Dual histone methyl reader ZCWPW1 facilitates repair of meiotic double strand breaks in male mice
- The Eunice Kennedy Shriver National Institute of Child Health and Human Development, NIH, United States
- Laboratory of Genome Integrity, National Cancer Institute, NIH, United States
- Immunology Graduate Group, University of Pennsylvania, United States
- Department of Epigenetics and Molecular Carcinogenesis, University of Texas MD Anderson Cancer Center, United States
Figures
Figure 1 with 2 supplements
ZCWPW1 tissue expression and methyl histone binding activity in vitro.
(A) Heatmap of the top 25 genes co-expressed with PRDM9 during meiosis in single cells (Chen et al., 2018). Rows (genes) are ordered based on PRDM9 correlation coefficient (rho), from Figure …
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Figure 1—source data 1
Correlation of expression between cellular genes and Prdm9 in single cell RNA-seq analysis.
- https://cdn.elifesciences.org/articles/53360/elife-53360-fig1-data1-v2.xlsx
Figure 1—figure supplement 1
Zcwpw1 transcript expression in human and mouse tissues.
ZCWPW1 transcript expression from different tissues in human (GTEx) (A), and mouse (Li et al., 2017) (B).
Figure 1—figure supplement 2
ZCWPW1 antibody specificity.
Testing ZCWPW1 antibody specificity for ZCWPW1 and potential cross-reactivity with ZCWPW2.
His-tagged recombinant ZCWPW1 (~70 kDa) and ZCWPW2 (~49 kDa) were loaded in SDS-PAGE gel and transferred to PVDF membrane. Membranes were then subjected for either Ponceau S staining (right) or immunoblotting with anti-ZCWPW1 rabbit polyclonal antibody (left).
Figure 2 with 4 supplements
Evolution of Zcwpw1 in vertebrates.
(A) ZCWPW1 protein sequences available from public databases (174 species) were aligned by CLC Genomics Workbench using MUSCLE algorithm (Edgar, 2004). Alignment conservation score is plotted as …
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Figure 2—source data 1
ZCWPW1 orthologs used in evolution analysis.
- https://cdn.elifesciences.org/articles/53360/elife-53360-fig2-data1-v2.xlsx
Figure 2—figure supplement 1
Phylogenetic analysis of potential ZCWPW1 orthologs discovered by BLAST.
Maximum likelihood phylogenetic tree was made to differentiate ZCWPW1 orthologs from ZCWPW2 orthologs among tentative sequences discovered by BLAST (see methods). Using CLC Genomics Workbench …
Figure 2—figure supplement 2
Amino acid variability in ZCWPW1 orthologs.
Plot for variability of amino acid residues in aligned sequences of ZCWPW1 orthologs in Figure 2A. Amino acid positions in mouse ZCWPW1 were used as reference for plotting. Shannon entropy analysis …
Figure 2—figure supplement 3
Alignment of SCP1 domains from mouse ZCWPW1 and SYCP1.
(A) Alignment of SCP1 domain from mouse ZCWPW1 (76–228 aa) with SCP1 domain from mouse SYCP1. Alignment is done by CLC Genomics Workbench using MUSCLE algorithm (Edgar, 2004). Alignment segments …
Figure 2—figure supplement 4
PRDM9 domain structure in bony fishes.
Comparison of PRDM9 domain structure in bony fish species with or without ZCWPW1 ortholog. PRDM9 information reproduced from Baker et al., 2017.
Figure 3 with 4 supplements
Mapping of ZCWPW1 chromatin biding in vivo using CUT&RUN in spermatocytes from B6/B6 mice.
(A) Heatmaps representing ZCWPW1, H3K4me3 and IgG (anti-GFP) CUT&RUN read coverage in B6/B6 mouse spermatocytes at hotspots determined by DMC1 SSDS (GSE99921). Signals are centered around PRDM9 …
Figure 3—figure supplement 1
Mapping of ZCWPW1 chromatin biding in vivo using CUT&RUN in spermatocytes from B6/B6 mice.
(A) Heatmaps representing ZCWPW1, H3K4me3 and IgG (anti-GFP) CUT&RUN read coverage in B6/B6 mice spermatocytes at SPO11 oligo summits (GSE84689). Y-axis of line plots in the top represents mean …
Figure 3—figure supplement 2
ZCWPW1 correlation with other metrics of meiotic recombination at DSB hotspots.
Correlation of ZCWPW1 CUT&RUN signal strength (left) and SPO11 oligo intensity (GSE84689) (right) with other metrics of recombination at DSB hotspots: DMC1 SSDS (GSE35498), H3K4me3 12dpp (GSE35498), …
Figure 3—figure supplement 3
ZCWPW1 binding at functional genomic sites.
