Multiple 9-1-1 complexes promote homolog synapsis, DSB repair, and ATR signaling during mammalian meiosis
- Department of Biomedical Sciences, Cornell University, United States
- Division of Mathematics and Natural Sciences, Elmira College, United States
- Department of Obstetrics and Gynecology, Brown University, United States
- Sex Chromosome Biology Laboratory, The Francis Crick Institute, United Kingdom
- Department of Molecular Biology and Genetics, Weill Institute for Cell and Molecular Biology, Cornell University, United States
- Department of Biochemistry and Immunology, FMRP, University of São Paulo, Brazil
- Division of Immunology, The Netherlands Cancer Institute, Netherlands
- Department of Human Genetics, University of Utah, United States
- Unidad de Investigación, Hospital Universitario de Canarias, Spain
- Instituto de Tecnologías Biomédicas, Universidad de La Laguna, Spain
- Universidad Fernando Pessoa Canarias, Spain
- Magee-Womens Research Institute, Department of Obstetrics, Gynecology and Reproductive Sciences, University of Pittsburgh, United States
Figures
Figure 1 with 1 supplement
Phylogenetic analysis of 9-1-1 complex subunits.
(A) Gene presence and absence matrix of human 9-1-1 subunit ortholog genes in 33 representative mammals. High confidence was determined if the genomic sequence had ≥50% of both target and query …
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Figure 1—source data 1
Phylogenetic analysis of the 9-1-1 complexes.
Data in this source file was used for Figure 1B.
- https://cdn.elifesciences.org/articles/68677/elife-68677-fig1-data1-v1.txt
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Figure 1—source data 2
Gene expression analysis in mouse testes.
Relative expression of 9-1-1 complex subunits and genes involved in ATR signaling. These data were used for Figure 1C. Original data are accessible through GEO series accession number GSE121904.
- https://cdn.elifesciences.org/articles/68677/elife-68677-fig1-data2-v1.xlsx
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Figure 1—source data 3
tSNE plots showing single-cell RNA expression of 9-1-1 complex subunits in testes.
These data were used to create tSNE plots for Figure 1—figure supplement 1B–D. Source code is available at https://github.com/nyuhuyang/scRNAseq-SSCs (copy archived at swh:1:rev:9e17b2b7a8871b9aa4f506fdb723f637fc9f0b2c, Hu, 2022).
- https://cdn.elifesciences.org/articles/68677/elife-68677-fig1-data3-v1.pdf
Figure 1—figure supplement 1
Expression of 9-1-1 complex subunits.
(A) Expression of 9-1-1 subunits in various human tissues. Data from the Genotype-Tissue Expression (GTEx) project was obtained in Expression Atlas – EMBL-EBI. Gene expression values are shown as …
Figure 2 with 2 supplements
Conditional knockout (CKO) of the 9-1-1 complex subunit RAD1 causes severe germ cell loss in testes.
(A) Evolutionary rate covariation analysis between 9-1-1 subunits. Lines depict significant covariance between 9-1-1 subunits. (B) Schematic showing putative meiotic 9-1-1 complexes: 9A-1-1, 9B-1-1, …
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Figure 2—source data 1
Control and Rad1 conditional knockout (CKO) testes weights.
- https://cdn.elifesciences.org/articles/68677/elife-68677-fig2-data1-v1.xlsx
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Figure 2—source data 2
Control and Rad1 conditional knockout (CKO) TUNEL+ cell counts.
- https://cdn.elifesciences.org/articles/68677/elife-68677-fig2-data2-v1.xlsx
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Figure 2—source data 3
Control and Rad1 conditional knockout (CKO) TRA98+ cell counts.
- https://cdn.elifesciences.org/articles/68677/elife-68677-fig2-data3-v1.xlsx
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Figure 2—source data 4
Control and Rad1 conditional knockout (CKO) LIN28+ cell counts.
- https://cdn.elifesciences.org/articles/68677/elife-68677-fig2-data4-v1.xlsx
Figure 2—figure supplement 1
RAD1 levels are reduced in juvenile testes.
Immunoblotting for RAD1 in control (n = 2) and Rad1 conditional knockout (CKO) (n = 4) whole testes lysates from 14-day-old mice. The last lane contains cell lysate from 293T cells transiently …
Figure 2—figure supplement 2
Rad1 inactivation in testis causes germ cell loss.
