Cyclindependent kinase promotes formation of ... - Wiley Online Library

Cyclin-dependent kinase promotes formation of the synaptonemal complex in yeast meiosis Zhihui Zhu1,2, Saori Mori1, Hiroyuki Oshiumi1a, Kenichiro Matsuzaki1,2, Miki Shinohara1,2 and Akira Shinohara1,2* 1

Institute for Protein Research, Osaka University, Suita, Osaka 565-0871, Japan Department of Biology, Graduate School of Science, Osaka University, Suita, Osaka 565-0871, Japan

2

Cyclin-dependent protein kinases (CDKs) are required for various cell cycle events both in mitosis and in meiosis. During the meiotic prophase of Saccharomyces cerevisiae, only one CDK, Cdc28, which forms a complex with B-type cyclins, Clb5 or Clb6, promotes not only the onset of premeiotic DNA replication but also the formation of meiotic double-strand breaks (DSBs). In this study, we showed that Cdc28 exhibits punctate staining on chromosomes during meiotic prophase I. Chromosomal localization of Cdc28, dependent on Clb5 and ⁄ or Clb6, is frequently observed in zygotene and pachytene, when formation of the synaptonemal complex (SC) occurs. Interestingly, the CDK localization is independent of DSB formation, but rather dependent on meiosis-specific chromosome components such as Red1, Hop1 and a cohesin subunit Rec8. Compromised CDK activity in meiotic prophase leads to defective SC formation without affecting DSB formation. These results suggest that CDK-dependent phosphorylation regulates meiotic chromosome morphogenesis.

Introduction Meiosis is a specialized form of cell division that produces haploid gametes from diploid cells. Faithful segregation of homologous chromosomes (homologues) at meiosis I requires reciprocal recombination between the homologues together with arm cohesion and monopolar spindle attachment (Marston & Amon 2004). In Saccharomyces cerevisiae, the molecular mechanism of meiotic recombination has been elucidated in detail. The recombination is initiated by the formation of double-strand breaks (DSBs), which is catalyzed by Spo11 (Bergerat et al. 1997; Keeney et al. 1997). A complex containing Mre11, Rad50 and Xrs2, together with Sae2 ⁄ Com1, processes DSB ends to produce a 3¢ single-strand DNA (ssDNA) at the DSB ends (McKee & Kleckner 1997; Prinz et al. 1997). The ssDNA is used by RecA homologues, Rad51 and Dmc1, as well as their accessory factors to Communicated by : Masayuki Yamamoto *Correspondence: [email protected] a Present address : Hokkaido University, School of Medicine, Hokkaido 060-8638, Japan. The first three authors contributed equally to the work.

capture homologous duplex DNA (Bishop et al. 1992; Shinohara et al. 1992; Hayase et al. 2004). Further processing generates two specialized intermediates in the recombination: single-end invasion and double-Holliday intermediates (Schwacha & Kleckner 1995; Hunter & Kleckner 2001). In the end of the recombination, the double-Holliday structures are resolved into products with a reciprocal exchange between parental DNAs. In most organisms, meiotic recombination is tightly coupled with development of the meiosis-specific chromosome structure (Zickler & Kleckner 1999). The most notable structure during meiosis is the synaptonemal complex (SC). SC, which pairs homologues with each other along with entire length, contains tripartite structures consisting of two thick filaments (lateral element; LE) of homologues and ladder-like structure (central element; CE) flanked by LEs. At leptotene, two sister chromosomes are folded into a thick filament with multiple chromatin loops, referred to as axial elements (AE; AE develops into the LE of SC later). In zygotene, an AE starts to pair with a homologous AE partner at a homologous region. A short SC is formed between the paired AEs. At pachytene, SC is extended along entire DOI: 10.1111/j.1365-2443.2010.01440.x

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2010 The Authors Journal compilation 2010 by the Molecular Biology Society of Japan/Blackwell Publishing Ltd.

