DNA damage response protein TOPBP1 regulates X chromosome silencing in the mammalian germ line

Significance

A fundamental step of meiosis is prophase I, when homologous chromosomes synapse and recombine to generate cross-overs that are accurate for chromosome segregation. In male mammals, chromosomes that are asynapsed undergo meiotic silencing, a process that may serve as a surveillance mechanism to prevent aneuploidy in offspring. We identify TOPBP1 as a factor involved in meiotic silencing. Deletion of TOPBP1 in male germ cells results in germ cell loss and failure to silence asynapsed chromosomes. We find that TOPBP1 acts at multiple stages in the silencing pathway. Our study highlights that TOPBP1 regulates sex chromosome expression in mammalian germ cells.

Abstract

Meiotic synapsis and recombination between homologs permits the formation of cross-overs that are essential for generating chromosomally balanced sperm and eggs. In mammals, surveillance mechanisms eliminate meiotic cells with defective synapsis, thereby minimizing transmission of aneuploidy. One such surveillance mechanism is meiotic silencing, the inactivation of genes located on asynapsed chromosomes, via ATR-dependent serine-139 phosphorylation of histone H2AFX (γH2AFX). Stimulation of ATR activity requires direct interaction with an ATR activation domain (AAD)-containing partner. However, which partner facilitates the meiotic silencing properties of ATR is unknown. Focusing on the best-characterized example of meiotic silencing, meiotic sex chromosome inactivation, we reveal this AAD-containing partner to be the DNA damage and checkpoint protein TOPBP1. Conditional TOPBP1 deletion during pachynema causes germ cell elimination associated with defective X chromosome gene silencing and sex chromosome condensation. TOPBP1 is essential for localization to the X chromosome of silencing “sensors,” including BRCA1, and effectors, including ATR, γH2AFX, and canonical repressive histone marks. We present evidence that persistent DNA double-strand breaks act as silencing initiation sites. Our study identifies TOPBP1 as a critical factor in meiotic sex chromosome silencing.

Meiosis is the process by which haploid gametes are generated from diploid germ cells (1, 2). It comprises a single phase of DNA replication followed by two successive cell divisions. The first division is reductional, separating homologous chromosomes, whereas the second is equational, segregating sister chromatids. Before the first meiotic division, during prophase I, homologous chromosomes synapse and recombine. These processes together result in the formation of cross-overs, which permit accurate chromosome segregation and generate genetic diversity in offspring.

At early pachynema in male mammals, homologous autosomes are fully synapsed. In contrast, the X and Y chromosomes synapse only at a region of limited sequence homology, the pseudoautosomal region (PAR) (3) (Fig. S1A). The remaining, asynapsed chromosome regions undergo meiotic sex chromosome inactivation (MSCI), resulting in silencing of XY genes and formation of the sex body (4–7) (Fig. S1A). MSCI is essential for spermatogenesis (8), and aberrations in this process contribute to infertility in sex chromosome aneuploid models, interspecific hybrids, and targeted mutants defective in synapsis or recombination (9–12).

Although a hallmark of spermatocytes, silencing of asynapsed chromosomes is confined neither to the X and Y chromosomes nor to males. Any chromosome region asynapsed in males or females is inactivated (13, 14) by a process referred to as meiotic silencing (15). Meiotic silencing is suggested to restrict double-strand break (DSB) formation (7), prevent nonhomologous recombination (4, 6, 15), and enable DSB repair (16) at sites of asynapsis. It may also serve a checkpoint function to eliminate germ cells with asynapsed chromosomes, thereby preventing aneuploidy in progeny (10). Finally, in males, MSCI may shield the asynapsed XY chromosome regions from a synapsis checkpoint (17).

Meiotic silencing involves multiple DNA damage response (DDR) proteins, which function in two key steps. First, asynapsis is detected by “sensors,” which localize to the axial elements (AEs). Subsequently, asynapsis signaling is transmitted to “effectors,” which reside in the associated chromatin loops and cause long-range gene silencing. Based on their localization to the inactive XY bivalent, many potential sensors and effectors have been identified (4–6, 15, 18). Among these are the sensors SYCP3 (19), HORMAD1 (20), HORMAD2 (21), and BRCA1 (22), and the effectors MDC1 (23) and histone H2AFX (24). A critical link between sensors and effectors is provided by the DNA damage kinase ATR (25). SYCP3, HORMAD1, HORMAD2, and BRCA1 first recruit ATR to asynapsed AEs. Subsequently, ATR, facilitated by MDC1, translocates along chromatin loops, effecting gene silencing via phosphorylation of H2AFX at serine 139 (γH2AFX).

