Genome-wide map of R-loops reveals its interplay with transcription and genome integrity during germ cell meiosis

Abstract

Introduction: The R-loop is a naturally formed three-strand nucleic acid structure that recently has been reported to participate in multiple biological processes and helped answer some previously unexplained scientific questions. Meiosis process involves multiple chromatin-related events such as DNA double-stranded breaks (DSB) formation, repairing and transcriptional dynamics.

Objectives: Explore the regulatory roles and physiological functions of R-loops in the mammalian meiosis process.

Methods: In our study, using genome-wide S9.6 CUT & Tag seq, we first mapped the genomic distribution and dynamic changes of R-loop during the meiotic process in mice, from spermatogonia to secondary spermatocytes. And we further explore the role of R-loop in physiological conditions by constructing conditional knockout mice of Rnaseh1, which deleted the R-loop endonuclease before meiosis entry.

Results: R-loop predominantly distributes at promoter-related regions and varies across different meiotic stages. By joint analysis with the corresponding transcriptome, we found that the R-loop was closely related to transcription during the meiotic process. The high frequency of promoter-related R-loop in meiotic cells is usually accompanied by high transcription activity, and we further verified this in the leptotene/zygotene to the pachytene transition process. Moreover, the lack of RNase H1 caused sterility in male mice with R-loop accumulation and abnormal DSB repair in spermatocytes. Further analysis showed that abnormal R-loop accumulation in the leptotene/zygotene stages influenced transcriptional regulation in the pachytene stage.

Conclusion: The mutual regulation of the R-loop and transcription plays an essential role in spermatogenesis. And R-loop is also important for the normal repair process of DSB during meiosis.

Keywords: DNA repair; Genome stability; Homologous recombination; Meiosis; R-loop; Spermatogenesis.

Graphical abstract

Fig. 1

Genomic distribution of R-loop peaks during spermatogenesis. A: Pie charts showing the genomic distribution variation of R-loop peaks during spermatogenesis. SPG, spermatogonia; L/Z, leptotene/zygotene; P/D, pachytene/diplotene; MII, secondary spermatocytes. B: Genomic metaplots showing relative signal intensity comparison of R-loops across the 2 kb window around gene bodies in spermatogonia and spermatocytes. R-loop signals of different stages were normalized based on library sizes and spike-in scaling factors. C: Alluvial plots showing the global dynamics of genes with R-loop and genes without R-loops during spermatocyte development. Each line represents an R-loop gene, defined as a gene-detected R-loop peak in at least one analyzed stage. D: Box plots showing the expression level comparison of different gene clusters grouped by R-loop variation between adjacent stages in meiotic progress. E: Scatter plots showing the comparison of signal intensities of R-loop peaks between leptotene/zygotene and pachytene/diplotene spermatocytes. F: Volcano plots showing differentially expressed genes between leptotene/zygotene and pachytene spermatocytes. Genes upregulated (FC > 2; FDR < 0.05) and downregulated (FC < 1/2; FDR < 0.05) in pachytene spermatocytes are colored red and blue, respectively. G: Venn diagrams showing the overlap of R-loop accumulated genes and transcriptionally upregulated R-loop genes in leptotene/zygotene to pachytene stage. H: Venn diagrams showing the overlap of R-loop decreased genes and transcriptionally downregulated R-loop genes in leptotene/zygotene to pachytene stage. I: Venn diagrams showing the overlap of H3K4me3 peaks in genes promoter regions and R-loop peaks in leptotene/zygotene spermatocytes (Fisher’s exact test; ***P < 0.001). J: Venn diagrams showing the overlap of H3K4me3 peaks in genes promoter regions and R-loop peaks in pachytene spermatocytes (Fisher’s exact test; ***P < 0.001). K: Example snapshot of the relative distribution of the R-loop, H3K4me3, and downstream transcriptional level. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 2

RNase H1 deletion in pre-meiotic germ cells causes spermatogenic defects. A: RNase H1 immunofluorescence staining on spermatocyte spreads from WT mice. DNA was counterstained with DAPI. Scale bar: 10 µm. B: Western blot of ribonuclease H1 (RNase H1) in spermatogenic cells isolated from WT mice. L/Z, leptotene/zygotene; P, pachytene; D, diplotene; M, meiotic metaphase; RS, round spermatids. C: S9.6 immunofluorescence staining on WT mice spermatocyte spreads. Scale bar: 10 µm. D: Western blot of RNase H1 in germ cells from WT and Rnaseh1^fl/−;Stra8-Cre mice. E-F: Left tri-color panel, Immunofluorescent co-staining of SYCP3 and RNase H1 in testes of 6-week-old WT (A) and Rnaseh1^fl/−;Stra8-Cre (B) mice. DNA was counterstained with DAPI. Scale bar: 50 µm. The right three panels show enlarged images of each channel. Meiotic cells of the lumen are divided out from spermatogonia and Sertoli cells with a white dotted line. G: Gross morphology of testes from Rnaseh1^fl/−;Stra8-Cre and WT littermate mice. Scale bar: 1 mm. H: Weights of testis from 6-week-old Rnaseh1^fl/−;Stra8-Cre and WT littermate mice. Error bars, s.e.m. ***P < 0.001 (two-tailed Student’s t-test). I-J: H&E staining results showing testis (F) and epididymis (G) histology of 6-week-old WT and Rnaseh1^fl/−;Stra8-Cre mice. Scale bar: 50 µm. K: MVH immunofluorescence staining in the testes of 6-week-old WT and Rnaseh1^fl/−;Stra8-Cre mice. DNA was counterstained with DAPI. Scale bar: 50 µm.

