The Fidelity of Synaptonemal Complex Assembly Is Regulated by A

View metadata, citation and similar papers at core.ac.uk brought to you by CORE

provided by Spiral - Imperial College Digital Repository

The fidelity of synaptonemal complex assembly is regulated by a

signaling mechanism that controls early meiotic progression

Nicola Silva1, Nuria Ferrandiz1, Consuelo Barroso1, Silvia Tognetti2, James

Lightfoot1,3, Oana Telecan1, Vesela Encheva4, 5, Peter Faull4, Simon Hanni6, Andre

Furger6, Ambrosius P. Snidjers4, 5, Christian Speck2, Enrique Martinez-Perez1,*

1Meiosis group, MRC Clinical Sciences Centre, Faculty of Medicine, Imperial College London, Du Cane Road, London W12 0NN, UK

2DNA replication group, Institute Clinical Sciences, Faculty of Medicine, Imperial College London, Du Cane Road, London W12 0NN, UK

3Current address: Max Planck Institute for Developmental Biology, 72076 Tubingen, Germany

4Proteomics facility, MRC Clinical Sciences Centre, Faculty of Medicine, Imperial College London, Du Cane Road, London W12 0NN, UK

5Current address: Protein analysis and proteomics, London Research Institute, South Mimms EN6 3LD, UK

6Department of Biochemistry, Oxford University, Oxford OX1 3QU, UK

* Corresponding author: Enrique Martinez-Perez phone: 44 (0)208 383 4314 [email protected]

Key words: Meiosis, homologue pairing, synapsis, HORMA domain, checkpoint

Running title: Quality control of SC assembly

1 Summary

Proper chromosome segregation during meiosis requires the assembly of the synaptonemal complex (SC) between homologous chromosomes. However, the SC structure itself is indifferent to homology and poorly understood mechanisms that depend on conserved HORMA-domain proteins prevent ectopic SC assembly.

Although HORMA-domain proteins are thought to regulate SC assembly as intrinsic components of meiotic chromosomes, here we uncover a key role for nuclear soluble

HORMA-domain protein HTP-1 in the quality control of SC assembly. We show that a mutant form of HTP-1 impaired in chromosome loading provides functionality of an

HTP-1-dependent checkpoint that delays exit from homology search-competent stages until all homologue pairs are linked by the SC. Bypassing of this regulatory mechanism results in premature meiotic progression and licensing of homology- independent SC assembly. These findings identify nuclear soluble HTP-1 as a regulator of early meiotic progression, suggesting parallels with the mode of action of

Mad2 in the spindle assembly checkpoint.

2 Introduction

The formation of euploid gametes requires pairing of homologous chromosomes during early meiotic prophase, a process involving homology search and stabilization of homologue interactions by a proteinaceous structure known as the synaptonemal complex (SC). However, the SC is intrinsically indifferent to homology and when homology recognition fails the SC can be assembled between non-homologous chromosomes (Baudat et al., 2000; Couteau and Zetka, 2005; Leu et al., 1998;

Martinez-Perez and Villeneuve, 2005). Although restricting SC assembly to occur only between correctly aligned homologue pairs is an essential aspect of meiosis, how improper SC assembly is prevented remains poorly understood.

The onset of meiotic prophase is marked by dramatic changes in chromosome structure that include the formation of an axial element, a proteinaceous structure that runs along the length of sister chromatids and contains cohesin as well as meiosis- specific proteins (Blat et al., 2002). Among these, a family of HORMA-domain proteins homologous to yeast Hop1 have been shown to be major regulators of meiotic chromosome function (Carballo et al., 2008; Couteau and Zetka, 2005; Daniel et al., 2011; Goodyer et al., 2008; Hollingsworth et al., 1990; Martinez-Perez et al.,

2008; Martinez-Perez and Villeneuve, 2005; Sanchez-Moran et al., 2007; Severson et al., 2009; Shin et al., 2010; Wojtasz et al., 2012; Zetka et al., 1999), and to participate in a series of surveillance mechanisms that provide quality control at different transitions of the meiotic program (MacQueen and Hochwagen, 2011). For example, yeast Hop1 and mouse HORMAD1 and HORMAD2 are required for the activity of the pachytene checkpoint, which triggers the arrest or culling of meiocytes with defects in recombination or incomplete SC assembly. In C. elegans, HTP-1 participates in earlier checkpoint-like mechanisms that operate to coordinate SC

3 assembly with homologue recognition and to regulate the timing of exit from early prophase stages characterized by active homology search (Couteau and Zetka, 2005;

Martinez-Perez and Villeneuve, 2005), but how HTP-1 participates in these regulatory mechanisms remains unknown. Furthermore, although meiotic HORMA- domain proteins are thought to exert their roles as components of axial elements, the mode of action of the best characterized HORMA-domain protein, the spindle assembly Mad2 protein, involves a soluble pool of Mad2 that interacts with Cdc20 to prevent the onset of anaphase until all kinetochores are attached to microtubules

(Lara-Gonzalez et al., 2012). Here we report evidence that HTP-1 acts as a nuclear soluble factor that regulates early meiotic progression to prevent the premature licensing of homology-independent SC assembly.