CDK on meiotic chromosomes

chromosomes, resulting in full synapsis between homologues. In the budding yeast, CEs in the SC consist of various proteins, including Zip1, a large coiled–coiled protein (Sym et al. 1993) and the small ubiquitin-like modifier (SUMO) chain (Cheng et al. 2006; Hooker & Roeder 2006). LEs ⁄ AEs contain various meiosis-specific proteins, Red1, Hop1 and Mre4 ⁄ Mek1, as well as a meiosis-specific cohesin complex with Rec8 (Rockmill & Roeder 1988, 1991; Hollingsworth et al. 1990; Klein et al. 1999). Hop1 and Mek1 ⁄ Mre4 are a DNA-binding protein and a serine ⁄ threonine protein kinase, respectively (Hollingsworth et al. 1990; Wan et al. 2004). Genetic and physical interactions are observed among Hop1, Red1 and Mek1 ⁄ Mre4 proteins (Hollingsworth & Ponte 1997). In a current model, it is believed that Hop1 recruits Red1 on chromosomes, and in turn Mek1 for its activation. A meiosis-specific cohesion containing Rec8 is a scaffold component on the Hop1-Red1-Mek1-assembly (Klein et al. 1999). Interestingly, most of components of SCs including Zip1, Hop1, Red1, Mek1 ⁄ Mre4 and Rec8 are a phosphoprotein. However, identity of kinase(s) responsible for the phosphorylation of these proteins as well as functional significance of the phosphorylation remains to be characterized. Cyclin-dependent kinases (CDKs) are involved in various cellular processes during meiosis as well as mitosis. In mouse spermatocytes, Cdk2 and Cdk4 associate with meiotic chromosomes as immunologically detectable foci (Ashley et al. 2001). Cdk2 and Cdk4 foci are often colocalized with a pro-crossover protein Mlh1 and an ssDNA-binding protein, replication protein-A (RPA), respectively (Baker et al. 1996; Plug et al. 1997). Furthermore, cdk2 knockout mice show defects in SC formation (Ortega et al. 2003). Only short SC structures are observed in cdk2) ⁄ ) spermatocytes. In the mutant nucleus, a LE component, SYCP3, forms an aggregate. These findings suggest a role for the mouse CDK in SC formation and ⁄ or meiotic recombination. The budding yeast bears only one CDK. Cdc28, a catalytic core of CDK, forms a complex with B-type cyclins, Clb1, -2, -3, -4, -5 and -6 as well as G1 cyclins, Cln1, -2 and -3. Both B-type and G1 cyclins show some redundancy in their function, although their expression patterns differ temporally. All Clns are expressed only in G1 and are necessary for G1–S transition. Clb5 and -6 are expressed from G1 to S phase, Clb3 and -4 from late S phase to G2 and Clb1 and -2 are mainly expressed at G2 ⁄ M phase. Five Btype cyclins, except for Clb2, are expressed not only

in mitosis but also in meiosis (Grandin & Reed 1993; Dahmann & Futcher 1995). Clb1, -3 and -4 are expressed around pachytene, and their expression is regulated by a meiosis-specific transcription factor, Ndt80 (Chu et al. 1998). Recently, a novel regulatory mechanism to restrict the translation of Clb1 and Clb3 in MI and MII, respectively, has been reported (Carlile & Amon 2008). Cdc28 bound to Clb5 and ⁄ or Clb6, referred as to S-CDK, plays a role in entry into the premeiotic S phase (Stuart & Wittenberg 1998). In contrast, a Cdc28 complex with Clb1, -3 or -4 facilitates exit from pachytene, as well as progression of MI and MII (Benjamin et al. 2003). It is shown that S-CDK regulates DSB formation by promoting priming phosphorylation of Mer2 protein, which is bound to Spo11 (Smith et al. 2001; Henderson et al. 2006). In addition, it is likely that S-CDK also controls ssDNA formation on DSB ends by phosphorylating Sae2 ⁄ Com1 endonuclease (Manfrini et al. 2010). In this study, we analyzed the role of CDK in recombination and chromosome structure during meiosis. Cdc28 was found to bind to chromosomes during meiotic prophase I. The localization of Cdc28 on meiotic chromosomes depends on its partner, Clb5 and Clb6. Meiosis-specific chromosomal proteins also promote assembly of Cdc28 foci on chromosomes. We also found that the inactivation of S-CDK activity after DSB formation leads to a defect in SC formation. These findings suggest that CDK-dependent phosphorylation plays a role in chromosome morphogenesis during meiosis.

