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; Topbp1B5/B5Mean = 26.43, SD = 2.28, n = 9) and C) appearance of Topbp1B5/B5and Topbp1+/+ littermate mice. D) Effect of full body IR (7 Gy) on changes in body mass of Topbp1B5/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; Topbp1B5/B5 Mean = 0.011, SD = 0.004, n = 9), and H) sperm count, of Topbp1B5/B5 and Topbp1+/+ littermate mice (Topbp1+/+ Mean = 2.1 x 107, SD = 6x 106; Topbp1B5/B5 Mean = 0.0, SD = 0.0, n = 9). I) H&E-stained histological testes sections displaying loss of cellularity in Topbp1B5/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; Topbp1B5/B5Mean = 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 unpaired t-test.

Topbp1B5/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 Topbp1B5/B5 and Topbp1+/+ littermate mice. The data from MEFs were performed using littermate pairs and validated using a second pair of Topbp1B5/B5 and Topbp1+/+ littermate mice. E) Immunofluorescence of MDC1 and phosphoH2AX_S139-stained nuclei from Topbp1B5/B5 and Topbp1+/+ MEFs treated with IR (7 Gy).

Markers of DNA repair, synapsis, sex body formation and TOPBP1 localization are mostly normal in Topbp1B5/B5spermatocytes.

A) Meiotic spreads showing Topbp1+/+ and Topbp1B5/B5 spermatocytes stained with SYCP3 and γH2AX, prepared as described in methods. B) Quantification of γH2AX-stained pachytene spreads, upper graph XY body (each dot represents one pachytene cell measured; Topbp1+/+ number of cells = 386, number of mice = 8; Topbp1B5/B5number of cells = 410, number of mice = 8; p-value = 0.3063), bottom graph autosomal chromosomes (each dot represents the average of signal from all autosomes in each mouse, Topbp1+/+ number of cells = 160, number of mice = 8; Topbp1B5/B5 number of cells = 161, number of mice = 8; p- value = 0.5081. C) Meiotic spreads showing Topbp1+/+and Topbp1B5/B5 pachytene spermatocytes stained with SYCP3 and RAD51. D) Quantification of RAD51 foci/cell of mid-pachytene meiotic spreads (each dot represents one pachytene cell measured; Topbp1+/+ number of cells = 149, number of mice = 3; Topbp1B5/B5 number of cells = 183, number of mice = 3; p-value = 0.2174). E) Meiotic spreads showing Topbp1+/+and Topbp1B5/B5 pachytene spermatocytes stained with SYCP3 and SYCP1. F) Meiotic spreads showing Topbp1+/+ and Topbp1B5/B5pachytene spermatocytes stained with SYCP3 and TOPBP1. G) Quantification of TOPBP1 on X and Y chromosome cores from F (each dot represents one pachytene cell measured; Topbp1+/+ number of cells = 246, number of mice = 3; Topbp1B5/B5 number of cells = 233, number of mice = 3; p-value = 0.8546). H) Quantification of TOPBP1 on X and Y chromatin loops from F (each dot represents one pachytene cell measured; Topbp1+/+number of cells = 246, number of mice = 3; Topbp1B5/B5 number of cells = 233, number of mice = 3; p-value = 0.6755). n = number of mice. p-Values were calculated using a linear mixed effect model (see Materials and Methods for details).

Topbp1B5/B5spermatocytes display normal localization of ATR, BRCA1, MDC1 and pMDC1-T4.