4 Results and discussion

HTP-1M127K limits SC assembly without loading to axial elements

During C. elegans meiosis, HTP-1 is required to prevent SC assembly until successful homologue recognition is achieved, and since HTP-1 is also required to promote homology search, worms lacking HTP-1 function display extensive non-homologous synapsis (Couteau and Zetka, 2005; Martinez-Perez and Villeneuve, 2005). This failure in regulating SC assembly impairs the formation of inter-homologue crossover events, as evidenced by the almost complete lack of chiasmata (physical connections between homologous chromosomes mediated by crossover events) in the diakinesis oocytes of mutants carrying the htp-1 null allele gk174 (Figure 1A). In contrast, oocytes of worms carrying the htp-1(fq8) allele, which we isolated in a genetic screen

(see Experimental Procedures), display a mixture of chromosome pairs attached by chiasmata and unattached chromosomes, suggesting that htp-1(fq8) retains partial competence for crossover formation (Figure 1A). The htp-1(fq8) mutation induces a single amino acid substitution (M127K) in the middle of the HORMA domain of

HTP-1, a region that is predicted to form a Beta fold and that aligns with the β5 region of Mad2 (Figure 1B), a structurally-characterized HORMA-domain protein that plays an essential role in the spindle assembly checkpoint (Luo et al., 2000).

In order to elucidate the effect of the M127K mutation on HTP-1 function, we first examined the localization of HTP-1M127K by creating transgenic worms homozygous for the htp-1(gk174) allele and for a single-copy transgene insertion that expressed HTP-1M127K::GFP using the regulatory elements of the htp-1 gene.

Surprisingly, HTP-1M127K::GFP was not detected on the axial elements at any stage of meiotic prophase in fixed germ lines or by live imaging, while wild-type HTP-

1::GFP, which rescued the chiasma defect of htp-1 (gk174) mutants, was clearly

5 loaded to axial elements (Figures 1C, S1A and S1B). However, western blot analysis of protein extracts made from transgenic worms expressing HTP-1M127K::GFP confirmed the presence of the HTP-1M127K protein, albeit in lower amounts than those observed for wild-type HTP-1::GFP (Figure 1D). We also detected HTP-1M127K expressed from the endogenous htp-1 locus carrying the fq8 mutation by using antibodies that react with both HTP-1 and its paralogue HTP-2, which shares 82% amino acid identity with HTP-1 (but cannot functionally substitute HTP-1) (Martinez-

Perez et al., 2008), in protein extracts from htp-1(fq8) htp-2 double mutants (Figure

1E). Moreover, using anti-HTP-1/2 antibodies we detected axis associated HTP-2 in germ lines of htp-1(gk174) mutants, but we failed to detect any axis-associated signal in htp-1(fq8) htp-2 double mutants (Figure S1C), despite the fact that similar amounts of HTP-2 and HTP-1M127K are present in these mutants (Figure 1E). Further evidence that HTP-1M127K fails to associate with axial elements came from quantifying polar body extrusion during the meiotic divisions in oocytes, a process that requires HTP-1 or HTP-2 bound to axial elements, where they protect a subset of cohesin complexes from removal during the first meiotic division (Martinez-Perez et al. 2008; Severson et al. 2009). While wild type, htp-1(gk174) and htp-1(fq8) embryos extrude two polar bodies, htp-1(fq8) htp-2 and htp-1(gk174) htp-2 double mutants extrude a single polar body, demonstrating a failure in preventing premature removal of cohesin from axial elements (Figures S1D and S1E ). These experiments show that although HTP-1M127K fails to load to axial elements, the protein is present in meiotic nuclei and retains some competence in promoting chiasma formation.

Next, we investigated if HTP-1M127K retained functionality in regulating SC assembly by monitoring loading of HIM-3, a HORMA-domain protein that loads to axial elements (Zetka et al., 1999), and of SYP-1, a central region component of the

6 SC (MacQueen et al., 2002). Wild-type controls and htp-1 null mutants demonstrated extensive SC formation in early meiotic prophase nuclei, a region of the germ line known as transition zone and corresponding to the leptotene and zygotene stages of meiotic prophase (Figure 1F). Although HIM-3 was loaded normally in transition zone nuclei of htp-1(fq8) mutants, few long tracks of SYP-1 were detected in transition zone and early pachytene regions of htp-1(fq8) mutant germ lines, and large chromosomal regions remained unsynapsed (Figure 1F). However, high levels of synapsis were observed in late pachytene nuclei of htp-1(fq8) mutants (Figure S1F), suggesting that SC assembly may be actively prevented during early prophase.

Transgenic worms expressing HTP-1M127K tagged with GFP or 6His also displayed delayed SC assembly (Figure 1F), confirming that HTP-1M127K is capable of restricting SC assembly during early prophase, despite its failure to associate with axial elements.

