1. Cell Biology
Download icon

A missense in HSF2BP causing primary ovarian insufficiency affects meiotic recombination by its novel interactor C19ORF57/BRME1

  1. Natalia Felipe-Medina
  2. Sandrine Caburet
  3. Fernando Sánchez-Sáez
  4. Yazmine B Condezo
  5. Dirk G de Rooij
  6. Laura Gómez-H
  7. Rodrigo Garcia-Valiente
  8. Anne Laure Todeschini
  9. Paloma Duque
  10. Manuel Adolfo Sánchez-Martin
  11. Stavit A Shalev
  12. Elena Llano
  13. Reiner A Veitia  Is a corresponding author
  14. Alberto M Pendás  Is a corresponding author
  1. Molecular Mechanisms Program, Centro de Investigación del Cáncer and Instituto de Biología Molecular y Celular del Cáncer (CSIC-Universidad de Salamanca), Spain
  2. Université de Paris, France
  3. Institut Jacques Monod, Université de Paris, France
  4. Reproductive Biology Group, Division of Developmental Biology, Department of Biology, Faculty of Science, Utrecht University, Netherlands
  5. Transgenic Facility, Nucleus platform, Universidad de Salamanca, Spain
  6. Departamento de Medicina, Universidad de Salamanca, Spain
  7. The Genetic Institute, "Emek" Medical Center, Israel
  8. Bruce and Ruth Rappaport Faculty of Medicine, Technion, Israel
  9. Departamento de Fisiología y Farmacología, Universidad de Salamanca, Spain
  10. Université Paris-Saclay, Institut de Biologie F. Jacob, Commissariat à l’Energie Atomique, France
Research Article
  • Cited 0
  • Views 528
  • Annotations
Cite this article as: eLife 2020;9:e56996 doi: 10.7554/eLife.56996

Abstract

Primary Ovarian Insufficiency (POI) is a major cause of infertility, but its etiology remains poorly understood. Using whole-exome sequencing in a family with three cases of POI, we identified the candidate missense variant S167L in HSF2BP, an essential meiotic gene. Functional analysis of the HSF2BP-S167L variant in mouse showed that it behaves as a hypomorphic allele compared to a new loss-of-function (knock-out) mouse model. Hsf2bpS167L/S167L females show reduced fertility with smaller litter sizes. To obtain mechanistic insights, we identified C19ORF57/BRME1 as a strong interactor and stabilizer of HSF2BP and showed that the BRME1/HSF2BP protein complex co-immunoprecipitates with BRCA2, RAD51, RPA and PALB2. Meiocytes bearing the HSF2BP-S167L variant showed a strongly decreased staining of both HSF2BP and BRME1 at the recombination nodules and a reduced number of the foci formed by the recombinases RAD51/DMC1, thus leading to a lower frequency of crossovers. Our results provide insights into the molecular mechanism of HSF2BP-S167L in human ovarian insufficiency and sub(in)fertility.

Introduction

The process of gametogenesis is one of the most complex and highly regulated differentiation programs. It involves a unique reductional cell division, known as meiosis, to generate highly specialized cells: the gametes. Indeed, the outcome of meiosis is the production of oocytes and spermatozoa, which are the most distinctive cells of an adult organism and are essential for the faithful transmission of the genome across generations.

The meiotic division is an orderly process that results in the pairing and synapsis of homologous chromosomes and crossover (CO) formation, which ultimately enable homologous chromosomes segregation (Hunter, 2015; Loidl, 2016; Zickler and Kleckner, 2015). In mammals, pairing of homologs is dependent on the repair of self-induced double-strand breaks (DSBs) during prophase I by homologous recombination (Handel and Schimenti, 2010) and it leads to the intimate alignment of homologous chromosomes (synapsis) through the zipper-like synaptonemal complex (SC) (Cahoon and Hawley, 2016). The SC is a proteinaceous tripartite structure that provides the structural framework for DSBs repair (Baudat et al., 2013), as epitomized by the tight association of the recombination nodules (RNs, multicomponent recombinogenic factories) and the axial elements of the SC (Zickler and Kleckner, 2015).

Meiotic DSBs repair is an evolutionarily conserved pathway that is highly regulated to promote the formation of at least one CO per bivalent. This chromosome connection between bivalents through chiasmata is required for a correct reductional division. As other DNA repair processes, proper meiotic recombination is essential for genome stability and alterations can result in infertility, miscarriage and birth defects (Geisinger and Benavente, 2017; Handel and Schimenti, 2010; Webster and Schuh, 2017).

Infertility refers to failure of a couple to reproduce and affects 10–15% of couples (Isaksson and Tiitinen, 2004). Infertility can be due to female factors, male factors, a combination of both or to unknown causes, each category representing approximately 25% of cases (Isaksson and Tiitinen, 2004; Matzuk and Lamb, 2008). There are several underlying causes and physiological, genetic and even environmental and social factors can play a role. Forward and reverse genetic analyses in model organisms have identified multiple molecular pathways that regulate fertility and have allowed to infer reasonable estimates of the number of protein-coding genes essential for fertility (de Rooij and de Boer, 2003; Schimenti and Handel, 2018).

Primary ovarian insufficiency (POI) is a major cause of female infertility and affects about 1–3% of women under 40 years of age. It is characterized by cessation of ovarian function before the age of 40 years. POI results from a depletion of the ovarian follicle pool and can be isolated or syndromic. Genetic causes of POI account for approximately 20% of cases (Rossetti et al., 2017). Although infertility-causing pathogenic variants are inherently unlikely to spread in a population, they can be observed within families, especially when there is consanguinity. Such cases provide crucial insights into the function of the genes and molecular mechanisms that they disrupt. Over the last decade, causative variants in several genes have been found using whole exome sequencing in ‘POI pedigrees’. In particular, pathogenic variants in genes involved in DNA replication, recombination or repair, such as STAG3, SYCE1, HFM1, MSH5 and MEIOB have been formally implicated in this condition by ourselves and others (Caburet et al., 2014; Caburet et al., 2019a; de Vries et al., 2014; Guo et al., 2017; Primary Ovarian Insufficiency Collaboration et al., 2014).

In this study, we have identified in a consanguineous family with POI the candidate S167L missense variant in HSF2BP, an essential yet poorly studied meiotic gene. HSF2BP encodes an interactor of the heat-shock response transcription factor HSF2 (Yoshima et al., 1998). During the course of this work and, in agreement with our results, two independent groups showed that HSF2BP is essential for meiotic recombination through its ability to interact with BRCA2 (Brandsma et al., 2019; Zhang et al., 2019). Here, we report that the introduction of the missense variant HSF2BP-S167L in mouse leads to subfertility and DNA repair defects during prophase I. In addition, we identified a protein complex composed of BRCA2, HSF2BP, and the as yet unexplored C19ORF57/BRME1 (meiotic double-stranded break BRCA2/HSF2BP complex associated protein) as a key component of the meiotic recombination machinery. Our studies show that a single substitution (S167L) in HSF2BP leads to a reduced loading of both BRME1 and HSF2BP at the RNs. Furthermore, our results suggest that meiotic progression requires a critical threshold level of HSF2BP/BRME1 for the ulterior loading of the recombinases to the RNs.

Results

Clinical cases

The parents are first-degree cousins of Israeli Arab origin. Of the five daughters, three are affected with POI and presented with early secondary amenorrhea. They had menarche at normal age (at 13–14) but with irregular menses that stopped around 25. Only one of the patients affected by POI could have a child with the help of a fertility treatment (see pedigree in Figure 1). In order to identify the genetic basis of this familial POI case, we performed whole exome sequencing on genomic DNA from two POI patients, III-2 and III-3, and their fertile sister III-10 (Supplementary file 1a). Variants were filtered on the basis of (i) their homozygosity in the patients, (ii) their heterozygosity or absence in the fertile sister, (iii) their absence in unrelated fertile in-house controls and (iv) a minor allele frequency (MAF) below 0.01 in all available databases (Supplementary file 1b). This filtering process led to the identification of a missense substitution located in the HSF2BP gene: rs200655253 (21:43630396 G > A, GRCh38). The variant lies within the sixth exon of the reference transcript ENST00000291560.7 (NM_007031.2:c.500C > T) and changes a TCG codon into a TTG (NP_008962.1:p.Ser167Leu). It is very rare (Variant Allele Frequency/VAF 0.0001845 in the GnomAD database and 0.0005 in the GME Variome dedicated to Middle-East populations) and absent in a homozygous state from all available databases. The variant was verified by Sanger sequencing and was found to segregate in a Mendelian fashion within the family: the affected twin III-1 was homozygous for the variant and both parents and fertile siblings were heterozygous carriers (Figure 1—figure supplement 1). Therefore, there was no homozygous males identified in this family, preventing the analysis of the impact of this variant on male fertility. Serine 167 is a highly conserved position and the S167L variant is predicted to be pathogenic or deleterious by 11 out of the 18 pathogenicity predictors available in dbNSFP 3.5. (Supplementary file 1c, Figure 1—figure supplement 2 and Figure 1—figure supplement 3).

Figure 1 with 3 supplements see all
Pedigree of the consanguineous family with the variant HSF2BP-S167L.

III-1 and III-2 are monozygotic twins, who appear phenotypically dizygotic. Clinical investigation confirmed POI, with normal 46, XX karyotype (500 bands and SKY spectral karyotyping). Year of birth and age of menarche are indicated when known. III-1 became amenorrheic at age 24 and III-2 at age 25, both after irregular menstruations since menarche. III-1 presents with a short stature (152 cm, within the 3–5 percentile), a normal neck, cubitus valgus and metacarpal shortening of 4–5. Ultrasound investigation showed normal uterus and ovaries. Her g-banding karyotyping was normal 46, XX (500 bands) and variants in FMR1 gene were ruled out. III-2 displays a normal secondary sexual development with no dysmorphic sign. Clinical investigation confirmed POI, with normal 46, XX karyotype (500 bands and SKY spectral karyotyping). The elder sister III-3 was also diagnosed with POI, with no further clinical information. She is 160.5 cm. She had one normal pregnancy with the help of ‘fertility treatment’, and a second unsuccessful attempt. The two fertile sisters, III-6 and III-10 had their menarche at 14–15 and 13–14 respectively, with regular menstruations ever since. They are respectively 150 cm and 151 cm, with no clinical sign, and each one had several children without difficulties. The fertile brother III-7 is 171 cm and shows no health or fertility problem. He developed frontal baldness since the age of 30. The genotype of each individual at the variant genomic position in HSF2BP is shown in red, as determined by Sanger sequencing for available DNAs (See Figure 1—figure supplement 1).

Mice with the HSF2BP S167L variant show a partial reduction of fertility

During the course of this work, two independent groups showed that HSF2BP is essential for meiotic recombination through its ability to interact with the armadillo repeats of BRCA2 (Zhang et al., 2019). Both groups showed that genetic disruption of Hsf2bp in mouse leads to the accumulation in the chromosomes axes of DNA repair proteins such as γH2AX (ATR-dependent phosphorylation of H2AX marks DSBs) and the single stranded-DNA binding protein RPA, a strong reduction of the recombinases DMC1 and RAD51 at the RNs and a lack of COs as labelled by MLH1 (Baker et al., 1996). The end result is male sterility (Brandsma, 2006; Brandsma et al., 2019; Zhang et al., 2019). However, loss of HSF2BP in female mice showed a milder meiotic phenotype (Zhang et al., 2019 and our own data, see below) and a weak albeit non-statistical significant reduction of fertility (Brandsma et al., 2019) despite all of the mutants are nulls though in different genetic backgrounds.

In order to confirm the causality of the S167L variant in this POI family, we generated a knock-in mouse Hsf2bpS167L/S167L by genome editing (Figure 2—figure supplement 1a). We also generated a loss-of-function model (Hsf2bp-/-) for direct comparison (Figure 2—figure supplement 1b–d). Hsf2bpS167L/S167L male and female mice were able to reproduce but females showed a significant reduction in the number of litters (Figure 2a), whilst males only showed a slight non-significant reduction in fertility (Figure 2a), suggesting that the S167L variant impacts murine fertility.

Figure 2 with 2 supplements see all
Mice carrying the HSF2BP S167L variant show a partial reduction of fertility.

(a) Fertility assessment of males and female Hsf2bpS167L/S167L and WT mice showing the number of litters per month and the number of pups per litter (see Materials and methods). Mice: Hsf2bp+/+n = 6 females/6 males, Hsf2bpS167L/S167Ln = 7 females/6 males. Two-tailed Welch’s t-test analysis: *p<0.05. (b) Hematoxylin and eosin stained sections of ovaries from adult (8 weeks) Hsf2bp+/+, Hsf2bpS167L/S167L and Hsf2bp-/- females. Hsf2bp-/- ovaries but not Hsf2bp+/+ and Hsf2bpS167L/S167L showed a strong depletion of follicles. Bar in panels 100 μm. (c) Quantification of the number of follicles (primordial, primary, secondary and antral follicles) per ovary in Hsf2bp+/+, Hsf2bpS167L/S167L and Hsf2bp-/- females at 5 dpp and 6 weeks of age showing no differences between Hsf2bp+/+ and Hsf2bpS167L/S167L but a strong reduction in the oocyte pool in Hsf2bp-/- females. Ovaries: five dpp/6 weeks = 5/5 ovaries from Hsf2bp+/+ and Hsf2bpS167L/S167L and 4/3 from Hsf2bp-/-. Two-tailed Welch’s t-test analysis: ns, no significant differences, **p<0.01, ***p<0.001. (d) Testis size of Hsf2bpS167L/S167L (left, 21% reduction) and Hsf2bp-/- mice (right, 70% reduction) in comparison with their WT counterparts. See Figure 2—figure supplement 2b for the quantification. (e) PAS and Hematoxylin stained testis sections. The S167L variant leads to a partial spermatogenic arrest with an elevated number of apoptotic meiotic divisions (blue asterisks) and a reduction of the number of spermatozoa in the epididymides in comparison with the WT control (Hsf2bp+/+). The null allele (Hsf2bp-/-) showed a complete spermatogenic arrest at epithelial stage IV and absence of spermatozoa. Massive apoptosis of spermatocytes is indicated (blue arrowheads). Bar: upper panels 10 μm, lower panels 20 μm. (St) Seminiferous tubules, (Ep) Epididymides. (f) Immunohistochemical detection of apoptotic cells by TUNEL staining showing an increase of apoptotic meiotic divisions in stage XII tubules from Hsf2bpS167L/S167L males (magnified panel). Plot under the panel represents the quantification. Mice: n = 3 adult mice for each genotype. Two-tailed Welch’s t-test analysis: ***p<0.001. Bar in panel, 25 μm. (g) Quantification of epididymal sperm in Hsf2bp+/+ and Hsf2bpS167L/S167L adult mice. Epididymides: n = 8 for each genotype. Two-tailed Welch’s t-test analysis: *p<0.05.

Histological analysis of Hsf2bpS167L/S167L ovaries revealed no apparent differences in the number of follicles in comparison to wild-type (WT) animals (Figure 2b–c and Figure 2—figure supplement 2a), in contrast with the drastic reduction of the follicle pool in Hsf2bp-/- ovaries (Figure 2b–c). Testes from Hsf2bpS167L/S167L mice displayed a reduced size (21% reduction compared to WT mice; testis/body weight ratio: S167L 0.26% ± 0.07 (n = 12) vs 0.33% ± 0.05 for WT controls (n = 14), **p<0,01, Figure 2d and Figure 2—figure supplement 2b) and this reduction was stronger in Hsf2bp-/- testes (70% reduction compared to WT, testis/body weight ratio: Hsf2bp-/- 0.10% ± 0.005 (n = 6) vs 0.33% ± 0.05 for WT controls (n = 14) ****p<0,001, Figure 2d and Figure 2—figure supplement 2b). Histological analysis of adult Hsf2bpS167L/S167L testes revealed seminiferous tubules with a partial arrest with apoptotic spermatocytes (meiotic divisions) and their epididymis exhibited scarcer spermatozoa (Figure 2e). Consistent with these results, Hsf2bpS167L/S167L males showed increased numbers of meiotic divisions positive for TUNEL staining (Figure 2f) and a reduction in the number of spermatozoa in the epididymis (3.3 × 106 in the Hsf2bpS167L/S167L mutant vs 4.3 × 106 in the WT; Figure 2g). During mouse spermatogenesis, the 12 stages of the epithelial cycle can be distinguished in seminiferous tubule sections by identifying groups of associated germ cell types (Ahmed and de Rooij, 2009). Following these criteria, the seminiferous epithelium of Hsf2bp-/- mice showed a stage IV arrest, characterized by a massive apoptosis of zygotene-like spermatocytes occurring at the same time that In spermatogonia divide into B spermatogonia (Figure 2e). The presence of spermatogonia, spermatocytes, Sertoli and Leydig cells was not altered in any of the mutants (Figure 2e). These results suggest that mice bearing the POI-causing variant only partially phenocopy the human disease.

Hsf2bpS167L/S167L meiocytes show an altered meiotic homologous recombination

To further characterize meiotic defects, Hsf2bpS167L/S167L meiocytes were first analyzed for the assembly/disassembly of the SC by monitoring the distribution of SYCP1 and SYCP3. We did not observe any difference in synapsis and desynapsis from leptotene to diplotene in both oocytes and spermatocytes (Figure 3—figure supplement 1a–b). However, we observed an elevated number of apoptotic meiotic divisions in Hsf2bpS167L/S167L males (Figure 2—figure supplement 2c). These results are consistent with the partial arrest observed in the histological analysis (Figure 2e). As expected, this phenotype was exacerbated in Hsf2bp-/- spermatocytes that were arrested at a zygotene-like stage (Figure 3—figure supplement 1c). Hsf2bp-/- oocytes showed a delay in prophase I progression with the majority of cells at zygotene stage in 17.5 days post-coitum (dpc) females, whilst the WT oocytes were mainly at pachytene stage. Additionally, we observed increased numbers of oocytes showing synapsis defects in the Hsf2bp-/- oocytes (Hsf2bp-/-: 45,5% ± 1,5 vs WT: 7,5% ± 1,5; n = 2 (both genotypes), **p<0,01, Figure 3—figure supplement 1d). These results strongly suggest that the POI variant S167L is a hypomorphic allele.

Next, we analyzed whether the POI-inducing variant affects the loading/stability of HSF2BP by immunolabeling meiocytes from Hsf2bpS167L/S167L mice. We observed a striking reduction of HSF2BP staining at the axes during prophase I in both spermatocytes and oocytes (Figure 3a–b). Western blot analysis of WT, Hsf2bpS167L/S167L and Hsf2bp-/- in whole testis extracts from 13 days post-partum (dpp) animals (Figure 3c) revealed that the reduced labeling observed by immunofluorescence correlated with a reduced protein expression level, suggesting that the mutation leads to a reduced expression and/or stability.

Figure 3 with 1 supplement see all
Meiocytes from Hsf2bpS167L/S167L mice show a decrease in the expression of HSF2BP.

(a–b) Double immunofluorescence of HSF2BP (green) and SYCP3 (red) in Hsf2bp+/+and Hsf2bpS167L/S167L (a) spermatocyte and (b) oocyte spreads showing a strong reduction in the labeling of HSF2BP at the chromosome axis. Plots under the panels show the quantification. Nuclei analyzed: 30 zygonemas and 40 pachynemas from two adult male mice of each genotype. In females 38/39 zygonemas and 37/35 pachynemas from two 17.5 dpc embryos of Hsf2bp+/+ and Hsf2bpS167L/S167L, respectively. Two-tailed Welch’s t-test analysis: ****p<0.0001. (c) Western blot analysis of protein extracts from 13 dpp WT, Hsf2bpS167L/S167L and Hsf2bp-/- testes using polyclonal antibodies against HSF2BP. Tubulin was used as loading control. Graph on the right represents the relative quantification of the immunoblotting. Mice: n = 2 Hsf2bp+/+, Hsf2bpS167L/S167L and Hsf2bp-/-. Two-tailed Welch’s t-test analysis: *p<0.05. Bar in panels a-c, 10 μm.

Given that HSF2BP is essential for DNA repair, we carried out a comparative staining analysis of γH2AX, the ssDNA-binding protein RPA, the recombinases RAD51 and DMC1, the ssDNA-binding protein SPATA22 (complexed to RPA during resection) and CO formation in meiocytes from Hsf2bpS167L/S167L, Hsf2bp-/- and WT animals (Figures 4, 5 and 6, Figure 4—figure supplement 1, Figure 5—figure supplement 1 and Figure 6—figure supplement 1). Our results revealed that Hsf2bpS167L/S167L spermatocytes showed an increased labeling of γH2AX at pachytene (Figure 4a), an accumulation of RPA at the chromosome axis (Figure 4b and Figure 4—figure supplement 1a), a reduction of the recombinases DMC1 and RAD51 staining (Figure 5a–b and Figure 5—figure supplement 1a–b), an accumulation of SPATA22 (Figure 6a and Figure 6—figure supplement 1a), and a decreased number of COs (measured as MLH1, Figure 6b and Figure 6—figure supplement 1b). In accordance with the reduction of COs, we observed the presence of univalents in the XY pair at pachynema as well as univalents in metaphase I spermatocytes from Hsf2bpS167L/S167L mice (Figure 6d and Figure 6—figure supplement 1c). These results would explain the elevated number of apoptotic metaphases observed (Figure 2e–f and Figure 2—figure supplement 2c).

