Rif1 inhibits replication fork progression and controls DNA copy number in Drosophila
- Vanderbilt University, United States
- Vanderbilt University School of Medicine, United States
Figures
Figure 1 with 2 supplements
The SNF2 domain is essential for SUUR function and replication fork localization.
(A) Schematic representation of the SUUR and SUURΔSNF proteins. (B) Illumina-based copy number profiles (Reads Per Million; RPM) of chr2L 1 – 20,000,000 from larval salivary glands. Black bars below …
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Figure 1—source data 1
ddPCR data for Figure 1D.
- https://doi.org/10.7554/eLife.39140.006
Figure 1—figure supplement 1
Genome-wide copy number profile of the SuURΔSNF mutant.
Illumina-based copy number profiles of all chromosome arms except the fourth for larval salivary glands of the indicated genotypes and wild type 0 – 2 hr embryos in which DNA is fully replicated. …
Figure 1—figure supplement 2
Quantification of SUUR and SUURΔSNF signal intensities at replication forks and heterochromatin.
(A) SUUR and SUURΔSNF mean signal intensities, normalized for background levels, at double bar structures from stage 12 egg chambers. Results are from two biological replicates. A two-tailed Welch’s …
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Figure 1—figure supplement 2—source data 1
SUUR signal intensity at double bar structures and heterochromatin - raw data.
- https://doi.org/10.7554/eLife.39140.005
Figure 2 with 2 supplements
SUUR associates with Rif1.
(A) Total spectrum counts of FLAG-SUUR, FLAG-SNF2 and Oregon R (no FLAG control) for three independent IP-mass spectrometry experiments (biological replicates). A Fisher’s Exact test of spectrum …
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Figure 2—source data 1
Results of SUUR IP-mass spec screen.
- https://doi.org/10.7554/eLife.39140.011
Figure 2—figure supplement 1
Western blot analysis of heat-shock inducible SUUR constructs.
(A) Full Western blots of images used in Figure 2B with total protein loading control. (B) Western blot analysis of FLAG-SUUR and FLAG-SNF2 heat-shocked 0 – 24 hr embryos with a total protein …
Figure 2—figure supplement 2
Verification of Rif1 mutants and validation of anti-Rif1 antibody.
(A) Western blot analysis of ovary extracts prepared from the indicated genotypes. Serum produced in guinea pigs was used at 1:1000 dilution. (B) Immunofluorescence of ovaries using affinity …
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Figure 2—figure supplement 2—source data 1
Raw data for hatch rate assay.
- https://doi.org/10.7554/eLife.39140.010
Figure 3 with 3 supplements
Rif1 is required for underreplication.
(A) Schematic representation of the Rif1 gene and CRISPR-induced Rif1 mutants. Lightning bolts represent the 5’ and 3’ gRNA positions. (B) Illumina-based copy number profiles of the chr2L from …
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Figure 3—source data 1
ddPCR data for Figure 3D.
- https://doi.org/10.7554/eLife.39140.017
Figure 3—figure supplement 1
Genome-wide copy number profile of the Rif1 mutant.
(A) Illumina-based copy number profiles of all chromosome arms except the fourth for larval salivary glands of the indicated genotypes. Black bars below each profile represent called underreplicated …
Figure 3—figure supplement 2
Rif1 mutant salivary gland cells display a pattern of late replication.
(A) Representative immunofluorescent images of 3rd instar salivary glands pulse labeled with EdU and stained with anti-HP1 to mark heterochromatin. Wild-type cells fail to incorporate EdU into …
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Figure 3—figure supplement 2—source data 1
Raw data for Figure 3—figure supplement 2B.
- https://doi.org/10.7554/eLife.39140.015
Figure 3—figure supplement 3
Rif1 mutant endo cycling cells have enlarged chromocenters.
Representative images of single nurse cell nuclei from stage 10 egg chambers. Egg chambers were stained with DAPI. Scale bar is 10 μm. Exposure times and scaling are equal in all images.
Figure 4 with 1 supplement
Rif1 regulates replication fork progression.