HTP-1M127K is a nuclear soluble protein and wild-type nuclei contain a large pool of HTP-1 that is not associated with chromatin

The observations described above suggest that the regulatory mechanisms that control

SC assembly during early meiotic prophase may involve a nuclear soluble pool of

HTP-1. This possibility is also conceptually attractive considering that the mode of action of Mad2 in the spindle assembly checkpoint involves a large pool of soluble

Mad2 that acts to amplify a signal that is initiated at unattached kinetochores, and that leads to formation of a soluble mitotic checkpoint complex containing Mad2 that prevents anaphase onset by blocking activation of the APC (Lara-Gonzalez et al.,

2012). Therefore, we sought to clarify if a pool of nuclear soluble HTP-1 is present in wild-type worms, and to confirm if HTP-1M127K is not found associated with

7 chromatin. To this end, we first prepared Triton soluble and insoluble protein fractions from whole worm extracts, and analyzed the distribution of HTP-1 by western blot using as controls anti-histone H3 antibodies (DNA bound fraction) and antibodies against HAL-2, a nuclear protein that is present throughout meiotic prophase and that is not thought to bind to DNA (Zhang et al., 2012). This demonstrated that 67.2 % of HTP-1 in wild-type nuclei is present in the soluble fraction and confirmed that HTP-1M127K is only found in the soluble fraction (Figures

2A, S2A and S2B). This analysis also revealed that the M127K mutation reduces the levels of soluble HTP-1 to 20.5 % of wild-type levels (n= 3, SD= 0.96). Using a different fractionation method, which involves the isolation of intact nuclei from whole worms (Haenni et al., 2012), we also observed that 68.9 % of HTP-1::GFP is present in the nuclear soluble protein fraction (Figures S2B and S2C). Interestingly, protein extracts from mouse testes also show that a large amount of HORMAD1 and

HORMAD2 accumulates in a Triton soluble fraction (Wojtasz et al., 2012). Thus, similar to Mad2 in the spindle assembly checkpoint, nuclear soluble pools of meiotic

HORMA-domain proteins may be important components of the surveillance mechanisms that provide quality control during meiotic prophase.

HTP-1 loading to axial elements requires a direct interaction with HTP-3

Loading of HTP-1 to axial elements depends on the cohesin complex and on

HORMA-domain protein HTP-3, but not on the two other meiotic HORMA-domain proteins HTP-2 and HIM-3 (Goodyer et al., 2008; Lightfoot et al., 2011; Martinez-

Perez et al., 2008; Severson et al., 2009). In addition, immunoprecipitation of the axis-associated protein LAB-1 identified the four C. elegans HORMA-domain proteins (HTP-1, HTP-2, HTP-3 and HIM-3) as LAB-1 interactors (Tzur et al., 2012).

8 These observations suggest that loading of HTP-1 to axial elements may involve a direct interaction with HTP-3, and since we failed to detect loading of HTP-1M127K to axial elements, we decided to investigate if the M127K mutation impairs HTP-1 loading by affecting a putative interaction with HTP-3. First, we sought to clarify if

HTP-1 and HTP-3 form a complex in vivo by immunoprecipitating HTP-1 from nuclear soluble and DNA-bound protein extracts made from worms expressing HTP-

1::GFP. Mass spectrometry analysis of these immunoprecipitation experiments identified a large number of peptides from HTP-1, HIM-3, HTP-2 and HTP-3, confirming that the four HORMA-domain proteins form a complex in vivo (Figure

2B). To clarify whether there is a direct physical interaction between HTP-1 and

HTP-3, we co-expressed HTP-1::FLAG and HTP-3::HA in bacteria.

Immunoprecipitation of HTP-1 followed by western blot analysis demonstrated a clear interaction between HTP-1 and HTP-3 (Figure 2C). However, when we coexpressed HTP-1M127K and HTP-3, the amount of HTP-3 that was co- immnoprecipitated with HTP-1M127K was clearly reduced, demonstrating that the

M127K mutation impairs the interaction between HTP-1 and HTP-3 (Figure 2C).

Although this experiment provides an explanation for the failure of HTP-1M127K in associating with axial elements, an alternative possibility is that loading of HTP-3 itself may be affected in htp-1(fq8) mutants. However, staining of htp-1(fq8) mutants with anti-HTP-3 antibodies demonstrated normal loading of HTP-3, ruling out this possibility (Figure 2D). Thus, a direct physical interaction connects HTP-1 with HTP-

3, and impairment of this interaction by the M127K mutation results in a defect in

HTP-1 loading.

9 HTP-1M127K is competent in promoting chromosome movement and nuclear reorganization at the onset of meiotic prophase

Since HTP-1 is required for the initial establishment of homologue pairing that precedes SC assembly (Couteau and Zetka, 2005; Martinez-Perez and Villeneuve,

2005), we investigated if HTP-1M127K was also capable of promoting homology search at early prophase. The onset of homologue pairing coincides with a clustering of chromosomes inside the nucleus and with motor-driven movement of chromosomal ends attached to SUN-1 aggregates on the nuclear envelope, which facilitate homologue encounters and promote faithful SC assembly (MacQueen and Villeneuve,

2001; Penkner et al., 2009; Sato et al., 2009). HTP-1 plays a role in these two processes, since htp-1 null mutant germ lines display reduced numbers of nuclei with clustered chromosomes as well as reduced chromosome mobility (Baudrimont et al.,

2010; Couteau and Zetka, 2005; Martinez-Perez and Villeneuve, 2005). In contrast to htp-1 null mutants, htp-1(fq8) germ lines contain large numbers of nuclei with clustered chromosomes in the transition zone of the germ line (Figure 1F). Moreover, analysis of SUN-1::GFP aggregate dynamics, which can be used as a proxy for chromosome-end movement on the nuclear envelope (Baudrimont et al., 2010), demonstrated vigorous movement in htp-1(fq8) mutants (Figure 3A, Supplemental