Figure 4 with 1 supplement see all
DNA repair in Hsf2bpS167L/S167L mice.

(a, c) Double labeling of γH2AX (green) and SYCP3 (red) in (a) spermatocyte and (c) oocyte spreads from WT, Hsf2bpS167L/S167L and Hsf2bp-/- mice. (a) Males display an accumulation of γH2AX patches in Hsf2bpS167L/S167L pachynemas and a strong accumulation in the whole nucleus in Hsf2bp-/- zygotene-like arrested cells. Plots on the right of the panel represent the percentage of pachynemas with γH2AX labeling (Nuclei: 364 Hsf2bp+/+ and 376 Hsf2bpS167L/S167L from three adult mice) and the quantification of γH2AX intensity on autosomes at early and mid-pachytene stages (Nuclei: 53 early and 60 mid pachynemas from three adult mice of each genotype). Two-tailed Welch’s t-test analysis: *p<0.05, ****p<0.0001. (c) In females there is an accumulation of γH2AX in Hsf2bpS167L/S167L pachynemas that is stronger in those from Hsf2bp-/- females. Nuclei: n = 21/20/19 pachynemas from 2 Hsf2bp+/+/Hsf2bpS167L/S167L/Hsf2bp-/- embryos (17.5 dpc). Two-tailed Welch’s t-test analysis: *p<0.05, ****p<0.0001. (b, d) Double immunolabeling of RPA1 (green) and SYCP3 (red) in (b) spermatocyte and (d) oocyte spreads from Hsf2bp+/+, Hsf2bp S167L/S167L and Hsf2bp-/-. (b) In males, RPA1 accumulates at early and mid-pachytene in S167L spermatocytes and in the zygotene-like arrested cells from Hsf2bp-/-. Plot on the right of the panel represents the quantification. Nuclei: n = 31/34/37 leptonemas, n = 29/29/37 zygonemas/zygonemas-like from three adult Hsf2bp+/+, Hsf2bp S167L/S167L and Hsf2bp-/- mice respectively, n = 33 early and 46 mid pachynemas from three adult Hsf2bp+/+ and Hsf2bp S167L/S167L mice. Two-tailed Welch’s t-test analysis: ns, no significant differences; **p<0.01, ****p<0.0001. (d) In females, RPA1 labeling is similar in Hsf2bp+/+, Hsf2bp S167L/S167L and Hsf2bp-/- oocytes at zygotene and pachytene. Plot on the right of the panel represents the quantification. Nuclei: Hsf2bp+/+/Hsf2bp S167L/S167L/ Hsf2bp-/- n = 42/41/23 zygonemas from two embryos (16.5 dpc) and n = 25/25/24 pachynemas from two embryos (17.5 dpc). Two-tailed Welch’s t-test analysis: ns, no significant differences. Bar in all panels, 10 μm. Extended panels for RPA1 figures in Figure 4—figure supplement 1.

Figure 5 with 1 supplement see all
The loading of recombinases is compromised in Hsf2bpS167L/S167L mice.

Double immunolabeling of (a, c) DMC1 or (b, d) RAD51 (green) and SYCP3 (red) in Hsf2bp+/+, Hsf2bp S167L/S167L and Hsf2bp-/- (a, b) spermatocytes and (c, d) oocytes showing a strong reduction (Hsf2bp-/-) and mild reduction (Hsf2bp S167L/S167L) in the number of foci in comparison with their WT counterparts. Plots on the right of the panels represent the quantification of foci on each genotype and stage. Male nuclei DMC1: Hsf2bp+/+/Hsf2bp S167L/S167L/Hsf2bp-/-, respectively, n = 43/38/37 early zygonemas, 41/43/37 late zygonemas/zygonemas-like and 44/37 early pachynemas from two adult mice of each genotype. Male nuclei RAD51: Hsf2bp+/+/Hsf2bp S167L/S167L/Hsf2bp-/- respectively n = 39 early zygonemas from all genotypes, 37/40/43 late zygonemas/zygonema-like and 37/39 early pachynemas from two adult mice of each genotype. Oocyte nuclei DMC1: Hsf2bp+/+/Hsf2bp S167L/S167L/Hsf2bp-/- n = 18/18/22 zygonemas from two embryos and n = 21/30/23 pachynemas from two embryos (17.5 dpc). Oocyte nuclei RAD51: Hsf2bp+/+/Hsf2bp S167L/S167L/Hsf2bp-/- n = 35/35/42 zygonemas and n = 42/42/40 pachynemas from two embryos (17.5 dpc). Two-tailed Welch’s t-test analysis: ns, no significant differences, **p<0.01, ***p<0.001, ****p<0.0001. Bar in all panels, 10 μm. Extended panels for these figures in Figure 5—figure supplement 1.

Figure 6 with 2 supplements see all
Recombination proficiency is decreased in Hsf2bpS167L/S167L mice.

(a) Double labeling of SPATA22 (green) and SYCP3 (red) in spermatocyte (upper panel) and oocyte (lower panel) spreads from WT, Hsf2bp-/- and Hsf2bpS167L/S167L mice. SPATA22 is accumulated in knock-out spermatocytes and oocytes and shows a milder accumulation in the Hsf2bpS167L/S167L spermatocytes. Hsf2bpS167L/S167L oocytes show a slight but not significant accumulation. Plots on the right of the panel represents the quantification of SPATA22 labeling. Males nuclei: n = 20 cells for each stage from two adult mice of each genotype. Females nuclei: Hsf2bp+/+/Hsf2bp S167L/S167L/Hsf2bp-/- n = 41/40/40 zygonemas from two embryos and n = 40/39/38 pachynemas from two embryos (17.5 dpc). Two-tailed Welch’s t-test analysis: ns, no significant differences, **p<0.01, ****p<0.0001. (b) Double immunofluorescence of MLH1 (green) and SYCP3 (red) in spermatocyte spreads from WT, Hsf2bpS167L/S167L and Hsf2bp-/-. MLH1 foci are significantly reduced in the Hsf2bpS167L/S167L spermatocytes and absent in the knock-out. The plot on the right shows the quantification. See also Figure 6—figure supplement 1b for the plot showing the percentage of bivalents without CO. Nuclei: n = 61 for Hsf2bp+/+, 89 for Hsf2bpS167L/S167L and 60 for Hsf2bp-/- from three adult mice of each genotype. Two-tailed Welch’s t-test analysis: ****p<0.0001. (c) Double labeling of CDK2 (green) and SYCP3 (red) in oocyte spreads from 17.5 dpc Hsf2bp+/+, Hsf2bpS167L/S167L and Hsf2bp-/- embryos. During meiotic prophase I, CDK2 localizes to the telomeres of chromosomes from leptotene to diplotene. However, around mid-pachytene additional interstitial CDK2 signals appear at CO sites, colocalizing with MLH1. As a measure of COs, just interstitial CDK2 foci (non-telomeric) have been counted. Hsf2bp-/- and Hsf2bpS167L/S167L females show a high and moderate reduction in the number of COs, respectively. Plot on the right of the panel show the quantification. See also Figure 6—figure supplement 1d for the plot showing the percentage of bivalents without CO. Nuclei: Hsf2bp+/+/Hsf2bp S167L/S167L/Hsf2bp-/- n = 79/67/46 from three embryos (17.5 dpc). Two-tailed Welch’s t-test analysis: ***p<0.0001, ****p<0.0001. (d) Double immunofluorescence of γH2AX (green) and SYCP3 (red) in spermatocyte spreads from WT and Hsf2bpS167L/S167L mice. At pachytene, γH2AX allows the identification of the XY bivalent. Diagram on the right represents the quantification of the pachynemas with unsynapsed sex chromosomes from Hsf2bpS167L/S167L and WT mice. Nuclei: n = 150 pachynemas from three adult mice of each genotype. Two-tailed Welch’s t-test analysis: *p<0.05. Bar in all panels, 10 μm.

Our analysis in females showed accumulation of γH2AX staining (Figure 4c) but no accumulation in RPA labeling in Hsf2bpS167L/S167L and Hsf2bp-/- oocytes (Figure 4d and Figure 4—figure supplement 1b). Similar to the spermatocytes, DMC1 and RAD51 staining showed a reduction in both Hsf2bpS167L/S167L and Hsf2bp-/- oocytes (Figure 5c–d and Figure 5—figure supplement 1c–d). SPATA22 labeling in females showed a clear accumulation in Hsf2bp-/- but only a trend towards accumulation in Hsf2bpS167L/S167L oocytes (Figure 6a and Figure 6—figure supplement 1a). In agreement with the lower presence of recombinases, the number of COs (measured as interstitial CDK2 foci) was also reduced in Hsf2bpS167L/S167L oocytes, and a stronger reduction was observed in Hsf2bp-/- oocytes (Figure 6c and Figure 6—figure supplement 1d). Overall, male and female Hsf2bpS167L/S167L mice share alterations in the meiotic recombination pathway although with different reproductive outcome.

We next sought to understand how the HSF2BP pathogenic variant was mediating the observed meiotic alteration. HSF2BP has been shown to bind BRCA2, an essential protein for meiotic homologous recombination (Martinez et al., 2016; Sharan et al., 2004), by a direct interaction that involves Arg200 in HSF2BP and the Gly2270-Thr2337 region within the C-terminal fragment of BRCA2 (Brandsma et al., 2019). Given the impossibility to detect endogenous BRCA2 by immunofluorescence in mouse spermatocytes, we carried out co-localization/interaction assays in a heterologous system by transfecting BRCA2-C (i.e. its C-term) and HSF2BP in U2OS/HEK293T. Our results showed that BRCA2-C co-immunoprecipitates with both HSF2BP-WT and HSF2BP-S167L in similar ways (Figure 6—figure supplement 2a). In single transfections, HSF2BP localized in the nucleus and cytoplasm whereas BRCA2-C showed nuclear localization. This pattern changed drastically to a nuclear dotted pattern when co-transfected (Figure 6—figure supplement 2b). This re-localization was independent of the HSF2BP variant, suggesting that the HSF2BP variant effects are not directly mediated by BRCA2 delocalization.

BRME1, a novel interactor of HSF2BP

In order to further understand the mechanism underlying the pathogenicity of the HSF2BP-S167L variant, we searched for proteins that interact with the murine HSF2BP through a yeast two hybrid (Y2H) screening. The analysis of the clones with putative interactors revealed that 19 out of 98 analyzed clones matched the uncharacterized gene 4930432K21Rik, which corresponds to human C19ORF57, hereby dubbed BRME1 for Break Repair Meiotic recombinase recruitment factor 1. This HSF2BP interactor consists of 600 amino acids with a high content of acidic residues, has no recognizable functional domains and is intrinsically disordered. The interaction was validated by transiently transfecting plasmids driving the expression of HSF2BP and BRME1. Both HSF2BP-S167L and WT interacted with BRME1 (Figure 7a). We further validated this interaction in vivo by co-immunoprecipitation (co-IP) of both proteins from mouse whole testis extracts (Figure 7b). To identify the regions required for this interaction, we split the BRME1 protein into three fragments (N-terminal, central region and C-terminal). We mapped the HSF2BP/BRME1-interacting domain to the C-term fragment of BRME1 (spanning residues 475–600 of the murine protein, Figure 7c). In line with this, the Brme1Δ142-472/Δ142-472 mutant mice, expressing the BRME1 protein devoid of its central part, were fertile and did not show defects in chromosome synapsis or an alteration of HSF2BP loading to axes, further indicating that a large fraction of the coding protein of BRME1 is not essential for BRME1/HSF2BP function in vivo (Figure 7—figure supplement 1).

Figure 7 with 6 supplements see all
BRME1, a novel HSF2BP interactor that colocalizes to the recombination nodules.

(a) HEK293T cells were transfected with Flag-HSF2BP (WT, upper panel; S167L, lower panel) and its novel interactor GFP-BRME1. Protein complexes were immunoprecipitated (IP: green text) with either anti-Flag or anti-EGFP or IgGs (negative control) and analysed by immunoblotting with the indicated antibody (WB: red text). Both HSF2BP variants (WT and S167L) co-immunoprecipitated similarly with BRME1. (b) IP of testis extracts with antibodies against BRME1, HSF2BP and IgGs as a negative control (IP: green text) and western blot with the indicated antibodies (WB: red text) (c) Schematic representation of full-length BRME1 protein and the corresponding deletion (Δ) constructs (filled boxes) generated to decipher the essential BRME1 region for interacting with HSF2BP (green positive interaction and red no interaction). Western blots under the scheme show the Co-IP experiments. HEK293T cells were transfected with GFP-HSF2BP and the different delta constructs of Flag-BRME1. The Δ475–600 abolishes the interaction, indicating that the C terminus of BRME1 is the essential region of interaction with HSF2BP. (d) Triple immunofluorescence of BRME1 (green), HSF2BP (red) and SYCP3 (blue) in WT spermatocyte spreads showing high colocalization between BRME1 and HSF2BP at late zygotene and pachytene (See Supplementary file 1d for quantification). Bar in panel, 10 μm. (e) Double immunolabeling of spermatocyte spread preparations with HSF2BP (green) and BRME1 (red) analyzed by Stimulated emission depletion (STED) microscopy. Bar in panel, 5 μm. (f–g) Double immunofluorescence of BRME1 (green) and SYCP3 (red) in (f) spermatocytes and (g) oocyte spreads from Hsf2bp+/+, Hsf2bpS167L/S167L and Hsf2bp-/- showing a strong reduction of BRME1 staining in the S167L mutant and absence in the Hsf2bp knock-out. Plots next to the panel represent the quantification. See also extended Figure 7—figure supplement 4a. Male nuclei: Hsf2bp+/+/Hsf2bpS167L/S167L/Hsf2bp-/-: n = 16/16/20 early and 15/15/16 late zygonemas, 16/16 /- pachynemas from two adult mice. Female nuclei: n = 18 pachynemas from two embryos (17.5 dpc) of each genotype. Two-tailed Welch’s t-test analysis: *p<0.05, ****p<0.0001. Bar in panels, 10 μm. (h) Western blot analysis of protein extracts from 13 dpp WT, Hsf2bpS167L/S167L and Hsf2bp-/- testes using an antibody against BRME1. Tubulin was used as loading control. Graph on the right represents the relative quantification of the immunoblotting. Mice: n = 2 Hsf2bp+/+, Hsf2bpS167L/S167L and Hsf2bp-/-. Two-tailed Welch’s t-test analysis: *p<0.05.

We also sought to characterize the involvement of BRME1 in meiosis through immunofluorescence. BRME1 localized to the chromosome axes of WT meiocytes from zygotene to pachytene with a pattern of discrete foci that mimics the RNs (Figure 7—figure supplement 2a–b). In agreement with the yeast two hybrid and co-IP results, BRME1 perfectly co-localized with HSF2BP on the chromosome axes (Figure 7d and Supplementary file 1d for quantification). This co-localization was verified by super-resolution microscopy (Figure 7e). In accordance with the tight association of BRME1 with HSF2BP and with a role in DSB repair, both HSF2BP and BRME1 colocalized with RPA and DMC1 foci. During prophase I, HSF2BP and BRME1 showed higher levels of spatio-temporal colocalization at the RNs with RPA than with DMC1 (Figure 7—figure supplement 3a–b and Supplementary files 1d-e for quantification). We also analyzed the HSF2BP-dependent localization of BRME1 in Hsf2bp-/- and Hsf2bpS167L/S167L mutants. Immunofluorescence analysis of meiocytes showed a complete lack of BRME1 staining in the absence of HSF2BP and a significant reduction of foci number in the presence of HSF2BP-S167L variant (35% reduction in males and 72% in females at pachytene; Figure 7f–g and Figure 7—figure supplement 4a). Western blot analysis also revealed a drastic reduction of BRME1 expression in Hsf2bpS167L/S167L and Hsf2bp-/- spermatocytes, suggesting an HSF2BP-dependent stabilization of BRME1 (Figure 7h).

To assess if HSF2BP and/or BRME1 had DNA-binding activity (targeting to DSBs), we carried out an in vitro binding assay using HSF2BP and BRME1 proteins expressed in a transcription and translation coupled reticulocyte system (TNT; Loregian et al., 2004; Souquet et al., 2013) in which there are no nuclear proteins and chromatin (Melton et al., 1984) and used RPA as positive control. Our results show that both proteins lacked direct DNA-binding abilities, in contrast to the strong activity of RPA (Figure 7—figure supplement 4b–c).

To determine the role of BRME1 in recombination and DNA repair, we analyzed its cytological distribution pattern in different mutants lacking synapsis/recombination-related proteins. These mutants were the meiotic cohesin REC8 (Bannister et al., 2004), the central element protein of the SC SIX6OS1 (Gómez-H et al., 2016), the E3 ligases involved in the stabilization of recombinogenic proteins RNF212 and HEI10 (Qiao et al., 2014), the spermatoproteasomal subunit PSMA8 (Gómez-H et al., 2019), and the nuclease SPO11 required for DSBs generation (Rnf212-/-, Hei10-/- and Spo11-/- mouse mutants are described in this work, see Materials and methods and Figure 7—figure supplement 5 and Figure 7—figure supplement 6a, Baudat et al., 2000). HSF2BP staining was also carried out for a direct comparison. We were able to show that none of the recombination-deficient mutants abrogate BRME1 labeling at zygotene (or the corresponding meiotic stage at which the mutant spermatocytes are arrested), in contrast to its absence of loading in SPO11-deficient mice (Figure 7—figure supplement 6b, left). These results are very similar to those obtained for HSF2BP in these mutants (Figure 7—figure supplement 6b, right) and indicate that SPO11-dependent DSBs are essential for targeting HSF2BP/BRME1 to the RNs, and that the heterocomplex can be positioned at early events soon after DSBs generation.

To functionally analyze the role of BRME1 in mouse fertility, we generated a Brme1-/- null mutant by genome editing (Figure 8—figure supplement 1a–d). Brme1-/- females, despite being fertile, showed a strong reduction of the follicle pool (Figure 8a). Male Brme1-/- mice were infertile, the average size of their testes was severely reduced (76% reduction compared to WT; testis weight/body weight ratio: Brme1-/- 0,08% ± 0004 (n = 6) vs 0,33% ± 0,05 for WT controls (n = 14), ****p<0.0001, Figure 8b and Figure 2—figure supplement 2b), and lacked spermatozoa (Figure 8c). Histological analysis showed a meiotic arrest at epithelial stage IV with apoptotic spermatocytes (Figure 8c). Double immunolabeling of SYCP3 and SYCP1 revealed that spermatocytes were partially synapsed and showed a partner-switch phenotype in which synapsis is not restricted to homologous pairs (Figure 8d). The arrest corresponds to a zygotene-like stage though a small fraction of cells (3,7% ± 1,9; n = 3) were able to escape this blockage reaching early pachytene. Brme1-/- oocyte spread analysis revealed the presence of a subset of fully synapsed pachynemas but an increased number of cells with different degree of asynapsis (47,9% ± 2,2 vs 12% ± 5,7 in the WT; n = 2 (both genotypes), *p<0,05 Figure 8e and Figure 8—figure supplement 1e). Given the interaction between HSF2BP and BRME1, we tested whether HSF2BP localization depended on BRME1 by immunolabeling of HSF2BP in Brme1-/- spermatocytes and oocytes. Our results showed a strong reduction of HSF2BP staining in BRME1-null spermatocytes (Figure 8f) and a total absence in oocytes (Figure 8g). Western blot analysis of HSF2BP in 13 dpp testis extracts from Brme1-/- mice showed a strong reduction in comparison with the WT control (Figure 8h), suggesting again that BRME1 is necessary for HSF2BP protein stabilization.

Figure 8 with 1 supplement see all
Brme1-/-mice show severe fertility defects.

(a) Hematoxylin+eosin stained sections of ovaries from adult Brme1-/- females showing a strong depletion of follicles. Plot on the right represents the quantification in 3 months-old females. Ovaries: n = 3 ovaries for each genotype. Two-tailed Welch’s t-test analysis: *p<0.05, ***p<0.001, ****p<0.0001. Bar in panels, 50 μm. (b) Testes from adult Brme1-/- males show a strong reduction of the testis size. See quantification of testis weight/body weight at Figure 2—figure supplement 2b. (c) Spermatogenesis is arrested at epithelial stage IV in Brme1-/- as shown in PAS+hematoxylin stained testis sections. Massive apoptosis of spermatocytes is indicated (red asterisks). The spermatogenic arrest leads to empty epididymides and non-obstructive azoospermia. (St) Seminiferous tubules. (Ep) Epididymides. Bar in panels, 10 μm. (d–e) Double labeling of (d) spermatocyte and (e) oocyte spreads from WT and Brme1-/- mice with SYCP3 (red) and SYCP1 (green). Brme1-/- spermatocytes arrest in a zygotene-like stage and show synapsis between non-homologous chromosomes. (e) Brme1-/- females showed a subset of fully-synapsed pachynemas (18.5 dpc) but increased numbers of synapsis-defective cells. See extended panel for females at Figure 8—figure supplement 1e. Bar in panels, 10 μm. (f–g) Double labeling with HSF2BP (green) and SYCP3 (red) of (f) spermatocyte and (g) oocyte spreads from Brme1-/- mice showing faint HSF2BP labeling in spermatocytes and total absence of labeling in oocytes. Plot on the right of (f) panel represents de quantification of HSF2BP foci in Brme1-/- spermatocytes. Nuclei: n = 30 zygonemas/zygonemas-like from two adult mice of each genotype (Brme1+/+ values from Figure 3a) Two-tailed Welch’s t-test analysis: ****p<0.0001. Bar in panels, 10 μm. (h) Western blot analysis of protein extracts from 13 dpp WT, Brme1-/- and Hsf2bp-/- testes with a specific antibody against HSF2BP. Tubulin was used as loading control. Graph on the right represents the relative quantification of the immunoblotting. Mice: n = 2 Brme1+/+, Brme1-/- and Hsf2bp-/-. Two-tailed Welch’s t-test analysis: *p<0.05.

