PerspectiveChromosome Dynamics

When degradation spurs segregation

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Science  27 Jan 2017:
Vol. 355, Issue 6323, pp. 349-350
DOI: 10.1126/science.aam5904

Each cell entering the meiotic divisions that ultimately generate eggs and sperm initiates a complex series of events that bring homologous chromosomes together to ensure their correct subsequent segregation. Anomalies in this process result in changes to chromosome number that are detrimental to life. Central to meiosis in most organisms is the exchange of DNA sequences between homologous chromosomes. These recombination events begin with the formation of programmed breaks in the DNA and can be repaired to form the chromosomal crossovers required for segregation. On pages 403 and 408 of this issue, Prasada Rao et al. (1) and Ahuja et al. (2), respectively, point to the unprecedented involvement of a chromosomally tethered proteasome in negotiating the transition of a recombination intermediate into the chromosomal crossovers required for segregation.

DNA double-strand breaks (DSBs) are catalyzed by a topoisomerase called sporulation-specific protein 11 (Spo11) (3). They can be repaired to generate intermediates that progress into the crossover pathway and create the necessary linkage between homologs. This typically occurs in the environment of the synaptonemal complex, a protein structure that forms between paired chromosomes (4). DSBs are typically produced in excess of the number of crossovers that appear, and an outstanding question is how the crossover-competent precursor is ushered into the crossover pathway, rather than the competing noncrossover pathway through which the majority of DSBs will be repaired (5).

The 26S proteasome is a highly conserved complex of at least 32 subunits that assembles in four to six rings to form the major cellular protease (6). How does the proteasome recognize a target? A conserved family of E3 ligases catalyzes the addition of small moieties onto proteins, which results in degradation of the modified targets. The canonical moiety is the small protein ubiquitin. However, transfer of the small ubiquitin-related modifier (SUMO) to a protein target can provoke a wide number of events, including the degradation (via SUMO-directed E3 ubiquitin ligases), stabilization, and relocalization of the target, as well as disruption of the target's protein-protein contacts (7).

Prasada Rao et al. and Ahuja et al. provide strong evidence that proteasome- and SUMO-directed events physically conspire at meiotic chromosomes to promote crossing over. Both studies show that the proteasome localizes to the meiotic chromosomes of mouse, budding yeast, and nematode model organisms. This suggests evolutionarily conserved functions for the complex during meiotic prophase, when pairs of duplicated chromosomes associate along their entire lengths and recombine. Prasada Rao et al. show that in mouse spermatocytes, chemical inhibitors that disrupt the proteasome, ubiquitylation, or SUMOylation pathways do not detectably affect chromosome structure or meiotic DSB formation, but do disrupt the accumulation of markers of crossover formation. By deleting the a subunit of the yeast proteasome core particle or chemically inhibiting the proteasome, Ahuja et al. arrive at the same conclusion as they molecularly monitored the appearance and kinetics of the DNA strand invasion events that lead to crossover formation. Prasada Rao et al. also used anti-ubiquitin or anti-SUMO antibodies to identify a robust population of modified proteins at mouse chromosome axes. Both studies collectively reveal a meiosis-specific chromosomal requirement for the proteasome and for SUMOylation in the emergence of crossover-competent intermediates that correlate with final events in recombination.

During meiosis, a conserved family of proteins sharing structural features common to SUMO E3 ligases has been implicated as key regulators in the crossover versus noncrossover decision. In mice, RING finger protein 212 (RNF212) forms numerous (∼150) discrete foci along chromosomes early in prophase. These foci then reduce to the few sites of crossover formation (8). Similarly, in budding yeast, localization of ZRT/IRT-like protein 3 (Zip3) also converges to crossover sites (9). Although the structures of RNF212 and Zip3 have implied functions as SUMO E3 ligases, the restriction of RNF212 to crossover sites depends on the proposed function of human enhancer of invasion 10 (HEI10) as an E3 ubiquitin ligase that recognizes SUMOylated proteins and targets them for destruction (8). Given that SUMO modification can stabilize targets, yet at the same time reveal them to the proteolytic machinery, the functional pairing of RNF212 and HEI10 in crossover formation sets in place a potential “race” between SUMO stabilization of a crossover-competent intermediate for crossover formation, and destabilization and noncrossover formation through proteasemediated degradation (see the figure).

Race to recombination

The model shows how SUMO modification may stabilize crossover sites between homologous chromosomes, but also makes these crossover-competent sites visible to the proteasome. Sites that are further ubiquitylated could become destabilized through protease-mediated degradation and are repaired as noncrossovers.

GRAPHIC: K. SUTLIFF/SCIENCE

To investigate the inferred protein activities of RNF212 and HEI10 in vivo, Prasada Rao et al. examined SUMO, ubiquitin, and proteasome localization in individual mouse mutants lacking either putative E3 ligase. The authors indeed found correlations consistent with a “race” model. Nevertheless, several critical questions persist, including the identity of the protein(s) recognized for stabilization by RNF212 and how RNF212 itself is stabilized at crossover sites given HEI10-dependent removal at others. However, not all organisms require the functions of both RNF212 and HEI10. Budding yeast lack an obvious HEI10-like protein, whereas plants (10, 11) and Sordaria fungi (12) lack an RNF212 counterpart and instead rely on a single HEI10 ortholog for crossover formation. Furthermore, Sordaria Hei10 incorporates functional characteristics of the mouse RNF212-HEI10 relay in that it mediates both the acquisition and depletion of SUMOylated chromosomal targets (12). These studies collectively suggest that the effectors of the SUMOylation and ubiquitylation pathways in different organisms may vary in number and biochemical competencies while negotiating the formation of mature crossovers.

Using the generous experimental tools of budding yeast, Ahuja et al. also identified a role for the proteasome in an earlier event in the crossover pathway—bringing the two chromosome homologs together in the first place to exchange their DNA. Although the molecular details of homolog recognition and chromosome pairing remain poorly understood, an emerging model is that the physical restriction of certain chromosome regions within the spatial confines of the nucleus promotes homolog engagement. For example, a widely adopted tactic is the tethering of telomeres of meiotic chromosomes to the nuclear envelope and restricting them to a small space (13). Another event in many species is the physical coupling of centromeres, which precedes pairing of chromosomal arms (14). In yeast, these centromere interactions require the synaptonemal complex component Zip1. Because synaptonemal complex proteins can assemble between nonhomologous sequences, they must be removed from nonhomologously paired centromeres to enable productive homologous pairing interactions. In the absence of a functional yeast proteasome, chromosomes display profound defects in pairing and allow centromere interactions between nonhomologs to inappropriately persist, and consequently disrupt homologous pairing.

Protein degradation has well-known roles in regulating meiotic chromosome segregation. The E3 ligase anaphase-promoting complex (APC) mediates degradation of securin. Securin inhibits separase, an enzyme that cleaves the cohesion complex. Thus, APC activity leads to the liberation of chromosomes for segregation (15). It is now evident from the work of Prasada Rao et al. and Ahuja et al. that the proteasome is also required for earlier events at chromosomes during prophase of meiosis I that facilitate the crossovers required for accurate chromosome segregation. Although the direct targets of the E3 ligases implicated in these studies are still unknown, our understanding of meiotic prophase transitions has been elaborated by the inclusion of timely protein stabilization and degradation as regulatory mechanisms.

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