Movies 1-3). The average displacement arc, which provides a measure of the area explored by SUN-1 aggregates on the nuclear envelope, was 78° in wild-type controls, 68° in htp-1(fq8) mutants and 55° in htp-1(gk174) mutants, while the percentage of SUN-1::GFP aggregates moving at high speeds (140 nm/s or higher) was 15.3% in wild-type controls, 12.1% in htp-1(fq8) mutants and 7.5% in htp-

1(gk174) mutants. Thus, HTP-1M127K supports near normal levels of chromosome mobility during early prophase, demonstrating that the role of HTP-1 in this process is

10 not simply contributing to the structural integrity of axial elements, but rather a regulatory role by which HTP-1 must be capable of affecting factors required to promote, or sustain, chromosome movement.

htp-1(fq8) mutants appear largely competent in the events associated with homology search, but display reduced levels of SC assembly during early prophase.

This suggests that HTP-1M127K may be deficient in licensing SC assembly (even when homology search is satisfied), a role that we previously proposed for HTP-1

(Martinez-Perez and Villeneuve, 2005). Analysis of mutants lacking SC central region components demonstrates that while the establishment of pairing is SC independent, the SC is required to maintain high levels of pairing throughout meiotic prophase (MacQueen et al., 2002), especially in regions away from the chromosomal end bearing the pairing center (PC), which promotes pairing locally in a synapsis- independent manner and is also required to promote SC assembly (Rog and Dernburg,

2013). We investigated the progression of pairing, and its dependency on SC assembly in htp-1(fq8) mutants using FISH. Pairing levels in htp-1(fq8) mutants never reached the values observed in wild-type controls, but were consistently higher than those observed in htp-1 null mutants, and appeared very similar to those seen in SC- deficient syp-2 mutants during early prophase (Figure 3B). However, by late pachytene pairing was higher in htp-1(fq8) mutants than in syp-2 mutants (Figure 3B), hinting that the limited SC assembly of htp-1(fq8) mutants may contribute to stabilize pairing. This prompted us to quantify pairing in htp-1(fq8) syp-2 double mutants. Mid and late pachytene nuclei of htp-1(fq8) syp-2 double mutants displayed a significant drop in the pairing levels of non-PC regions compared to htp-1(fq8) mutants (Figure

3B). Furthermore, PC regions of chromosome III and the X chromosome also showed a dramatic drop in pairing in late pachytene nuclei of htp-1(fq8) syp-2 double mutants.

11 Therefore, the limited synapsis observed in htp-1(fq8) mutants contributes to stabilize homologue pairing during pachytene, while the high levels of SC assembly seen in htp-1 null mutants induce extensive non-homologous synapsis (Couteau and Zetka,

2005; Martinez-Perez and Villeneuve, 2005).

HTP-1M127K is competent in inducing delayed exit from early meiotic prophase in response to synapsis defects

Defects in SC assembly induce persistence of chromosome clustering and of dynamic

SUN-1 aggregates into the pachytene region of the germ line (Baudrimont et al.,

2010; MacQueen et al., 2002; Phillips and Dernburg, 2006; Phillips et al., 2005), suggesting the existence of a synapsis surveillance mechanism that triggers a delay in early meiotic progression in response to synapsis defects (Martinez-Perez and

Villeneuve, 2005; Smolikov et al., 2007; Woglar et al., 2013; Zhang et al., 2012).

HTP-1 plays an active role in mediating this delay, since removal of HTP-1 from SC- deficient mutants triggers rapid exit from chromosome clustering (Martinez-Perez and

Villeneuve, 2005). However, DAPI staining demonstrated that chromosome clustering persisted in germ lines of htp-1(fq8) mutants (Figures 1F and 4A), suggesting that HTP-1M127K retains competence in delaying meiotic progression in response to the presence of unsynapsed regions in early prophase nuclei. In order to test this possibility, we investigated meiotic progression in htp-1(fq8) mutants using the presence of PLK-2 (Polo Like Kinase 2), which is recruited to PC regions at meiotic onset to induce the formation of SUN-1 aggregates and robust chromosome movement (Harper et al., 2011; Labella et al., 2011; Woglar et al., 2013), as a molecular marker of active homology search. In wild-type germ lines, 40 % of prophase nuclei displayed PLK-2 aggregates on the nuclear envelope, while in htp-

12 1(fq8) mutants this number increased to 65 %, demonstrating a delay in meiotic progression (Figures 4A and 4B). In contrast, only 11% of prophase nuclei in htp-

1(gk174) displayed PLK-2 aggregates, demonstrating premature exit from early prophase. Furthermore, while removing HTP-1 from syp-2 mutants suppressed the accumulation of nuclei with multiple PLK-2 aggregates seen in syp-2 mutants, htp-

1(fq8) syp-2 double mutants showed extensive accumulation of nuclei with PLK-2 aggregates (Figure 4B). Thus HTP-1M127K is capable of triggering delayed meiotic progression when synapsis defects are present, suggesting that the signaling mechanism that regulates early meiotic progression involves a nuclear-soluble pool of

HTP-1.