Immunostaining of Brme1-/- spermatocytes for γH2AX, RPA, the recombinases RAD51 and DMC1 or SPATA22 revealed an accumulation of γH2AX and RPA on zygonema-like spermatocytes (Figure 9—figure supplement 1a–b), a drastic reduction of RAD51/DMC1 foci in early and late zygonema (Figure 9a and c, Figure 9—figure supplement 2a and c) and a strong accumulation of SPATA22 (Figure 9e and Figure 9—figure supplement 2e). According to the meiotic arrest at the zygotene-like stage, MLH1 staining revealed a total absence of COs (Figure 9g). As in males, Brme1-/- oocytes showed an accumulation of γH2AX (Figure 9—figure supplement 1c) and SPATA22 (Figure 9f and Figure 9—figure supplement 2f) and a reduced staining of DMC1 and RAD51 leading to a reduced number of COs (measured as interstitial CDK2; Figure 9b, d and h, Figure 9—figure supplement 2b and d). However, Brme1-/- oocytes did not show RPA accumulation (Figure 9—figure supplement 1d). These results are similar to the phenotypes described for HSF2BP mutants (Figures 4, 5 and 6, Figure 4—figure supplement 1, Figure 5—figure supplement 1, Figure 6—figure supplement 1 and (Brandsma et al., 2019) see Supplementary file 1f for a complete comparison among mutants and their meiotic alterations). Thus, both HSF2BP and BRME1 mutant mice show a highly similar phenotype including sexual dimorphism.

Figure 9 with 2 supplements see all
BRME1 is essential for meiotic recombination.

(a–b) Double immunofluorescence of DMC1(green) and SYCP3 (red) in Brme1+/+ and Brme1-/- (a) spermatocytes and (b) oocytes showing a reduction in the number of DMC1 foci. Plots under each panel represent the quantification. See also extended panels on Figure 9—figure supplement 2a–b. Male nuclei for DMC1: Brme1+/+/Brme1-/- n = 43/50 early and 41/52 late zygonemas/zygonemas like from two adult mice of each genotype (Brme1+/+ values from Figure 5a). Female nuclei for DMC1: Brme1+/+/Brme1-/- n = 18/30 zygonemas and 21/31 pachynemas from two embryos (17.5 dpc) of each genotype (Brme1+/+ values from Figure 5c). Two-tailed Welch’s t-test analysis: ***p<0.001, ****p<0.0001. Bar in panels, 10 μm. (c–d) Double immunofluorescence of RAD51 (green) and SYCP3 (red) in Brme1+/+ and Brme1-/- (c) spermatocytes and (d) oocytes showing a reduction in the number of RAD51 foci in the Brme1-/- in comparison to the WT. Plots under each panel represent the quantification. See also extended panels on Figure 9—figure supplement 2c–d. Male nuclei for RAD51: Brme1+/+/Brme1-/- n = 39/39 early and 37/45 late zygonemas/zygonemas like from two adult mice of each genotype (Brme1+/+ values from Figure 5b). Female nuclei for RAD51: Brme1+/+/Brme1-/- n = 35/31 zygonemas and 42/40 pachynemas from two embryos (17.5 dpc) of each genotype (Brme1+/+ values from Figure 5d). Two-tailed Welch’s t-test analysis: ****p<0.0001. (e–f) Quantification of SPATA22 intensity in (e) spermatocytes and (f) oocyte spreads from Brme1+/+ and Brme1-/-. See immunofluorescences in Figure 9—figure supplement 2e–f. Nuclei: Males, n = 20 cells from two adult mice of each genotype (Brme1+/+ from Figure 6a). Females, Brme1+/+/Brme1-/- n = 41/40 zygonemas and 40/40 pachynemas from two embryos of each genotype (17.5 dpc) (Brme1+/+ from Figure 6a). Two-tailed Welch’s t-test analysis: ****p<0.0001. (g) Double immunofluorescence of MLH1 (green) and SYCP3 (red) in Brme1+/+ and Brme1-/- spermatocytes showing the absence of MLH1 labeling in the knock-out. (h) Double labeling of CDK2 (green) and SYCP3 (red) in oocyte spreads from 17.5 dpc Brme1+/+ and Brme1-/- embryos. During meiotic prophase I, CDK2 localizes to the telomeres of chromosomes from leptotene to diplotene. However, around mid-pachytene additional interstitial CDK2 signals appear at CO sites, colocalizing with MLH1. As a measure of COs, just interstitial CDK2 foci (non-telomeric) have been counted. Brme1-/- females show a strong reduction in the number of COs. Plot under the panel show the quantification. Nuclei: Brme1+/+/Brme1-/- n = 79/49 from three embryos (17.5 dpc) in WT and two embryos in Brme1-/- (Brme1+/+ from Figure 6c). Two-tailed Welch’s t-test analysis: ***p<0.0001. Bar in all panels, 10 μm.

BRME1 and HSF2BP form a multimeric complex with PALB2 and BRCA2

To further delineate the interactome of BRME1, we immuno-precipitated BRME1 from testis extracts coupled to mass-spectrometry. We identified as expected HSF2BP as the main interactor, but also BRCA2, PALB2, RAD51 and RPA, strongly suggesting that they form a large multimeric complex (Supplementary files 1g-1h). For validation, we transfected the corresponding expression plasmids in HEK293T cells for co-IP analysis. BRME1 co-immunoprecipitated with BRCA2 and HSF2BP when they were all co-transfected, but importantly BRME1 alone did not co-immunoprecitate with BRCA2 (Figure 10a–b). The reciprocal co-IP of BRCA2 with HSF2BP and BRME1 was also positive. We also observed modest but positive co-IP of HSF2BP with RPA, PALB2 and RAD51; and of BRME1 with RAD51 and RPA but not with PALB2 (Figure 10—figure supplement 1a). These interactions were further analyzed in a cell-free TNT system coupled to co-immunoprecipitation assays. We observed an absence of direct interaction between any of them, with the exception of BRME1 and HSF2BP, as expected from the Y2H analysis (Figure 10—figure supplement 1b). These results suggest that these proteins belong to a complex (or complexes) in vivo (likely through BRCA2) and that the HSF2BP-S167L variant could be altering BRME1 interaction with partners of major BRCA2-containing recombination complexes.

Figure 10 with 1 supplement see all
BRME1 forms a complex with BRCA2 and HSF2BP and stabilizes HSF2BP.

(a–b) HEK293T cells were co-transfected with GFP-BRME1, Flag-BRCA2-C and HA-HSF2BP. Protein complexes were immunoprecipitated (IP: green text) with either an anti-Flag, anti-EGFP, anti-HA or IgGs, and analyzed by western blot with the indicated antibody (WB: red text). (a) BRME1 does not co-immunoprecipitate with BRCA2-N, BRCA2-M or BRCA2-C. (b) In the presence of HA-HSF2BP (triple co-transfection) BRCA2-C and BRME1 coimmunoprecipitate (co-IPs between HSF2BP and BRCA2-C are shown in Figure 6—figure supplement 2a). (c) Transfected U2OS cells with plasmids encoding Flag-HSF2BP (WT or S167L) and EGFP-BRME1 alone or together were immuno-detected with antibodies against Flag (red) and EGFP (green). Transfected HSF2BP (WT and S167L) labels the whole cell (S167L less intense) whereas BRME1 shows nuclear localization. When co-expressed, BRME1 and HSF2BP change their patterns and form nuclear invaginations that resemble nucleoplasmic reticulum. This phenotype is milder in the presence of HSF2BP-S167L than with the WT (graph under the panel: quantification of the number of cells showing a nucleoplasmic reticulum pattern). n > 400 cells from two independent transfections of each condition. Two-tailed Welch’s t-test analysis: *p<0.05. Bar in panel, 20 μm. (d) HEK293T cells were transfected with Flag-HSF2BP (WT and S167L) alone or with GFP-BRME1. Additionally, cells transfected with Flag-HSF2BP were treated with the proteasome inhibitor (MG132, 10 μM) and analyzed by western blot. Cherry was used as transfection efficiency control. HSF2BP-WT was expressed at higher levels than HSF2BP-S167L and their detection (both the WT and the S167L variant) was increased when co-transfected with BRME1. The increase was greater for the HSF2BP-S167L variant in comparison with the WT. Incubation with MG132 increased the detection levels of transfected HSF2BP mimicking the effect of co-transfecting BRME1. n = 3 independent transfections for each condition. Two-tailed Welch’s t-test analysis: **p<0.01, ***p<0.001, ****p<0.0001.

Finally, given the interaction of BRME1 and HSF2BP, we analysed their interdependence in U2OS cells. Transfected HSF2BP was localized diffusely in the cytoplasm and the nucleus (Figure 10c). However, when HSF2BP was co-overexpressed with BRME1, its pattern changed to an intense nucleoplasm staining with nuclear invaginations that resemble nucleoplasmic reticulum (Figure 10cMalhas et al., 2011). Interestingly, such invaginations were reduced when BRME1 was co-transfected with HSF2BP-S167L (Figure 10c). In addition, the intensity of the fluorescence signal of HSF2BP-S167L was lower than for HSF2BP-WT and both intensities increased when HSF2BP was co-expressed with BRME1 (Figure 10c). Western blot analysis indicated a reduced protein stability of the S167L variant (Figure 10d), in agreement with the results observed in vivo (Hsf2bpS167L/S167L mutant, Figure 3). Interestingly, the protein expression level of transfected HSF2BP increased when co-transfected with BRME1 and was partially dependent on proteasome degradation (Figure 10d), indicating a role of BRME1 in stabilizing HSF2BP. Taken altogether and given the low protein expression of BRME1 in the Hsf2bpS167L/S167L, these results suggest a functional interdependence between BRME1 and HSF2BP that leads to their lower protein stability/expression in mutant meiocytes, which might induce recombination defects.

Discussion

Using exome sequencing, we identified the S167L missense variant in HSF2BP in a consanguineous family with three cases of POI with secondary amenorrhea. All affected family members are homozygous for the variant, and the healthy relatives are heterozygous carriers. The causality of the HSF2BP-S167L variant is supported by the meiotic phenotype and the subfertility observed in Hsf2bpS167L/S167L female mice. Furthermore, the DNA repair defects in murine Hsf2bpS167L/S167L meiocytes, displayed by the reduced number of RAD51/DMC1 foci on DSBs and the subsequent reduction in the number of COs, provide evidence that this missense variant alters meiotic recombination. This conclusion was further supported by the comparative analysis of the S167L allele with the Hsf2bp null allele, which revealed that the missense variant can be considered as a hypomorphic allele. This is in agreement with the secondary amenorrhea observed in the patients, and the residual (medically-assisted) fertility in one of the affected sisters. Our identification of HSF2BP as a gene implicated in POI is in line with recent reports of POI-causing variants in genes that are required for DNA repair and recombination, such as MCM8, MCM9, SYCE1, MSH4, PSMC3IP, FANCM or NBN (AlAsiri et al., 2015; Carlosama et al., 2017; de Vries et al., 2014; Fouquet et al., 2017; He et al., 2018; Tenenbaum-Rakover et al., 2015; Tucker et al., 2018; Wood-Trageser et al., 2014; Zangen et al., 2011).

Meiotic mouse mutants often exhibit sexually dimorphic phenotypes (Cahoon and Libuda, 2019). These differences can have a structural basis, given that the organization of the axial elements is known to be different between sexes. This is supported by the difference in length of the axes and by the essential role that the meiotic cohesin subunit RAD21L plays in males but not in females (Herrán et al., 2011). In general, meiotic recombination mutants appear to proceed further in female than in male, because in males the asynapsis of the sex bivalent leads to a loss of silencing of the Y chromosome, and perhaps also because of the presence of less stringent checkpoints in oogenesis (Hunt and Hassold, 2002). A sexually dimorphic phenotype is also observed here in our HSF2BP-S167L mutant mice. Both sexes show a significant decrease in the number of COs and consequently an increase in the frequency of meiocytes showing bivalents without CO. This leads to a significant reduction of spermatozoa in the epididymis, while the number of oocytes and their distribution in the follicular pool are not affected in females. Our failure to detect any significant impact of the variant on male fertility could be due to the high variability in litter frequency and size in our study. However, it is known that a strong reduction of the spermatozoa count (up to 60%) does not affect male mouse fertility (Schürmann et al., 2002), which would explain the normal fertility of male mice bearing the HSF2BP-S167L variant despite the presence of overt meiotic alterations. By contrast, female mice with the HSF2BP-S167L variant show a mild sub-fertility phenotype with a reduction of litter frequency. This can be due to the very much fewer gametes available for fertilization in females in comparison to males but also to molecular differences in the meiotic recombination process in both sexes (Cahoon and Libuda, 2019), as displayed by the absence of RPA accumulation or the more pronounced decrease in DMC1 foci observed in HSF2BP-S167L oocytes.

Although we cannot exclude that a POI-like phenotype would appear over time in HSF2BP-S167L female mice, the sub-fertility observed in HSF2BP-S167L females appears to be milder in comparison to the phenotype of the human patients. This could be explained by a lower sensitivity of mice to hypomorphic alleles compared to humans, in a similar manner to the known lower gene dosage sensitivity of the former in the context of genes that are haploinsufficient in human (Veitia, 2003). In addition, the initial events of human female meiosis appear to be more error-prone than in mice, or even than human males, as evidenced by the increased incidence of synaptic defects in the human oocytes or the fact that MLH1 foci appear much earlier in prophase I (Hassold et al., 2007). Furthermore, it has recently been shown that human oocytes exhibit a specific CO maturation inefficiency (Wang et al., 2017). Indeed, despite a higher total number of COs in women, the frequency of bivalents without CO is paradoxically higher in women than in men. Altogether, these observations could explain the stronger phenotype of the human POI patients compared to the Hsf2bpS167L/S167L female mice.

The absence of homozygous male carriers in the consanguineous family studied here prevents the direct comparison of the impact of the HSF2BP-S167L variant on fertility phenotypes between human and mouse males. Although future studies might identify infertile men homozygous for the S167L variant, it is interesting to note that another variant of HSF2BP (G224*) was shown to affect recombination rate in males and that two siblings homozygous for this HSF2BP variant in the analyzed Icelandic population were healthy but without descendants, suggesting they were infertile (Halldorsson et al., 2019). This reinforces a conserved function of HSF2BP in human male fertility.

We have shown through a biochemical analysis that BRME1 immunoprecipitates mainly with its partner HSF2BP (constituting or belonging to a complex) but also with PALB2, RAD51, RPA and BRCA2 (in testis extracts). These interactions could possibly be mediated by the multidomain hub protein BRCA2 (Siaud et al., 2011) as HSF2BP interacts directly with BRCA2 (Brandsma et al., 2019) and with BRME1 (this work). In addition, BRCA2 also directly interacts with the DSBs recruiter PALB2, with the recombinases RAD51 and DMC1 (through different specific domains), and with DNA (Siaud et al., 2011). This BRCA2-containing complex participates in the orderly orchestration of events at DSBs such as the initial binding of RPA to the resected DNA, the exchange of RPA by RAD51/DMC1, and the loading of the MEIOB-SPATA22 complex to the RPA complexes (Martinez et al., 2016; Zhao et al., 2015). Interestingly, genes with recently identified variants in POI patients are implicated in the repair of induced DSBs at the early stages of meiosis and encode BRCA2-interacting factors, such as MEIOB, DMC1 or BRCA2 itself (Caburet et al., 2019a; Caburet et al., 2020; Caburet et al., 2019b; He et al., 2018). This highlights the crucial importance and the high sensitivity of this particular meiotic step, and the hub role of BRCA2 as a tightly regulated platform for correct meiotic recombination.

We have also shown by several complementary approaches that the proteins HSF2BP/BRME1 constitute in vivo a functional complex in which both subunits are essential for meiotic recombination and for their mutual protein expression and/or stability in vivo. Accordingly, the genetic depletion of BRME1 or HSF2BP leads to similar if not identical phenotypes in which oogenesis is altered with severe defects in chromosome synapsis that promotes premature loss of ovarian follicles and spermatogenesis is arrested at zygotene-like stage resulting in a lack of spermatozoa. These meiocytes are not able to load the recombinases RAD51/DMC1, impairing the proper repair of DSBs leading to the generation of no COs or very few in males and females, respectively. As a consequence, zygonema-like spermatocytes accumulate the single strand binding proteins SPATA22 and RPA, whereas oocytes accumulates only SPATA22. During the course of the reviewing of this work, three Brme1 knockouts have been described (Zhang et al., 2020; Shang et al., 2020; Takemoto et al., 2020). All the described male mutants show strong fertility defects and similar molecular alterations although with different severity. In females, the two works that address their analysis (Shang et al. and Takemoto et al.), describe normal fertility which is in contrast with the strong reduction in the follicle pool and meiotic defects observed in our Brme1-/- females. The higher severity of our male and female mutants could be explained on the basis of the different genetic background of the mice given that all of them are apparently similar.

The S167L human recessive POI variant behave as a hypomorphic allele in mice, which results in a reduction of the protein expression/stability of itself and of its partner BRME1 in vivo and in transfected cells. As a consequence, both male and female Hsf2bpS167L/S167 mice show a similar but milder phenotype than that of the Hsf2bp-/-or Brme1-/-, consisting in a reduction in the spermatozoa count while being fertile and a subtle reduction in female fertility. Molecularly, the reduction observed in the meiocytes of the mutant Hsf2bpS167L/S167L mice of RAD51/DMC1, the reduction of COs (both in males and females), the accumulation of RPA (only in males) and SPATA22 (in males and females) are also weaker than in the null mutant. The observed accumulation of RPA in males is likely to occur at the early stages of recombination because SPATA22 loading to the DSBs is also increased in the mutants of HSF2BP and BRME1. RPA, as part of a trimeric replication protein complex (RPA1-RPA2-RPA3), binds and stabilizes ssDNA intermediates that form during DNA repair. In meiosis, RPA is also forming a complex with two other essential meiotic players MEIOB (homologue of RPA) and SPATA22. However, the loading of this complex to DSBs is RPA-independent (Shi et al., 2019). It has been postulated that RPA functions in meiosis at two different stages; (i) during the early recombination stages when the DSBs ends are resected by the MRN complex and (ii) during the strand invasion into the homologous duplex that is carried out by RAD51/DMC1 and ssDNA is generated at the displacement loops (Shi et al., 2019). The observed lack of DNA-binding ability of HSF2BP/BRME1 points towards a model in which the absence of the complex HSF2BP/BRME1 through a direct interaction with BRCA2 impairs the replacement of RPA by RAD51/DMC1 in the foci that form on the DSBs of the spermatocytes. Similarly, the reduced expression at the protein level of HSF2BP/BRME1 as a consequence of the POI variant, which does not affect their heterodimerization, would make them less proficient in replacing RPA in the spermatocytes by the recombinases RAD51/DMC1 leading to a lower frequency of COs. Given the unknown function that RPA plays in vivo during oogenesis (Shi et al., 2019), it is tempting to speculate that the role of RPA in mediating the replacement of RAD51/DMC1 in female meiosis would be carried out by another protein complex such as SPATA22/MEIOB in a HSF2BP/BRME1-dependent manner.

Very recently, a high-resolution genome-wide recombination map revealed novel loci involved in the control of meiotic recombination and highlighted genes involved in the formation of the SC (SYCE2, RAD21L, SYCP3, SIX6OS1) and the meiotic machinery itself as determinants of COs (Halldorsson et al., 2019). Within the second category, variants of the SUMO ligase RNF212 and the ubiquitin ligase HEI10 have been largely documented as genetic determinants of the recombination rate in humans and, importantly, so were variants of HSF2BP. Consequently, gene dosage of RNF212 and HEI10 affects CO frequency through their activity in CO designation and maturation (Lake and Hawley, 2013; Reynolds et al., 2013). We found that both BRME1 and HSF2BP localization are unaffected in the loss-of-function mouse mutants of Rnf212 and Hei10 (Ccnb1ip1). This observation together with the proper co-localization of BRME1/HSF2BP with RPA allows us to map these proteins upstream in the recombination pathway.