Fig. 3

RNase H1 deletion in pre-meiotic germ cells causes pachytene arrest with increased R-loops. A: Immunofluorescent co-staining of H1T and γH2AX in the testes of 6-week-old WT and Rnaseh1^fl/−;Stra8-Cre mice. DNA was counterstained with DAPI. Scale bars: 50 µm. B: S9.6 immunofluorescent staining in the testes of 6-week-old WT and Rnaseh1^fl/−;Stra8-Cre mice. DNA was counterstained with DAPI. Scale bars: 50 µm. The below panels are enlarged images. C: S9.6 immunofluorescence staining on spermatocyte spreads made from WT and Rnaseh1^fl/−;Stra8-Cre mice. Scale bars: 10 µm. D: Spermatocyte stage proportion in WT and Rnaseh1^fl/−;Stra8-Cre mice based on SYCP3 and γH2AX immunofluorescence staining of spermatocyte spreads. The number of analyzed spermatocytes is indicated (n). Error bars, s.e.m.. n.s., non-significant. **P < 0.01. ***P < 0.001 (two-tailed Student’s t-test). The experiment was repeated at least 3 times.

Fig. 4

Rnaseh1 deletion impairs the DSB repair process in spermatogenic cells. A: Immunofluorescence staining of SYCP3 and γH2AX on WT and Rnaseh1^fl/−;Stra8-Cre spermatocyte spreads. Scale bars: 10 µm. B: The proportion of autosomes DSB repaired and unrepaired ratio in WT and Rnaseh1^fl/−;Stra8-Cre spermatocytes. Error bars, s.e.m. **P < 0.01 (two-tailed Student’s t-test). The experiment was repeated at least 3 times. C-E: Immunofluorescence staining of DMC1 (A), RAD51 (B), and RPA2 (C) on spermatocyte spreads made from WT and Rnaseh1^fl/−;Stra8-Cre mice. Scale bars: 10 µm. F: Western blot of DSB repairing-related proteins in spermatogenic cells isolated from adult WT and Rnaseh1^fl/−;Stra8-Cre mice. L/Z, leptotene/zygotene; P, pachytene. G-I: Signal quantification of DMC1 (E), RAD51 (F), and RPA2 (G) foci number per cell at different meiotic stages. Error bars, s.e.m. ***P < 0.001 (two-tailed Student’s t-test). n.s., non-significant. Above experiments were repeated at least 3 times.

Fig. 5

R-loops accumulate in Rnaseh1^fl/−;Stra8-Cre spermatocytes. A: Pie charts showing the genomic distribution of R-loops in leptotene/zygotene spermatocytes of WT and Rnaseh1^fl/−;Stra8-Cre mice.B: Genomic metaplots showing relative signal intensity comparison of R-loops from the transcription start sites (TSSs) to transcription end sites (TESs) in WT and Rnaseh1^fl/−;Stra8-Cre leptotene/zygotene spermatocytes. C: Venn diagram showing the overlap of R-loop peaks in WT and Rnaseh1^fl/−;Stra8-Cre leptotene/zygotene spermatocytes (Fisher’s exact test; ***P < 0.001). D: Average R-loop read density and heatmap of WT and Rnaseh1^fl/−;Stra8-Cre leptotene/zygotene spermatocytes in the 1 kb window around the center of cKO-only peaks (D) and shared R-loop peaks. Loci are ordered by the descending R-loop intensities in cKO samples. E: Average R-loop read density and heatmap of WT and Rnaseh1^fl/−;Stra8-Cre leptotene/zygotene spermatocytes in the 1 kb window around the center of shared R-loop peaks. Loci are ordered by the descending R-loop intensities in cKO samples. F: Snapshots of accumulated R-loops on example genes. G: Gene ontology analysis of R-loop accumulated genes in Rnaseh1-null spermatocytes.

Fig. 6

RNA-seq analyses of leptotene/zygotene and pachytene spermatocytes derived from WT and Rnaseh1^fl/−;Stra8-Cre mice. A-B: Volcano plots showing differentially expressed genes of leptotene/zygotene and pachytene spermatocytes. Genes upregulated (FC > 2; FDR < 0.05) and downregulated (FC < 1/2; FDR < 0.05) in spermatocytes are colored red and blue, respectively. C: Dynamic profiling of the 3,102 downregulated genes in Rnaseh1^fl/−;Stra8-Cre pachytene spermatocytes. D: Dynamic profiling of DNA damage repair and homologous recombination-related genes across different stages in WT and Rnaseh1^fl/−;Stra8-Cre spermatocytes. E: Gene ontology analysis of differentially expressed genes in Rnaseh1-null pachytene spermatocytes. F: Venn diagram showing the overlap of genes with accumulated R-loops in Rnaseh1-null leptotene/zygotene spermatocytes and down-regulated R-loop genes in pachytene spermatocytes. L/Z, leptotene/zygotene; P, pachytene. G: Example snapshots of the increased R-loops and downregulated transcription in Rnaseh1-null spermatocytes. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)