We reasoned that if htp-1(fq8) mutants display a delay in meiotic progression, early meiotic events such as the formation of DSBs should be affected. In fact, htp-1 null mutants display a severe reduction in the number of recombination intermediates labeled by the RAD-51 recombinase (Couteau and Zetka, 2005; Martinez-Perez and

Villeneuve, 2005), an observation that could be explained, at least in part, considering that premature exit from early prophase in htp-1 mutants reduces the time that nuclei spend in a stage competent for DSB formation. In agreement with this interpretation, htp-1(fq8) mutants display a substantial increase in the number of RAD-51 foci compared to htp-1(gk174) mutants (Figure S3). Furthermore, htp-1(fq8) germ lines also show a dramatic increase in the number of nuclei positive for DSB-2, a protein required for DSB formation and the presence of which can be used as an indicator of competence for DSB formation (Rosu et al., 2013) (Figures S4A and S4B). These observations confirm the presence of an early prophase arrest in htp-1(fq8) mutants, demonstrating that HTP-1M127K is capable of regulating meiotic progression.

13 HIM-8, a protein that binds the X chromosome pairing center, is required for the prophase arrest of htp-1(fq8) mutants

The best evidence for HTP-1 playing part in a quality-surveillance mechanism that responds to synapsis defects by delaying early meiotic progression is that removal of

HTP-1 bypasses the arrest of syp-2 mutants, in which SC assembly is completely abolished. We investigated if HTP-1 is also required for the persistent chromosome clustering seen when the X chromosomes fail to synapse due to impaired function of

HIM-8, a PC-binding protein that is specifically required to promote SC assembly between the X chromosomes (Phillips et al., 2005). Removal of HTP-1 from him-8 mutants suppressed the accumulation of nuclei with PLK-2 aggregates, confirming that HTP-1 also mediates meiotic delay when a single chromosome pair fails to synapse (Figure 4B). Next, we asked whether HTP-1M127K could also mediate delayed meiotic progression in him-8 mutants, as it did in syp-2 mutants. Surprisingly, although PLK-2 aggregates were formed in transition zone nuclei of htp-1(fq8) him-8 double mutants, they were lost by early pachytene (Figures 4B and S4C). Thus, although wild-type HTP-1 can support prophase arrest independently of HIM-8, the delayed meiotic progression of htp-1(fq8) mutants is HIM-8 dependent.

Having found that impairing HIM-8 function bypasses the delayed meiotic progression seen in htp-1(fq8) mutants, we asked if the ability of HTP-1M127K to prevent improper SC assembly was coupled to delayed meiotic progression. In contrast with the reduced synapsis seen in early pachytene of htp-1(fq8) mutants, extensive SC assembly was observed in the same region of htp-1(fq8) him-8 mutant germ lines (Figure 4C). Furthermore, htp-1(fq8) him-8 double mutants displayed a significant drop in the levels of homologue pairing compared to htp-1(fq8) single mutants (Figure 4D). This, together with the high levels of SC assembly seen in htp-

14 1(fq8) him-8 double mutants, suggests that extensive non-homologous synapsis occurs in these mutants. These observations demonstrate that the delay in meiotic progression seen in htp-1(fq8) mutants contributes to promote homologue pairing and to prevent improper SC assembly, suggesting that the ability to couple SC assembly to homology recognition is lost upon exit from zygotene, and that entrance into pachytene licenses homology-independent SC assembly. Recent findings in mice are consistent with the active inhibition of non-homologous synapsis during early prophase (Kauppi et al., 2013). Thus, coupling of homology-regulated SC assembly to meiotic progression may be a general feature of the meiotic program, and HORMA- domain proteins are key regulators of this coupling mechanism.

15 Conclusions

Our findings suggest that soluble HTP-1 is a component of the early prophase checkpoint that regulates exit from homology search-competent stages until interactions between all homologue pairs are stabilized by loading of the SC. In this context, HTP-1 appears to transmit a “wait signal” that prevents removal of PLK-2 from pairing centers, thereby maintaining homology search. Interestingly, the ability of HTP-1M127K to provide checkpoint function depends on the X chromosome pairing center-binding protein HIM-8, suggesting that the signal transmitted by HTP-1M127K must emanate from, or being integrated at X chromosome pairing centers. This model bears striking parallels with the mode of action of HORMA-domain protein Mad2 in the spindle assembly checkpoint, in which signals emanating from unattached kinetochores are transmitted and amplified by nuclear soluble Mad2 to prevent the onset of anaphase until all kinetochores are bound by microtubules. Future studies should address how signals emanating from individual chromosomes at the time of

SC assembly are transmitted and integrated to regulate the transition between zygotene and pachytene.

16 Experimental Procedures

Isolation of htp-1(fq8) mutants.

EMS mutagenesis was performed by incubating L4 animals in M9 buffer containing

0.5 % EMS for four hours as described in Martinez-Perez and Villeneuve (2005). fq8 mutants were isolated due to the presence of univalents in diakinesis oocytes. SNP mapping and sequencing of the htp-1 locus in fq8 mutants confirmed the presence of a single base pair substitution (T to A) in the third exon that resulted in a M127K substitution.

Preparation of protein extracts and Western blotting

Whole worm extracts were prepared by resuspending 70 young adult hermaphrodites in 1x Laemmli buffer. Protein fractionation for HTP-1::GFP immunoprecipitation experiments was performed from large C. elegans cultures, and 100-300 worms were used to produce Triton soluble and insoluble protein fractions. Protein extracts were run in 10% acrylamide gels, transferred onto nitrocellulose membranes and incubated with the corresponding antibodies. Full details of these methods can be found in

Supplemental Experimental Procedures.