(A) Illumina-based copy number profile of sites of follicle cell gene amplification. DNA was extracted from wild type and Rif1 mutant stage 13 egg chambers and compared to DNA extracted from 0 to 2 …
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Figure 4—source data 1
Data for Figure 4B.
- https://doi.org/10.7554/eLife.39140.021
Figure 4—figure supplement 1
The developmental window of gene amplification is not affected by loss of Rif1 function.
Egg chamber distribution from wild-type and Rif1 mutant ovaries fattened for 2 days on wet yeast. Bars represent the fraction of a given egg chamber stage relative to the total population of counted …
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Figure 4—figure supplement 1—source data 1
Raw data for egg chamber distribution assay.
- https://doi.org/10.7554/eLife.39140.020
Figure 5 with 1 supplement
Rif1 acts downstream of SUUR.
Localization of replication forks (EdU) and SUUR in a wild-type and Rif1 mutant follicle cell nuclei. A single representative stage 13 follicle cell nucleus is shown. Scale bars are 2 μm. Arrowheads …
Figure 5—figure supplement 1
Quantification of SUUR signal intensity at replication forks in the presence and absence of Rif1.
SUUR mean signal intensity at double bar structures, normalized for background levels, from stage 12 egg chambers. Results are from two biological replicates. A two-tailed Welch’s t test was used to …
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Figure 5—figure supplement 1—source data 1
SUUR signal intensity at double bar structures - raw data.
- https://doi.org/10.7554/eLife.39140.024
Figure 6 with 2 supplements
SUUR is necessary to retain Rif1 at replication forks.
Localization of active replication forks (EdU) and Rif1 in a wild-type and SuUR mutant follicle cell nuclei. Single representative follicle cell nuclei are shown for each stage. Scale bars are 2 μm. …
Figure 6—figure supplement 1
Quantification of Rif1 signal intensity at replication forks in the presence and absence of SUUR.
Rif1 mean signal intensity at double bar structures from stage 10B, 11, 12 and 13 egg chambers. Results from two biological replicates are shown independently. A two-tailed Welch’s t test was used …
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Figure 6—figure supplement 1—source data 1
Rif1 signal intensity at amplificaiton loci - stages 10B-13 - raw data.
- https://doi.org/10.7554/eLife.39140.027
Figure 6—figure supplement 2
Rif1 localizes to replication forks in cultured cells.
(A) Quantification of protein intensity from iPOND mass spec data in cultured S2 cells. Intensities represent the summed MS1 spectrum for each peptide identified for Histone H1, PCNA or Rif1. Each …
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Figure 6—figure supplement 2—source data 1
Data for Figure 6—figure supplement 2B.
- https://doi.org/10.7554/eLife.39140.029
Figure 7 with 2 supplements
The Rif1 PP1 interaction motif is necessary to promote underreplication.
(A) Illumina-based copy number profiles of chr2L 1 - 20,000,000 from larval salivary glands. Black bars below each profile represent underreplicated regions identified by CNVnator. Rif1PP1/CyO was …
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Figure 7—source data 1
ddPCR data for Figure 7C.
- https://doi.org/10.7554/eLife.39140.033
Figure 7—figure supplement 1
The Rif1PP1 protein expression level is similar to wild-type Rif1.
(A) Western blot analysis of ovary extracts from Rif1PP1/Rif11 and Rif11/+adults. Serum was produced in guinea pigs and used at 1:1000 dilution.
Figure 7—figure supplement 2
Genome-wide copy number profile of the Rif1PP1 mutant.
