A TOPBP1 Allele Causing Male Infertility Uncouples XY Silencing Dynamics From Sex Body Formation

Carolline F. R. Ascencao
Jennie R. Sims
Alexis Dziubek
William Comstock
Elizabeth A. Fogarty
Jumana Badar
Raimundo Freire
Andrew Grimson
Robert S. Weiss
Paula E. Cohen
Marcus Smolka author has email address

Department of Molecular Biology and Genetics, Cornell University, United States;
Fundación Canaria del Instituto de Investigación Sanitaria de Canarias (FIISC), Unidad de Investigación, Hospital Universitario de Canarias, La Laguna, Santa Cruz de Tenerife, Spain;
Instituto de Tecnologías Biomédicas, Universidad de La Laguna, 38200 La Laguna, Spain;
Universidad Fernando Pessoa Canarias, Las Palmas de Gran Canaria, Spain;
Department of Biomedical Sciences, Cornell University, United States

https://doi.org/10.7554/eLife.90887.1

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Figures and data

A new TOPBP1 mutant separating its role in fertility from organismal viability.
A) Schematic showing mutations in the Topbp1 B5 allele. B) Body mass (Topbp1^+/+ Mean = 25.26, SD = 2.38; Topbp1^B5/B5 Mean = 26.43, SD = 2.28, n = 9) and C) appearance of Topbp1^B5/B5 and Topbp1 ^+/+ littermate mice. D) Effect of full body IR (7 Gy) on changes in body mass of Topbp1^B5/B5 and Topbp1^+/+ littermate mice E) Breeding scheme and resulting litters. F) and G) Comparison of testes size (Topbp1 ^+/+ Mean = 0.038, SD = 0.006; Topbp1^B5/B5 Mean = 0.011, SD = 0.004, n = 9), and H) sperm count, of Topbp1^B5/B5 and Topbp1+/+ littermate mice (Topbp1+/+ Mean = 2.1 x 107, SD = 6x 106; Topbp1^B5/B5 Mean = 0.0, SD = 0.0, n = 9). I) H&E-stained histological testes sections displaying loss of cellularity in Topbp1^B5/B5. Green arrow = spermatogonia, Red arrow = healthy spermatocyte, Blue arrow = dying spermatocyte. J) and K) TUNEL assay performed on histological testes sections (Topbp1+/+ Mean = 0.36, SD = 0.15; Topbp1^B5/B5 Mean = 4.80, SD = 0.43, n = 3). L) Meiotic spreads stained for SYCP3, SYCP1 and H1t. **** = p< 0.0001, n = number of mice. p-Values were calculated using Welch’s unpaired t-test in GraphPad.

Topbp1^B5/B5 MEFs display no detectable DDR defects.
A) MEFs obtained from littermate mice of indicated genotypes were subjected to clonogenic survival assay in the indicated concentrations of hydroxyurea, or C) phleomycin. B) and D) Quantification of clonogenic survival assays from A and C, respectively, performed in biological and experimental triplicates. D) Western blot for indicated DDR markers in MEFs obtained from Topbp1^B5/B5 and Topbp1^+/+ littermate mice. The data from MEFs were performed using littermate pairs and validated using a second pair of Topbp1^B5/B5 and Topbp1^+/+ littermate mice. E) Immunofluorescence of MDC1 and phosphoH2AX_S139-stained nuclei from Topbp1^B5/B5 and Topbp1^+/+ MEFs treated with IR (7 Gy).

Markers of DNA repair, synapsis, sex body formation and TOPBP1 localization are mostly normal in Topbp1^B5/B5 spermatocytes.
A) Meiotic spreads showing Topbp1^+/+ and Topbp1^B5/B5 spermatocytes stained with SYCP3 and γH2AX, prepared as described in methods. B) Quantification of γH2AX-stained pachytene spreads, upper graph autosomal chromosomes (each dot represents the average of signal from all autosomes in each mouse) and bottom, XY body. C) Meiotic spreads showing Topbp1^+/+ and Topbp1^B5/B5 pachytene spermatocytes stained with SYCP3 and RAD51. D) Quantification of RAD51 foci/cell of mid-pachytene meiotic spreads. E) Meiotic spreads showing Topbp1^+/+ and Topbp1^B5/B5 pachytene spermatocytes stained with SYCP3 and SYCP1. F) Meiotic spreads showing Topbp1^+/+ and Topbp1^B5/B5 pachytene spermatocytes stained with SYCP3 and TOPBP1. Quantification of TOPBP1-stained pachytene spreads G) X and Y chromosome cores, and H) X and Y chromatin loops. n = number of mice. p-Values were calculated using Welch’s unpaired t-test in GraphPad.