Immunostaining and fluorescence in situ hybridization

Germ lines from young adult hermaphrodites were dissected, fixed and processed as described in Martinez-Perez and Villeneuve (2005). All images were acquired using a

Delta Vision Deconvolution system equipped with an Olympus 1X70 microscope.

Details of these methods, including primary and secondary antibodies used can be found in Supplemental Experimental Procedures.

C. elegans genetics, Plotting of SUN-1 aggregate movement,

Immunoprecipitation, Mass spectrometry and Protein expression methods

Please see Supplemental Experimental Procedures for detail description

18 Acknowledgements

We thank Anne Villeneuve, Monique Zetka, Verena Jantsch and Rueyling Lin for kindly providing antibodies and strains. This work was supported by a BBSRC David

Phillips Fellowship and an MRC core-funded grant to E.M-P.

19 References

Baudat, F., Manova, K., Yuen, J.P., Jasin, M., and Keeney, S. (2000). Chromosome synapsis defects and sexually dimorphic meiotic progression in mice lacking Spo11. Mol. Cell 6, 989-998.

Baudrimont, A., Penkner, A., Woglar, A., Machacek, T., Wegrostek, C., Gloggnitzer, J., Fridkin, A., Klein, F., Gruenbaum, Y., Pasierbek, P., et al. (2010). Leptotene/zygotene chromosome movement via the SUN/KASH protein bridge in Caenorhabditis elegans. PLoS Genet. 6, e1001219.

Blat, Y., Protacio, R.U., Hunter, N., and Kleckner, N. (2002). Physical and functional interactions among basic chromosome organizational features govern early steps of meiotic chiasma formation. Cell 111, 791-802.

Carballo, J.A., Johnson, A.L., Sedgwick, S.G., and Cha, R.S. (2008). Phosphorylation of the axial element protein Hop1 by Mec1/Tel1 ensures meiotic interhomolog recombination. Cell 132, 758-770.

Couteau, F., and Zetka, M. (2005). HTP-1 coordinates synaptonemal complex assembly with homolog alignment during meiosis in C. elegans. Genes Dev. 19, 2744-2756.

Daniel, K., Lange, J., Hached, K., Fu, J., Anastassiadis, K., Roig, I., Cooke, H.J., Stewart, A.F., Wassmann, K., Jasin, M., et al. (2011). Meiotic homologue alignment and its quality surveillance are controlled by mouse HORMAD1. Nat. Cell Biol. 13, 599-610.

Goodyer, W., Kaitna, S., Couteau, F., Ward, J.D., Boulton, S.J., and Zetka, M. (2008). HTP-3 links DSB formation with homolog pairing and crossing over during C. elegans meiosis. Dev. Cell 14, 263-274.

Haenni, S., Ji, Z., Hoque, M., Rust, N., Sharpe, H., Eberhard, R., Browne, C., Hengartner, M.O., Mellor, J., Tian, B., et al. (2012). Analysis of C. elegans intestinal gene expression and polyadenylation by fluorescence-activated nuclei sorting and 3'-end-seq. Nucleic Acids Res. 40, 6304-6318.

Harper, N.C., Rillo, R., Jover-Gil, S., Assaf, Z.J., Bhalla, N., and Dernburg, A.F. (2011). Pairing centers recruit a Polo-like kinase to orchestrate meiotic chromosome dynamics in C. elegans. Dev. Cell 21, 934-947.

Hollingsworth, N.M., Goetsch, L., and Byers, B. (1990). The HOP1 gene encodes a meiosis-specific component of yeast chromosomes. Cell 61, 73-84.

Kauppi, L., Barchi, M., Lange, J., Baudat, F., Jasin, M., and Keeney, S. (2013). Numerical constraints and feedback control of double-strand breaks in mouse meiosis. Genes Dev. 27, 873-886.

20 Labella, S., Woglar, A., Jantsch, V., and Zetka, M. (2011). Polo kinases establish links between meiotic chromosomes and cytoskeletal forces essential for homolog pairing. Dev. Cell 21, 948-958.

Lara-Gonzalez, P., Westhorpe, F.G., and Taylor, S.S. (2012). The spindle assembly checkpoint. Curr. Biol. 22, R966-980.

Leu, J.Y., Chua, P.R., and Roeder, G.S. (1998). The meiosis-specific Hop2 protein of S. cerevisiae ensures synapsis between homologous chromosomes. Cell 94, 375- 386.

Lightfoot, J., Testori, S., Barroso, C., and Martinez-Perez, E. (2011). Loading of meiotic cohesin by SCC-2 is required for early processing of DSBs and for the DNA damage checkpoint. Curr. Biol. 21, 1421-1430.

Luo, X., Fang, G., Coldiron, M., Lin, Y., Yu, H., Kirschner, M.W., and Wagner, G. (2000). Structure of the Mad2 spindle assembly checkpoint protein and its interaction with Cdc20. Nat. Struct. Biol. 7, 224-229.

MacQueen, A.J., Colaiacovo, M.P., McDonald, K., and Villeneuve, A.M. (2002). Synapsis-dependent and -independent mechanisms stabilize homolog pairing during meiotic prophase in C. elegans. Genes Dev. 16, 2428-2442.

MacQueen, A.J., and Hochwagen, A. (2011). Checkpoint mechanisms: the puppet masters of meiotic prophase. Trends Cell Biol. 21, 393-400.