Illumina-based copy number profiles of all chromosome arms except the fourth for larval salivary glands of the indicated genotypes. Black bars below each profile represent called underreplicated …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Gene (Drosophila melanogaster) | Suppressor of Underreplication (SuUR) | NA | FBgn0025355 | |
| Gene (D. melanogaster) | Rap1 interacting factor 1 (Rif1) | NA | FBgn0050085 | |
| Strain, strain background (D. melanogaster) | WT: Oregon R | |||
| Strain, strain background (D. melanogaster) | SuUR | (Makunin et al., 2002) PMID: 11901119 | w118; SuURES | |
| Strain, strain background (D. melanogaster) | SuURΔSNF | This paper | SuURES, PBac{w+ SuURΔSNF} | |
| Strain, strain background (D. melanogaster) | hs > FLAG-SUUR | This paper | w118; hs > FLAG-SUUR | |
| Strain, strain background (D. melanogaster) | hs > FLAG-SNF2 | This paper | w118; hs > FLAG-SNF2 | |
| Strain, strain background (D. melanogaster) | Rif11 | This paper | w118; Rif11 | |
| Strain, strain background (D. melanogaster) | Rif12 | This paper | w118; Rif12 | |
| Strain, strain background (D. melanogaster) | Rif1- | This paper | w118; Rif11/Rif12 | |
| Strain, strain background (D. melanogaster) | Rif1PP1 | This paper | w118; Rif1PP1 | |
| Cell line (D. melanogaster) | S2-DGRC | Drosophila Genomics Resource Center (DGRC) | embryo derived | isolate of S2 used for RNAi in the DRSC modENCODE line |
| Antibody | anti-SUUR (Guinea pig, polyclonal) | (Nordman et al., 2011) PMID: 25437540) | ||
| Antibody | anti-Rif1 (Guinea pig, polyclonal) | This paper | (1:200) | |
| Antibody | anti-Rif1 (Rabbit, polyclonal) | This paper | (1:1000) | |
| Antibody | HRP-anti-FLAG (Mouse, monoclonal) | Sigma-Aldrich | A8592 | (1:1000) |
| Antibody | anti-HP1 (Mouse, monoclonal) | The Developmental Studies Hybridoma Bank (DSHB) | C1A9 | (1:1000) |
| Antibody | anti-biotin (Mouse, moncolonal) | Sigma-Aldrich | SAB4200680 | (1:20,000) |
| Antibody | anti-biotin (rabbit, polyclonal) | Bethyl | A150-109A | (1:3,000) |
| Antibody | HRP-secondaries | Jackson ImmunoResearch | (1:20,000) | |
| Recombinant DNA reagent | pCaSpeR-hs | (Thummel and Pirrotta, V.)Drosophila Genomics Resource Center | ||
| Recombinant DNA reagent | pStinger | (Barolo et al., 2000) PMID: 11056799 | ||
| Recombinant DNA reagent | CHORI-322 (CH322-163L18) | BACPAC Resources | ||
| Recombinant DNA reagent | pET17b | Millipore-Sigma | 69663 | |
| Recombinant DNA reagent | pET17b-Rif1 (694–1094) | This paper | Progenitors:PCR, pET17b | |
| Peptide, recombinant protein | Rif1(694–1094) | This paper | Ni-NTA purified | |
| Commercial assay or kit | PLA probes | Duolink Sigma | ||
| Commercial assay or kit | PLA probemaker | Duolink Sigma | DUO92010 | |
| Commercial assay or kit | PLA Detection Reagents | Duolink Sigma | DUO92008 | |
| Chemical compound, drug | Alexa Fluor Azide 555 | Life Technologies | A20012 | |
| Chemical compound, drug | Biotin-TEG Azide | Berry and Associates | BT 1085 | |
| Chemical compound, drug | EdU (5-ethynyl-2- deoxyuridine) | Life Technologies | A10044 | |
| Software, algorithm | Sequest | Thermo Scientific | ||
| Software, algorithm | Scaffold 4.3.4 | Proteome Software | ||
| Software, algorithm | Skyline version 4.1 | Schilling et al. (2012) (PMID:22454539) | ||
| Software, algorithm | deepTool 2.5.0 | Ramírez et al. (2016) (PMID:27079975) | ||
| Software, algorithm | CNVnator 0.3.3 | Abyzov et al., 2011 (PMID:21324876) | ||
| Other |
Additional files
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Supplementary file 1
Underreplicated regions called by CNVnator.
- https://doi.org/10.7554/eLife.39140.034
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Supplementary file 2
Half-max copy number analysis.
- https://doi.org/10.7554/eLife.39140.035
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Transparent reporting form
- https://doi.org/10.7554/eLife.39140.036