It is worth noting that some of the genes affecting the recombination rate have also been described as ‘fertility genes’, such as SYCP3, HFM1 and HSF2BP (Geisinger and Benavente, 2017; Primary Ovarian Insufficiency Collaboration et al., 2014 and this work). Altogether, we propose that different variants of the same meiotic gene (alleles responsible for mild or strong phenotypes) can give rise to either an altered genome-wide recombination rate with no detrimental effect, or cause infertility when the decreased recombination rate falls below the lower limit of one COs per bivalent. In the present POI family, the S167L variant in HSF2BP seems to be below that limit. To our knowledge, HSF2BP is one of the very few human genes with variants known to affect both the genome-wide recombination rate in the human population and meiotic chromosome missegregation (fertility) through a reduction of the recombination rate (Halldorsson et al., 2019). Along similar lines, it is conceivable that variants with additive effects (Schimenti and Handel, 2018) can lead to a genome-wide reduction of the recombination rate and thus to aneuploidy and infertility. Specifically, variants in genes involved in meiotic recombination and SC constituents could be responsible for a large fraction of genetic infertilities. These variants should be under purifying selection and would be removed or substantially reduced from the population. However, this is not the case for genes with sexual phenotypic dimorphism (Gershoni and Pietrokovski, 2014) as is apparent for a wide number of meiotic genes (Cahoon and Libuda, 2019), including HSF2BP and BRME1, where individuals of one of the sexes are fertile carriers.

In summary, we describe for the first time a human family where POI co-segregates with a genetic variant in HSF2BP (S167L) in a Mendelian fashion. Humanized mice reveal that the HSF2BP variant is a hypomorphic allele that promotes the lower protein expression and/or stability of the HSF2BP/BRME1 complex and phenocopy in a milder manner the meiotic defects observed in mice lacking either HSF2BP or its direct interactor BRME1.

Materials and methods

Whole exome sequencing

Request a detailed protocol

Written informed consent was received from participants prior to inclusion in the study and the institutions involved. Genomic DNA was extracted from blood samples by standards protocols.

For individuals III-3 and III-10, library preparation, exome capture, sequencing and initial data processing were performed by Beckman Coulter Genomics (Danvers, USA). Exon capture was performed using the hsV5UTR kit target enrichment kit. Libraries were sequenced on an Illumina HiSEQ instrument as paired-end 100 bp reads. For individual III-2, library preparation, exome capture, sequencing and data processing were performed by IntegraGen SA (Evry, France) according to their in-house procedures. Target capture, enrichment and elution were performed according to manufacturer’s instructions and protocols (SureSelect Human All Exon Kits Version CRE, Agilent). The library was sequenced on an Illumina HiSEQ 2500 as paired-end 75 bp reads. Image analysis and base calling was performed using Illumina Real Time Analysis (RTA 1.18.64) with default parameters.

Bioinformatic analysis

Request a detailed protocol

For the three individuals, sequence reads were mapped onto the human genome build (hg38/GRCh38) using the Burrows-Wheeler Aligner (BWA) tool. Duplicated reads were removed using sambamba tools. Whole exome sequencing metrics are provided in Supplementary file 1a. Variant calling, allowing the identification of SNV (Single Nucleotide Variations) and small insertions/deletions (up to 20 bp) was performed via the Broad Institute GATK Haplotype Caller GVCF tool (3.7). Ensembl VEP (Variant Effect Predictor, release 87) program was used for initial variant annotation. This tool considers data available in dbSNP (dbSNP147), the 1000 Genomes Project (1000G_phase3), the Exome Variant Server (ESP6500SI-V2-SSA137), the Exome Aggregation Consortium (ExAC r3.0), and IntegraGen in-house databases. Additional annotation data was retrieved using dbNSFP (version 3.5, https://sites.google.com/site/jpopgen/dbNSFP) and Varsome (https://varsome.com/). Minor allele frequencies were manually verified on GnomAD (http://gnomad.broadinstitute.org), ISB Kaviar (http://db.systemsbiology.net/kaviar/), and Great Middle Eastern variant database GME Variome (http://igm.ucsd.edu/gme/).

Variant filtering was performed on the following criteria:

  • minimum depth at variant position of 10,

  • correct segregation in the family, on the basis of homozygosity by descent: variants should be homozygous in both affected sisters III-2 and III-3, and heterozygous or homozygous for Reference allele in the fertile sister III-10,

  • absence in unrelated in-house fertile controls,

  • Minor Allele Frequency (MAF) below 1% in global and in each population in the GnomAD database,

  • presence in the coding sequence (i.e not in UTRs, introns, intergenic,.)

  • high predicted functional impact on the protein. Impact was evaluated based on the predictors included in dbNSFP3.5 (Cahoon and Libuda, 2019; Carlosama et al., 2017; Cox and Mann, 2008) (considered as pathogenic when the majority of the predictors agreed).

The number of variants fulfilling those criteria is provided in Supplementary file 1b. Visual inspection of the variant was performed using the IGV viewer.

Sanger sequencing analysis

Request a detailed protocol

To confirm the presence and segregation of the variant, direct genomic Sanger DNA sequencing of HSF2BP was performed in the patients, the parents and non-affected siblings using specific primers: HSF2BP-ex6F: 5’-ctagaatcttctgtatcctgca-3’ and HSF2BP-ex6R2: 5’-ggtctggaagcaaacaggcaa-3’. The resulting chromatograms are shown in Figure 1—figure supplement 1.

Predictions of pathogenicity and sequence conservation

Request a detailed protocol

The S167L variant was predicted to be pathogenic or deleterious and highly conserved by 11 out of the 18 pathogenicity predictors available in dbNSFP 3.5 (Supplementary file 1c). Upon verification, it appears that the conflicting interpretation of this variant might stem from the single occurrence of a Leu at this position in zebrafish. As the change in zebrafish is the variant that we have in the human family, we checked all the available sequences (Ensembl Release 99, January 2020, removing the one-to-many relationships). Ser167 is very highly conserved in mammals, birds and reptiles and fish and is present in 208 of 212 orthologous sequences (Figure 1—figure supplement 2 and Figure 1—figure supplement 3).

Generation of CRISPR/Cas9-edited mice

Request a detailed protocol

For developing all the mutant mice models (Hsf2bp-/-, Hsf2bpS167L/S167L, Brme1Δ142-472/Δ Δ142-472, Brme1-/-, Spo11-/-, Rnf212-/- and Hei10-/-) the different crRNAs were predicted at https://eu.idtdna.com/site/order/ designtool/index/CRISPR_CUSTOM. The crRNAs, the tracrRNA and the ssODNs were produced by chemical synthesis at IDT (crRNAs and ssODNs sequences are listed in Supplementary files 1i-1j). For the Hsf2bpS167L we introduced a mutation in the mouse counterpart residue (p.Ser171Leu) of the POI mutation found in the clinical case (p.Ser167Leu). However, for the shake of simplicity, on this manuscript we refer to the mutant allele by the acronym of the human mutation (S167L). The ssODN contains the mutation on the corresponding position of the mouse sequence (c.512C > T, p.Ser171Leu, see character in red in Supplementary file 1j) and the PAM mutations avoiding amino acid changes (see characters in bold in the Supplementary file 1j). For the Spo11-/- mice generation, the ssODN contains the mutations in the active site (TACTAC >TTCTTC p.YY137-138FF, see Supplementary file 1j) and the PAM mutations (bold characters in Supplementary file 1j). In all cases the crRNA and tracrRNA were annealed to obtain the mature sgRNA. A mixture containing the sgRNAs, recombinant Cas9 protein (IDT) and the ssODN (30 ng/μl Cas9, 20 ng/μl of each annealed sgRNA and 10 ng/μl ssODN) were microinjected into B6/CBA F2 zygotes (hybrids between strains C57BL/6J and CBA/J) (Singh et al., 2015) at the Transgenic Facility of the University of Salamanca. Edited founders were identified by PCR amplification (Taq polymerase, NZYtech) with primers flanking the edited region (see Supplementary file 1k for primer sequences). The PCRs products were direct sequenced or subcloned into pBlueScript (Stratagene) followed by Sanger sequencing, selecting the founders carrying the desired alelles. The selected founders were crossed with wild-type mice to eliminate possible unwanted off-targets. Heterozygous mice were re-sequenced and crossed to give rise to edited homozygous. Genotyping was performed by analysis of the PCR products produced from genomic DNA extracted from tail biopsies. The primers and the expected amplicon sizes are listed in the Supplementary file 1k. Mouse mutants for Rec8, Six6os1 and Psma8 have been previously described (Bannister et al., 2004; Gómez-H et al., 2019; Gómez-H et al., 2016).

Ethics statement

All the experiments were approved by the Ethics Committee for Animal Experimentation of the University of Salamanca (USAL) and the Ethics committee of the Spanish Research Council (CSIC) under protocol #00–245. Accordingly, all the mouse protocols used in this work have been approved by the Animal Experimentation committees mentioned above. Specifically, mice were always housed in a temperature-controlled facility (specific pathogen free, spf) using individually ventilated cages, standard diet and a 12 hr light/dark cycle, according to EU law (63/2010/UE) and the Spanish royal law (53/2013) at the “Servicio de Experimentación Animal, SEA. In addition, animal suffering was always minimized, and we made every effort to improve animal welfare during the life of the animals. The mice analysed were between 2 and 4 months of age, except in those experiments where the age is indicated.

Histology

Request a detailed protocol

For histological analysis, after the necropsy of the mice their testes or ovaries were removed and fixed in Bouin´s fixative or formol 10%, respectively. They were processed into serial paraffin sections and stained with haematoxylin-eosin (ovaries) or Periodic acid–Schiff (PAS) and hematoxylin (testes). The samples were analysed using a microscope OLYMPUS BX51 and images were taken with a digital camera OLYMPUS DP70. For TUNEL assay, sections were deparaffinized and apoptotic cells were detected with the In Situ Cell Death Detection Kit (Roche) and counterstained with DAPI.

Follicle counting

Request a detailed protocol

The inner third of each ovary was serially sliced into 5 μm thick sections and follicles were counted every five sections and classified into four stages (primordial, primary, secondary and antral). Only those follicles in which the nucleus of the oocyte was clearly visible were counted.

Epididymal sperm count

Request a detailed protocol

The epididymides were removed, minced and incubated in 1,5 ml of KSOM for 30 min at 37°C to release sperm into the medium. The suspension was incubated for 10 min at 60°C and the total sperm count was quantified by using a hemacytometer.

Fertility assessment

Request a detailed protocol

Hsf2bp+/+ and Hsf2bpS167L/S167L males and females (8 weeks old) were mated with WT females and males, respectively, over the course of 4–12 months. six mice per genotype (seven mice for Hsf2bp S167L/S167L females) were crossed. The presence of copulatory plug was examined daily and the number of pups per litter was recorded.

Immunocytology and antibodies

Request a detailed protocol

Testes were detunicated and processed for spreading using a conventional ‘dry-down’ technique or for squashing (Gómez-H et al., 2016). Oocytes from fetal ovaries (E16.5, E17.5 and E19.5 embryos) were digested with collagenase, incubated in hypotonic buffer, disaggregated and fixed in paraformaldehyde. Rabbit polyclonal antibodies against HSF2BP and BRME1 were developed by ProteintechTM against a fusion protein of poly-His with full length HSF2BP or BRME1 (pUC57 vector) of mouse origin. Two antibodies (named R1 and R2) were generated against each protein (HSF2BP or BRME1) by immunization of two different host rabbits. Rabbit polyclonal antibody against DMC1 was developed by ProteintechTM against a DMC1 peptide (EESGFQDDEESLFQDIDLLQKHGINMADIKKLKSVGICTIKG). The primary antibodies used for immunofluorescence were rabbit αHSF2BP R2 (1:30, ProteintechTM), rabbit αBRME1 R2 (1:100, ProteintechTM), mouse αSYCP3 IgG sc-74569 (1:100, Santa Cruz), rabbit α-SYCP3 serum K921 (provided by Dr. José Luis Barbero, Centro de Investigaciones Biológicas, Spain), rabbit αSYCP1 IgG ab15090 (1:200, Abcam), rabbit anti-γH2AX (ser139) IgG #07–164 (1:500, Millipore), mouse αMLH1 51-1327GR (1:20, BD Biosciences), mouse αCDK2 (1:20; Santa Cruz Sc-6248) rabbit αRAD51 PC130 (1:50, Calbiochem), rabbit αRPA1 serum ¨Molly¨ (1:30, provided by Dr. Edyta Marcon, Medical Research University of Toronto, Canada), rat αRPA2 2208S (1:100, Cell Signaling), rabbit αDMC1 (1:500, ProteintechTM), rabbit αSPATA22 16989–1-AP (1:60, Proteintech), mouse αFlag IgG (1:100; F1804, Sigma-Aldrich).

Image acquisition and analysis

Request a detailed protocol

Slides were visualized at room temperature using a microscope (Axioplan 2; Carl Zeiss, Inc) with 63 × objectives with an aperture of 1.4 (Carl Zeiss, Inc). Images were taken with a digital camera (ORCA-ER; Hamamatsu) and processed with OPENLAB 4.0.3 and Photoshop (Adobe). The slides from the different genotypes used for comparative analyses were all freshly prepared in parallel and immunofluorescence were also carried out in parallel with the same freshly prepared cocktail of antibodies. Slides were not frozen to avoid differences in the background and antigen reactivity. All the images acquired were taken with constant exposure times for comparison. Quantification of foci and fluorescence intensity were performed using Image J software. Only the axis-associated foci were counted. For colocalization analysis, the same nucleus was quantified without rotation (experiment) and after rotating 90 degrees one of the images. This condition allows to determine non-specific colocalization (random). Background was subtracted for intensity quantification. Squashed preparations were visualized with a Delta vision microscopy station. Stimulated emission depletion (STED) microscopy (SP8, Leica) was used to generate the super-resolution images. Secondary antibodies for STED imaging were conjugated to Alexa 555 and 488 (Invitrogen) and the slides were mounted in Prolong Antifade Gold without DAPI.

Generation of plasmids

Request a detailed protocol

Full-length cDNAs encoding HSF2BP, BRME1 (full length and delta constructs), RPA1, BRCA2 (N, M and C constructs), PALB2, RAD51, and PSMA8 were RT-PCR amplified from murine testis RNA. The cDNAs were cloned into the EcoRV pcDNA3-2XFlag, SmaI pcDNA3-2XHA or SmaI pEGFP-C1 expression vectors under the CMV promoter. In frame cloning was verified by Sanger sequencing.

Y2H assay and screening

Y2H assay was performed using the Matchmaker Gold Yeast Two-Hybrid System (Clontech) according to the manufacturers' instructions. Mouse Hsf2bp cDNA was subcloned into the vector pGBKT7 and was used as bait to screen a mouse testis Mate and Plate cDNA library (Clontech Laboratories Inc). Positive clones were initially identified on double dropout SD (synthetic dropout)/–Leu /– Trp/X-α-Gal/Aureobasidin A plates before further selection on higher stringency quadruple dropout SD /–Ade /– His /– Leu /– Trp/X-α-Gal/Aureobasidin A plates. Pray plasmids were extracted from the candidate yeast clones and transformed into Escherichia coli. The plasmids from two independent bacteria colonies were independently grown, extracted and Sanger sequenced.

DNA pull-down assay

Request a detailed protocol

ssDNA/dsDNA pull down assays were performed using the protocol previously described by Souquet et al., 2013. A HPLC-purified biotinylated oligonucleotide was used for the DNA pull down assays: ss60-mer F: 5′-GAT CTG CACGACGCACACCGGACGTATCTGCTATCGCTCATGTCAACCGCTCAAGCTGC/3’BiotinTEG/ (IDT) and ss60-mer R (No biotinylated): 5′- GCAGCTTGAGCGGTTGACATGAGCGATAGCAGATACGTCCGGTGTGCGTCGTGCAGATC-3’. Double-stranded DNA annealing was carried out in 50 mM NaCl, 25 mM Tris-HCl, pH 7.5 buffer with complementary sequences at molecular equivalence by a denaturing step (5 min at 95°C) and a slow return to room temperature. DNA was immobilized onto Dynabeads M-280 Streptavidin (Dynal) following the manufacturer instructions (0.2 pmol per 1 µg of beads). Protein extracts were obtained from in vitro coupled transcription/translation systems (TNT T7 Coupled Reticulocyte Lysate Systems, Promega) according to manufacturer’s protocol. 15 µl of Flag-tagged proteins from TNT assays were pre-incubated on ice for 10 min in modified DBB (DBB: 50 mM Tris HCl, 100 mM NaCl, 10% (w/v) glycerol, Complete Protease inhibitor, 1 mM 2-mercaptoethanol pH 7,4 modified with 25 mM Tris-HCl, 1 mM EDTA plus 5 mg/ml BSA). After this preincubation 500 µg Dynabeads with immobilized ss- or ds-DNA were added and incubated for 1 hr at 4°C under agitation. Then the beads were washed three times (5 min rotating at RT) in 700 µl of modified DBB without BSA, before being washed once in 700 µl of rinsing buffer (modified DBB with 150 mM NaCl). Finally, DNA-binding proteins were eluted by resuspending the beads in 30 µl of Laemmli buffer boiling the samples for 5 min. The samples were analyzed by western blot.

Cell lines and transfections

Request a detailed protocol

HEK293T and U2OS cell lines were obtained from the ATCC and transfected with Jetpei (PolyPlus) according to the manufacturer protocol. Cell lines were tested for mycoplasma contamination using the Mycoplasma PCR ELISA (Sigma).

Immunoprecipitation and western blotting

Request a detailed protocol

HEK293T cells were transiently transfected and whole cell extracts were prepared in a 50 mM Tris-HCl pH 7,4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100 buffer supplemented with protease inhibitors. Those extracts were cleared with protein G Sepharose beads (GE Healthcare) for 1 hr. The corresponding antibodies were incubated with the extracts for 2 hr and immunocomplexes were isolated by adsorption to protein G-Sepharose beads o/n. After washing, the proteins were eluted from the beads with 2xSDS gel-loading buffer 100 mM Tris-Hcl (pH 7), 4% SDS, 0.2% bromophenol blue, 200 mM β-mercaptoethanol and 20% glycerol, and loaded onto reducing polyacrylamide SDS gels. The proteins were detected by western blotting with the indicated antibodies. Immunoprecipitations were performed using mouse αFlag IgG (5 µg; F1804, Sigma-Aldrich), mouse αGFP IgG (4 µg; CSB-MA000051M0m, Cusabio), ChromPure mouse IgG (5 µg/1 mg prot; 015-000-003). Primary antibodies used for western blotting were rabbit αFlag IgG (1:2000; F7425 Sigma-Aldrich), goat αGFP IgG (sc-5385, Santa Cruz) (1:3000), rabbit αMyc Tag IgG (1:3000; #06–549, Millipore), rabbit αHSF2BP R2 (1:2000, ProteintechTM), rabbit αBRME1 R1 (1:3000, ProteintechTM), rat αRPA2 (1:1000, Cell Signaling (Cat 2208S)). Secondary horseradish peroxidase-conjugated α-mouse (715-035-150, Jackson ImmunoResearch), α-rabbit (711-035-152, Jackson ImmunoResearch), α-goat (705-035-147, Jackson ImmunoResearch) or α-rat (712-035-150, Jackson ImmunoResearch) antibodies were used at 1:5000 dilution. Antibodies were detected by using Immobilon Western Chemiluminescent HRP Substrate from Millipore. Secondary DyLight conjugated α-mouse (DyLight 680, 35518 Thermo-Scientific) and α-rabbit (DyLight 800, 35571 Thermo-Scientific) were used at 1:10,000 dilution. Antiboides were detected using a LI-COR Oddysey fluorescent Imager.

Testis immunoprecipitation

Request a detailed protocol

Testis extracts were prepared in 50 mM Tris-HCl (pH8), 500 mM NaCl, 1 mM EDTA 1% Triton X100. 4 mg of protein were incubated with 10 µg of the specific antibody against the protein to be immunoprecipitated for 2 hr at 4°C rotating. Then 50 µl of sepharose beads (GE Healthcare) were added to the protein-Ab mixture and incubated overnight at 4°C with rotation. After that, the protein-bounded beads were washed four times with 500 µl of the extraction buffer by centrifugating 1 min at 10,000 rpm and 4°C. Finally, the co-immunoprecipitated proteins were eluted from the beads by resuspending the beads in 50 µl Laemmli buffer and boiling for 5 min. The samples were analyzed by western blot.

Testis immunoprecipitation coupled to mass spectrometry analysis

Request a detailed protocol

200 µg of antibodies R1 and R2 against BRME1 (two independent IPs) and IgG from rabbit (negative control) were crosslinked to 100 ul of sepharose beads slurry (GE Healthcare). Testis extracts were prepared in 50 mM Tris-HCl (pH8), 500 mM NaCl, 1 mM EDTA 1% Triton X100. 20 mg of protein extracts were incubated o/n with the sepharose beads. Protein-bound beads were packed into columns and washed in extracting buffer for three times. Proteins were eluted in 100 mM glycine pH3 and analysed by Lc-MS/MS shotgun in LTQ Velos Orbitrap at the Proteomics facility of Centro de Investigación del Cáncer (CSIC/University of Salamanca).

Mass spectrometry data analysis

Request a detailed protocol

Raw data were analysed using MaxQuant v 1.6.2.6 (Cox and Mann, 2008) against SwissProt Mouse database (UP000000589, Oct, 2019) and MaxQuant contaminants. All FDRs were of 1%. Variable modifications taken into account were oxidation of M and acetylation of the N-term, while fixed modifications included considered only carbamidomethylation of C. The maximum number of modifications allowed per peptide was of 5. Proteins were quantified using iBAQ (Schwanhäusser et al., 2011). Potential contaminants, reverse decoy sequences and proteins identified by site were removed. Proteins with less than two unique peptides in the R1 and R2 groups were not considered for ulterior analysis. Proteins with less than two unique peptides in the control group and more than two in both groups R1 and R2 were selected as high-confidence candidates (group R1 and R2 only). An additional group of putative candidates was selected for those proteins with two or more unique peptides in one of the R1 or R2 groups and no unique peptides in the control sample (groups R1 only and R2 only, respectively).

