP-MS experiment shown in Figure 4A. (B) Enrichment plot showing the fold-change of PICTS complex proteins and Piwi in the 3xFLAG-Ctp IP vs control (n = 3). (C) …
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Figure 4—figure supplement 1—source data 1
Source data for volcano plot in Figure 4—figure supplement 1K.
- https://cdn.elifesciences.org/articles/65557/elife-65557-fig4-figsupp1-data1-v1.csv
Figure 5 with 1 supplement
Ctp induces silencing via Panx.
(A) Western blot analyses showing the relative level of Piwi, Panx, and Ctp in S2 cells compared to OSCs. Tubulin was used as a loading control. (B) Bar graphs showing the level of HA-ZsGreen …
Figure 5—figure supplement 1
Repression by Ctp is mediated via Panx.
(A), (C), and (E) western blot analyses from S2 cell lysates showing the expression of the indicated LacI-tagged construct and the level of the HA-ZsGreen protein used for the quantification in Figur…
Figure 6 with 1 supplement
Ctp promotes higher order assembly of PICTS through dimerisation of Panx.
(A) Western blot analysis for the indicated FLAG- and HA-tagged constructs following FLAG immunoprecipitation from OSCs. IN=input (1x), UB=unbound (1x), and IP=immunoprecipitate (10x). (B) Western …
Figure 6—figure supplement 1
Dimerisation of Panx is essential for TGS.
(A) Western blot analysis for the indicated FLAG- and HA-tagged constructs following FLAG immunoprecipitation from OSCs. IN=input (1x), UB=unbound (1x), and IP=immunoprecipitate (10x). (B) Bar graph …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Gene (Drosophila melanogaster) | ctp | FlyBase | FBgn0011760 | |
| Gene (Drosophila melanogaster) | panx | FlyBase | FBgn0034617 | |
| Gene (Drosophila melanogaster) | nxf2 | FlyBase | FBgn0036640 | |
| Gene (Drosophila melanogaster) | nxt1 | FlyBase | FBgn0028411 | |
| Gene (Drosophila melanogaster) | piwi | FlyBase | FBgn0004872 | |
| Gene (Drosophila melanogaster) | egg | FlyBase | FBgn0086908 | |
| Cell line (D. melanogaster) | Ovarian somatic cells (OSC) | DOI:10.1038/nature08501 | RRID:CVCL_IY73 | |
| Cell line (D. melanogaster) | S2 cells | Thermo Fisher Scientific | Cat#: R69007 RRID:CVCL_Z232 | |
| Antibody | Anti-FLAG (Mouse monoclonal) magnetic beads | Sigma-Aldrich | Cat# M8823, RRID:AB_2637089 | |
| Antibody | Anti-GFP (Chicken polyclonal) | Abcam | Cat# ab13970, RRID:AB_300798 | IF(1:1,000) |
| Antibody | Anti-Piwi (Rabbit polyclonal) | DOI: 10.1016/j.cell.2007.01.043 | IF(1:500) WB(1:2,500) | |
| Antibody | Anti-HA (Mouse monoclonal) | Abcam | Cat# ab18181, RRID:AB_444303 | IF(1:1,000) |
| Antibody | Anti-FLAG (Rabbit monoclonal) | Cell Signaling Technology | Cat# 14793, RRID:AB_2572291 | IF(1:1,000) |
| Antibody | Anti-lamin (Mouse monoclonal) | Developmental Studies Hybridoma Bank | Cat# adl67.10, RRID:AB_528336 | IF(1:200) WB(1:200) |
| Antibody | Anti-FLAG (Mouse monoclonal) | Sigma-Aldrich | Cat# F1804, RRID:AB_262044 | WB(1:2,500) |
| Antibody | Anti-HA (Rabbit monoclonal) | Cell Signaling Technology | Cat# 3724, RRID:AB_1549585 | WB(1:2,500) |
| Antibody | Anti-tubulin (Rabbit polyclonal) | Abcam | Cat# ab18251, RRID:AB_2210057 | WB(1:2,500) |
| Antibody | Anti-tubulin (Mouse monoclonal) | Abcam | Cat# ab44928, RRID:AB_2241150 | WB(1:2,500) |