Results Localization of CDK on meiotic chromosomes

It is shown that mouse CDK binds to meiotic chromosomes in spermatocytes (Ashley et al. 2001). To analyze localization of the budding yeast CDK during meiosis, the endogenous CDC28 gene encoding a catalytic subunit of CDK was tagged with three influenza hemagglutinin (HA) tags at the C-terminus of its open reading frame. The CDC28-3HA cell grows normally at 30 C in the vegetative stage and does not show any temperature sensitivity in mitotic growth (data not shown). CDC28-3HA homozygous diploid cells form spores very efficiently, with a wild-type level of spore viability (98%) at 30 C. This indicates that HA-tagged Cdc28 is functional in meiosis as well as mitosis. To examine the association of Cdc28 with meiotic chromosomes, meiotic nuclear spread of CDC28-3HA


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diploid cells was probed with anti-HA antibody. Cdc28-3HA shows punctate staining on meiotic chromosomes (Fig. 1A). This staining was not observed for cells without the HA-tag on Cdc28 (Fig. 1A). Cdc28 foci are predominantly observed in meiotic prophase I, but not in meiosis I and -II or the mitotic phase. Foci start to appear after 2.5-h

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incubation with sporulation medium (SPM), peak at 4 h and then disappear after 8-h incubation (Fig. 1B). An average number of Cdc28 foci per a focus-positive nucleus (foci >5) from 3 to 5 h is 18.1 ± 10.5 (n = 155), and the maximum number is 56. Binding of Cdc28 to chromosomes is not attributed to an increased amount of Cdc28 protein

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Figure 1 Cyclin-dependent kinase localization on meiotic chromosomes. (A) Meiotic nuclei with or without CDC28-3HA were spread at different time points and stained with anti-HA antibodies and DAPI. Cdc28-3HA and DNA are shown in green and blue, respectively. Representative images at each time point are shown. White bars, one micrometer. (B) Kinetics of Cdc28-focus formation. Nuclei containing more than five Cdc28 (green) or Rad51 (red) foci were counted and plotted at each time point (green). (C) Expression of Cdc28-3HA protein during meiosis was analyzed by Western blotting using the anti-HA antibody. Meiotic cells of wild type without the tag (4 h) were also analyzed. (D) Spread nuclei of CDC28-3HA cells were stained with anti-HA and anti-Rad51 antibodies. Cdc28, Rad51 and DNA were shown in green, red and blue, respectively. White bars, one micrometer. (E) Nuclear spreads of CDC28-3HA cells were stained with anti-HA (green) and anti-Zip1 (red) antibodies. Nuclei were classified into three groups based on Zip1 staining pattern. Class I, Zip1 dotty foci (i); Class II, short linear Zip1 and dots (ii); Class III, fully-elongated linear Zip1 (iii). (F) Nuclear spreads of CDC28-3HA were stained with anti-HA (green) and antiRed1 (red) antibodies. (G) Spread nuclei of CDC28-3HA cells were stained with anti-HA (green) and anti-Zip3 (red) antibodies.