Statistics

In order to compare counts between genotypes, we used the Two-tailed Welch’s t-test (unequal variances t-test), which was appropriate as the count data were not highly skewed (i.e., were reasonably approximated by a normal distribution) and in most cases showed unequal variance. We applied a two-sided test in all the cases. Asterisks denote statistical significance: *p-value<0.05, **p-value<0.01, ***p-value<0.001 and ****p-value<0.0001.

Appendix 1

Key resources table

Appendix 1—key resources table
Reagent type
(species) or resource
DesignationSource or referenceIdentifiersAdditional information
Genetic reagent (M. musculus)Hsf2bp-/-This paperMaterials and methods section
Figure 2—figure supplement 1
Available from the authors upon request
Dr. Alberto M. Pendás
(amp@usal.es)
Genetic reagent (M. musculus)Hsf2bpS167L/S167LThis paperMaterials and methods section
Figure 2—figure supplement 1
Available from the authors upon request
Dr. Alberto M. Pendás
(amp@usal.es)
Genetic reagent (M. musculus)Brme1-/-This paperMaterials and methods section
Figure 8—figure supplement 1
Available from the authors upon request
Dr. Alberto M. Pendás
(amp@usal.es)
Genetic reagent (M. musculus)Brme1Δ142-472/Δ142-472This paperMaterials and methods section
Figure 7—figure supplement 1
Available from the authors upon request
Dr. Alberto M. Pendás
(amp@usal.es)
Genetic reagent (M. musculus)Rnf212-/-This paperMaterials and methods section
Figure 7—figure supplement 5
Available from the authors upon request
Dr. Alberto M. Pendás
(amp@usal.es)
Genetic reagent (M. musculus)Hei10-/-This paperMaterials and methods section
Figure 7—figure supplement 5
Available from the authors upon request
Dr. Alberto M. Pendás
(amp@usal.es)
Genetic reagent (M. musculus)Spo11-/-This paperMaterials and methods section
Figure 7—figure supplement 6
Available from the authors upon request
Dr. Alberto M. Pendás
(amp@usal.es)
Genetic reagent (M. musculus)Psma8-/-PMID:31437213
Genetic reagent (M. musculus)Six6os1-/-PMID:27796301
Genetic reagent (M. musculus)Rec8-/-PMID:15515002Dr. John C. Schimenti
(Cornell university)
Cell line (H. sapiens)U2OSATCCHTB-96
Cell line (H. sapiens)HEK293TATCCCRL-11268
Recombinant DNA reagentpEGFP-C1ClontechCatalog:
6084–1
Recombinant DNA reagentpcDNA3InvitrogenA-150228
Recombinant DNA reagentpcDNA3-2xFlagThis paper
Generated from pcDNA3
Materials and methods section
Figure 6—figure supplement 2
Available from the authors upon request
Dr. Alberto M. Pendás
(amp@usal.es)
Recombinant DNA reagentpEGFP-C1
HSF2BP
This paperMaterials and methods section
Figure 7
Available from the authors upon request
Dr. Alberto M. Pendás
(amp@usal.es)
Recombinant DNA reagentpcDNA3 2xFlag
HSF2BP
This paperMaterials and methods section
Figure 7
Available from the authors upon request
Dr. Alberto M. Pendás
(amp@usal.es)
Recombinant DNA reagentpcDNA3 2xFlag
HSF2BP-S167L
This paperMaterials and methods section
Figure 7
Available from the authors upon request
Dr. Alberto M. Pendás
(amp@usal.es)
Recombinant DNA reagentpcDNA3 2xHA HSF2BPThis paperMaterials and methods section
Figure 10—figure supplement 1
Available from the authors upon request
Dr. Alberto M. Pendás
(amp@usal.es)
Recombinant DNA reagentpEGFP-C1
BRME1
This paperMaterials and methods section
Figure 10—figure supplement 1
Available from the authors upon request
Dr. Alberto M. Pendás
(amp@usal.es)
Recombinant DNA reagentpcDNA3 2xFlag
BRME1
This paperMaterials and methods section
Figure 10—figure supplement 1
Available from the authors upon request
Dr. Alberto M. Pendás
(amp@usal.es)
Recombinant DNA reagentpcDNA3 2xFlag
BRME1Δ142–472
This paperMaterials and methods section
Figure 7
Available from the authors upon request
Dr. Alberto M. Pendás
(amp@usal.es)
Recombinant DNA reagentpEGFP-C1
HSF2BP
This paperMaterials and methods section
Figure 10
Available from the authors upon request
Dr. Alberto M. Pendás
(amp@usal.es)
Recombinant DNA reagentpEGFP-C1
HSF2BP-S167L
This paperMaterials and methods section
Figure 10
Available from the authors upon request
Dr. Alberto M. Pendás
(amp@usal.es)
Recombinant DNA reagentpEGFP-C1 BRCA2-CThis paperMaterials and methods section
Figure 6—figure supplement 2
Available from the authors upon request
Dr. Alberto M. Pendás
(amp@usal.es)
Recombinant DNA reagentpcDNA3 2xFlag BRCA2-CThis paperMaterials and methods section
Figure 10
Available from the authors upon request
Dr. Alberto M. Pendás
(amp@usal.es)
Recombinant DNA reagentpcDNA3 2xFlag BRCA2-MThis paperMaterials and methods section
Available from the authors upon request
Dr. Alberto M. Pendás
(amp@usal.es)
Recombinant DNA reagentpcDNA3 2xFlag BRCA2-NThis paperMaterials and methods section
Available from the authors upon request
Dr. Alberto M. Pendás
(amp@usal.es)
Recombinant DNA reagentpcDNA3 2xFlag RPA1This paperMaterials and methods section
Figure 10—figure supplement 1
Available from the authors upon request
Dr. Alberto M. Pendás
(amp@usal.es)
Recombinant DNA reagentpcDNA3 2xFlag RAD51This paperMaterials and methods section
Figure 10—figure supplement 1
Available from the authors upon request
Dr. Alberto M. Pendás
(amp@usal.es)
Recombinant DNA reagentpEGFP-C1 PALB2This paperMaterials and methods section
Figure 10—figure supplement 1
Available from the authors upon request
Dr. Alberto M. Pendás
(amp@usal.es)
Recombinant DNA reagentpcDNA 2xHA PALB2This paperMaterials and methods section
Figure 10—figure supplement 1
Available from the authors upon request
Dr. Alberto M. Pendás
(amp@usal.es)
AntibodyAnti-HSF2BP-R2
(rabbit polyclonal)
This paper
(ProteintechTM)
Materials and methods section
IF (1:30)
WB (1:2000)
Available from the authors upon request
Dr. Alberto M. Pendás
(amp@usal.es)
AntibodyAnti-BRME1-R1
(rabbit polyclonal)
This paper
(ProteintechTM)
Materials and methods section
WB (1:3000)
Available from the authors upon request
Dr. Alberto M. Pendás
(amp@usal.es)
AntibodyAnti-BRME1-R2
(rabbit polyclonal)
This paper
(ProteintechTM)
Materials and methods section
IF (1:100)
Available from the authors upon request
Dr. Alberto M. Pendás
(amp@usal.es)
AntibodyAnti-DMC1
(rabbit polyclonal)
This paper
(ProteintechTM)
Materials and methods section
IF (1:500)
Available from the authors upon request
Dr. Alberto M. Pendás
(amp@usal.es)
AntibodyAnti-SYCP3
(mouse monoclonal)
Santa cruzsc-74569IF (1:100)
AntibodyAnti-SYCP3
(rabbit polyclonal)
PMID:27796301K921Dr. José Luis Barbero
(Centro de Investigaciones Biologicas)
IF (1:60)
AntibodyAnti-SYCP1
(rabbit polyclonal)
Abcamab15090IF (1:200)
Antibodyanti-γH2AX (ser139)
(rabbit polyclonal)
Millipore#07–164IF (1:500)
AntibodyAnti-MLH1
(mouse monoclonal)
BD Biosciences51-1327GRIF (1:20)
AntibodyAnti-CDK2
(mouse monoclonal)
Santa Cruzsc-6248IF (1:20)
AntibodyαRAD51
(rabbit polyclonal)
CalbiochemPC130IF (1:50)
AntibodyαRPA1 serum
(rabbit polyclonal)
¨Molly¨Dr. Edyta Marcon (Medical Research University of Toronto)
IF (1:30)
AntibodyαRPA2
(rat monoclonal)
Cell Signalling2208SIF (1:100)
WB (1:1000)
AntibodyAnti-SPATA22
(rabbit polyclonal)
Proteintech Europe16989–1-APIF (1:60)
AntibodyAnti-Flag
(mouse monoclonal)
Sigma-AldrichF1804IF (1:100)
IP (5 µg)
AntibodyAnti-GFP
(mouse monoclonal)
CusabioCSB-MA000051M0mIP (5 µg)
AntibodyAnti-HA
(mouse monoclonal)
BioLegendMMS-101PIP (5 µg)
AntibodyMouse IgGs
(mouse polyclonal)
Jackson Immunoresearch015-000-003IP (5 µg)
AntibodyAnti-Flag
(rabbit polyclonal)
Sigma-AldrichF7425WB (1:2000)
AntibodyAnti-GFP
(goat polyclonal)
Santa Cruzsc-5385WB (1:3000)
AntibodyAnti-GFP
(rabbit polyclonal)
Life technologiesA-11122WB (1:3000)
AntibodyAnti-HA
(rabbit polyclonal)
Sigma-AldrichH6908WB (1:3000)
AntibodyGoat α-mouse Alexa555
(goat polyclonal)
ThermoFisherA-32727IF (1:200)
AntibodyGoat α-mouse Alexa488
(goat polyclonal)
ThermoFisherA-11001IF (1:200)
AntibodyDonkey α-rabbit
Alexa555
(donkey polyclonal)
ThermoFisherA-31572IF (1:200)
AntibodyGoat α-rabbit
Alexa488
(goat polyclonal)
ThermoFisherA-32731IF (1:200)
AntibodyGoat α-rabbit
Alexa488 Fab
(goat polyclonal)
Jackson Immunoresearch111-547-003IF (1:100)
AntibodyGoat α-mouse
AMCA
(goat polyclonal)
Jackson Immunoresearch115-155-146IF (1:100)
AntibodyDonkey α-rabbit
AMCA
(donkey polyclonal)
Jackson Immunoresearch711-155-152IF (1:100)
AntibodyGoat α-rat
Alexa488
(goat polyclonal)
ThermoFisherA-11006IF (1:200)
AntibodySecondary horseradish peroxidase-conjugated α-mouse
(donkey polyclonal)
Jackson Immunoresearch715-035-150WB (1:5000)
AntibodySecondary horseradish peroxidase-conjugated α-rabbit
(donkey polyclonal)
Jackson Immunoresearch711-035-152WB (1:5000)
AntibodySecondary horseradish peroxidase-conjugated α-goat
(donkey polyclonal)
Jackson Immunoresearch705-035-147WB (1:5000)
AntibodySecondary horseradish peroxidase-conjugated α-rat
(donkey polyclonal)
Jackson Immunoresearch712-035-150WB (1:5000)
AntibodySecondary DyLightTM 680 conjugated α-mouse
(goat polyclonal)
Thermo Scientific35518WB (1:10000)
AntibodySecondary DyLightTM 800 conjugated α-rabbit
(goat polyclonal)
Thermo Scientific35571WB (1:10000)
Sequence-based reagentsgRNA1
Hsf2bp
This paper
(IDT)
CRISPR-Cas9
crRNA
Materials and methods section
Supplementary file 1i
Figure 2—figure supplement 1
5’-TCACAAAACTCTCCATCGTC-3’
Sequence-based reagentsgRNA2
Hsf2bp
This paper
(IDT)
CRISPR-Cas9
crRNA
Materials and methods section
Supplementary file 1i
Figure 2—figure supplement 1
5’-ATTGGATGGGGATGTCAAGG-3’
Sequence-based reagentsgRNA3
Brme1
This paper
(IDT)
CRISPR-Cas9
crRNA
Materials and methods section
Supplementary file 1i
Figure 7—figure supplement 1
5’-AACCTCAGGGACTCTCTCTG-3’
Sequence-based reagentsgRNA4
Brme1
This paper
(IDT)
CRISPR-Cas9
crRNA
Materials and methods section
Supplementary file 1i
Figure 7—figure supplement 1
5’-GAAGTCTAGTTCCATTGCTG-3’
Sequence-based reagentsgRNA5
Spo11
This paper
(IDT)
CRISPR-Cas9
crRNA
Materials and methods section
Supplementary file 1i
Figure 7—figure supplement 6
5’-TATGTCTCTATGCAGATGCA-3’
Sequence-based reagentsgRNA6
Spo11
This paper
(IDT)
CRISPR-Cas9
crRNA
Materials and methods section
Supplementary file 1i
Figure 7—figure supplement 6
5’- ACACTGACAGCCAGCTCTTT-3’
Sequence-based reagentsgRNA7
Rnf212
This paper
(IDT)
CRISPR-Cas9
crRNA
Materials and methods section
Supplementary file 1i
Figure 7—figure supplement 5
5’- ACCCACGTGAGACTCGCGCG-3’
Sequence-based reagentsgRNA8
Rnf212
This paper
(IDT)
CRISPR-Cas9
crRNA
Materials and methods section
Supplementary file 1i
Figure 7—figure supplement 5
5’- CCTCAAAGGTCCGCGTATTC-3’
Sequence-based reagentsgRNA9
Hei10
This paper
(IDT)
CRISPR-Cas9
crRNA
Materials and methods section
Supplementary file 1i
Figure 7—figure supplement 5
5’- GAAAGGGTACTGTTGCAAGC-3’
Sequence-based reagentssODN
Hsf2bpS167L/S167L
This paper
(IDT)
Materials and methods section
Supplementary file 1j
Figure 2—figure supplement 1
5’CTTTGGAAAGATGTGACAGTTCTATCTTTTTTATCTTTCAGGACAAAGCATTGAAGTTTTTCAACATAACTGGACAGACGATGGAGAGTTTTGTGAAGTTATTGGATGGGGATGTCAAGGAAGTTGATTCTGATGAAAATCAATTTGTCTTTGCACTGGCTGGAATTGTAACAAGTAGGTAACTTTTCAGATACAGCGCT3’
Sequence-based reagentssODN
Brme1Δ142-472 /Δ142-472
This paper
(IDT)
Materials and methods section
Supplementary file 1j
Figure 7—figure supplement 1
5’CTTCAGAGTGCTTGCTTATTGAAGGCCAGGACTGAATCTTCTTTTTCCACAGGAAACAAGGCCAGAGCTGGGAGCCCTCAAAGCAGCCAGCCAGCCACAGGCAATGGAACTAGACTTCCTGCCTGACAGCCAGATACAGGATGCCCTGGATGCCACTAACATGGAGCAGGTAAGAGCTTTCTGTACTCAAATGTACACCC3’
Sequence-based reagentssODN
Spo11-/-
This paper
(IDT)
Materials and methods section
Supplementary file 1j
Figure 7—figure supplement 6
5’GTTTCCTGCGGTATGTGTTCTCTGCCGTGGTCTGTGTTTGTCACCGTCCAGGAGCAATGCTCATTCTGTGTTGAGCTTGCATCTGCATAGAGACATATTCTTCACTGACAGCCAGCTCTTTGGCAACCAGGCTGCGGTGGACAGCGCCATCGATGACATTTCCTGTATGCTGAAAGTGCCCAGGAGGAGTCTGCACGTGG-3’
Sequence-based reagentHSF2BP-F1This paperPCR primerMaterials and methods section
Supplementary file 1j
Figure 2—figure supplement 1
5’-TTCTTTGGAAAGATGTGACAGTTC-3’
Sequence-based reagentHSF2BP-R1This paperPCR primerMaterials and methods section
Supplementary file 1j
Figure 2—figure supplement 1
5’-ACCTGGGTTTCCTTTAGATCAGTTA-3’
Sequence-based reagentBRME1- F2This paperPCR primerMaterials and methods section
Supplementary file 1j
Figure 7—figure supplement 1
5’-GAAAGTTCTTCAGAGTGCTTGCT-3’
Sequence-based reagentBRME1- R2This paperPCR primerMaterials and methods section
Supplementary file 1j
Figure 7—figure supplement 1
5’-AGCCCTATCTTGTCACCTAAAG-3’
Sequence-based reagentBRME1- F3This paperPCR primerMaterials and methods section
Supplementary file 1j
Figure 7—figure supplement 1
5’-CCCAGCAGATGCCTCTCTTAT-3’
Sequence-based reagentBRME1- R3This paperPCR primerMaterials and methods section
Supplementary file 1j
Figure 7—figure supplement 1
5’-CTCAGCAGAGTTCCAATGCAG-3’
Sequence-based reagentSPO11-F4This paperPCR primerMaterials and methods section
Supplementary file 1j
Figure 7—figure supplement 6
5’- AGAGCCCCCAGTGCTCTTAAC-3’
Sequence-based reagentSPO11-R4This paperPCR primerMaterials and methods section
Supplementary file 1j
Figure 7—figure supplement 6
5’- GGCAGACCCCTCTACCTCTGT-3’
Sequence-based reagentRNF212-F5This paperPCR primerMaterials and methods section
Supplementary file 1j
Figure 7—figure supplement 5
5’- TTTCTTTGCCTCCGTACTTTTGG-3’
Sequence-based reagentRNF212-R5This paperPCR primerMaterials and methods section
Supplementary file 1j
Figure 7—figure supplement 5
5’- CCCAGGCTTTACTTCAACAACAA −3’
Sequence-based reagentHEI10-F6This paperPCR primerMaterials and methods section
Supplementary file 1j
Figure 7—figure supplement 5
5’- CTGCCTGTTCTCACATCTTC-3’
Sequence-based reagentHEI10-R6This paperPCR primerMaterials and methods section
Supplementary file 1j
Figure 7—figure supplement 5
5’- AGCTTTCCAGAAAGGGTACTG −3’
Sequence-based reagentss60-mer FPMID:24068956DNA Binding assay primerMaterials and methods section
Figure 7—figure supplement 4
5′-GAT CTG CACGACGCACACCGGACGTATCTGCTATCGCTCATGTCAACCGCTCAAGCTGC/3’BiotinTEG/
Sequence-based reagentss60-mer RPMID:24068956DNA Binding assay primerMaterials and methods section
Figure 7—figure supplement 4
5′- GCAGCTTGAGCGGTTGACATGAGCGATAGCAGATACGTCCGGTGTGCGTCGTGCAGATC-3’
Sequence-based reagentHSF2BP-EX6FThis PaperSanger sequencing primerMaterial and methods section
Figure 1—figure supplement 1
5’-CTAGAATCTTCTGTATCCTGCA-3’
Sequence-based reagentHSF2BP-EX6R2This PaperSanger sequencing primerMaterial and methods section
Figure 1—figure supplement 1
5’-GGTCTGGAAGCAAACAGGCAA-3’
Commercial assay or kitTNT T7 Coupled Reticulocyte Lysate SystemsPromegaL4610Figure 7—figure supplement 4
Commercial assay or kitIn Situ Cell Death Detection KitRoche11684795910Figure 2
Commercial assay or kitMatchmaker Gold Yeast Two-Hybrid SystemClontech630489Materials and methods section
Commercial assay or kitMouse testis Mate and Plate cDNA libraryClontech638852Materials and methods section
Commercial assay or kitJetpeiPolyPlus101–40NMaterials and methods section
Commercial assay or kitGammaBind G SepharoseGE Healthcare17-0885-02Materials and methods section
Commercial assay or kitDynabeads M-280 StreptavidinThermo Fisher11205DMaterials and methods section
Figure 7—figure supplement 4
Chemical compound, drugMG132Sigma-Aldrich
PMDI:28059716
M8699Figure 10
OtherDAPI stainInvitrogenD1306Materials and methods section
OtherVectashield Mounting MediumVector LaboratoriesH1000Materials and methods section
OtherProLong Gold antifade reagentInvitrogenP10144Materials and methods section

References

  1. 1
  2. 2
  3. 3
  4. 4
  5. 5
  6. 6
  7. 7
    Balancing Pathways in DNA Double Strand Break Repair
    1. I Brandsma
    (2006)
    Erasmus Rotterdam University.
  8. 8
  9. 9
  10. 10
  11. 11
  12. 12
  13. 13
  14. 14
  15. 15
  16. 16
  17. 17
  18. 18
  19. 19
  20. 20
  21. 21
  22. 22
  23. 23
  24. 24
  25. 25
  26. 26
  27. 27
  28. 28
  29. 29
  30. 30
  31. 31
  32. 32
  33. 33
  34. 34
  35. 35
  36. 36
  37. 37
  38. 38
  39. 39
  40. 40
  41. 41
  42. 42
  43. 43
  44. 44
  45. 45
  46. 46
  47. 47
  48. 48
  49. 49
  50. 50
  51. 51
  52. 52
  53. 53
  54. 54
  55. 55
  56. 56
  57. 57
  58. 58
  59. 59
  60. 60
  61. 61
  62. 62
  63. 63
  64. 64
  65. 65

Decision letter

  1. Bernard de Massy
    Reviewing Editor; CNRS UM, France
  2. Kevin Struhl
    Senior Editor; Harvard Medical School, United States
  3. Bernard de Massy
    Reviewer; CNRS UM, France
  4. Maria Jasin
    Reviewer; Memorial Sloan-Kettering Cancer Center, United States

In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.

