Shaping meiotic prophase chromosomes: cohesins and synaptonemal complex proteins
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- Revenkova, E. & Jessberger, R. Chromosoma (2006) 115: 235. doi:10.1007/s00412-006-0060-x
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Recent progress in elucidating the function of synaptonemal complex (SC) proteins and of cohesins in meiocytes made possible, in particular, through the analysis of mice deficient in SC or cohesin proteins has significantly enriched our understanding of how meiotic chromosome architecture is determined. Cohesins and the SC proteins act together in generating the characteristic axis-loop structure of meiotic chromosomes, their pairing into bivalents, their ability to recombine, and to be properly segregated. This minireview attempts to summarize the current knowledge with a focus on higher eukaryotic systems and to ask questions that ought to be addressed in the future.
This special issue of Chromosoma dedicated to the synaptonemal complex (SC) provides thorough insights and discussion of many aspects of the SC. Therefore, no introduction into the SC itself is being attempted in this chapter. Cohesins, however, need to be introduced briefly. For a more detailed description, recent reviews may be consulted (Firooznia et al. 2005; Hagstrom and Meyer 2003; Hirano 2005; Jessberger 2002).
Unfortunately, in the literature, the term “cohesin” is sometimes used as a synonym for “the” cohesin complex and/or for individual cohesin complex subunits. This creates confusion because the impression may be generated that individual cohesin subunits, such as REC8, represent all of cohesins in a meiocyte, which certainly is wrong. Thus, we recommend to clearly emphasize what is being discussed: an individual cohesin subunit, a specific cohesin complex, or the sum of all cohesin complexes in a meiocyte.
As their name implies, cohesin complexes serve to ensure sister chromatid cohesion, which is established during S-phase, maintained until metaphase, and dissolved at the metaphase/anaphase transition to allow orderly segregation of the chromatids. It was recently discovered that yeast cohesin can also be loaded in the G2 phase at the sites of double-strand breaks and sister chromatid cohesion can be established de novo (Strom et al. 2004; Strom and Sjogren 2005; Unal et al. 2004). In mitotically dividing mammalian cells, accumulation of cohesin at the sites of double-strand breaks was also observed (Kim et al. 2002). It is not yet entirely clear whether in meiocytes cohesin complexes can be loaded onto chromosomes also after S phase or whether there is replacement of a particular cohesin complex with a different one.
“Mitotic” cohesin proteins such as SMC1α, RAD21, and STAG2 can be detected in rodent meiocytes by immunofluorescence and their localization to chromosomes strongly suggests a role in meiotic chromosome dynamics (Eijpe et al. 2000; Parra et al. 2004; Prieto et al. 2002; Revenkova et al. 2001; Xu et al. 2004). The presence of sister chromatid cohesion—albeit impaired—in mice deficient in meiosis-specific cohesin proteins SMC1β or REC8 (Bannister et al. 2004; Revenkova et al. 2004; Revenkova and Jessberger 2005; Xu et al. 2005), indicates partial functional overlap of mitotic and meiosis-specific cohesins.
Meiosis-specific SMC1β can be detected on chromosomes by immunofluorescence from leptotene up to metaphase II (Revenkova et al. 2001). In mouse, it is associated both with the chromosome arms and centromeres during prophase I. SMC1β starts to visibly disappear from the chromosome arms and concentrate at the centromeres in diplotene. At metaphase I, very little of SMC1β can be detected by immunofluorescence in the arms but a significant amount is present at the centromeres up to metaphase II (Kouznetsova et al. 2005; Revenkova et al. 2001).
Mitotic SMC1α localizes to chromosomes in prophase I (Eijpe et al. 2000) but in contrast to SMC1β, SMC1α does not accumulate at the centromeres in diplotene (Revenkova et al. 2001). REC8 and STAG3 coimmunoprecipitate with SMC1α from testis nuclear extracts (Revenkova et al. 2004) indicating that complexes of SMC1α and meiosis-specific non-SMC cohesin proteins exist and may play a role in sister chromatid cohesion, mostly in the chromosome arms. While immunofluorescence staining with anti-SMC1α antibodies was not detected beyond prophase I, SMC1β-based complex(es) remains present until metaphase/anaphase II (Kouznetsova et al. 2005; Revenkova et al. 2001). It is interesting to note that in rat spermatocytes, REC8 behaves partly independent of SMC1β and SMC3 for it appears earlier in preleptotene before premeiotic S-phase, does not localize to bridges that appear in diplotene between the homologs, are supposedly sites of chiasmata, and disappears later than SMC1β and SMC3 from the chromosome arms (Eijpe et al. 2003). However, like the SMC1β/SMC3, it stays at the centromeres until metaphase/anaphase II (Eijpe et al. 2003). Localization pattern of RAD21 is similar to the one of SMC1β and SMC3 but evidence for the presence of RAD21 at metaphase II centromeres is controversial (Parra et al. 2004; Xu et al. 2004). Thus, it is possible that the SMC1β/SMC3 dimer forms two complexes with REC8 or RAD21 and both localize to the arms and the centromeres, but only the RAD21 complex localizes to the chromosome bridges (Fig. 1).