(A) Heatmaps representing ZCWPW1, H3K4me3 and IgG (anti-GFP) CUT&RUN read coverage in B6/B6 mouse spermatocytes at indicated regions. Y-axis of line plots in the top represents mean coverage …
Figure 3—figure supplement 4
ZCWPW1 binding at chromosome X pseudoautosomal region.
Read coverage plots for ZCWPW1, H3K4me3 and IgG (anti-GFP) CUT&RUN, and DMC1 SSDS hotspots from WT and PRDM9 KO (GSE35498) in chromosome X pseudoautosomal region (PAR) or at PAR-adjacent …
Figure 4 with 1 supplement
Mapping of ZCWPW1 chromatin biding in vivo using CUT&RUN in spermatocytes from F1 B6/CAST hybrid mice.
(A) Comparisons of de novo discovered motifs for PRDM9Dom2, PRDM9Cast and ZCWPW1. For PRDM9Dom2 and PRDM9Cast, PRDM9 ChIP-seq peaks (GSE93955) for either allele were queried by HOMER to identify the …
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Figure 4—source data 1
Enrichment of known motifs at ZCWPW1 binding sites in B6/B6 mice.
- https://cdn.elifesciences.org/articles/53360/elife-53360-fig4-data1-v2.xlsx
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Figure 4—source data 2
De novo motifs discovery at ZCWPW1 binding sites in B6/B6 and F1 B6/CAST hybrid mice.
- https://cdn.elifesciences.org/articles/53360/elife-53360-fig4-data2-v2.xlsx
Figure 4—figure supplement 1
Mapping of ZCWPW1 chromatin biding in vivo using CUT&RUN in spermatocytes from F1 B6/CAST hybrid mice.
(A) Upset plot showing intersections of ZCWPW1 CUT&RUN peaks (from B6/CAST F1 hybrid spermatocytes, n = 8,976 peaks) with DMC1 SSDS hotspots (GSE75419) from (i) B6/B6, (ii) CAST/CAST and (iii) …
Figure 5 with 1 supplement
Zcwpw1 KO mice are azoospermic and display hallmarks of defective synapsis and DSB repair.
(A) Hematoxylin and Eosin staining of paraffin embedded tissue sections from testes of Zcwpw1 WT or Zcwpw1 KO. Scale bar is 100 µm. (B) Percentage distribution for meiotic prophase I stages in …
Figure 5—figure supplement 1
Phenotyping of Zcwpw1 KO spermatocytes.
(A) Western blot with ZCWPW1 specific polyclonal antibody in tissues from WT (blue lanes) or KO male mouse (red lanes) tissues. The arrow indicates ZCWPW1 band (B) Photo comparing size of testes …
Figure 6
Double Strand Break mapping by END-seq in spermatocytes from adult Zcwpw1 WT and Zcwpw1 KO mice.
(A) Venn diagram showing END-seq peak overlap between Zcwpw1 KO and Zcwpw1 WT (left) or between Zcwpw1 KO END-seq peaks and DMC1 SSDS hotspots (GSE35498) (right). (B) END-seq read coverage heatmaps …
Author response image 1
ZCWPW1 peaks overlapping hotspots are stronger than non-hotspots.
Left panel: B6, Right panel B6xCAST.
Author response image 2
ZCWPW1 CUT&RUN shows very little enrichment at functional sites in the genome.
Sequencing reads (q > 30) at each set of genomic intervals were counted for our four CUT&RUN experiments as well as for H3K4me3 ChIP-Seq data in 12dpp testis. The genomic intervals used are TSS and …
Author response image 3
ZCWPW1 CUT&RUN shows very little enrichment at functional sites in the genome.
Only sequencing reads with a mapping quality of >30 were used. The top and second-to-top panels represent DSB hotspots with / without a called ZCWPW1 peak, respectively. Data here are from B6 x CAST …
Author response image 4
ZCWPW1 CUT&RUN signal correlates with other metrics of recombination at DSB hotspots.
Data are shown for ZCWPW1 CUT&RUN in B6 mice. For all panels, only autosomal hotspots that coincide with a ZCWPW1 peak were used. Strength for SSDS and H3K4me3 12dpp are taken from Brick et al., …
Author response image 5
This figure is simply to provide a comparison point to Author response image 3.
Spo11-oligo signal correlates with other metrics of recombination. For all panels, only autosomal hotspots that coincide with a ZCWPW1 peak were used. Spo11 signal correlates best with DMC1-SSDS. …