Thank you for submitting your article "A missense in HSF2BP causing Primary Ovarian Insufficiency affects meiotic recombination by its novel interactor MIDAP" for consideration by eLife. Your article has been reviewed by three peer reviewers, including Bernard de Massy as the Reviewing Editor and Reviewer #1, and the evaluation has been overseen by Kevin Struhl as the Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Maria Jasin (Reviewer #2).

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

As the editors have judged that your manuscript is of interest, but as described below that additional experiments are required before it is published, we would like to draw your attention to changes in our revision policy that we have made in response to COVID-19 (https://elifesciences.org/articles/57162). First, because many researchers have temporarily lost access to the labs, we will give authors as much time as they need to submit revised manuscripts. We are also offering, if you choose, to post the manuscript to bioRxiv (if it is not already there) along with this decision letter and a formal designation that the manuscript is "in revision at eLife". Please let us know if you would like to pursue this option. (If your work is more suitable for medRxiv, you will need to post the preprint yourself, as the mechanisms for us to do so are still in development.)

Summary:

This paper presents a primary ovarian insufficiency (POI) case study, proband and affected family members with a unique genetic variant in HSF2BP. To examine the functional consequences of this variant, they generated a mouse mutant with the equivalent allele (S167L). Despite ostensibly normal numbers of follicles, Hsf2bp- S167L female mice showed reduced fertility in females. The authors also identify an interaction partner of HSF2BP named MIDAP and provide evidence that these two proteins function interdependently to facilitate BRCA2-mediated assembly of RAD51/DMC1 strand-exchange complexes.

Essential revisions:

Overall, while there are a lot of data in this paper, the quality of some experiments is questionable and lack rigorous quantification. The text is often inaccurate, sometimes not correct and requires major clarification and improvement.

Below are mentioned essential points and important point to revise, that include new data, formatting figures, text modification and reorganization.

1) One essential issue is to validate the conclusion that the case of human POI is caused by a variant of HSF2BP. Thus, the mouse mutant Hsf2bp167L requires additional analysis.

2) Another major problem is order and clarity of data presentation.

a) When presenting the phenotype of the Hsf2bp point mutant, the authors should clarify first the already published phenotype of the null mutant. They can add the data of their own null mutant as comparison.

b) Order of data:

Males and female data are not presented in a consistent way for the analysis of Hsf2bp mutant (Figure 3, Figure 4 and Figure 5): Best would be to present male and female in parallel: Figure 3 (RPA), Figure 4 (DMC1, RAD51), Figure 5 (Mlh1). RAD51 data for female is required.

MIDAP localization in Hsf2bp mutants should be presented along with its colocalization with HSF2BP in wt.

Analysis of SPATA22 should be moved to main figures.

3) Removing poorly informative data

The in vitro assay fails to detect DNA binding activity of MIDAP or HSF2BP. This negative result obtained with non-purified proteins of undetermined concentration and molar ratio with the substrate do not allow to conclude on the DNA binding property of those proteins.

Only part of the co-transfection analysis (Figure 10) should be maintained (see details below).

4) The localization of HSF2BP/MIDAP at DSB repair foci is not shown in this study. This should be clarified either by referring to the previous study or by doing colocalization analysis (with RPA or DMC1 or RAD51).

5) Clarifying interpretations.

Many issues in the text are reported below.

Importantly the protein levels of HSF2BP and MIDAP is the mutants (Midap and Hsfp2bp) should be tested (in extracts from juvenile to avid confounding effect of cellular composition). Any coIP and western blot analysis from mouse testis to validate the interactions? and also the one with BRCA2? This would be expected given the Mass/spec data.

Figure 1

Fertility and rigorous follicle (at different stages) counting for Hsf2bp-S167L mice at different ages. One prediction is that oocyte reserve should decreased at least at advanced ages.

Mutants with decreased ovarian reserves generally activate normal numbers of follicles and thus can look normal, but pools will deplete faster. Oocyte counts using immunohistochemistry of ovaries from mice of different ages with an appropriate marker (e.g. MVH) is required.

1(D) – please provide quantification of testis weights corrected for animal body weight (mean {plus minus} standard deviation).

1(F) – Epididymis histology for Hsf2bp-S167L requires quantification. An easier approach would be to present sperm counts using standard methods. Scale bars in Figure 1B and 1C should be the same size to facilitate comparison. Also, it is difficult to see follicles, especially in (B). Immunostaining should be performed to identify follicles.

1(H). Spreads are not appropriate to quantify apoptotic cells. How was apoptosis monitored? The legend is incorrect: "(H).…spread preparations from Hsf2bp-/- and Hsf2bpS167L/S167L mice." It should be +/+ and S167L/S167L.

Figure 1 legend is much too long.

Figure 2

2 (A) – HSF2BP staining intensity -- were graphs showing HSF2BP staining intensity normalized against Sycp3 intensity? Were exposure times constant between images and samples? Please provide more details regarding image acquisition. This comment applies to other figures as well.

There is no information about image analysis in the Materials and methods section? How were foci counted? Was background subtracted? Which software? Which parameters?

2 (A,B). Which test was used, meaning of stars?

2 (C). A non-specific band of unknown origin cannot be used as loading control. Please add a proper loading control. The extracts should be from juvenile to interpret the result since wt and mutant do not have the same testicular cell composition.

What is the mouse background for the -/- and S167L/S167L mice? Are they the same? Differences in strain background (especially mixed versus inbred) can affect the penetrance of meiotic mutants.

Figure 3

H2AX plots Y axis: add γ

Figure 4

(A) – right hand side graph is missing a y-axis label. Labels should be more explicit, e.g. "number of DMC1 foci".

(B) In the right graph there is extraneous text ("S167L") under the data points.

Figure 5

(a) The evidence for XY univalent is weak. One may indeed consider the long univalent axis as X, but the smaller one could be any chromosome. This is only a deduction (On Figure 1G, impossible to tell). This observation needs further analysis: is the recombination defect (CO defect and univalent) mostly impacting the X and Y? Please, analyze the distributions of MLH1 foci, specifically the fraction of pachytene SCs that lack MLH1 foci.

(B,C) – Please comment on the observation that RPA counts are similar between control and mutant, while DMC1 counts are significantly lower. RAD51 counts should be analyzed in S167L females.

SPATA22 which appears to give a strong phenotype in MIDAP mutant should be tested in male and female Hsf2bp mutants.

Identification and analysis of MIDAP

The acronym MIDAP is not the best choice: It's unclear how MIDAP is an acronym for "meiotic double- stranded break BRCA2/HSF2BP complex associated protein". Where is the "I" from, and how is BRCA2/HSF2BP part of the acronym? What is the expression profile of MIDAP? And if the gene is not meiotic-specific it should not be labeled as such. Moreover, it's not clear that its major function is through BRCA2. Please indicate that C19ORF57 is the name of the human not the mouse gene (thus in the yeast two hybrid screen the gene identified was not C19ORF57; was the cDNA overlapping with the domain identified as interacting by CoIP?).

One learns very little from the study of the MIDAP internal deletion. Thus, this could be removed or added as supplementary information. In addition, this mutant allele does not show that the interaction domain is sufficient as the mutant protein is much larger than the interaction domain.

Midap∆/∆: we would suggest adding a number such as ∆6. Otherwise, it can be easily thought that this allele is a full deletion.

Figure 6:

(A): Add MW markers (and check this for all western blots)

(C) – IP with Δ475-600 – please provide evidence that IP worked – western blot for protein used for setting up IP reaction must be also provided in all cases. Which fraction of input, of IP was loaded? (these comments apply to all CoIPs).

(D) – The authors cannot conclude on a perfect colocalization without quantification. Please quantify. Squares indicating areas being magnified are not being reproduced properly. Also, these areas are not magnified very much- please make them larger! The insets are shifted, this should be mentioned.

Figure 7. Depletion of follicle pool in Midap-/- females should be quantified.

Males: provide evidence for the stage IV arrest; in the text explain what is stage IV for non specialist. Asterisks are barely visible, change color.

(E): 5 nuclei are shown. The four upper nuclei show various levels of synapsis defects. It is not clear what information is provided with the nucleus in the lower part which does not seem to be a pachytene but more likely a diplotene nucleus. Unless a specific conclusion is reached from this nucleus, it could be removed.

Figure 8:

(A) – Absence of HSF2BP staining in the Midap-/- mutant could be due to protein destabilization. See comment above; legend, Quantification is HSF2BP foci not MIDAP.

Figure 9. The absence of MIDAP foci should also be quantified in the Hsf2bp null mutant even if the number of foci is close to 0.

Figure 10:

(A) – and in general and for all western blots, the labeling of IPs is confusing and

ambiguous. It is unclear what the arrows are supposed to highlight. Best is to indicate which antibody was used to probe the blots, and to add arrows to identify proteins when required.

The comparison of Hsf2bp wt and S167L does not make sense in such overexpression system since the property of S167L reported above was a potential lower stability and/or level. Transfection with Hsf2bpS167L should be removed.

Overall, the information gained is the CoIP between the three proteins, the nuclear localization and stabilization of Hsf2Bp in the presence of MIDAP. Note that there is no evidence for a multimeric complex from this study. This would require performing sequential IP.

The one and half page paragraph should be reduced to half a page, which would improve the quality of the paper.

The legend should be reduced.

Figure 1: Please include the genotypes for the sequenced individuals. From the text, it appears as if the brother is heterozygous, so there does not appear to be a male homozygote to comment on human male fertility. This should be clearly stated early in the manuscript (see comment below).

Figure 2—figure supplement 1(A) legend: "Thin (non-coding) and thick (coding sequences) lines under exons represent the expected transcript derived from wild-type and Hsf2bp edited allele." There are no such lines in panel (A).

Figure 7—figure supplement 6 – What stage are these WT spermatocytes and approximate stage-matched mutants? Late zygonema?

Figure 8—figure supplement 1C. Please show the whole membrane. Any truncated protein detected? Where are the epitopes of the antibody?

Interpretation and discussion of the Hsf2bpS167L mutant

Subsection “Mice with the HSF2BP S167L variant show a partial reduction of fertility” "male and female mice were able to reproduce but females showed a significant reduction in the number and size of litters (Figure 1A) whilst males only showed a slight non-significant reduction in fertility (Figure 1A).This is in agreement with the fertility defects observed in Hsf2bp-/- mice (Figure 1C,E-F and (Brandsma et al., 2019; Zhang et al.,2019)), and suggests that the S167L variant might impact more specifically female fertility."

There are several problems with this statement:

a) This is not in agreement with the two published papers, but rather the opposite, since the published papers describe a more pronounced defect in males. Thus, this text needs to be changed.

b) However, importantly also, the authors appear to have a more pronounced phenotype in their female knockouts compared to the published reports. This needs to be discussed, included how the knockout alleles vary for the 3 knockouts that have now been generated.

c) Males with S167L have more variability in litter frequency and size, and so with the small numbers it is difficult to draw a conclusion about fertility. Moreover, testis weights and histology of tubules, which show a clear male defect.

Related to this in Subsection “Mice with the HSF2BP S167L variant show a partial reduction of fertility”: "These results suggest that mice bearing the POI-causing variant only partially phenocopy the human disease and fits well with the exacerbated sexual phenotypic dimorphism observed in the Hsf2bp-/- mice which might be less pronounced or absent in humans."

As above, no conclusion can be made about human male fertility. The lack of follicle counts/breeding over time in females makes it difficult to conclude about POI in mice. Moreover, what sexual dimorphism in -/- mice is this referring to? Both males and females are completely devoid of gametes in the HSF2BP-null.

Subsection “Mice with the HSF2BP S167L variant show a partial reduction of fertility”: "Overall, male and female Hsf2bpS167L/S167L mice show similar molecular alterations although with different severity".

There are obvious differences in molecular alterations between males and females:

- There is a dramatic increase in RPA foci in males during pachynema while no difference in RPA foci in females during pachynema.

- DMC1 is virtually unchanged in males (perhaps slight reduction in EZ), but greatly reduced in females.

- MLH1 is reduced 20-30% in males, but only ~5% (and not significant) in females.

- γ-H2AX is greatly increased during pachynema in males, but these data do not exist for females.

- RAD51 is decreased through zygonema to early pachynema with the biggest decrease seen in late zygonema, but these data don't exist for females.

- The only similarity is with DMC1, but even this is misleading as the defect in males is quite mild, but quite severe in females.

Supplementary file 1G, Supplementary file 1H

What are the two antibodies (Ab1 and Ab2) used for mass spec? What is the control sample (IP with IgG?). The Materials and methods section indicates the IP was performed with two antibodies R1 and R2 (against HSF2BP and MIDAP)? This is very confusing.

Please can you specify how many peptides of MIDAP identified in this screen.

With the large number of figures and different mutants examined, a summary table should be provided.

Comments on the Discussion section

The discussion is unfocused with no clear link back to the POI of the human family. Also, the data are generally over-interpreted into specific molecular mechanisms. This should be toned down and the authors should clarify when they are speculating.

Where is the evidence for a supramolecular complex? In the absence of validation this should also be removed from the abstract ("the higher order macromolecular structure").

Where is the evidence for direct interaction between HSF2BP and BRCA2 and MIDAP ?

Where is the evidence that the supramolecular (?) complex participate to the binding of RPA?

The known roles of DSS1 should be introduced noting that meiotic functions are not characterized.

"strongly suggesting a parallelism in the early recombination pathway where HSF2BP/MIDAP work" – this is unclear. Please rephrase.

"In general, meiotic recombination mutants seem to proceed further in female than in male meiosis perhaps because of the presence of less stringent checkpoints (Hunt and Hassold, 2002)."

But also, recombination mutants often show more severity in males due to X-Y asynapsis.

"importantly also of HSF2BP" – please rephrase.

https://doi.org/10.7554/eLife.56996.sa1

Author response

Essential revisions:

Overall, while there are a lot of data in this paper, the quality of some experiments is questionable and lack rigorous quantification. The text is often inaccurate, sometimes not correct and requires major clarification and improvement.

Below are mentioned essential points and important point to revise, that include new data, formatting figures, text modification and reorganization.

1) One essential issue is to validate the conclusion that the case of human POI is caused by a variant of HSF2BP. Thus, the mouse mutant Hsf2bp167L requires additional analysis.

We thank the referees for the comment. We have carried out additional analyses of the human mutation in both male and female mice including a comparative sperm count, TUNEL analysis of histological sections of mutant testis, follicle counts of mutant ovaries (5 dpp, 6 weeks, and 13 months), analysis of the distribution of COs along the autosomes and XY bivalents, analysis of the COs in a higher number of female mice and cells and a supplementary analysis of γH2AX as well as RAD51 and SPATA22 foci in mutant oocytes. Taking together, our new analysis corroborates and validates that the introduction of the point mutation in the Hsf2bp mouse gene found in the POI family leads to meiotic defects in the mouse germline.

2) Another major problem is order and clarity of data presentation.

a) When presenting the phenotype of the Hsf2bp point mutant, the authors should clarify first the already published phenotype of the null mutant. They can add the data of their own null mutant as comparison.

We have included in the revised version of the MS the phenotype of the published null mutant at the beginning of the Results as suggested by the reviewers. We have also incorporated the data of our null mutant for a straight comparison. The added paragraph is:

“During the course of this work, two independent groups showed that HSF2BP is essential for meiotic recombination through its ability to interact with the armadillo repeats of BRCA2 (Zhang et al., 2019; Brandsma et al., 2019). Both groups showed that genetic disruption of Hsf2bp in mouse leads to the accumulation in the chromosomes axes of DNA repair proteins such as γH2AX (ATR-dependent phosphorylation of H2AX marks DSBs) and the single stranded-DNA binding protein RPA, a strong reduction of the recombinases DMC1 and RAD51 at the RNs and a lack of COs as labelled by MLH1 (Baker et al., 1996). The end result is male sterility (Brandsma et al., 2019; Zhang et al., 2019). However, loss of HSF2BP in female mice showed a milder meiotic phenotype (Zhang et al., 2019 and our own data, see below) and a weak albeit non statistical significant reduction of fertility (Brandsma et al., 2019) despite all of the mutants are nulls though in different genetic backgrounds.”

b) Order of data:

Males and female data are not presented in a consistent way for the analysis of Hsf2bp mutant (Figure 3, Figure 4 and Figure 5): Best would be to present male and female in parallel: Figure 3 (RPA), Figure 4 (DMC1, RAD51), Figure 5 (Mlh1). RAD51 data for female is required.

MIDAP localization in Hsf2bp mutants should be presented along with its colocalization with HSF2BP in wt.

We thank the referees for the comment. We have followed their advice and we have reorganized the data accordingly to improve the manuscript. Thus, we have presented in this revised version male and female in parallel corresponding to the reorganized Figure 4, Figure 5 and Figure 6.

We have also incorporated new data involving the analysis of RAD51 foci in Hsf2bp-/- and Hsf2bpS167L/S167L females and their corresponding quantification and comparison with the wild type (Figure 5D and Figure 5—figure supplement 1D).

Following the reviewers´ comments, we have placed MIDAP localization in Hsf2bp mutants in Figure 7 (7F-G and extended panel in Figure 7—figure supplement 4A) along with the colocalization of HSF2BP and MIDAP (Figure 7D-E).

Analysis of SPATA22 should be moved to main figures.

Following this suggestion, we have moved the SPATA22 analysis to the main figures for Hsf2bp mutants (Figure 6A) and also moved the SPATA22 analysis in Midap mutants to the main Figure 9E-F.

3) Removing poorly informative data

The in vitro assay fails to detect DNA binding activity of MIDAP or HSF2BP. This negative result obtained with non-purified proteins of undetermined concentration and molar ratio with the substrate do not allow to conclude on the DNA binding property of those proteins.

We understand this comment. However, our experiments have been performed with purified protein extracts obtained from in vitro transcription and translation (TNT) and the protein concentration on each condition was estimated by loading the same amount of protein (estimated from the western blot signal) to avoid substantial differences on the molar ratio with the substrate. We have included the well-known DNA-binding activity of RPA as a positive control. This assay based on TNT produced proteins has been validated previously in several publications (Souquet et al., 2013 ; Loregian et al., 2004). We thus consider that these results point towards HSF2BP and MIDAP lack DNA binding activity.

Only part of the co-transfection analysis (Fig10) should be maintained (see details below)

We have carried out the modification (see below).

4) The localization of HSF2BP/MIDAP at DSB repair foci is not shown in this study. This should be clarified either by referring to the previous study or by doing colocalization analysis (with RPA or DMC1 or RAD51).

We agree with this commentary. We have carried out a new co-localization experiment by IF analysis of HSF2BP and MIDAP with RPA and DMC1 as well as the corresponding quantification. The new results have been included in a new Figure 7—figure supplement 3A-B and Supplementary files 1D and 1E.

5) Clarifying interpretations.

Many issues in the text are reported below.

Importantly the protein levels of HSF2BP and MIDAP is the mutants (Midap and Hsfp2bp) should be tested (in extracts from juvenile to avid confounding effect of cellular composition). Any coIP and western blot analysis from mouse testis to validate the interactions? and also the one with BRCA2? This would be expected given the Mass/spec data.

Following this criticism, we have analysed the protein levels of HSF2BP and MIDAP in juvenile mutants as suggested by the editor/referees and replaced the former WBs by the new ones (Figure 3C, Figure 7H and Figure 8H). The new results have been included in the MS: “Western blot analysis of WT, Hsf2bpS167L/S167L and Hsf2bp-/- in whole testis extracts from 13 days post-partum (dpp) animals (Figure 3C) revealed that the reduced labelling observed by immunofluorescence correlated with a reduced protein expression level, suggesting that the mutation leads to a reduced expression and/or stability.”

We have analysed the protein levels of MIDAP in the Hsf2bp mutants (S167L variant and knock-out) revealing a drastic reduction of MIDAP expression in Hsf2bpS167L/S167L and Hsf2bp-/-spermatocytes. The new results have been included in the MS: “Western blot analysis also revealed a drastic reduction of MIDAP expression in Hsf2bpS167L/S167L and Hsf2bp-/-spermatocytes, suggesting an HSF2BP-dependent stabilization of MIDAP (Figure 7H).”

We also have analysed the protein level of HSF2BP in MIDAP mutants by Western blot analysis of 13 dpp testis extracts. The new results have been included in the manuscript: “Western blot analysis of HSF2BP in 13 dpp testis extracts from Midap-/- mice showed a strong reduction in comparison with the WT control (Figure 8H), suggesting again that MIDAP is necessary for HSF2BP protein stabilization.”

Additionally, we have carried out Co-IP experiments from mouse testis between HSF2BP and MIDAP to demonstrate the interaction in vivo. The results show positive co-IP and have been included in the MS: “We further validated this interaction in vivo by co-immunoprecipitation (co-IP) of both proteins from mouse whole testis extracts (Figure 7B).”