The exact kinetics, composition, and localization of each cohesin complex throughout meiosis, however, is unknown.
How do SC proteins and cohesin complexes act together to promote the fascinating events that happen with chromosomes during meiosis?
The role of cohesins in assembly of the SC
The SYCP3 gene can be seen expressed during embryogenesis (Chuma and Nakatsuji 2001), but its function at this stage is unclear. In meiosis, SYCP3 and SYCP2 proteins can be detected in leptotene and SCP1 appears at leptotene/zygotene transition (Eijpe et al. 2003). In mice and rats, the kinetics of expression and chromosome association of SYCP3 is generally very similar to that of cohesin complex(es) based on SMC1β and SMC3. REC8 is an exception because it appears earlier (Eijpe et al. 2003). In Schizosaccharomyces pombe, REC8 is expressed and associates with the chromosomes during premeiotic S phase (Watanabe and Nurse 1999). Little is known about the chromosome association of the SMC1α/SMC3/RAD21/SA1 or SA2 complex during the premeiotic S phase in mice. The meiosis-specific SMC1β and STAG3 cohesins can hardly be detected during early leptotene but are detectable on chromatin in late leptotene, paralleling axial element appearance (Prieto et al. 2001). On the XY pair, only STAG3 and SYCP3, but no SYCP1, can be detected.
The largely parallel assembly of cohesins and SC proteins on the meiotic chromosomes raises the question of whether there is a requirement for cohesins in the assembly of the SC, i.e., the association of SC proteins with the chromatin.
In lower organisms such as Saccharomyces cerevisiae (Klein et al. 1999) or Caenorhabditis elegans (Pasierbek et al. 2001), the presence of REC8 is critical for SC formation. Drosophila melanogaster does not have an obvious REC8 homologue. However, C(2)M, a protein distantly related to REC8, localizes in fruit flies to lateral elements (LEs) close to the central region (Anderson et al. 2005). It is not involved in sister chromatid cohesion but interacts with SMC3 and is required for SC formation (Heidmann et al. 2004; Manheim and McKim 2003). The C(2)M localizes very closely to the position of C-terminal domain of the transverse filament (TF) protein C(3)M (Anderson et al. 2005) suggesting that an interaction of these two proteins may link TFs to LEs.
The variety and redundancy of cohesin complexes present in meiotic cells is clearly larger in vertebrates, allowing the SC to form in the absence of particular meiosis-specific cohesins, for example, in SMC1β−/− or REC8−/− mice (Bannister et al. 2004; Revenkova et al. 2004; Xu et al. 2005). In these mice, SCs are formed with normal kinetics. Their structure, however, is strikingly altered. Most obviously, they are of only half the length as in wild-type meiocytes. It also appears as if in the absence of SMC1β or REC8, more “gaps” in SCs that are otherwise clearly stainable with anti-SYCP3 exist.
This is unexpected given the high degree of lateral compaction, which requires SC proteins. Should the extreme compaction cause dissociation of SYCPs at certain regions of the axes? And is a balance between SYCP-mediated compaction and limited ability of SYCPs to associate on hypercompacted axes one of the parameters limiting compaction? In wild-type cells, the presence of SMC1β cohesin apparently limits longitudinal compaction, but the mechanism remains to be shown.
Because in cohesin-deficient meiocytes chromosome axes are shortened, the question arises on what happens to the chromatin that is normally packaged into those axes. Is there less chromatin included to axial structures or is it simply more condensed? Measuring the size of chromatin loops that emerge from the axes in SMC1β−/− mutant first provided indications that there may be less chromatin in the axes because the loop chromatin maximally extends almost twice as far in the absence of SMC1β as in a wild-type situation (Revenkova et al. 2004)(Fig. 2). However, because compaction within the loop chromatin itself could have also changed in SMC1β−/− meiocytes—although in an opposite fashion as in the axes, i.e., extended—it is not yet clear whether reduced packaging in the axes is indeed the only reason for the altered axis to loop ratio in these mutant animals.