(A) Representative images of zygotene/pachytene-stage TUNEL-positive cells from 4 week-old and 12 week-old Rad1 conditional knockout (CKO) testes. (B, C) Representative images of TRA98-positive …
Figure 3 with 2 supplements
Testis-specific RAD1 loss disrupts 9-1-1 complex localization and causes defects in homolog synapsis and DNA damage signaling.
(A-C) Meiotic spreads from 12-week-old control and Rad1 conditional knockout (CKO) mice stained for RAD1 (A), RAD9B (B), or RAD9A (C). (D) Co-staining for SYCP1 and SYCP3 in meiotic spreads from …
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Figure 3—source data 1
RAD1 foci counts in control and Rad1 conditional knockout (CKO) spermatocytes.
- https://cdn.elifesciences.org/articles/68677/elife-68677-fig3-data1-v1.xlsx
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Figure 3—source data 2
Quantification of synapsed chromosomes in control and Rad1 conditional knockout (CKO) spermatocytes.
- https://cdn.elifesciences.org/articles/68677/elife-68677-fig3-data2-v1.xlsx
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Figure 3—source data 3
Total MLH1 foci in control and Rad1 conditional knockout (CKO) spermatocytes.
- https://cdn.elifesciences.org/articles/68677/elife-68677-fig3-data3-v1.xlsx
Figure 3—figure supplement 1
Rad1 conditional knockout (CKO) spermatocytes vary in the extent of RAD1 loss and meiotic defects.
(A) Total RAD1 foci counts in mid-pachytene control and Rad1 CKO cells. Approximately equal numbers of Rad1 CKO cells with apparently normal homolog synapsis (normal) or with synapsis defects …
Figure 3—figure supplement 2
RAD1-deficient spermatocytes have synapsis defects and do not progress to mid-pachynema.
(A) Examples of SYCP1/3 co-staining in control and Rad1 conditional knockout (CKO) meiotic spreads (three control mice; n = 156 cells; three CKO mice; n = 131 cells). (B) Total synapsed chromosomes …
Figure 4 with 1 supplement
Double-strand break (DSB) repair is compromised in the absence of 9-1-1 complexes.
(A, B) Representative images (A) and quantification (B) of RPA2 staining of meiotic spreads from 12-week-old control and Rad1 conditional knockout (CKO) mice (three mice per genotype analyzed; n = …
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Figure 4—source data 1
Total foci counts for RPA, MEIOB, and RAD51 in spermatocytes from control and Rad1 conditional knockout (CKO) mice, as well as RAD51 and RPA in spermatocytes from irradiated control and Rad1 CKO mice.
- https://cdn.elifesciences.org/articles/68677/elife-68677-fig4-data1-v1.xlsx
Figure 4—figure supplement 1
The 9-1-1 complexes are critical for proper localization of meiotic double-strand break (DSB) repair proteins.
(A) Representative images of wild-type spermatocytes co-stained for RAD1 and RPA2 (three mice analyzed, n = 107 cells). (B) Representative images of control and Rad1 conditional knockout (CKO) …
Figure 5 with 2 supplements
Key ATR phosphorylation events for double-strand break (DSB) repair and cohesion are dependent upon 9-1-1 complexes.
(A) ATR localization in meiotic spreads from control and Rad1 conditional knockout (CKO) 12-week-old mice (three control mice, n = 171 cells; three CKO mice, n = 146). (B) Representative images of …
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Figure 5—source data 1
Evolutionary rate covariation (ERC) calculations for 9-1-1 complex subunits and meiosis I-related proteins.
Data in this source file were used to generate Figure 2A, Figure 5—figure supplement 1B and C, and Figure 5—figure supplement 2A and B.
- https://cdn.elifesciences.org/articles/68677/elife-68677-fig5-data1-v1.txt
Figure 5—figure supplement 1
Phosphorylation of CHK1 and SMC3 is reduced in the absence of 9-1-1 complexes.
(A) Representative immunoblots for phosphorylated pCHK1 (S317 and S345) in whole testis lysates from 8-week-old control and Rad1 conditional knockout (CKO) mice. Arrowheads denote the pCHK1 band. (B,…
Figure 5—figure supplement 2
Evolutionary rate covariation (ERC) network of meiosis I proteins.