A) Meiotic spreads showing Topbp1+/+ and Topbp1B5/B5 pachytene spermatocytes stained with SYCP3 and ATR. B) Quantification of ATR in pachytene spreads from A. Left: ATR on X and Y chromosome cores (each dot represents one pachytene cell measured; Topbp1+/+ number of cells = 127, number of mice = 3; Topbp1B5/B5 number of cells = 127, number of mice = 3; p-value = 0.4068). Right: ATR on X and Y chromatin loops (each dot represents one pachytene cell measured; Topbp1+/+number of cells = 127, number of mice = 3; Topbp1B5/B5 number of cells = 127, number of mice = 3; p-value = 0.9396). C) Meiotic spreads showing Topbp1+/+ and Topbp1B5/B5 pachytene spermatocytes stained with SYCP3 and BRCA1. D) Quantification of BRCA1 in pachytene spreads from C (each dot represents one pachytene cell measured; Topbp1+/+ number of cells = 152, number of mice = 3; Topbp1B5/B5 number of cells = 140, number of mice = 3; p-value = 0.6509). E) Meiotic spreads showing Topbp1+/+ and Topbp1B5/B5pachytene spermatocytes stained with SYCP3 and MDC1. F) Quantification of MDC1 in pachytene spreads from E (each dot represents one pachytene cell measured; Topbp1+/+ number of cells = 696, number of mice = 8; Topbp1B5/B5number of cells = 988, number of mice = 8; p-value = 0.3603). G) Meiotic spreads showing Topbp1+/+ and Topbp1B5/B5 pachytene spermatocytes stained with SYCP3 and pCHK1-S317. H) Quantification of pCHK1- S317 in pachytene spreads from G (each dot represents one pachytene cell measured; Topbp1+/+ number of cells = 223, number of mice = 3; Topbp1B5/B5 number of cells = 254, number of mice = 3; **p-value = 0.0023). p-Values were calculated using a linear mixed effect model (see Materials and Methods for details). I) Table summarizing the normal or disrupted ATR and TOPBP1-dependent events during male fertility accessed in Topbp1B5/B5. n = number of mice.

Defective SETX phosphorylation and localization in Topbp1B5/B5 spermatocytes.

A) Scatter plot of phosphoproteomic datasets corresponding to Topbp1+/+/Topbp1B5/B(Y axis) and Topbp1+/+(vehicle)/Topbp1+/+(AZ20) (X axis) from whole testes of mice. B) SETX phosphopeptides identified in the Topbp1+/+/Topbp1B5/Bphosphoproteomic experiment shown in A. Red: reduced in Topbp1B5/B mutant; Blue: unchanged. C) Meiotic spreads showing pachytene spermatocytes from Topbp1+/+and Topbp1B5/B5 mice stained with SYCP3 and SETX in regular immunofluorescence. D) 3D-SIM analysis of meiotic spreads described in C. E) Quantification of SETX on X and Y chromatin loops in pachytene spreads from C (each dot represents one pachytene cell measured; Topbp1+/+ number of cells = 152, number of mice = 4; Topbp1B5/B5 number of cells = 174, number of mice = 4; *p-value = 0.0452). F) Quantification of SETX on X and Y chromosome cores in pachytene spreads from C (each dot represents one pachytene cell measured; Topbp1+/+ number of cells = 152, number of mice = 4; Topbp1B5/B5 number of cells = 174, number of mice = 4; p-value = 0.5987). p-Values were calculated using a linear mixed effect model (see Materials and Methods for details).

Single cell RNAseq reveals that Topbp1B5/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, representing all cells captured for both, Topbp1+/+ and Topbp1B5/B5. 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 Topbp1B5/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 Topbp1B5/B5 cells. The level of X-genes expression in Spermatocyte 3 is significantly higher in Topbp1B5/B5 cells when compared to Topbp1+/+ cells, with a p-value of 1.5e-178 using a two-sided Wilcoxon rank sum test.

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 and respective p-values above each graph (the p-values were calculated using the Fisher’s exact test). C) Examples of genes with altered silencing dynamics in the Topbp1B5/B5, red = reduced silencing, blue = unaltered silencing and green = increased silencing D) Scatter plot showing the difference in RNA level between Topbp1+/+ and Topbp1B5/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 Topbp1B5/B5 (blue) at SP3. F) Graph plotting expression levels of X-chromosome genes, normalized to pre-leptotene levels, in Topbp1+/+ (Y axis) and Topbp1B5/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 Topbp1B5/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.