| Antibody | Anti-His3 (Mouse monoclonal) | Abcam | Cat# ab10799, RRID:AB_470239 | WB(1:1,000) |
| Antibody | Anti-DYNLL1 (Rabbit monoclonal) | Abcam | Cat# ab51603, RRID:AB_2093654 | WB(1:1,000) |
| Antibody | Anti-Panx (Mouse monoclonal) | DOI: 10.1101/gad.271908.115 | WB(1:20) | |
| Antibody | Anti-H3K9me3 (rabbit polyclonal) | Active Motif | Cat# 39161, RRID:AB_2532132 | |
| Antibody | Anti-H3K4me2 (rabbit polyclonal) | Merck Millipore | Cat# 07–030, RRID:AB_310342 | |
| Commercial assay or kit | TALON Metal Affinity Resin | Takara Bio | Cat# 635502 | |
| Commercial assay or kit | NEBNext Poly(A) mRNA magnetic Isolation Module | New England Biolabs | Cat# E7490L | |
| Commercial assay or kit | NEBNext Ultra Directional Library Prep Kit for Illumina | New England Biolabs | Cat# E7420L | |
| Commercial assay or kit | CleanTag Small RNA Library Preparation Kit | TriLink | Cat# L-3206 | |
| Commercial assay or kit | NEBNext Ultra II DNA Library Prep Kit for Illumina | New England Biolabs | Cat# E7645 | |
| Strain, strain background (Escherichia coli) | Rosetta (DE3) | Sigma-Aldrich | Cat# 70954 | |
| Recombinant DNA reagent | pET-24d(+) | Sigma-Aldrich | Cat# 69752 | |
| Recombinant DNA reagent | pVALIUM20 | DOI:10.1038/nmeth.1592 | ||
| Software, algorithm | Origin 7.0 | OriginLab | ||
| Software, algorithm | XPLOR-NIH | DOI:10.1002/pro.3248 | https://nmr.cit.nih.gov/xplor-nih/ | |
| Software, algorithm | Bowtie | DOI:10.1186/gb-2009-10-3-r25 | RRID:SCR_005476 | |
| Software, algorithm | STAR | DOI:10.1093/bioinformatics/bts635 | RRID:SCR_015899 | |
| Software, algorithm | deepTools | DOI:10.1093/nar/gkw257 | RRID:SCR_016366 | |
| Software, algorithm | DESeq2 | DOI:10.1186/s13059-014-0550-8 | RRID:SCR_015687 | |
| Software, algorithm | Trim Galore! | Felix Krueger | RRID:SCR_011847 | |
| Software, algorithm | LC8Pred | DOI:10.26508/lsa.201900366 | http://lc8hub.cgrb.oregonstate.edu/LC8Pred.php | |
| Software, algorithm | Image Studio Lite | LI-COR | RRID:SCR_013715 |
Additional files
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Supplementary file 1
List of all TQT-containing proteins enriched in Ctp IP-MS (log2FC > 2 and p<0.01).
The sequence of each TQT motif was identified using LC8Pred and is shown alongside gene names, number of motifs per protein and the corresponding LC8Pred amino acid score. Both TQT motifs in Panx are highlighted in green.
- https://cdn.elifesciences.org/articles/65557/elife-65557-supp1-v1.xlsx
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Supplementary file 2
Thermodynamic parameters of Ctp-Panx interactions.
PanxTQT#1 refers to the construct in which the first TQT motif is mutated but the second site is intact (and vice versa).
- https://cdn.elifesciences.org/articles/65557/elife-65557-supp2-v1.xlsx
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Supplementary file 3
List of siRNA sequences, qPCR primers, and fly stocks used in this study.
- https://cdn.elifesciences.org/articles/65557/elife-65557-supp3-v1.xlsx
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Supplementary file 4
List of ChIP-seq, RNA-seq, and small RNA-seq libraries used in this study.
- https://cdn.elifesciences.org/articles/65557/elife-65557-supp4-v1.xlsx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/65557/elife-65557-transrepform-v1.pdf