To fulfill its DNA repair and checkpoint functions, ATR must first be activated by proteins harboring an ATR activation domain (AAD). Two AAD-containing proteins, ETAA1 (26–29) and TOPBP1 (30, 31), act via independent pathways to activate ATR during mitosis (32–37). In contrast, the identity of the AAD-containing protein that activates ATR to initiate meiotic silencing is not known. ETAA1 expression during meiosis has not been examined. TOPBP1 is observed on the XY bivalent during pachynema (38, 39). However, the role of TOPBP1 in MSCI is unclear because mice carrying homozygous Topbp1 deletions or AAD point mutations die during early embryogenesis (40–42).

By using a conditional deletion strategy, we now identify TOPBP1 as the ATR partner in meiotic silencing. We find that TOPBP1 directs assembly of multiple sensors and effectors at the XY pair. Thus, in addition to its roles in DNA damage repair and checkpoint signaling, TOPBP1 is an epigenetic regulator of sex chromosome expression in the mammalian germ line.

Results

To assess potential roles of Topbp1 and Etaa1 in MSCI, we studied their RNA expression and immunolocalization during spermatogenesis. We first examined RNA-sequencing (RNA-seq) datasets from isolated mouse spermatogonia, spermatocytes, and spermatids (43). The expression profile of Topbp1 was similar to that of established MSCI effectors Atr, Mdc1, and H2afx, with transcript levels highest in spermatocytes (Fig. S1B). In contrast, Etaa1 expression was relatively low in all germ cell subtypes and was decreased in spermatocytes relative to spermatogonia (Fig. S1B). By using combined antibody staining for TOPBP1 and SYCP3 on spermatocyte spreads, we confirmed previous reports that TOPBP1 localizes to the XY bivalent and is absent at the PAR (38, 39) (Fig. S1C; legend provides quantification). We also found that the dynamics of sex chromosome TOPBP1 localization matched that of ATR (Fig. S1C). TOPBP1 and ATR colocalized on the asynapsed regions of the XY pair during early, mid-, and late pachynema before disappearing at diplonema. In contrast, no localization of ETAA1 to the XY pair was observed (Fig. S1D). TOPBP1 was therefore a superior candidate to ETAA1 for regulating MSCI.

To determine whether TOPBP1 is required for XY silencing, we generated male mice carrying one Topbp1 floxed and one Topbp1 null allele (Topbp1^flox/−) (44), together with an Ngn3 Cre transgene that drives Cre recombinase expression in germ cells from postnatal day (P) 7 (breeding scheme presented in Materials and Methods) (45, 46). P42 testis weights in resulting Topbp1^flox/− Ngn3 Cre males [hereafter Topbp1 conditional KO (cKO)] were reduced fivefold relative to those in Topbp1^flox/+Ngn3 Cre males (hereafter control; Fig. 1A), whereas body weights were similar between these genotypes (Fig. S1E). TOPBP1 protein expression was markedly lower in Topbp1 cKO than in control testes (Fig. 1B). Topbp1 cKO seminiferous tubules exhibited a deficiency of spermatogonia and spermatocytes, and spermatids were rarely seen (Fig. 1C). Germ cell elimination in MSCI-deficient mutants occurs at midpachynema, or stage IV of the seminiferous cycle (47). As accurate tubule staging requires the presence of spermatogonia, which, in our model, were present in low numbers, we used an alternative approach to determine whether spermatocytes deficient in TOPBP1 were eliminated at midpachynema. TOPBP1 XY immunolocalization was compared between Topbp1 cKO spermatocytes at early and late pachynema. At early pachynema, 60% of Topbp1 cKO spermatocytes showed complete or partial loss of TOPBP1 from the XY bivalent, whereas the remaining 40% exhibited grossly unaffected TOPBP1 localization (Fig. 1D). However, at late pachynema, 100% of Topbp1 cKO spermatocytes showed normal TOPBP1 XY localization (Fig. S1F). Spermatocytes exhibiting complete or partial loss of XY-associated TOPBP1 are therefore eliminated during midpachynema. As noted previously (8), we also observed in Topbp1 cKO males loss of unaffected germ cells as a result of a bystander effect (SI Results).

Fig. 1.

