Roberto J. Pezza

Synaptonemal Complex Components Persist at Centromeres and Are Required for Homologous Centromere Pairing in Mouse Spermatocytes

Recent studies in simple model organisms have shown that centromere pairing is important for ensuring high-fidelity meiotic chromosome segregation. However, this process and the mechanisms regulating it in higher eukaryotes are unknown. Here we present the first detailed study of meiotic centromere pairing in mouse spermatogenesis and link it with key events of the G2/metaphase I transition. In mouse we observed no evidence of the persistent coupling of centromeres that has been observed in several model organisms. We do however find that telomeres associate in non-homologous pairs or small groups in B type spermatogonia and pre-leptotene spermatocytes, and this association is disrupted by deletion of the synaptonemal complex component SYCP3. Intriguingly, we found that, in mid prophase, chromosome synapsis is not initiated at centromeres, and centromeric regions are the last to pair in the zygotene-pachytene transition. In late prophase, we first identified the proteins that reside at paired centromeres. We found that components of the central and lateral element and transverse filaments of the synaptonemal complex are retained at paired centromeres after disassembly of the synaptonemal complex along diplotene chromosome arms. The absence of SYCP1 prevents centromere pairing in knockout mouse spermatocytes. The localization dynamics of SYCP1 and SYCP3 suggest that they play different roles in promoting homologous centromere pairing. SYCP1 remains only at paired centromeres coincident with the time at which some kinetochore proteins begin loading at centromeres, consistent with a role in assembly of meiosis-specific kinetochores. After removal of SYCP1 from centromeres, SYCP3 then accumulates at paired centromeres where it may promote bi-orientation of homologous centromeres. We propose that, in addition to their roles as synaptonemal complex components, SYCP1 and SYCP3 act at the centromeres to promote the establishment and/or maintenance of centromere pairing and, by doing so, improve the segregation fidelity of mammalian meiotic chromosomes.

A SUMO-ubiquitin relay recruits proteasomes to chromosome axes to regulate meiotic recombination.

Proteasomes and SUMO wrestle chromosomes Meiosis is the double cell division that generates haploid gametes from diploid parental cells. Pairing of homologous chromosomes during the first meiotic division ensures that each gamete receives a complete set of chromosomes. The proteasome, on the other hand, is a molecular machine that degrades proteins tagged for destruction within the cell (see the Perspective by Zetka). Ahuja et al. show that the proteasome is also involved in ensuring that homologous chromosomes pair with each other during meiosis. Rao et al. show that the SUMO (small ubiquitin-like modifier) protein, ubiquitin, and the proteasome localize to the axes between homologous chromosomes. In this location, they help mediate chromosome pairing and recombination between homologs. Science, this issue p. 349, p. 408; see also p. 403 The cellular proteostasis machinery helps direct chromosome pairing and recombination during germ cell division. Meiosis produces haploid gametes through a succession of chromosomal events, including pairing, synapsis, and recombination. Mechanisms that orchestrate these events remain poorly understood. We found that the SUMO (small ubiquitin-like modifier)–modification and ubiquitin-proteasome systems regulate the major events of meiotic prophase in mouse. Interdependent localization of SUMO, ubiquitin, and proteasomes along chromosome axes was mediated largely by RNF212 and HEI10, two E3 ligases that are also essential for crossover recombination. RNF212-dependent SUMO conjugation effected a checkpointlike process that stalls recombination by rendering the turnover of a subset of recombination factors dependent on HEI10-mediated ubiquitylation. We propose that SUMO conjugation establishes a precondition for designating crossover sites via selective protein stabilization. Thus, meiotic chromosome axes are hubs for regulated proteolysis via SUMO-dependent control of the ubiquitin-proteasome system.

Mechanistic insights into the role of Hop2–Mnd1 in meiotic homologous DNA pairing

The Hop2–Mnd1 complex functions with the DMC1 recombinase in meiotic recombination. Hop2–Mnd1 stabilizes the DMC1-single-stranded DNA (ssDNA) filament and promotes the capture of the double-stranded DNA partner by the recombinase filament to assemble the synaptic complex. Herein, we define the action mechanism of Hop2–Mnd1 in DMC1-mediated recombination. Small angle X-ray scattering analysis and electron microscopy reveal that the heterodimeric Hop2–Mnd1 is a V-shaped molecule. We show that the protein complex harbors three distinct DNA binding sites, and determine their functional relevance. Specifically, the N-terminal double-stranded DNA binding functions of Hop2 and Mnd1 co-operate to mediate synaptic complex assembly, whereas ssDNA binding by the Hop2 C-terminus helps stabilize the DMC1-ssDNA filament. A model of the Hop2-Mnd1-DMC1-ssDNA ensemble is proposed to explain how it mediates homologous DNA pairing in meiotic recombination.

Solution Structure and DNA-binding Properties of the Winged Helix Domain of the Meiotic Recombination HOP2 Protein.

