Figures and data
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Znhit1 deletion in spermatocytes disrupts spermatogenesis.
(A) Schematic representation of the male germline development in mice. (B) Heatmap showing the clustering of 47 epigenetic factors based on the transcription levels in leptotene and zygotene spermatocytes. (C) Znhit1 in situ hybridization in postnatal day 60 (P60) testis sections. Scale bar, 20 μm. Sg, spermatogonia; pL, preleptotene; L, leptotene; Z, zygotene; P, pachytene; D, diplotene, MI, metaphase; R, round spermatids; E, elongating spermatids. (D) Schematic diagram showing the onset of Stra8-cre expression in the male germline development. (E) Znhit1 in situ hybridization in P42 testis sections. Scale bar, 50 μm. (F) Histological testicular sections in control or Znhit1-sKO testis sections at the indicated times. Scale bar, 50 μm. (G) Immunostaining of PNA in testis sections from control or Znhit1-sKO mice at the indicated times. rST, round spermatids; eST, elongating spermatids. Scale bar, 20 μm. All images are representative of n = 3 mice per genotype.
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Znhit1 deletion leads to meiotic pachytene arrest.
(A) Immunostaining of SYCP3 and HSPA2 in testis sections of P35 control or Znhit1-sKO mice. Scale bar, 20 μm. (B) Immunostaining of SYCP3 and pH3 in testis sections of P35 control or Znhit1- sKO mice. Scale bar, 20 μm. (C-E) Immunostaining of SYCP3 in spermatocyte chromosome spreads of control (C) or Znhit1-sKO mice (D). Quantitative data are shown in (E). Scale bar, 10 μm. (F and G) TUNEL staining in testis sections from P14 control or Znhit1-CKO mice. Quantitative data are shown in (G). Scale bar, 40 μm. All images are representative of n = 3 mice per genotype. Data are presented as the mean ± s.d. *** p < 0.001.
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Znhit1 controls meiotic recombination.
(A and B) Immunostaining of SYCP3 and SYCP1 in spermatocyte chromosome spreads of control or Znhit1-sKO mice. X, X chromosome; Y, Y chromosome; PAR, pseudoautosomal region. Quantitative data are shown in (B). Scale bar, 10 μm. (C) Immunostaining of SYCP3 and γH2AX in spermatocyte chromosome spreads of control or Znhit1-sKO mice. Scale bar, 10 μm. (D) Immunostaining of SYCP3 and pATM in spermatocyte chromosome spreads of control or Znhit1-sKO mice. Scale bar, 10 μm. (E and F) Immunostaining of SYCP3 and RAD51 in spermatocyte chromosome spreads of control or Znhit1-sKO mice. Quantitative data are shown in (F). Scale bar, 10 μm. (G and H) Immunostaining of SYCP3 and MLH1 in spermatocyte chromosome spreads of control or Znhit1-sKO mice. Quantitative data are shown in (H). Scale bar, 10 μm. All images are representative of n = 3 mice per genotype. Data are presented as the mean ± s.d. *** p < 0.001.
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Spermatocyte transcriptome analysis using scRNA-seq.
(A) UMAP plot showing the annotated germ cells captured from both control and Znhit1-sKO testicular cells. (B) Expression of selected markers identifying major somatic cell types and germ cell types on the UMAP plot. UMAP plot showing the expression patterns of Znhit1 in control or Znhit1-sKO testicular cells. (C) Heatmap showing the top marker genes of each cell cluster from scRNA-seq data. U-Sg, undifferentiated spermatogonia; D-Sg, differentiating spermatogonia; PreL, preleptotene; Lep, leptotene; Zyg, zygotene; Pac, pachytene. (D) Top: UMAP plot showing the annotated germ cells captured from control or Znhit1-sKO testicular cells. Down: UMAP plot showing the expression patterns of Znhit1 in control or Znhit1-sKO testicular cells. (E) Bar plot showing the percentage of major spermatocyte types for control or Znhit1-sKO testicular cells. (F) UMAP plot showing the expression patterns of Pou5f2 in control or Znhit1-sKO germ cells.
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Znhit1 deletion impairs meiotic transcriptional activation.
(A) Line graph showing DEGs (upregulation or downregulation) in two consecutive spermatocyte stages. PreL, preleptotene; Lep, leptotene; Zyg, zygotene; Pac, pachytene. (B) Volcano plot showing the distribution of upregulated and downregulated DEGs for each cell type in spermatocytes between control or Znhit1-sKO testicular cells. (C) Cluster annotation on the basis of the expression of pachytene-activated genes in control or Znhit1-sKO testicular cells. (D) Venn plots showing the number of shared genes between pachytene- activated genes and DEGs in Znhit1-sKO pachytene spermatocytes. (E) Representative shared Gene Ontology (GO) terms of upregulated (upper) and downregulated (lower) DEGs at consecutive spermatocyte stages. (F) Volcano plot showing differentially expressed genes (DEGs) in bulk RNA-seq following Znhit1 deletion (n = 2). (G) Heatmap depicting the expression of the indicated genes at the zygotene and pachytene stages in the scRNA-seq data. (H) Cluster annotation on the basis of the expression of downregulated genes in bulk RNA-seq between control and Znhit1-sKO testicular cells. (I) GSEA of RNA-seq data for the control and Znhit1-sKO testicular cells. Selected gene sets encoded products related to homologous recombination. (J) Violin plots showing downregulated DEGs of Znhit1-deficient zygotene cells and pachytene cells related to homologous recombination. (K) Immunostaining of SYCP3 and SUMO2/3 in spermatocyte chromosome spreads of control or Znhit1-sKO mice. Scale bar, 10 μm. cluster genes. ** p < 0.01, *** p < 0.001, **** p < 0.0001.