MacQueen, A.J., and Villeneuve, A.M. (2001). Nuclear reorganization and homologous chromosome pairing during meiotic prophase require C. elegans chk-2. Genes Dev. 15, 1674-1687.

Martinez-Perez, E., Schvarzstein, M., Barroso, C., Lightfoot, J., Dernburg, A.F., and Villeneuve, A.M. (2008). Crossovers trigger a remodeling of meiotic chromosome axis composition that is linked to two-step loss of sister chromatid cohesion. Genes Dev. 22, 2886-2901.

Martinez-Perez, E., and Villeneuve, A.M. (2005). HTP-1-dependent constraints coordinate homolog pairing and synapsis and promote chiasma formation during C. elegans meiosis. Genes Dev. 19, 2727-2743.

Penkner, A.M., Fridkin, A., Gloggnitzer, J., Baudrimont, A., Machacek, T., Woglar, A., Csaszar, E., Pasierbek, P., Ammerer, G., Gruenbaum, Y., et al. (2009). Meiotic chromosome homology search involves modifications of the nuclear envelope protein Matefin/SUN-1. Cell 139, 920-933.

Phillips, C.M., and Dernburg, A.F. (2006). A family of zinc-finger proteins is required for chromosome-specific pairing and synapsis during meiosis in C. elegans. Dev. Cell 11, 817-829.

21 Phillips, C.M., Wong, C., Bhalla, N., Carlton, P.M., Weiser, P., Meneely, P.M., and Dernburg, A.F. (2005). HIM-8 binds to the X chromosome pairing center and mediates chromosome-specific meiotic synapsis. Cell 123, 1051-1063.

Rog, O., and Dernburg, A.F. (2013). Chromosome pairing and synapsis during Caenorhabditis elegans meiosis. Curr. Opin. Cell Biol. 25, 349-356.

Rosu, S., Zawadzki, K.A., Stamper, E.L., Libuda, D.E., Reese, A.L., Dernburg, A.F., and Villeneuve, A.M. (2013). The C. elegans DSB-2 protein reveals a regulatory network that controls competence for meiotic DSB formation and promotes crossover assurance. PLoS Genet. 9, e1003674.

Sanchez-Moran, E., Santos, J.L., Jones, G.H., and Franklin, F.C. (2007). ASY1 mediates AtDMC1-dependent interhomolog recombination during meiosis in Arabidopsis. Genes Dev. 21, 2220-2233.

Sato, A., Isaac, B., Phillips, C.M., Rillo, R., Carlton, P.M., Wynne, D.J., Kasad, R.A., and Dernburg, A.F. (2009). Cytoskeletal forces span the nuclear envelope to coordinate meiotic chromosome pairing and synapsis. Cell 139, 907-919.

Severson, A.F., Ling, L., van Zuylen, V., and Meyer, B.J. (2009). The axial element protein HTP-3 promotes cohesin loading and meiotic axis assembly in C. elegans to implement the meiotic program of chromosome segregation. Genes Dev. 23, 1763-1778.

Shin, Y.H., Choi, Y., Erdin, S.U., Yatsenko, S.A., Kloc, M., Yang, F., Wang, P.J., Meistrich, M.L., and Rajkovic, A. (2010). Hormad1 mutation disrupts synaptonemal complex formation, recombination, and chromosome segregation in mammalian meiosis. PLoS Genet. 6, e1001190.

Smolikov, S., Eizinger, A., Schild-Prufert, K., Hurlburt, A., McDonald, K., Engebrecht, J., Villeneuve, A.M., and Colaiacovo, M.P. (2007). SYP-3 restricts synaptonemal complex assembly to bridge paired chromosome axes during meiosis in Caenorhabditis elegans. Genetics 176, 2015-2025.

Tzur, Y.B., Egydio de Carvalho, C., Nadarajan, S., Van Bostelen, I., Gu, Y., Chu, D.S., Cheeseman, I.M., and Colaiacovo, M.P. (2012). LAB-1 targets PP1 and restricts Aurora B kinase upon entrance into meiosis to promote sister chromatid cohesion. PLoS Biol. 10, e1001378.

Woglar, A., Daryabeigi, A., Adamo, A., Habacher, C., Machacek, T., La Volpe, A., and Jantsch, V. (2013). Matefin/SUN-1 phosphorylation is part of a surveillance mechanism to coordinate chromosome synapsis and recombination with meiotic progression and chromosome movement. PLoS Genet. 9, e1003335.

Wojtasz, L., Cloutier, J.M., Baumann, M., Daniel, K., Varga, J., Fu, J., Anastassiadis, K., Stewart, A.F., Remenyi, A., Turner, J.M., et al. (2012). Meiotic DNA double- strand breaks and chromosome asynapsis in mice are monitored by distinct HORMAD2-independent and -dependent mechanisms. Genes Dev. 26, 958-973.

Zetka, M.C., Kawasaki, I., Strome, S., and Muller, F. (1999). Synapsis and chiasma formation in Caenorhabditis elegans require HIM-3, a meiotic chromosome core component that functions in chromosome segregation. Genes Dev. 13, 2258- 2270.