TOPBP1 deletion causes germ cell loss. (A) Mean P42 testis weights ± SEM: 70.49 ± 4.60 mg for control and 14.40 ± 0.75 mg for Topbp1 cKO. Each symbol represents the mean testis weight from one male. Significance was determined by unpaired t test. (B) Western blot for TOPBP1 in control and Topbp1 cKO testes. Tubulin is used as loading control. (C) Periodic acid-Schiff–stained seminiferous tubule sections showing normal spermatogenesis in control and loss of spermatogonia and spermatocytes in Topbp1 cKO males. A, apoptotic spermatocyte; SC, spermatocyte; SG, spermatogonia. (Inset) Presence of spermatids in some tubules from Topbp1 cKO males. (D) Topbp1 cKO XY bivalents at early pachynema showing complete TOPBP1 loss (Left), partial TOPBP1 loss (defined as regions of TOPBP1 discontinuity on the asynapsed XY axes; Middle), and unaffected TOPBP1 localization (Right; n = 3 males, n = 186 cells). (Scale bars, C, 50 µm; D, 5 µm.)

We next used immunofluorescence to investigate relationships between TOPBP1 and meiotic silencing sensors SYCP3, HORMAD1, and HORMAD2 in early pachytene (EP) Topbp1 cKO spermatocytes. We focused on cells with normal autosomal synapsis because asynapsed autosomes can indirectly disrupt MSCI by titrating silencing factors from the XY bivalent (9). Inclusion of an antibody to TOPBP1 allowed us to determine the localization of each sensor in cells with partial TOPBP1 loss (see Figs. 1–5), as well as in cells with complete TOPBP1 loss (see Figs. S1–S7). Accumulation of SYCP3 (Fig. S2A), HORMAD1 (Fig. S2A), and HORMAD2 (Fig. S2B) to XY AEs was unaffected by TOPBP1 depletion. These findings demonstrate that TOPBP1 lies downstream of SYCP3, HORMAD1, and HORMAD2 in the meiotic silencing pathway.

Fig. 5.

TOPBP1 is required for MSCI. (A, Top) Mouse X chromosome showing the location of Utx, Zfx, and Scml2. RNA FISH images for each gene in control, Topbp1 cKO, and H2afx^−/− EP spermatocytes. Dotted circles enclose the X chromosome, for which γH2AFX immunostaining is shown (Insets). Arrowheads indicate Utx, Zfx, and Scml2 RNA FISH signals (n = 2 males). (B) Graphs showing the proportion of EP spermatocytes expressing each gene in each genotype, with corresponding numbers of cells indicated. (Scale bars, 5 µm.)

SYCP3 and HORMAD2 also label asynapsed autosomes. This localization pattern was unaffected in Topbp1 cKO cells. We therefore used SYCP3/HORMAD2 immunostaining to determine whether TOPBP1 regulates synapsis. Addition of a TOPBP1 antibody again allowed us to compare phenotypes in cells with different extents of XY-associated TOPBP1 depletion. Normal synapsis occurred in 95% of Topbp1 cKO cells with unaffected XY TOPBP1 localization, a frequency similar to that observed in controls (91%; Fig. S3). In contrast, normal synapsis was observed in only 46% and 52% of Topbp1 cKO cells with partial and complete XY-associated TOPBP1 loss, respectively (Fig. S3). In the remaining cells, asynapsis affected the XY pair or the autosomes. TOPBP1 is therefore required for autosomal and XY synapsis.

Next, we examined XY localization of silencing factors ATR and BRCA1. Interestingly, ATR localization to the XY pair was compromised in Topbp1 cKO cells. We observed a strict correspondence between TOPBP1 and ATR localization patterns: in cells with partial TOPBP1 depletion, regions of the XY AEs positive for TOPBP1 were positive for ATR, whereas regions lacking TOPBP1 lacked ATR (Fig. 2A). In cells with complete TOPBP1 loss, ATR was undetectable (Fig. S2C). Similarly, in TOPBP1-depleted cells, loading of BRCA1 to the XY bivalent was deficient except at regions where presumptive TOPBP1 was located (Fig. 2B and Fig. S2D; note that ATR acts as a TOPBP1 proxy in this experiment, as detailed in the legend to Fig. 2). XY localization of RBBP8, a partner of TOPBP1 (48) and BRCA1 (49, 50), was also defective in TOPBP1-depleted cells (Fig. 2C and Fig. S2E). Furthermore, serine-271 phosphorylation of HORMAD2 (pHORMAD2^Ser271), which is mediated by ATR (25), was compromised in Topbp1 cKO cells (Fig. 2D and Fig. S2F), consistent with TOPBP1 acting as a cofactor of ATR in silencing. TOPBP1 is therefore essential for region-by-region assembly of ATR, BRCA1, RBBP8, and pHORMAD2^Ser271 at asynapsed XY axes.