(A) Heatmap of proteins found under Gene Ontology term meiosis I (GO:0007127) that showed ERC values ≥ 0.4 for each subunit. Pink labeling denotes proteins with no significant ERC values (p>0.05). …
Figure 6
9-1-1 complexes are required for ATR-mediated meiotic sex chromosome inactivation.
(A, B) Representative images of HORMAD1 (A) and HORMAD2 (B) localization in meiotic spreads from 12-week-old control and Rad1 conditional knockout (CKO) mice (three control mice, n = 146 cells; …
Tables
Table 1
Analysis of epididymal sperm counts and fertility in Rad1 conditional knockout (CKO) and control mice.
| Genotype | No. males | Epididymal sperm count(×106) | No. matings | No. copulatory plugs | No. pregnancies | Total viable pups |
|---|---|---|---|---|---|---|
| Control | 3 | 16.6 ± 4.5 | 12 | 12 | 10 | 66 |
| Rad1 CKO | 3 | 0.0 ± 0 | 15 | 15 | 0 | 0 |
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Male Rad1 CKO mice at 8-12 weeks of age were bred to 6-week-old wild-type FVB female mice.
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Antibody | Anti-RAD1; HM454(rabbit polyclonal) | Lyndaker et al., 2013a | IF (1:100) WB (1:1000) | |
| Antibody | Anti-RAD9A; HM456(rabbit polyclonal) | Lyndaker et al., 2013b | IF (1:100) | |
| Antibody | Anti-RAD9B(rabbit polyclonal) | Pérez-Castro and Freire, 2012 | IF (1:100) | |
| Antibody | Anti-phospho-histone H2A.X (Ser139) antibody, clone JBW301 (mouse monoclonal) | Millipore | Cat# 05-636;RRID:AB_309864 | IF (1:1000) |
| Antibody | SCP3 antibody [Cor 10G11/7](mouse polyclonal) | Abcam | Cat# ab97672;RRID:AB_10678841 | IF (1:1000) |
| Antibody | Anti-SYCP3(rabbit polyclonal) | Lenzi et al., 2005 | IF (1:1000) | |
| Antibody | Rabbit anti-SCP1 polyclonal antibody, unconjugated | Abcam | Cat# ab15090;RRID:AB_301636 | IF (1:1000) |
| Antibody | Anti-Rad51 (Ab-1) rabbit pAb antibody(rabbit polyclonal) | Millipore | Cat# PC130;RRID:AB_2238184 | IF (1:1000) |
| Antibody | Anti-RPA2; UP2436(rabbit polyclonal) | Shi et al., 2019 | IF (1:500) | |
| Antibody | Anti-MEIOB; UP2327(rabbit polyclonal) | Luo et al., 2013 | IF (1:500) | |
| Antibody | Anti-replication protein A, clone RPA34-20(mouse monoclonal) | Millipore | Cat# MABE285;RRID:AB_11205561 | IF (1:100) |
| Antibody | ATR antibody(rabbit polyclonal) | Cell Signaling | Cat# 2790;RRID:AB_2227860 | IF (1:100) |
| Antibody | Anti-TOBP1(rabbit polyclonal) | Rendtlew Danielsen et al., 2009 | IF (1:500) | |
| Antibody | Anti-phospho-Chk1 (ser317) (D12H3) XP(rabbit monoclonal) | Cell Signaling | Cat# 12302;RRID:AB_2783865 | IF (1:100) |
| Antibody | Anti-MLH1(mouse monoclonal) | BD Biosciences | Cat# 550838;RRID:AB_2297859 | IF (1:1000) |
| Antibody | Anti-H1T(guinea pig polyclonal) | Inselman et al., 2003 | IF (1:500) | |
| Antibody | Anti-HORMAD2; AB324(rabbit polyclonal) | Wojtasz et al., 2009 | IF (1:500) | |
| Antibody | Anti-HORMAD1; AB211(rabbit polyclonal) | Wojtasz et al., 2009 | IF (1:500) | |