Topbp1^B5/B5 spermatocytes display normal localization of ATR, BRCA1, MDC1 and pMDC1-T4.
A-H) Meiotic spreads stained for indicated proteins and related quantification in Topbp1^+/+ and Topbp1^B5/B5 pachytene spermatocytes. I) Table summarizing the normal or disrupted ATR and TOPBP1-dependent events during male fertility accessed in Topbp1^B5/B5. n = number of mice. p-Values were calculated using Welch’s unpaired t-test in GraphPad.

Defective SETX phosphorylation and localization in Topbp1^B5/B5 spermatocytes.
A) Scatter plot of phosphoproteomic datasets corresponding to Topbp1^+/+/Topbp1^B5/B (Y axis) and Topbp1^+/+(vehicle)/Topbp1^+/+(AZ20) (X axis) from whole testes of mice. B) SETX phosphopeptides identified in the Topbp1^+/+/Topbp1^B5/B phosphoproteomic experiment shown in A. Red: reduced in Topbp1^B5/B mutant; Blue: unchanged. C) Meiotic spreads showing pachytene spermatocytes from Topbp1^+/+ and Topbp1^B5/B5 mice stained with SYCP3 and SETX in regular immunofluorescence, and D) 3D-SIM analysis of meiotic spread described in C. E) and F) Quantification of the intensity of SETX chromatin loops and cores of the XY chromosomes, respectively. * p < 0.05, n = number of mice. p-Values were calculated using Welch’s unpaired t-test in GraphPad.

Single cell RNAseq reveals that Topbp1^B5/B5 spermatocytes initiate MSCI but fail to promote full silencing.
A) scRNAseq workflow for isolation and purification of single cells for RNA-seq. B) UMAP analysis of sub-clusters captured in the scRNAseq. C) UMAP analysis of sub-clusters captured in the scRNAseq of Topbp1^+/+. D) UMAP analysis of sub-clusters captured in the scRNAseq of Topbp1^+/+ (gray) and Topbp1^B5/B5 (blue). E) Violin plots displaying the ratio of the average expression of X chromosome genes by the average expression of chromosome 9 genes at different stages of spermatogenesis for Topbp1^+/+ and Topbp1^B5/B5 cells.

Topbp1 regulates silencing dynamics of X genes at the Spermatocyte 3 stage.
A) Illustration of the gene markers used to define spermatocyte 1 as leptotene, spermatocyte 2 as zygotene, and spermatocyte 3 as pachytene; hypothetical examples illustrating the categorization of transitions in silencing dynamics between the stages of pre-leptotene (PL), spermatocyte 1 (SP1), spermatocyte 2 (SP2), spermatocyte 3 (SP3). B) Number of genes in each of the categories described in A, during the different stage transitions. C) Examples of genes with altered silencing dynamics in the Topbp1^B5/B5, red = reduced silencing, blue = unaltered silencing and green = increased silencing D) Scatter plot showing the difference in RNA level between Topbp1^+/+ and Topbp1^B5/B5 for each of the indicated stage transitions. E) Scatter plot showing expression level of X-chromosome genes, normalized to pre-leptotene levels, in Topbp1^+/+ (gray) and Topbp1^B5/B5 (blue) at SP3. F) Graph plotting expression levels of X-chromosome genes, normalized to pre-leptotene levels, in Topbp1^+/+ (Y axis) and Topbp1^B5/B5 (X axis) and split in three categories based on the severity of silencing defect. G) Box plot showing PL expression levels of X-chromosome genes in each of the categories of silencing defect severity shown in F.

A new TOPBP1 mutant separates XY silencing from sex body formation.
Schematic of sub-stages of meiotic prophase I. In Wild-type mice, MSCI initiates following the accumulation of the DDR proteins at the XY chromosomes. During mid-pachytene, the XY body is fully formed, and transcription is restricted to the autosomes. In Atr or Topbp1 CKOs, the sex body is not formed, and the DDR proteins are not sequestered to the XY. Asynapsis events and transcription of toxic genes at the sex chromosomes are observed, triggering mid-pachytene arrest. In Topbp1^B5/B5, MSCI initiates, the sex body is normally formed with normal recruitment of DDR proteins to the X and Y chromosomes, yet cells fail in the reinforcement/maintenance of silencing. Cells progress through mid-pachytene but not into diplotene.

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