Fig. 2.

TOPBP1 regulates localization of silencing sensors at XY AEs. (A–D) Immunostaining of meiotic chromosome spreads at early pachynema for SYCP3 (gray), TOPBP1 (green), and indicated silencing sensors (magenta) in control (Left; n = 2 males, n = 50 cells for each silencing sensor) and Topbp1 cKO spermatocytes with partial TOPBP1 loss (Right; n = 2 males, n = 55, 52, 60, and 45 cells for ATR, BRCA1, RBBP8, and pHORMAD2^Ser271, respectively). Arrowheads indicate regions of XY AEs that are depleted for TOPBP1. Colocalization of green and magenta appears in white. (B–D) ATR is used as TOPBP1 proxy, as TOPBP1, BRCA1, RBBP8, and pHORMAD2^Ser271 antibodies are raised in the same species and therefore cannot be used in combined immunofluorescence. (Scale bars, 5 µm.)

We subsequently assessed XY localization of silencing effectors MDC1 and γH2AFX. In control EP cells, both factors formed a cloud marking the XY chromatin loops (Fig. 3 A and B). However, in Topbp1 cKO cells, formation of MDC1 and γH2AFX clouds was impaired. Chromatin loops arising from TOPBP1-positive AE stretches were positive for MDC1 and γH2AFX, whereas those arising from TOPBP1-deficient AE stretches were not (Fig. 3 A and B). XY bivalents without TOPBP1 were devoid of both effectors (Fig. S4). These findings show that TOPBP1 connects the sensor and effector arms of the silencing response. Furthermore, they demonstrate that spreading of silencing effectors into chromatin loops initiates from multiple independent sites along the length of asynapsed AEs.

Fig. 3.

TOPBP1 regulates localization of silencing effectors at XY chromatin loops. (A–F) Immunostaining of meiotic chromosome spreads at early pachynema using SYCP3 (gray), TOPBP1 (green), and indicated silencing effectors (magenta) in control (Left; n = 2 males, n = 50 cells for each silencing effector) and Topbp1 cKO spermatocytes with partial TOPBP1 loss (Right; n = 2 males, n = 51, 53, 50, 47, 50, and 59 cells for MDC1, γH2AFX, USP7, H3K9me3, polyub, and SUMO-1, respectively). Arrowheads indicate regions of XY AEs that are depleted for TOPBP1. Colocalization of green and magenta appears in white. In C, D, and F, ATR is used as TOPBP1 proxy because TOPBP1, USP7, H3K9me3, and SUMO-1 antibodies are raised in the same species and therefore cannot be used in combined immunofluorescence. (Scale bars, 5 µm.)

Following acquisition of MDC1 and γH2AFX, XY chromatin undergoes further remodeling involving removal of histone H2A lysine-119 monoubiquitination (H2AK119ub) by USP7 (51, 52), acquisition of histone H3 lysine-9 trimethylation (H3K9me3) (53), and conjugation of unidentified targets with polyubiquitination (polyub) (54) and sumoylation (SUMO-1) (55, 56). We performed immunostaining for USP7, H3K9me3, polyub, and SUMO-1 to determine which, if any, of these pathways is TOPBP1-dependent. In Topbp1 cKO cells, all four factors were undetectable, even at regions where TOPBP1 (or its proxy ATR) was preserved (Fig. 3 C–F). Absence of these components at TOPBP1-positive locations may be the result of antibody inefficiencies, or because high levels of TOPBP1 are required for successful recruitment of these components. Either way, TOPBP1 lies upstream of USP7, H3K9me3, polyub, and SUMO-1 in the XY chromatin-remodeling pathways.