| Antibody | Rabbit anti-SMC3 antibody, affinity purified(rabbit polyclonal) | Bethyl | Cat# A300-060A; RRID:AB_67579 | IF (1:100) WB (1:1000) |
| Antibody | Rabbit anti-phospho SMC3 (S1083) IHC antibody(rabbit polyclonal) | Bethyl | Cat# IHC-00070;RRID:AB_2255076 | IF (1:100) WB (1:1000) |
| Antibody | Anti-mouse TRA98 monoclonal antibody, unconjugated(mouse monoclonal) | BioAcademia | Cat# 73-003; RRID:AB_1056334 | IF (1:100) |
| Antibody | Rabbit anti-Lin28 polyclonal antibody, unconjugated(rabbit polyclonal) | Abcam | Cat# ab63740; RRID:AB_1310410 | IF (1:100) |
| Antibody | GAPDH monoclonal antibody (6C5)(mouse monoclonal) | Thermo Fisher Scientific | Cat# AM4300; RRID:AB_2536381 | WB (1:5000) |
| Antibody | β-Actin antibody(rabbit polyclonal) | Cell Signaling | Cat# 4967; RRID:AB_330288 | WB (1:5000) |
| Antibody | Goat anti-rabbit IgG (H + L) highly cross-adsorbed secondary antibody, Alexa Fluor 488(rabbit polyclonal) | Thermo Fisher Scientific | Cat# A-11034; RRID:AB_2576217 | IF (1:1000) |
| Antibody | Goat anti-mouse IgG (H + L) antibody, Alexa Fluor 488 conjugated(mouse polyclonal) | Thermo Fisher Scientific | Cat# A-11017; RRID:AB_143160 | IF (1:1000) |
| Antibody | Goat anti-rabbit IgG (H + L) antibody, Alexa Fluor 594 conjugated(rabbit polyclonal) | Thermo Fisher Scientific | Cat# A-11012; RRID:AB_141359 | IF (1:1000) |
| Antibody | Goat anti-mouse IgG (H + L) highly cross-adsorbed secondary antibody, Alexa Fluor Plus 594(mouse polyclonal) | Thermo Fisher Scientific | Cat# A32742; RRID:AB_2762825 | IF (1:1000) |
| Antibody | Goat anti-guinea pig IgG (H + L) highly cross-adsorbed secondary antibody, Alexa Fluor 647(guinea pig polyclonal) | Thermo Fisher Scientific | Cat# A-21450; RRID:AB_141882 | IF (1:1000) |
| Sequence-based reagent | Cre ic318R | Lyndaker et al., 2013a | PCR primers | AGGGACACA GCATTGGAGTC |
| Sequence-based reagent | Cre ic202F | Lyndaker et al., 2013b | PCR primers | GTGCAAGCT GAACAACAGGA |
| Sequence-based reagent | Rad1 G1F | Wit et al., 2011 | PCR primers | AGGTACGTC AGTGCGATTACCCT |
| Sequence-based reagent | Rad1 G3R | Wit et al., 2011 | PCR primers | CCCTCAAGAT GTAACCTC ATCTAC |
| Sequence-based reagent | Hus1 3.107 | Lyndaker et al., 2013a | PCR primers | GGGCTGATGC GGAGGGTG CAGGTT |
| Sequence-based reagent | Hus1 Neo1 | Lyndaker et al., 2013b | PCR primers | GCTCTTTACT GAAGGCTCTTTAC |
| Sequence-based reagent | Hus1 5-OSMCS2 | Lyndaker et al., 2013a | PCR primers | GCGAAGACGG AATTGATCA GGCCACG |
| Sequence-based reagent | Hus1 5.-20 | Lyndaker et al., 2013b | PCR primers | CCGTCGGCCT GGTATCC GCCATGA |
| Sequence-based reagent | Hus1 3.159 | Lyndaker et al., 2013b | PCR primers | CTCACAACTGCT ACAAGGTTAGGC |
| Commercial assay or kit | ApopTag Plus Peroxidase In Situ Apoptosis Kit | Millipore | Sigma-Aldrich: S7101 | |
| Chemical compound, drug | AZ20, ATR inhibitor | Selleckchem | Selleckchem: S7050 | |
| Software, algorithm | GraphPad Prism 9 | GraphPad | RRID:SCR_002798 |