The role of SC proteins in the association of cohesins with chromosomes
Even in SYCP3-deficient spermatocytes that show no axial elements (AEs), cohesins assemble in core-like structures as shown by immunolocalization of SMC1α, SMC3, and STAG3 (Pelttari et al. 2001). In the absence of SYCP3, SYCP2 does not localize to chromosomes, but the TF protein SYCP1 still forms short stretches, which colocalize with cohesin cores, i.e., filamentous cohesin structures that run alongside the SC in wild-type cells (Pelttari et al. 2001). Cohesin cores in SYCP3−/− spermatocytes are two to fourfold longer than those of wild type (Kolas et al. 2004). In SYCP3−/− oocytes, the extended SYCP1 fibers formed along cohesin cores are also two times longer than in wild type (Yuan et al. 2002)(Fig. 2). This striking feature of meiotic chromosomes formed in the absence of SYCP3 reveals a role of AE proteins in chromosome compaction, which also directly affects the cohesin core.
SYCP3 also stabilizes the association of cohesins with chromatin because the absence of SYCP3 causes premature disassembly of cohesin core as seen in diplotene oocytes (Kouznetsova et al. 2005).
The absence of the TF protein SYCP1 does not impede the assembly of AEs in spermatocytes and apparently does not have an effect on the association of cohesin with chromatin (de Vries et al. 2005). In SYCP1−/− spermatocytes, homologous chromosomes align along their entire length and connect by axial associations but do not synapse. SYCP1-deficiency significantly affects double-strand break repair and blocks crossover formation (de Vries et al. 2005), but it remains to be clarified whether and how the SYCP1 and cohesins act together in recombination, as mice deficient in either show recombination failures.
How do cohesins and SC proteins act together?
The recent description of mice deficient in either SC proteins such as SYCP3 or SYCP1 or in the meiosis-specific cohesins SMC1β or REC8 shed light on how these proteins affect various aspects of chromosome structure. Their effect on the axes to loop ratio and on compaction of chromosomes was already mentioned.
Another meiosis-specific chromosomal event affected by the absence of SYCP3 or SMC1β cohesin is telomere attachment to nuclear periphery and bouquet formation. In late leptotene, telomeres attach to the nuclear envelope and at leptotene/zygotene transition, form a cluster so that chromosomes acquire a bouquet-like configuration. The fraction of SYCP3−/− spermatocytes that shows telomere bouquets is increased about threefold compared to the wild type (Liebe et al. 2004). This can be caused by extended duration of the bouquet stage, but indicates that probably, SYCP3−/− meiocytes have no deficiency in formation of bouquets. Like in the wild type, in the absence of SYCP3, all telomeres localize to the nuclear envelope from leptotene onward. In contrast, SMC1β−/− spermatocyte nuclei at leptotene and zygotene contain on average of four telomeres, which appear unattached to the nuclear envelope (Revenkova et al. 2004). This can be interpreted as a failure to attach or a failure to maintain attachment for the normal period of time. An explanation for the absence of full attachment can be seen in the reduced length of SCs in SMC1β−/− meiocytes: The shortest SCs may simply be too short to attach on both ends. Experiments to test this hypothesis are ongoing but preliminary results indicate that the attachment failure is not a direct consequence of chromosome length (R. Jessberger, unpublished data). Alternatively, SMC1β may have a telomere-specific function, which needs to be explored. Recently, deletion of REC8 protein was reported to cause prolongation of the bouquet stage in yeast (Trelles-Sticken et al. 2005). It is important to note that in mouse telomere proteins TRF1, TRF2, and RAP1 and telomere repeats colocalize with meiosis-specific cohesin STAG3 and with SYCP2 (Liebe et al. 2004).
There are many and, indeed, central open questions that concern the relationship between SCPs and individual cohesins and their complexes.
This implicates the question on how the loop to axis ratio is codetermined by SCPs and cohesins and how these two factors interplay with other proteins and complexes that certainly also shape the meiotic chromosomes. Not only questions relating to the axes, but also to the structure of the loops themselves, should be addressed. How is the structure of chromatin within the loops affected by cohesins and what other proteins do they interact with there?
Furthermore, it is not yet clear whether centromeric cohesins are required for SCP association with the centromeres, or vice versa, or whether there is no interdependence at all.
Recent data provide evidence that the SMC1β cohesin is required for meiotic recombination and stabilization of chiasma positions (Hodges et al. 2005). Do SCPs collaborate with cohesins in the process of crossover formation and if so, how? Finally, the apparent telomere-specific role of SMC1β cohesin begs the question on how the structure SC at the telomeres and its transition into the attachment plates depend on particular cohesins.
For many of those questions to be answered it ought to be revealing to determine chromosome behavior in meiocytes that are deficient in both, SYCP3 and SMC1β. Would this result in axes of normal length or would the cores be twice longer like in the SYCP3−/− cells, indicative of a dominant effect of SYCP3? Would normal levels of telomere attachment be restored or would a putative telomere-specific function of SMC1β be dominant? Because double mutant mice are currently being created, the answers to these questions are on the horizon and will further clarify the complex interplay between cohesin complexes and the SC.
Work in the authors’ laboratory is supported by grants from the NIH and from the DFG.