At early pachynema, the asynapsed X chromosome contains multiple persistent meiotic DSBs. These DSBs may act as initiation sites for silencing factor spreading (15, 57). Before investigating the potential relationship between DSBs and silencing, we first assessed whether global DSB abundance was perturbed in Topbp1 cKO males. Leptotene focus counts for the DSB markers RPA subunit 2 (RPA2; Fig. S5A) and RAD51 (Fig. S5B) were similar to those in controls, indicating that DSB numbers in Topbp1 cKO males were grossly unaffected. Focusing on EP Topbp1 cKO cells with partial TOPBP1 loss, we then examined spatial proximity between the TOPBP1 proxy ATR and RPA2 foci along the X chromosome AE (Fig. S6). We noticed a striking colocalization between these DDR factors: 98% of AE stretches that were positive for ATR contained RPA2 foci, whereas only 3% of AE stretches that were negative for ATR contained RPA2 foci. Conversely, 99% of all X chromosome RPA2 foci resided within AE stretches that were positive for ATR. These findings support the hypothesis that DNA damage is the initiating lesion for meiotic silencing.

During MSCI, the X and Y chromosomes condense to form the sex body, in which their centromeres come into close proximity. Given the defects in localization of silencing factors to the XY pair in Topbp1 cKO males, we suspected that sex body formation in these mice would be perturbed. To test this possibility, we used an established method (23) to compare the mean X-to-Y centromere distance between EP Topbp1 cKO cells with differing extents of TOPBP1 depletion (Fig. 4). Topbp1 cKO cells with unaffected TOPBP1 localization exhibited a mean X-to-Y centromere distance indistinguishable from that in controls, suggesting that they had achieved sex chromosome condensation. However, Topbp1 cKO cells with complete TOPBP1 loss had a mean X-to-Y centromere distance threefold higher than controls. Interestingly, the mean X-to-Y centromere distance in Topbp1 cKO cells with partial TOPBP1 loss was similar to that in cells with complete TOPBP1 loss. TOPBP1 colocalization to the XY bivalent must therefore exceed a threshold to initiate sex chromosome condensation.

Fig. 4.

Sex chromosome condensation is defective in Topbp1 cKO spermatocytes. (A) Conformation of XY chromosome axes at early pachynema assessed in control spermatocytes and in Topbp1 cKO spermatocytes with unaffected TOPBP1 localization, partial TOPBP1 loss, and complete TOPBP1 loss: SYCP3 (gray), TOPBP1 (green), centromeres (magenta). (B) Plots of linear distances between X and Y centromeres. The distance is normalized to the nuclear diameter. P values were calculated by one-way ANOVA. (Scale bars, 5 µm.)

Finally, we used RNA FISH to examine whether TOPBP1 is required for silencing of X genes at pachynema. We examined in Topbp1 cKO and controls expression of genes located near the centromere (Utx), the center (Zfx), and the PAR (Scml2) of the X chromosome (Fig. 5A). Following RNA FISH, we performed immunostaining for HORMAD2 and γH2AFX. XY HORMAD2 localization, which is unaffected by TOPBP1 depletion (Fig. S2B), was used to identify pachytene cells by a method described previously (58, 59). Among the Topbp1 cKO pachytene population, cells with TOPBP1 depletion were identified by virtue of having defective XY γH2AFX acquisition. We observed silencing of Utx, Zfx, and Scml2 in almost all pachytene cells from control males (Fig. 5). However, expression of all three X genes persisted in TOPBP1-depleted pachytene cells (Fig. 5). Notably, the proportion of TOPBP1-deficient pachytene cells misexpressing Utx, Zfx, and Scml2 was equivalent to that observed in an established MSCI mutant, the H2afx-null male (60) (Fig. 5). We conclude that meiotic X chromosome silencing requires TOPBP1.

Discussion

Aside from its well-established roles in DNA replication, DNA repair, and checkpoint control, TOPBP1 can interact with chromatin-remodeling complexes and transcription factors to influence gene expression (61). Here we show that TOPBP1 is also critical for meiotic silencing (Fig. S7), thereby supporting a role for this protein in epigenetic regulation. Although, in other contexts, TOPBP1 has functions that are independent of ATR (61), the meiotic silencing defects in TOPBP1-deficient males resemble those in ATR-deficient males (25). In both models, localization of early sensor components SYCP3, HORMAD1, and HORMAD2 to the XY pair is unaffected, whereas subsequent acquisition of BRCA1 and of the ATR-phosphortarget pHORMAD2Ser²⁷¹ is perturbed. Furthermore, the same downstream effector pathways that enforce X-gene silencing are disrupted. Based on these phenotypic similarities, we suggest that TOPBP1 imparts its silencing functions principally as an ATR cofactor.