Zhang, W., Miley, N., Zastrow, M.S., MacQueen, A.J., Sato, A., Nabeshima, K., Martinez-Perez, E., Mlynarczyk-Evans, S., Carlton, P.M., and Villeneuve, A.M. (2012). HAL-2 promotes homologous pairing during Caenorhabditis elegans meiosis by antagonizing inhibitory effects of synaptonemal complex precursors. PLoS Genet. 8, e1002880.

23 Figure Legends

Figure 1. HTP-1M127K is not detected at axial elements but regulates SC assembly. (A) Chiasmata are formed between all homologue pairs in WT diakinesis oocytes, but not in htp-1(gk174). Arrowheads in htp-1(fq8) point to two pairs of chromosomes linked by chiasmata. Differences between htp-1(gk174) and htp-1(fq8) are statistically significant by a two-tailed Mann-Whitney test (p< 0.05). (B) Predicted structure of HTP-1 (Phyre2 protein folding prediction) compared to human Mad2

(sequence alignment shows region around HTP-1 M127). (C) Projections of pachytene nuclei demonstrating that HTP-1::GFP, but not HTP-1M127K ::GFP, is loaded to axial elements. (D) Western blot of whole-worm protein extracts showing the presence, at decreased levels, of HTP-1M127K::GFP. (E) Western blot of whole- worm protein extracts probed with anti-HTP-1/2 antibodies. Comparison between htp-2 and htp-1(fq8) htp-2 lanes demonstrates a band corresponding to HTP-1M127K in htp-1(fq8) htp-2. htp-1 htp-2 double mutants were used as a negative control and asterisk indicates a non specific band detected by the HTP-1/2 antibodies. (F) Early prophase nuclei of htp-1(fq8) mutants display reduced SC assembly (see SYP-1 panels) compared to WT and htp-1(gk174). Scale bar = 5 µm in all panels. See also

Figure S1.

Figure 2. HTP-1M127K is a soluble protein that shows impaired interaction with

HTP-3. (A). Western blots of triton soluble and insoluble (DNA bound) protein fractions showing that HTP-1M127K is only found in the soluble fraction (htp-1(fq8) htp-2 lanes), and that a large pool of wild-type HTP-1 accumulates in the soluble fraction (htp-2 lanes). Extracts from htp-1(fq8) htp-2 mutants were prepared from 230 worms, while htp-2 extracts contain 115 worms. HAL-2 was used as a control for the soluble fraction and asterisks indicate a non specific band recognized by HTP-1/2

24 antibodies. (B) Number of peptides of the indicated proteins identified by mass spectrometry following the immunoprecipitation of HTP-1::GFP. The posterior error probability (PEP) for protein identification was: HTP-1 (7.6E-90), HTP-2 (1.7E-116),

HTP-3 (2.1E-122) and HIM-3 (8.2E-269). (C) Immunoprecipitation of HTP-1::FLAG or

HTP-1M127K::FLAG from Escherichia coli that also expressed HTP-3::HA. The amount of HTP-3::HA that is co-immunoprecipitated with HTP-1M127K is reduced compared to wild-type HTP-1. Immunoprecipitation using anti-MBP antibodies was used as a negative control. (D) Projections of transition zone nuclei demonstrating normal loading of HTP-3 in htp-1(fq8) mutants. Scale bar = 5 µm. See also Figure S2.

Figure 3. HTP-1M127K induces chromosome movements and promotes homologue interactions. (A) SUN-1::GFP aggregate movement in transition zone nuclei. Left- hand side column: displacement tracks of SUN-1::GFP aggregates in a single nucleus over 15 minutes. Middle column: each arc represents the distance traveled by a SUN-

1::GFP aggregate inside a nucleus, with larger angles indicating larger distance traveled. Right-hand side column: speeds of SUN-1 aggregates, each line represents the distribution of projected speeds for all SUN-1::GFP aggregates inside a nucleus over 15 minutes. Note that displacement tracks, traveled distance, and speed distribution of htp-1(fq8) mutants are similar to WT controls. (B) Analysis of homologue pairing using FISH probes for regions in chromosomes III and V (left- hand side panels) and anti-HIM-8 antibodies (X chromosome PC region).

Quantification demonstrates higher levels of pairing in htp-1(fq8) than in htp-

1(gk174). Germ line regions correspond to: 1, premeiotic nuclei; 2, premeiotic nuclei and transition zone nuclei; 3 to 6 early to late pachytene. Differences between htp-

1(fq8) and syp-2 in zone 6 are statistically significant (p< 0.005) by a two-tailed

Fisher’s exact test. Scale bar = 5 µm. See also movies S1, S2 and S3.

25 Figure 4. HTP-1M127K regulates meiotic progression by a pathway that requires

HIM-8. (A) PLK-2 aggregates persist in mid pachytene nuclei of htp-1(fq8) mutants.

(B) Quantification of the percentage of nuclei in leptotene-zygotene, as indicated by the presence of more than one PLK-2 aggregate. Note the accumulation of PLK-2- positive nuclei in htp-1(fq8) and htp-1(fq8) syp-2 mutants, but not in htp-1(fq8) him-8.

(C) Early pachytene nuclei of htp-1(fq8) him-8 double mutants display increased SC assembly compared to htp-1(fq8) single mutants. (D) htp-1(fq8) him-8 double mutants display a significant (p< 0.005 by a two-tailed Fisher’s exact test) reduction in the levels of pairing compared to htp-1(fq8). See also Figures S3 and S4.