Background: HOP2 protein promotes recombination and is required for meiotic chromosome synapsis. Results: The N terminus of HOP2 has a winged head DNA binding structure. Conclusion: The solution structure of the winged head DNA binding domain integrates biochemical and functional aspects of HOP2 recombinational function. Significance: Determining the three-dimensional structure of HOP2 is crucial to understand the mechanism of HOP2 action. The HOP2 protein is required for efficient double-strand break repair which ensures the proper synapsis of homologous chromosomes and normal meiotic progression. We previously showed that in vitro HOP2 shows two distinctive activities: when it is incorporated into a HOP2-MND1 heterodimer, it stimulates DMC1 and RAD51 recombination activities, and the purified HOP2 alone is proficient in promoting strand invasion. The structural and biochemical basis of HOP2 action in recombination are poorly understood; therefore, they are the focus of this work. Herein, we present the solution structure of the amino-terminal portion of mouse HOP2, which contains a typical winged helix DNA-binding domain. Together with NMR spectral changes in the presence of double-stranded DNA, protein docking on DNA, and mutation analysis to identify the amino acids involved in DNA coordination, our results on the three-dimensional structure of HOP2 provide key information on the fundamental structural and biochemical requirements directing the interaction of HOP2 with DNA. These results, in combination with mutational experiments showing the role of a coiled-coil structural feature involved in HOP2 self-association, allow us to explain important aspects of the function of HOP2 in recombination.

Genome instability and embryonic developmental defects in RMI1 deficient mice.

RMI1 forms an evolutionarily conserved complex with BLM/TOP3α/RMI2 (BTR complex) to prevent and resolve aberrant recombination products, thereby promoting genome stability. Most of our knowledge about RMI1 function has been obtained from biochemical studies in vitro. In contrast, the role of RMI1 in vivo remains unclear. Previous attempts to generate an Rmi1 knockout mouse line resulted in pre-implantation embryonic lethality, precluding the use of mouse embryonic fibroblasts (MEFs) and embryonic morphology to assess the role of RMI1 in vivo. Here, we report the generation of an Rmi1 deficient mouse line (hy/hy) that develops until 9.5 days post coitum (dpc) with marked defects in development. MEFs derived from Rmi1(hy/hy) are characterized by severely impaired cell proliferation, frequently having elevated DNA content, high numbers of micronuclei and an elevated percentage of partial condensed chromosomes. Our results demonstrate the importance of RMI1 in maintaining genome integrity and normal embryonic development.

Heterochromatin and the Synaptonemal Complex Maintain Homologous Centromere Interactions in Mouse Spermatocytes

Summary In meiosis, crossovers between homologous chromosomes link them together. This enables them to attach to microtubules of the meiotic spindle as a unit, such that the homologs will be pulled away from one another at anaphase I. Homologous pairs can sometimes fail to become linked by crossovers. In some organisms, these non-exchange partners are still able segregate properly. In several organisms, associations between the centromeres of non-exchange partners occur in meiotic prophase. These associations have been proposed to promote segregation in meiosis I. But how centromere pairing could promote subsequent proper segregation is unclear. Here we report that meiotic centromere pairing if chromosomes in mouse spermatocytes allows the formation of an association between chromosome pairs. We find that peri-centromeric heterochromatin connections tether the centromeres of chromosome pairs after dissolution of centromere paring. Our results suggest that, in mouse spermatocytes, heterochromatin maintains the association of chromosome centromeres in the absence crossing-over.How achiasmate chromosomes become aligned and segregate during meiotic prophase is an important issue in eukaryotic genetics. We show that chromosomes are frequently transmitted properly in the absence of a chiasma in mouse spermatocytes, suggesting the existence of back-up systems aiding achiasmate chromosome segregation. Here, we report mechanisms that maintain the alignment of homologous pairs prior to disjunction, which may enable achiasmate chromosomes to segregate from each other in meiosis I. We present direct physical evidence showing centromere interactions provide a stable physical link between chromosome pairs in the absence of recombination and that pericentromeric heterochromatin tethers the centromeres of chromosome pairs after dissolution of synaptonemal complex-mediated centromere paring. Our results establish for the first time in a mammal a role for heterochromatin in maintaining the alignment of chromosomes in the absence of euchromatic synapsis and suggest that homologous recognition may lead to segregation, even in the absence of recombination. Highlights Non-recombination based systems promote achiasmate chromosome disjunction in mice. Two novel mechanisms maintain homologous pairs alignment just before segregation. One is directed by SYCP1 at centromeres; the other by centromeric heterochromatin. A comprehensive analysis of pre- and post-synaptic centromere interactions.

Shugoshin protects centromere pairing and promotes segregation of non-exchange partner chromosomes in meiosis

Faithful chromosome segregation during meiosis I depends upon the formation of connections between homologous chromosome pairs. For most chromosome pairs a connection is provided by crossovers. The crossover creates a link that allows the pair to attach to the meiotic spindle as a unit, such that at anaphase I, the partners will migrate away from one another. Recent studies have shown that some chromosome pairs that fail to experience crossovers become paired at their centromeres in meiotic prophase, and in some organisms, this pairing has been shown to promote proper segregation of the partners, later, at anaphase I. Centromere pairing is mediated by synaptonemal complex (SC) proteins that persist at the centromere when the SC disassembles. Here, using experiments in mouse and yeast model systems, we tested the role of shugoshin in promoting meiotic centromere pairing by protecting centromeric synaptonemal components from disassembly. The results show that shugoshin protects centromeric SC in meiotic prophase and also promotes the proper segregation in anaphase of partner chromosomes that are not joined by a crossover.