Notably, partial TOPBP1 depletion was sufficient to abolish sex chromosome condensation and silencing of X-linked genes. This finding suggests that MSCI is sensitive to the levels of silencing components. In support of this hypothesis, MSCI is perturbed by an increasing dose of asynapsed autosomes, which titrate silencing factors from the XY bivalent (9, 62). TOPBP1, like other silencing effectors ATR (63) and H2AFX (64), exhibits higher expression in testis than in somatic tissues (65). We suggest that elevated levels of silencing effectors TOPBP1, ATR, and H2AFX within spermatocytes reflect the unique requirement of these cells to inactivate hundreds of X and Y chromosome genes.

The existence in our Topbp1 cKO mutant of pachytene cells with partial TOPBP1 depletion was fortuitous, as it allowed us to observe silencing initiation sites along the X chromosome AE. Two resulting observations were noteworthy. First, spreading of silencing factors initiates from multiple sites along the X chromosome axis. This mechanism contrasts with that observed during female somatic X chromosome inactivation, in which silencing components spread from a single site termed the X inactivation center (66). Second, on the meiotic X chromosome, silencing initiation sites coincide with DSB sites. Despite involving multiple DDR factors, meiotic silencing was originally thought to be DSB-independent because it also occurs in spermatocytes lacking the meiotic DSB-inducing enzyme SPO11 (64, 67, 68). However, DNA damage foci, the origin of which is unknown, have since been observed in Spo11-null pachytene spermatocytes, where they colocalize with silencing domains (57). Our findings build on these observations and provide compelling evidence that DSBs are meiotic silencing initiation sites.

We also show that TOPBP1 is essential for completion of autosomal and XY synapsis. The asynapsis phenotype in Topbp1 cKO males could result from defective recombination, synaptonemal complex (SC) formation, or both. Although DSB abundance is unaffected in Topbp1 cKO males at leptonema, we were unable to establish the extent of TOPBP1 depletion in individual spermatocytes at this stage. Further investigation of the role of TOPBP1 in recombination will therefore require a superior Cre driver. We find that TOPBP1 is required for serine-271 phosphorylation of the SC component HORMAD2. TOPBP1 may facilitate phosphorylation of other known ATR SC targets, as well as SC components with (S/T)Q cluster domains (69). Finally, we note that localization of ATR to the XY pair during meiosis is TOPBP1-dependent. This observation is intriguing, because assembly of ATR at sites of DNA damage during mitosis is TOPBP1-independent (70). The extent to which interrelationships between TOPBP1 and ATR differ in meiosis and mitosis is therefore an interesting avenue for future study.

Materials and Methods

Mice.

All mice were maintained according to UK Home Office Regulations at the National Institute for Medical Research and the Francis Crick Institute Mill Hill laboratory. To generate Topbp1^flox/− Ngn3 Cre males, we first mated conditional Topbp1^flox/+ females (44) to males carrying the Ngn3 Cre transgene (45, 46). In the germ line of resulting Topbp1^flox/+ Ngn3 Cre sons, Cre recombinase converts Topbp1^flox alleles into Topbp1⁻ (i.e., null) alleles. Mating these males to Topbp1^flox/flox females generates Topbp1^flox/− Ngn3 Cre sons and Topbp1^flox/+ Ngn3 Cre control brothers. Topbp1^flox and Ngn3 Cre were maintained on a C57BL/6 background. H2afx^−/− males (60) were maintained on an MF1 background.

RNA FISH, Immunofluorescence, and Western Blotting.

RNA FISH digoxigenin-labeled probes were prepared from BAC DNA (from CHORI, Scml2, RP24-204O18; Zfx, RP24-204018; Utx, gift from Mike Mitchell, University Aix-Marseille, Marseille, France) as described previously (71). Immunofluorescence experiments were carried out as described previously (13) (SI Materials and Methods lists antibodies). Western blotting was carried out as described previously (72) with TOPBP1 and tubulin (T9026; Sigma) antibodies used at 1:1,000 and 1:14,000, respectively.

Microscopy.

Imaging was performed by using an Olympus IX70 inverted microscope with a 100-W mercury arc lamp. An Olympus UPlanApo 100×/1.35 N.A. oil-immersion objective was used for meiotic chromosome spread and RNA FISH imaging. An Olympus UPlanApo 40×/0.75 N.A. objective was used for imaging testis sections. A DeltaVision RT computer-assisted Photometrics CoolSnap HQ CCD camera with an ICX285 Progressive scan CCD image sensor was used for capturing images. Fiji software was used to process 8- or 16-bit (512 × 512 or 1,024 × 1,024 pixels) captured images.