In the case of BRCA2, we would like to point out that we have not been able to detect endogenous BRCA2 with any antibody neither by immunofluorescence (this is a universal problem) nor by WB. Thus, in our hands, such an experiment is not possible.

Figure 1

Fertility and rigorous follicle (at different stages) counting for Hsf2bp-S167L mice at different ages. One prediction is that oocyte reserve should decreased at least at advanced ages.

Mutants with decreased ovarian reserves generally activate normal numbers of follicles and thus can look normal, but pools will deplete faster. Oocyte counts using immunohistochemistry of ovaries from mice of different ages with an appropriate marker (e.g. MVH) is required.

We thank the referees for the comment and accordingly we have included oocyte counts at 5 dpp, 6 weeks and 13 months from Hsf2bpS167L/S167L mice. The new results have been included in the MS. “Histological analysis of Hsf2bpS167L/S167L ovaries revealed no apparent differences in the number of follicles in comparison to wild-type (WT) animals (Figure 2B-C and Figure 2—figure supplement 2A), in contrast with the drastic reduction of the follicle pool in Hsf2bp-/- ovaries (Figure 2B-C).” However, we consider that staining with a marker is not necessary since follicles and oocytes can be clearly identified just with a Hematoxylin-eosin staining of the histological sections as shown in Figure 2B and Figure 8A.

Alternative to the hypothesis of the erosion of the follicle pool, subfertility can be also caused by a defect in the quality of the secondary oocytes (i.e. absence of at least one COs per bivalent). Indeed, we have increased the number of cells and female mice analysed for interstitial CDK2 foci (as a measure of COs) and we have found a statistically significant reduction in the total number of COs and also an increased number of cells showing bivalents without CO. The new results have been included in the manuscript: “In agreement with the lower presence of recombinases, the number of COs (measured as interstitial CDK2 foci) was also reduced in Hsf2bpS167L/S167Loocytes, and a stronger reduction was observed in Hsf2bp-/-oocytes (Figure 6C and Figure 6—figure supplement 1D)”. These differences could explain the subfertility of our Hsf2bpS167L/S167L females. We used interstitial CDK2 staining instead of MLH1 due to our anti-MLH1 antibody was finished and we could not get a new one due to the COVID-19 lockdown, so we decided to do this analysis using CDK2 for female analysis which is well accepted to be equivalent.

1(D) – please provide quantification of testis weights corrected for animal body weight (mean {plus minus} standard deviation).

We have modified these data following the advice of the referees and we have included a new graph showing the new data (Figure 2—figure supplement 2B). The sentences have been modified as follows:

“Testes from Hsf2bpS167L/S167L mice displayed a reduced size (21% reduction compared to WT mice; testis/body weight ratio: S167L 0,26% ± 0,07 (n=12) vs 0,33% ± 0,05 for WT controls (n=14), **p<0,01, Figure 2D and Figure 2—figure supplement 2B) and this reduction was stronger in Hsf2bp-/- testes (70% reduction compared to WT, testis/body weight ratio: Hsf2bp-/- 0,10% ± 0,005 (n=6) vs 0,33% ± 0,05 for WT controls (n=14) **** p<0,001, Figure 2D and Figure 2—figure supplement 2B).”

“Male Midap-/- mice were infertile, the average size of their testes was severely reduced (76% reduction compared to WT; testis weight/body weight ratio: Midap-/- 0,08% ± 0,004 (n=6) vs 0,33% ± 0,05 for WT controls (n=14), ****p<0.0001, Figure 8B and Figure 2—figure supplement 2B),”

1(F) – Epididymis histology for Hsf2bp-S167L requires quantification. An easier approach would be to present sperm counts using standard methods.

We have performed sperm counts of our S167L mutants following the referees’ advice and we show these data in the new Figure 2G. The new results have been included in the MS: “and a reduction in the number of spermatozoa in the epididymis (3.3x106 in the Hsf2bpS167L/S167L mutant vs 4.3x106 in the WT; Figure 2G)”

Scale bars in Figure 1B and 1C should be the same size to facilitate comparison. Also, it is difficult to see follicles, especially in (B). Immunostaining should be performed to identify follicles.

We thank the referees for the comments. We have included new pictures (Figure 2B) where the follicles can be clearly distinguished by standard H&E staining which we are convinced is sufficient to show them clearly and unambiguously. On these new figures the scale bars are the same size, as requested by the reviewers.

1(H). Spreads are not appropriate to quantify apoptotic cells. How was apoptosis monitored? The legend is incorrect: "(H).…spread preparations from Hsf2bp-/- and Hsf2bpS167L/S167L mice." It should be +/+ and S167L/S167L.

Figure 1 legend is much too long.

have carried out a TUNEL assay on histological sections (new panel on Figure 2F). The new results have been included in the MS: “Consistent with these results, Hsf2bpS167L/S167L males showed increased numbers of meiotic divisions positive for TUNEL staining (Figure 2F)”.

We have also corrected the legend and shortened it by reducing it to a plain description. However, the need to incorporate the number of mice used in each experiment and the new results corresponding to the follicle count, TUNEL and the sperm count (new panels 2C, 2F and 2G) lead to a net increase of 23 words.

Figure 2

2 (a) – HSF2BP staining intensity -- were graphs showing HSF2BP staining intensity normalized against Sycp3 intensity? Were exposure times constant between images and samples? Please provide more details regarding image acquisition. This comment applies to other figures as well.

There is no information about image analysis in the Materials and methods section? How were foci counted? Was background subtracted? Which software? Which parameters?

As recommended by the referees, we have provided more details regarding image acquisition and analysis/software. We would like to mention that chromosome spreads were prepared from mutants and controls at the same time by the same person (matched controls) and that at least two different slides from each genotype were used for direct IF comparison. Slides were not frozen to avoid differences in the background and antigen reactivity. The time acquisition was always constant among slides (different genotypes) for the same marker to allow direct comparison. We have included this new subsection “Image acquisition and analysis”:

Slides were visualized at room temperature using a microscope (Axioplan 2; Carl Zeiss, Inc) with 63 × objectives with an aperture of 1.4 (Carl Zeiss, Inc). […] Secondary antibodies for STED imaging were conjugated to Alexa 555 and 488 (Invitrogen) and the slides were mounted in Prolong Antifade Gold without DAPI.”

2 (A,B). Which test was used, meaning of stars?

All statistical analyses have been carried out using the Two-tailed Welch´s t-test. As the meaning of the asterisks is the same in all graphs of a Figure, we usually add the interpretation only once at the end of the legend for all of them. However, we have now included it on each individual graph.

2 (C). A non-specific band of unknown origin cannot be used as loading control. Please add a proper loading control. The extracts should be from juvenile to interpret the result since wt and mutant do not have the same testicular cell composition.

What is the mouse background for the -/- and S167L/S167L mice? Are they the same? Differences in strain background (especially mixed versus inbred) can affect the penetrance of meiotic mutants.

We thank the referee for the comment. We have performed the experiment using juvenile extracts to overcome this limitation (new panel in Figure 3C). We have also included new loading controls using anti-tubulin in the western blots (new panels in Figure 7—figure supplement 1C, Figure 7H and Figure 8H).

We would like to note that all the mutants generated in this work have the same mixed genetic background Bl6/CBA.

Figure 3

H2AX plots Y axis: add γ

Thanks for the comment, we have added the corresponding symbol (Figure 4A).

Figure 4

(a) – right hand side graph is missing a y-axis label. Labels should be more explicit, e.g. "number of DMC1 foci".

(b) In the right graph there is extraneous text ("S167L") under the data points.

Thanks for the suggestions and remarks and apologies for the mistakes, we have corrected them and used more explicit labels.

Figure 5

(a) The evidence for XY univalent is weak. One may indeed consider the long univalent axis as X, but the smaller one could be any chromosome. This is only a deduction (On Figure 1G, impossible to tell). This observation needs further analysis: is the recombination defect (CO defect and univalent) mostly impacting the X and Y? Please, analyze the distributions of MLH1 foci, specifically the fraction of pachytene SCs that lack MLH1 foci.

We thank the referees. We have included new analyses to address this point:

1) We have quantified the percentage of pachytene spermatocytes showing unsynapsed XY chromosomes by SYCP3 and γH2AX (sex body) labelling (Figure 6D) to assure the designation.

2) We have also carried out the quantification of the number of pachynemas showing XY/autosomic bivalents without COs (MLH1 staining) (new Figure 6B and Figure 6—figure supplement 1B).

These analyses show that in Hsf2bpS167L/S167L males appear a significant number of spermatocytes with bivalents without CO (that will be eliminated) and this impacts more on the XY. The new data were included in the text: “and a decreased number of COs (measured as MLH1, Figure 6b and Figure 6—figure supplement 1B). In accordance with the reduction of COs, we observed the presence of univalents in the XY pair at pachynema as well as univalents in metaphase I spermatocytes from Hsf2bpS167L/S167L mice (Figure 6D and Figure 6—figure supplement 1C).

(B,C) – Please comment on the observation that RPA counts are similar between control and mutant, while DMC1 counts are significantly lower.

We have incorporated a new section in the refocused Discussion section about RPA and recombinases. Specifically, we have included de following text dealing with the sex differences in RPA foci: “Given the unknown function that RPA plays in vivo during oogenesis (Shi et al., 2019), it is tempting to speculate that the role of RPA in mediating the replacement of RAD51/DMC1 in female meiosis would be carried out by another protein complex such as SPATA22/MEIOB in a HSF2BP/MIDAP-dependent manner.”

RAD51 counts should be analyzed in S167L females.

SPATA22 which appears to give a strong phenotype in MIDAP mutant should be tested in male and female Hsf2bp mutants.

We thank the referees’ comments. We have now included new counts of RAD51 foci (included in the new Figure 5D and Figure 5—figure supplement 1D) as well as SPATA22 analysis in females (included in the new Figure 6A and Figure 6—figure supplement 1A). The results for SPATA22 in male Hsf2bp mutants were already shown in former Figure S18 and have been moved to main Figure 6A and Figure 6—figure supplement 1A.

Identification and analysis of MIDAP

The acronym MIDAP is not the best choice: It's unclear how MIDAP is an acronym for "meiotic double- stranded break BRCA2/HSF2BP complex associated protein". Where is the "I" from, and how is BRCA2/HSF2BP part of the acronym? What is the expression profile of MIDAP? And if the gene is not meiotic-specific it should not be labeled as such. Moreover, it's not clear that its major function is through BRCA2. Please indicate that C19ORF57 is the name of the human not the mouse gene (thus in the yeast two hybrid screen the gene identified was not C19ORF57; was the cDNA overlapping with the domain identified as interacting by CoIP?).

We have indicated in the Y2H screen the name of the mouse RIKEN following referees’ advice indicating that C19ORF57 is the name of the human ORF.

The name of MIDAP was initially proposed and strongly suggested by the gene nomenclature committee (Dr. Ruth Seal <hgnc@genenames.org>). After receiving the reviewing report of the present MS, we have contacted again with the HGNC committee. To date, we have not received a definitive response to our query asking for a final name for the gene C19ORF57/4930432K21Rik. We are thus moving forward with the approved name decided by the authorized HGNC committee.

In relation with the Y2H analysis, all the clones identified and sequenced overlapped with the domain identified by CoIP. We only sequenced the full insert of a fraction of the positive clones and these included the domain identified in the CoIP. The remaining positive clones were only partially sequenced (due to their length) to determine the identity of the gene.

One learns very little from the study of the MIDAP internal deletion. Thus, this could be removed or added as supplementary information. In addition, this mutant allele does not show that the interaction domain is sufficient as the mutant protein is much larger than the interaction domain.

Midap∆/∆: we would suggest adding a number such as ∆6. Otherwise, it can be easily thought that this allele is a full deletion.

We thank the referees for the comments. We have shortened this section and included a supplementary figure (Figure 7—figure supplement 1). We have also changed the nomenclature of the mutant to Midap∆142-472/∆142-472 to avoid any misunderstanding. It is clear that the mutant δ allele (lacking from 142 to 472) does include the interaction domain and also the N-terminal domain (1 to 141). Thus, one cannot formally prove that the interaction domain is sufficient for MIDAP function in vivo from this mutant. However, i) the unstructured nature of the MIDAP protein, ii) the lack of recognizable domains, iii) the lack of meiotic phenotype of the Midap∆142-472/∆142-472 , iv) the retained ability of interaction of the MIDAP-∆142-472 with HSF2BP,v) and the relatively proper loading of HSF2BP in the Midap∆142-472/∆142-472spermatocytes suggest that MIDAP carry out most of its meiotic function through a minimum portion of its sequence (270 residues out of the 600 residues of the wild type protein) which includes its interacting domain with HSF2BP (125 residues).

This part is now as follows:

“We mapped the HSF2BP/MIDAP-interacting domain to the C-term fragment of MIDAP (spanning residues 475-600 of the murine protein, Figure 7C). In line with this, the MidapΔ142-472/Δ142-472 mutant mice, expressing the MIDAP protein devoid of its central part, were fertile and did not show defects in chromosome synapsis or an alteration of HSF2BP loading to axes, further indicating that a large fraction of the coding protein of MIDAP is not essential for MIDAP/HSF2BP function in vivo (Figure 7—figure supplement 1).”

Figure 6:

(A): Add MW markers (and check this for all western blots)

(C) – IP with Δ475-600 – please provide evidence that IP worked – western blot for protein used for setting up IP reaction must be also provided in all cases. Which fraction of input, of IP was loaded? (these comments apply to all CoIPs).

Following the advice of the referees, we have included in all the western blots the MW markers. We have also included in all the co-IPs the lane demonstrating that the IP works as well as the % of input loaded.

(D) – The authors cannot conclude on a perfect colocalization without quantification. Please quantify. Squares indicating areas being magnified are not being reproduced properly. Also, these areas are not magnified very much- please make them larger! The insets are shifted, this should be mentioned.

We have included the corresponding quantification of the colocalization between HSF2BP and MIDAP (Supplementary file 1D) and following the recommendations of the referees we have made the insets larger and properly reproduced. We have mentioned that the insets are shifted (Figure 7D-E).

Figure 7. Depletion of follicle pool in Midap-/- females should be quantified.

Males: provide evidence for the stage IV arrest; in the text explain what is stage IV for non specialist. Asterisks are barely visible, change color.

As suggested by the referees we have quantified the pool of follicles in Midap-/- females (Figure 8A).

-We have also explained the term stage IV of the epithelium cycle (referred to Hsf2bp-/- arrest) as follows:

“During mouse spermatogenesis, the twelve stages of the epithelial cycle can be distinguished in seminiferous tubule sections by identifying groups of associated germ cell types (Ahmed and de Rooij, 2009). Following these criteria, the seminiferous epithelium of Hsf2bp-/- mice showed a stage IV arrest, characterized by a massive apoptosis of zygotene-like spermatocytes occurring at the same time that in spermatogonia divide into B spermatogonia (Figure 2E)”

The color of asterisks has been changed to red following the editor/reviewer’s advice.

(E): 5 nuclei are shown. The four upper nuclei show various levels of synapsis defects. It is not clear what information is provided with the nucleus in the lower part which does not seem to be a pachytene but more likely a diplotene nucleus. Unless a specific conclusion is reached from this nucleus, it could be removed.

We have removed the non-informative nuclei from this figure. Additionally, we have reduced the panel of SYCP1 and SYCP3 (in oocytes) in the main Figure 8E and we have added a supplementary Figure 8—figure supplement 1E showing the remaining nuclei.

Figure 8:

(A) – Absence of HSF2BP staining in the Midap-/- mutant could be due to protein destabilization. See comment above; legend, Quantification is HSF2BP foci not MIDAP.

We have carried out western blot analysis of HSF2BP in Midap-/- 13 dpp testis extracts to determine whether the absence of HSF2BP labelling in the chromosome axes is due to the absence of MIDAP or due to a lower protein expression/stabilization. Our new WB results show that in Midap null mutant testis extracts there is also (as with the IF analysis) a strong reduction of HSF2BP protein expression suggesting lower protein stabilization. These results have been included in the manuscript: “Western blot analysis of HSF2BP in 13 dpp testis extracts from Midap-/- mice showed a strong reduction in comparison with the WT control (Figure 8H), suggesting again that MIDAP is necessary for HSF2BP protein stabilization.

Figure 9. The absence of MIDAP foci should also be quantified in the Hsf2bp null mutant even if the number of foci is close to 0.

We have included the requested quantification (Figure 7F). The figures showing these results have been relocated to the Figure 7 following the reviewers’ recommendation (moreover the Figure 7—figure supplement 4A shows the extended panel for MIDAP staining in Hsf2bp mutant males).

Figure 10:

(A) – and in general and for all western blots, the labeling of IPs is confusing and

ambiguous. It is unclear what the arrows are supposed to highlight. Best is to indicate which antibody was used to probe the blots, and to add arrows to identify proteins when required.

The comparison of Hsf2bp wt and S167L does not make sense in such overexpression system since the property of S167L reported above was a potential lower stability and/or level. Transfection with Hsf2bpS167L should be removed.

We have modified the WB labelling for the IPs. We agree with this consideration and accordingly we have removed the triple transfection with the Hsf2bp-S167L as suggested.

Overall, the information gained is the CoIP between the three proteins, the nuclear localization and stabilization of Hsf2Bp in the presence of MIDAP. Note that there is no evidence for a multimeric complex from this study. This would require performing sequential IP.

The one and half page paragraph should be reduced to half a page, which would improve the quality of the paper.

The legend should be reduced.

Regarding to the absence of evidence for a multimeric complex between BRCA2, MIDAP and HSF2BP, we consider that the sequential IP is not necessary (see the reasoning below) since triple transfections is equivalent in this case. We have provided direct evidence that this complex is indeed formed from the double transfections and triple transfection experiments in HEK293 cell:

Double transfections:

- BRCA2-C + HSF2BP: BRCA2 Co-IP with HSF2BP (Figure 6—figure supplement 2A).

- HSF2BP + MIDAP: HSF2BP Co-IP with MIDAP (Figure 7A).

- BRCA2 + MIDAP: BRCA2 does not Co-IP with MIDAP (Figure 10A). This negative result is essential for the conclusion (see below).

When we transfect the three proteins (BRCA2, HSF2BP and MIDAP):

HSF2BP co-IP with BRCA2-C (Figure 10B).

HSF2BP co-IP with MIDAP (Figure 10B).

BRCA2-C co-IP with MIDAP (but this Co-IP is not observed when we co-transfect of BRCA2 and MIDAP alone, see Figure 10A).

-Altogether, these Co-IP results can only be explained if the three proteins form a multimeric complex. The sequential IP experiment would be required if all pairwise Co-IPs would have been observed (which is not the case).

Please see (Liu et al., 2014) for a sequential IP example because Bag6 can interact with both gp78 and USP13 (subsection “MIDAP is a novel interactor of HSF2BP” and Figure 4F).

However, we agree that these results were too scattered in different figures which could lead to a difficult interpretation. We have now displayed the single negative co-IPs of BRCA2 with MIDAP together with the Triple co-IP (Figure 10A-B).

We agree that the manuscript would be improved by reducing this paragraph. We have shortened this paragraph (from 535 to 385 words).

In relation with the size of the legend, we have shortened the text. Despite that we have included new panels for clarity (scattered in the initial version) and that we also have incorporated the number of cells analysed and replicates (following the reviewers advice), the final number of words has been shortened from 332 to 286.

Figure 1: Please include the genotypes for the sequenced individuals. From the text, it appears as if the brother is heterozygous, so there does not appear to be a male homozygote to comment on human male fertility. This should be clearly stated early in the manuscript (see comment below).

We agree with the referees that this point should be clearer. We have added the genotypes on the pedigree in Figure 1 (former Figure S1), as requested (and modified the legend accordingly). We have added a sentence to highlight the fact that no homozygous male is available early in the Results section: "Therefore, there was no homozygous males identified in this family, preventing the analysis of the impact of this variant on male fertility."

Figure 2—figure supplement 1(A) legend: "Thin (non-coding) and thick (coding sequences) lines under exons represent the expected transcript derived from wild-type and Hsf2bp edited allele." There are no such lines in panel (A).

We have corrected the mistake.

Figure 7—figure supplement 6 – What stage are these WT spermatocytes and approximate stage-matched mutants? Late zygonema?

We have corrected the panel Figure 7—figure supplement 6B and we have indicated the stage at which the wild type and mutant spermatocytes are. We have also included a new description in the text: “We were able to show that none of the recombination-deficient mutants abrogate MIDAP labelling at zygotene (or the corresponding meiotic stage at which the mutant spermatocytes are arrested), in contrast to its absence of loading in SPO11-deficient mice (Figure 7—figure supplement 6B, left).”

Figure 8—figure supplement 1C. Please show the whole membrane. Any truncated protein detected? Where are the epitopes of the antibody?

We appreciate the referees for the comment, but no truncated protein was detected in the Midap-null mutant. The antibodies are rabbit polyclonal and have been raised against the full-length murine protein.

