Znhit1 deletion in spermatocytes disrupts spermatogenesis.

(A) Schematic representation of the male germline development in mice. (B) Heatmap showing the clustering of 47 chromatin 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.

Znhit1 deletion leads to meiotic pachytene arrest.

(A-C) Immunostaining of SYCP3 in spermatocyte chromosome spreads of control (A) or Znhit1-sKO mice (B). Quantitative data are shown in (C). Scale bar, 10 μm. (D and E) TUNEL staining in testis sections from P14 control or Znhit1-sKO mice. Quantitative data are shown in (E). Scale bar, 40 μm. All images are representative of n = 3 mice per genotype. (F) UMAP plot showing the annotated cells captured from P35 control and Znhit1-sKO testicular cells. (G) UMAP plot showing the expression of Znhit1 and Pou5f2 in control P35 control and Znhit1-sKO testicular cells. Data are presented as the mean ± s.d. *** p < 0.001.

Znhit1 deletion impairs meiotic recombination.

(A and B) Immunostaining of SYCP3 and SYCP1 in spermatocyte chromosome spreads of control or Znhit1-sKO mice. Arrows indicate autosomal regions with abnormal synapsis. 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 and E) Immunostaining of SYCP3 and RAD51 in spermatocyte chromosome spreads of control or Znhit1-sKO mice. Quantitative data are shown in (E). Scale bar, 10 μm. (F) Immunostaining of SYCP3 and MLH1 in spermatocyte chromosome spreads of control or Znhit1-sKO mice. 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.

Znhit1 deletion impairs meiotic transcriptional programs.

(A) UMAP plot showing the annotated cells captured from P16 control and Znhit1-sKO testicular cells. (B) Violin plots showing the average expression level of upregulated or downregulated genes between two consecutive spermatocyte stages. PreL, preleptotene; Lep, leptotene; Zyg, zygotene; Pac, pachytene. (C) Volcano plot showing the distribution of upregulated and downregulated DEGs for each cell type in spermatocytes between control and Znhit1-sKO testicular cells. (D and E) Representative shared Gene Ontology (GO) terms of upregulated and downregulated DEGs at consecutive spermatocyte stages. (F) Volcano plot showing differentially expressed XY-linked genes in scRNA-seq data following Znhit1 deletion.

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.

ZNHIT1/H2A.Z/A-MYB axis regulates chromatin state 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 Mybl1-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) 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.

Znhit1 expression during meiotic progression and spermatocyte-conditional 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 scRNA-seq data (Chen et al, 2018). (D) Znhit1 in situ hybridization in P14 testis sections. Scale bar, 20 μm. (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. Scale bar, 50 μm.

Spermatocyte development in Znhit1-sKO testes.

(A) Immunostaining of SYCP3 and HSPA2 in testis sections of control or Znhit1-sKO mice at P35. Scale bar, 20 μm. (B) Immunostaining of SYCP3 and pH3 in testis sections of control or Znhit1-sKO mice at P35. Scale bar, 20 μm. (C) Immunostaining of H1T in testis sections from control and Znhit1-sKO mice at P35. Scale bar, 20 μm.

ZNHIT1 is required for meiotic recombination.

(A and B) Immunostaining of SYCP3 and RPA2 in spermatocyte chromosome spreads of control or Znhit1-sKO mice. Quantitative data are shown in (B). Scale bar, 10 μm. (C) Plots showing quantitative data of MLH1 foci of control or Znhit1-sKO mice. Data are presented as the mean ± s.d. *** p < 0.001

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) UMAP plots showing the normalized expression of the indicated genes in scRNA-seq data.

Transcriptomic analysis of testicular cells.

(A) Heatmap showing differentially expressed genes (DEGs) in P14 testicular cells between control and Znhit1-sKO mice. (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. (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.

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.

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.

KAS-seq analysis.

(A) Mean plots of KAS-seq showing signal changes within indicated genes.

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.