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Znhit1 regulates H2A.Z binding on promoter and enhancer regions.
(A) Immunostaining of SYCP3 and H2A.Z in spermatocyte chromosome spreads of control or Znhit1-sKO mice. The dashed circle indicates X-Y chromosomes. Scale bar, 10 μm. (B) Representative tracks of END-seq, DMC1-SSDS, and H2A.Z ChIP-seq in wild-type, control, or Znhit1-sKO testicular cells. (C) Heatmaps of chromatin states produced by ChromHMM and showing different enrichment for H2A.Z in control or Znhit1-sKO testicular cells. (D) Heatmaps and mean plots of H2A.Z ChIP-seq signal in control or Znhit1-sKO testicular cells (n = 2). (E) Venn diagram showing the overlap between decreased H2A.Z-bound genes and downregulated genes in Znhit1-sKO testicular cells. (F) Heatmaps and mean plots of downregulated H2A.Z ChIP-seq signals surrounding different types of promoters and enhancers.
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Znhit1/H2A.Z/A-MYB axis regulates enhancer activation and gene expression.
(A) Motif analysis of active promoters or active enhancers with decreased H2A.Z binding for putative transcription factor (TF)-binding sites using the HOMER database. (B) Ridge map showing the A-MYB activity (AUC) at each cell type. (C) Scatter plot with marginal histograms comparing the fold change of DEGs from A-MYB- related RNA-seq data with Znhit1-related RNA-seq data in P14 testicular cells. (D) GSEA of RNA-seq data for the control and Znhit1-sKO testicular cells. Selected gene sets encoded products related to A-MYB target genes. (E) Violin plots showing the expression of A-MYB target genes at consecutive spermatocyte stages. (F) Histogram showing H2A.Z and A-MYB binding sites. (G) Pie chart showing the distribution of H2A.Z and A- MYB co-binding sites. (H) Representative H2A.Z ChIP-seq tracks, KAS-seq signals, and RNA-seq signals of the Ccnb1ip1 locus in the control and Znhit1-sKO testicular cells, compared with A-MYB ChIP-seq peaks. (I and J) Heatmaps and mean plots of H2A.Z ChIP-seq, KAS-seq, and ATAC-seq showing signal changes within A-MYB and H2A.Z co-binding active promoters or active enhancers. **** p < 0.0001.
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Znhit1 expression during meiotic progression and spermatocyte-specific deletion of Znhit1 with Stra8-cre.
(A and B) Venn diagram and heatmaps showing chromatin regulators highly expressed during leptotene and zygotene spermatocytes. (C) Violin plot showing Znhit1 transcription level during spermatogenesis. Gene expression profiles derived from published data (Chen et al, 2018). (D) Znhit1 in situ hybridization in P14 testis sections. (E) Testis size comparison between control and Znhit1-sKO mice at P35. (F) Hematoxylin and eosin (H&E) - stained testis sections from control and Znhit1-sKO mice at P60.
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H1T+ pachytene spermatocytes are detected in Znhit1-sKO testes.
(A) Immunostaining of H1T in testis sections from control and Znhit1-sKO mice at P35. Scale bar, 20 μm.
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Znhit1 controls meiotic recombination.
(A) Immunostaining of SYCP3 and RPA2 in spermatocyte chromosome spreads of control or Znhit1-sKO mice. Scale bar, 10 μm.
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Single-cell RNA-seq of testicular cells.
(A) Dot plot showing the relative expression and the percentage of cells expressing selected markers across scRNA-seq clusters. (B) Violin plot showing the normalized expression of Znhit1 in control and Znhit1-sKO spermatocytes at consecutive stages.
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Single-cell transcriptomic analysis of meiotic genome activation.
(A) Violin plots showing the average expression level of upregulated or downregulated genes between two consecutive spermatocyte stages. (B) Violin plots showing the average expression level of upregulated or downregulated genes between P14 control and Znhit1- sKO testicular cells. PreL, preleptotene; Lep, leptotene; Zyg, zygotene; Pac, pachytene.
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differentially expressed genes (DEGs) in P14 testicular cells between control and Znhit1-sKO mice. (B) Gene ontology (GO) analysis of DEGs showing changed biological processes. (C and D) Bar charts showing HR-related gene expression in P14 control and Znhit1-sKO testes. Data from bulk RNA-seq are shown in (C). Validated data from RT-qPCR are shown in (D). Ctrl, control; sKO, Znhit1-sKO.
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H2A.Z is enriched at meiotic gene promoters and enhancers.
(A) Schematic representation of the components of the SRCAP chromatin remodeling complex. (B) Heatmaps showing H2A.Z ChIP-seq signals surrounding different types of promoters. (C) Heatmaps showing H2A.Z ChIP-seq signals surrounding different types of enhancers.
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Znhit1 deletion downregulates the expression of A-MYB target genes.
(A) Violin plot showing the normalized expression of Mybl1 in control and Znhit1-sKO spermatocytes at consecutive stages. (B) Cluster annotation on the basis of the expression of A-MYB target genes in control or Znhit1-sKO testicular cells.
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Reproducibility of replicates in this study.
(A) Scatter plots showing H2A.Z enrichments in P14 control or Znhit1-sKO testes for two independent H2A.Z ChIP-seq replicates. Pearson correlation coefficients are indicated. (B) Scatter plots showing KAS- seq signals in P14 control or Znhit1-sKO pachytene cells for two independent H2A.Z ChIP-seq replicates. Pearson correlation coefficients are indicated. (C) Scatter plots showing ATAC-seq signals in P14 control or Znhit1-sKO pachytene cells for two independent H2A.Z ChIP-seq replicates. Pearson correlation coefficients are indicated.