Author response image 1

Interpretation and discussion of the Hsf2bpS167L mutant

Subsection “Mice with the HSF2BP S167L variant show a partial reduction of fertility” "male and female mice were able to reproduce but females showed a significant reduction in the number and size of litters (Figure 1A) whilst males only showed a slight non-significant reduction in fertility (Figure 1A).This is in agreement with the fertility defects observed in Hsf2bp-/- mice (Figure 1C,E-F and (Brandsma et al., 2019; Zhang et al., 2019)), and suggests that the S167L variant might impact more specifically female fertility."

There are several problems with this statement:

a) This is not in agreement with the two published papers, but rather the opposite, since the published papers describe a more pronounced defect in males. Thus, this text needs to be changed.

We apologize for this error in this text. The fertility defects of the Hsf2bpS167L/S167L and the Hsf2bp-/- are not in agreement and they are the opposed (only the meiotic phenotype is in agreement and this is very early in the text to make any mention and is now included in the Discussion section). We have accordingly modified the text in the new revised version of the manuscript: “Hsf2bpS167L/S167L male and female mice were able to reproduce but females showed a significant reduction in the number of litters (Figure 2A) whilst males only showed a slight non-significant reduction in fertility (Figure 2A), suggesting that the S167L variant impacts murine fertility.”

b) However, importantly also, the authors appear to have a more pronounced phenotype in their female knockouts compared to the published reports. This needs to be discussed, included how the knockout alleles vary for the 3 knockouts that have now been generated.

We have incorporated the description of the 2 additional knockouts and their corresponding references. However, we would like also to highlight that our knockout is phenotypically similar to the one already published by Zhang et al., 2019 (males are arrested and females are subfertile) and stronger than the one published by Brandsma et al., 2019 (males are arrested but females show a non-significant reduction of fertility, only a trend). We have included it in the new manuscript: “However, loss of HSF2BP in female mice showed a milder meiotic phenotype (Zhang et al., 2019 and our own data, see below) and a weak albeit non statistical significant reduction of fertility (Brandsma et al., 2019) despite all of the mutants are nulls though in different genetic backgrounds.”

c) Males with S167L have more variability in litter frequency and size, and so with the small numbers it is difficult to draw a conclusion about fertility. Moreover, testis weights and histology of tubules, which show a clear male defect.

We have also the same impression. However, we have not been able to perform new crosses during the lockdown period because it was strictly forbidden in the animal facility. We have however carried out sperm count to analyse the reduction of the number of spermatozoa. The results show a 25% of reduction which is in the limit to have any physiological consequence in terms of fertility. This aspect has been analysed in more detailed in the Discussion section: “Our failure to detect any significant impact of the variant on male fertility could be due to the high variability in litter frequency and size in our study. […] This can be due to the very much fewer gametes available for fertilization in females in comparison to males but also to molecular differences in the meiotic recombination process in both sexes (Cahoon and Libuda, 2019), as displayed by the absence of RPA accumulation or the more pronounced decrease in DMC1 foci observed in HSF2BP-S167L oocytes.”

Related to this in Subsection “Mice with the HSF2BP S167L variant show a partial reduction of fertility”: "These results suggest that mice bearing the POI-causing variant only partially phenocopy the human disease and fits well with the exacerbated sexual phenotypic dimorphism observed in the Hsf2bp-/- mice which might be less pronounced or absent in humans."

As above, no conclusion can be made about human male fertility. The lack of follicle counts/breeding over time in females makes it difficult to conclude about POI in mice. Moreover, what sexual dimorphism in -/- mice is this referring to? Both males and females are completely devoid of gametes in the HSF2BP-null.

We would like to highlight that Hsf2bp-null females from our lab and from the other two published works (Zhang et al., 2019 and Brandsma et al., 2019) are able to reproduce and have gametes. However, we agree that the sentences were not very descriptive. We have rephrased the text and reduced it to avoid misinterpretation.

“These results suggest that mice bearing the POI-causing variant only partially phenocopy the human disease.”

We have restricted the sexual dimorphism in the mice to the Discussion section “By contrast, female mice with the HSF2BP-S167L variant show a mild sub-fertility phenotype with a reduction of litter frequency. This can be due to the very much fewer gametes available for fertilization in females in comparison to males but also to molecular differences in the meiotic recombination process in both sexes (Cahoon & Libuda, 2019), as displayed by the absence of RPA accumulation or the more pronounced decrease in DMC1 foci observed in HSF2BP-S167L oocytes.”

We have also addressed the comparison between mouse and human infertility in a new restructured Discussion section. “Although we cannot exclude that […]. This reinforces a conserved function of HSF2BP in human male fertility.”

Subsection “Mice with the HSF2BP S167L variant show a partial reduction of fertility”: "Overall, male and female Hsf2bpS167L/S167L mice show similar molecular alterations although with different severity".

There are obvious differences in molecular alterations between males and females:

- There is a dramatic increase in RPA foci in males during pachynema while no difference in RPA foci in females during pachynema.

- DMC1 is virtually unchanged in males (perhaps slight reduction in EZ), but greatly reduced in females.

- MLH1 is reduced 20-30% in males, but only ~5% (and not significant) in females.

- γ-H2AX is greatly increased during pachynema in males, but these data ds not exist for females.

- RAD51 is decreased through zygonema to early pachynema with the biggest decrease seen in late zygonema, but these data don't exist for females.

- The only similarity is with DMC1, but even this is misleading as the defect in males is quite mild, but quite severe in females.

We have reconsidered this sentence following the reviewers’ advice. Accordingly, we have changed the statement to: “Overall, male and female Hsf2bpS167L/S167L mice share alterations in the meiotic recombination pathway although with different reproductive outcome.”

With the new experiments carried out, the meiotic alterations and their comparison between males and females and between genotypes has been summarized into a new Supplementary file 1f. The emerging picture make use of the expanded analysis (including now MLH1/CDK2 and the new RAD51 and SPATA22 analysis) show a similar trend in males and females for all the markers in all the mutants though with different intensity, with the exception of the RPA accumulation in males (HSF2BP KO=MIDAP KO>HSF2BPS167L) but not in females. Please see the new Supplementary file 1F. This has been addressed specifically in the Discussion section (as mentioned above).

Supplementary file 1G, Supplementary file 1H

What are the two antibodies (Ab1 and Ab2) used for mass spec? What is the control sample (IP with IgG?). The Materials and methods section indicates the IP was performed with two antibodies R1 and R2 (against HSF2BP and MIDAP)? This is very confusing.

Please can you specify how many peptides of MIDAP identified in this screen.

We thank the referees for the comment. We have immunized two independent rabbits with HSF2BP (HSF2BP-R1 and HSF2BP-R2) and two additional rabbits with MIDAP (MIDAP-R1 and MIDAP-R2). For the mass spec analysis, we performed two IP (one with MIDAP-R1 and another with MIDAP-R2). The control sample was IgGs from rabbit. We have explained this point as follows:

Subsection “Immunocytology and antibodies”:

“Rabbit polyclonal antibodies against HSF2BP and MIDAP were developed by ProteintechTM against a fusion protein of poly-His with full length HSF2BP or MIDAP (pUC57 vector) of mouse origin. Two antibodies (named as R1 and R2) were generated against each protein (HSF2BP-R1/2 or MIDAP-R1/2) by immunization of two different host rabbits.”

Subsection “Testis immunoprecipitation coupled to mass spectrometry analysis”:

“200 µg of antibodies R1 and R2 against MIDAP (two independent IPs) and IgG from rabbit (negative control) were crosslinked to 100 ul of sepharose beads slurry (GE Healthcare).”

The number of peptides identified for MIDAP (and all the putative interactors founded) are included in Supplementary files 1G and 1H.

With the large number of figures and different mutants examined, a summary table should be provided.

We have included a new table including a brief description of all the figures (Supplementary file 1L). We have included the title and all the panels that appear on each main figure. We have only included the title of the supplementary figures and its cross-relationship with the main figures for simplicity.

Additionally, we have also included a new table showing the comparative alterations found in the different mutants analysed and between genders (Supplementary file 1F) in a visual fashion so that allows for a rapid comparison.

Comments on the Discussion section

The discussion is unfocused with no clear link back to the POI of the human family. Also, the data are generally over-interpreted into specific molecular mechanisms. This should be toned down and the authors should clarify when they are speculating.

We have rewritten the Discussion section and toned down the conclusions. We have also paid particular attention to make it clear in the text when we are speculating. In brief, at first, we link our results of the mutant mice of HSF2BP (null and knock-in) to the human POI and discuss why the human HSF2BP-S167L variant is more severe in humans than in mouse attending to the fertility severity. We then discuss how the fertility defects in the mutant mice can be explained by the role of HSF2BP, together with its new identified partner MIDAP, in meiotic DSB repair and their relationship with other meiotic players involved in the recombination pathway. Finally, we speculate how HSF2BP gene variants, one of them recently known to modify human meiotic recombination rate genome wide (Halldorson et al., 2019), could also cause infertility by decreasing the recombination rate much below the lower limit of one CO per bivalent.

Where is the evidence for a supramolecular complex? In the absence of validation this should also be removed from the Abstract ("the higher order macromolecular structure").

We have removed this concept from the abstract given its speculative nature which is based on the fact that the proteins identified in the IP coupled to mass spec experiments have in common its known interaction with BRCA2 and the lack of direct interaction with MIDAP.

Where is the evidence for direct interaction between HSF2BP and BRCA2 and MIDAP ?

The evidence for the direct interaction between HSF2BP and BRCA2 comes from previous work (Brandsma et al., 2019) and also by our co-IP experiments (Figure 6—figure supplement 2A). Our evidence of direct interaction between HSF2BP and MIDAP comes from our experiments (Y2H, co-IP Figure 7A and testis IP Figure 7B). We have not detected direct interaction between MIDAP and BRCA2 (Figure 10A). However, we have observed that BRCA2 and MIDAP co-immunoprecipitates (Figure 10B) when HSF2BP is present.

Where is the evidence that the supramolecular (?) complex participate to the binding of RPA?

We have already replied to this point at the beginning of this question.

The known roles of DSS1 should be introduced noting that meiotic functions are not characterized.

We eliminated any mention relative to DSS1 given that nothing is known about its meiotic function.

"strongly suggesting a parallelism in the early recombination pathway where HSF2BP/MIDAP work" – this is unclear. Please rephrase.

We have restructured this part of the Discussion section following your advice and the comparison with TEX15 mutant is now out of focus after expanding the analysis of RAD51/DMC1 to females showing that foci formation follows the same trend than in males.

"In general, meiotic recombination mutants seem to proceed further in female than in male meiosis perhaps because of the presence of less stringent checkpoints (Hunt and Hassold, 2002)."

But also, recombination mutants often show more severity in males due to X-Y asynapsis.

We agree and appreciate the comment since it is very well known the role that X-Y asynapsis play in the severity of the meiotic arrest in males. We have included this concept in the text: “In general, meiotic recombination mutants appear to proceed further in female than in male, because in males the asynapsis of the sex bivalent leads to a loss of silencing of the Y chromosome, and perhaps also because of the presence of less stringent checkpoints in oogenesis (Hunt and Hassold, 2002).”

"importantly also of HSF2BP" – please rephrase.

This has been corrected.

https://doi.org/10.7554/eLife.56996.sa2

Article and author information

Author details

  1. Natalia Felipe-Medina

    Molecular Mechanisms Program, Centro de Investigación del Cáncer and Instituto de Biología Molecular y Celular del Cáncer (CSIC-Universidad de Salamanca), Salamanca, Spain
    Contribution
    Resources, Formal analysis, Supervision, Investigation, Methodology, Writing - review and editing
    Contributed equally with
    Sandrine Caburet
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-6975-2524
  2. Sandrine Caburet

    1. Université de Paris, Paris Cedex, France
    2. Institut Jacques Monod, Université de Paris, Paris, France
    Contribution
    Formal analysis, Investigation, Methodology, Writing - original draft, Writing - review and editing
    Contributed equally with
    Natalia Felipe-Medina
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-7404-8213
  3. Fernando Sánchez-Sáez

    Molecular Mechanisms Program, Centro de Investigación del Cáncer and Instituto de Biología Molecular y Celular del Cáncer (CSIC-Universidad de Salamanca), Salamanca, Spain
    Contribution
    Formal analysis, Methodology
    Competing interests
    No competing interests declared
  4. Yazmine B Condezo

    Molecular Mechanisms Program, Centro de Investigación del Cáncer and Instituto de Biología Molecular y Celular del Cáncer (CSIC-Universidad de Salamanca), Salamanca, Spain
    Contribution
    Formal analysis, Methodology
    Competing interests
    No competing interests declared
  5. Dirk G de Rooij

    Reproductive Biology Group, Division of Developmental Biology, Department of Biology, Faculty of Science, Utrecht University, Utrecht, Netherlands
    Contribution
    Formal analysis, Investigation
    Competing interests
    No competing interests declared
  6. Laura Gómez-H

    Molecular Mechanisms Program, Centro de Investigación del Cáncer and Instituto de Biología Molecular y Celular del Cáncer (CSIC-Universidad de Salamanca), Salamanca, Spain
    Contribution
    Formal analysis, Methodology
    Competing interests
    No competing interests declared
  7. Rodrigo Garcia-Valiente

    Molecular Mechanisms Program, Centro de Investigación del Cáncer and Instituto de Biología Molecular y Celular del Cáncer (CSIC-Universidad de Salamanca), Salamanca, Spain
    Contribution
    Formal analysis
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-0444-5587
  8. Anne Laure Todeschini

    1. Université de Paris, Paris Cedex, France
    2. Institut Jacques Monod, Université de Paris, Paris, France
    Contribution
    Formal analysis, Investigation
    Competing interests
    No competing interests declared
  9. Paloma Duque

    Molecular Mechanisms Program, Centro de Investigación del Cáncer and Instituto de Biología Molecular y Celular del Cáncer (CSIC-Universidad de Salamanca), Salamanca, Spain
    Contribution
    Formal analysis, Methodology
    Competing interests
    No competing interests declared
  10. Manuel Adolfo Sánchez-Martin

    1. Transgenic Facility, Nucleus platform, Universidad de Salamanca, Salamanca, Spain
    2. Departamento de Medicina, Universidad de Salamanca, Salamanca, Spain
    Contribution
    Resources, Methodology
    Competing interests
    No competing interests declared
  11. Stavit A Shalev

    1. The Genetic Institute, "Emek" Medical Center, Afula, Israel
    2. Bruce and Ruth Rappaport Faculty of Medicine, Technion, Haifa, Israel
    Contribution
    Resources
    Competing interests
    No competing interests declared
  12. Elena Llano

    1. Molecular Mechanisms Program, Centro de Investigación del Cáncer and Instituto de Biología Molecular y Celular del Cáncer (CSIC-Universidad de Salamanca), Salamanca, Spain
    2. Departamento de Fisiología y Farmacología, Universidad de Salamanca, Salamanca, Spain
    Contribution
    Conceptualization, Formal analysis, Investigation, Writing - original draft, Writing - review and editing
    Competing interests
    No competing interests declared
  13. Reiner A Veitia

    1. Université de Paris, Paris Cedex, France
    2. Institut Jacques Monod, Université de Paris, Paris, France
    3. Université Paris-Saclay, Institut de Biologie F. Jacob, Commissariat à l’Energie Atomique, Fontenay aux Roses, France
    Contribution
    Conceptualization, Funding acquisition, Investigation, Writing - original draft, Writing - review and editing
    For correspondence
    reiner.veitia@ijm.fr
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-4100-2681
  14. Alberto M Pendás

    Molecular Mechanisms Program, Centro de Investigación del Cáncer and Instituto de Biología Molecular y Celular del Cáncer (CSIC-Universidad de Salamanca), Salamanca, Spain
    Contribution
    Conceptualization, Formal analysis, Supervision, Funding acquisition, Investigation, Methodology, Writing - original draft, Writing - review and editing
    For correspondence
    amp@usal.es
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-9264-3721

Funding

Ministerio de Economía y Competitividad

  • Alberto M Pendás

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

We thank Dr. Barbero and Dr. Edyta Marcon, for providing antibodies against SYCP3 and RPA, respectively, Dr. Emmanuelle Martini for helpful advice with the DNA binding assay,Dr. Fabien Fauchereau for the genetic analysis of the family and Dr. Alex N Zelenskyy for helpful discussion. This study was supported by Université Paris Diderot and the Fondation pour la Recherche Médicale (Labelisation Equipes DEQ20150331757, SC, A-LT and RAV). This work was supported by MINECO (BFU2017-89408-R) and by Junta de Castilla y León (CSI239P18). NFM, FSS and LGH are supported by European Social Fund/JCyLe grants (EDU/310/2015, EDU/556/2019 and EDU/1083/2013). YBC is funded by a grant from MINECO (BS-2015–073993). The proteomic analysis was performed in the Proteomics Facility of Centro de Investigación del Cáncer, Salamanca, Grant PRB3(IPT17/0019 - ISCIII-SGEFI/ERDF). CIC-IBMCC is supported by the Programa de Apoyo a Planes Estratégicos de Investigación de Estructuras de Investigación de Excelencia cofunded by the Castilla–León autonomous government and the European Regional Development Fund (CLC–2017–01). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Ethics

Animal experimentation: All the experiments were approved by the Ethics Committee for Animal Experimentation of the University of Salamanca (USAL) and the Ethics committee of the Spanish Research Council (CSIC) under protocol #00-245. Accordingly, all the mouse protocols used in this work have been approved by the above mentioned Animal Experimentation committees. Specifically, mice were always housed in a temperature-controlled facility (specific pathogen free, spf) using individually ventilated cages, standard diet and a 12 h light/dark cycle, according to EU law (63/2010/UE) and the Spanish royal law (53/2013) at the "Servicio de Experimentación Animal, SEA. In addition, animal suffering was always minimized and we made every effort to improve animal welfare during the life of the animals.

Senior Editor

  1. Kevin Struhl, Harvard Medical School, United States

Reviewing Editor

  1. Bernard de Massy, CNRS UM, France

Reviewers

  1. Bernard de Massy, CNRS UM, France
  2. Maria Jasin, Memorial Sloan-Kettering Cancer Center, United States

Publication history

  1. Received: March 17, 2020
  2. Accepted: August 26, 2020
  3. Accepted Manuscript published: August 26, 2020 (version 1)
  4. Version of Record published: September 17, 2020 (version 2)

Copyright

© 2020, Felipe-Medina et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

Metrics

  • 528
    Page views
  • 133
    Downloads
  • 0
    Citations

Article citation count generated by polling the highest count across the following sources: Crossref, PubMed Central, Scopus.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Download citations (links to download the citations from this article in formats compatible with various reference manager tools)

Open citations (links to open the citations from this article in various online reference manager services)

Further reading

    1. Biochemistry and Chemical Biology
    2. Cell Biology
    İbrahim Avşar Ilik et al.
    Research Article

    The nucleus of higher eukaryotes is a highly compartmentalized and dynamic organelle consisting of several biomolecular condensates that regulate gene expression at multiple levels (Banani et al., 2017; Shin and Brangwynne, 2017). First reported more than 100 years ago by Ramón y Cajal, nuclear speckles (NS) are among the most prominent of such condensates (Spector and Lamond, 2011). Despite their prevalence, research on the function of NS is virtually restricted to colocalization analyses, since an organizing core, without which NS cannot form, remains unidentified (Chen and Belmont, 2019; Galganski et al., 2017). The monoclonal antibody SC35, which was raised against a spliceosomal extract, is a frequently used reagent to mark NS since its debut in 1990 (Fu and Maniatis, 1990). Unexpectedly, we found that this antibody has been misidentified and the main target of SC35 mAb is SRRM2, a large (~300 kDa), spliceosome-associated (Jia and Sun, 2018) protein with prominent intrinsically disordered regions (IDRs) that sharply localizes to NS (Blencowe et al., 1994). Here we show that, the core of NS is likely formed by SON and SRRM2, since depletion of SON leads only to a partial disassembly of NS as reported previously (Ahn et al., 2011; Fei et al., 2017; Sharma et al., 2010), in contrast, combined depletion of SON together with SRRM2, but not other NS associated factors, or depletion of SON in a cell line where IDRs of SRRM2 are genetically deleted, leads to a near-complete dissolution of NS. This work, therefore, paves the way to study the role of NS under diverse physiological and stress conditions.

    1. Cell Biology
    2. Biochemistry and Chemical Biology
    Melissa V Gammons et al.
    Research Article Updated

    Feedback control is a universal feature of cell signaling pathways. Naked/NKD is a widely conserved feedback regulator of Wnt signaling which controls animal development and tissue homeostasis. Naked/NKD destabilizes Dishevelled, which assembles Wnt signalosomes to inhibit the β-catenin destruction complex via recruitment of Axin. Here, we discover that the molecular mechanism underlying Naked/NKD function relies on its assembly into ultra-stable decameric core aggregates via its conserved C-terminal histidine cluster (HisC). HisC aggregation is facilitated by Dishevelled and depends on accumulation of Naked/NKD during prolonged Wnt stimulation. Naked/NKD HisC cores co-aggregate with a conserved histidine cluster within Axin, to destabilize it along with Dishevelled, possibly via the autophagy receptor p62, which binds to HisC aggregates. Consistent with this, attenuated Wnt responses are observed in CRISPR-engineered flies and human epithelial cells whose Naked/NKD HisC has been deleted. Thus, HisC aggregation by Naked/NKD provides context-dependent feedback control of prolonged Wnt responses.