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during meiosis, as the amount of Cdc28-3HA protein is constant during meiosis (Fig. 1C). To study the location of Cdc28 foci on chromosomes, colocalization Cdc28 with a RecA homologue, Rad51, was examined. Foci containing Rad51 mark sites of ongoing recombination (Bishop 1994; Miyazaki et al. 2004). Rad51 foci appear and disappear during meiotic prophase. Double staining analysis showed that most Cdc28-focus-positive nuclei contain Rad51 foci (Fig. 1D). Kinetics of Cdc28-focus-positive nuclei are similar to those of Rad51-focus-positive nuclei, although Rad51 dissociates from slightly earlier than Cdc28 (Fig. 1B). These findings support the idea that Cdc28 localizes on meiotic chromosomes at prophase I when meiotic recombination occurs. Cdc28 foci show some colocalization with Rad51. However, the colocalization is not extensive with a frequency of only 34% (per Cdc28 foci). This is significantly higher than random colocalization in controls (approximately 13%). Spatial relationship of Cdc28 foci with Zip1, a CE component of the SC, was analyzed by double staining using anti-HA and anti-Zip1 antibodies (Fig. 1E). Zip1 staining was classified into three classes, Class I (dots), -II (partial lines with dots) and –III (lines), which corresponds with leptotene, zygotene and pachytene, respectively (Sym et al. 1993). More than half (54%) of Class I nuclei contain Cdc28-3HA foci, but Cdc28-3HA foci do not colocalize with Zip1 foci in this class. At this time, Cdc28 foci are faint and the number is fewer compared to later stages. In Class II nuclei, Cdc28 foci are much brighter than those in Class I stage, and 95% of Class II nuclei are positive for Cdc28 focus. Cdc28 tends to colocalize with Zip1 in Class II nuclei, and many Cdc28 foci are located in the vicinity of short Zip1 lines (Fig. 1Eii). A large majority of Class III nuclei (86%) contain Cdc28 foci. Cdc28 foci are often located on or near Zip1 lines at this stage (Fig. 1Eiii). Consistent with this colocalization with SC, Cdc28 foci are localized on line-like staining of Red1, which is an axial element (AE) component of the SC (Fig. 1F). An aggregate of Zip1, called polycomplex (PC), is frequently observed in mutants deficient in SC formation. Zip1 PC is also seen in wild type, but with a much reduced frequency. Strong Cdc28 staining is also observed in Zip1 PC (Fig. 1Eii, arrow) only in the HA-tagged strain. These findings suggest that Cdc28 is a component of SCs. Zip1 elongation depends on a protein complex called ZMM or SIC (synaptic initiation complex;

Zip1, Zip2, Zip3, Mer3, Msh4, Msh5, Spo22 ⁄ Zip4, Spo16 (Borner et al. 2004; Shinohara et al. 2008). Zip3 protein, a member of ZMM ⁄ SIC, which marks initiation sites of chromosome synapsis, forms foci on chromosomes from leptotene to pachytene (Agarwal & Roeder 2000). Colocalization of Cdc28 with Zip3 proteins showed that a total of 57% of Cdc28 foci (per Cdc28) were juxtaposed or colocalized with Zip3 foci (Fig. 1G). This suggests that Cdc28 may work with Zip3 and also with the other ZMM ⁄ SIC components necessary for SC elongation as well as meiotic recombination. CDK-focus formation requires meiotic chromosome components

To determine the genetic requirement of Cdc28focus formation, localization of Cdc28-3HA was studied in various mutants defective in meiosis (Fig. 2). Spo11 is a catalytic subunit of a protein complex that mediates DSB formation. Substitution of tyrosine 135 with phenylalanine, the spo11-Y135F mutation, completely eliminates DSB formation (Bergerat et al. 1997). Interestingly, Cdc28 foci were still observed in the spo11-Y135F mutant as in wild type. Kinetics of Cdc28 foci in the mutant were similar to those in wild type, but at a slightly reduced frequency (Fig. 2A,B), suggesting that Cdc28 loading on chromosomes does not depend on DSB formation, i.e. meiotic recombination. Meiosis-specific components are incorporated into chromosomes in a DSB-independent manner. Next, we examined the effect of mutations in genes that encode a component of meiosis-specific chromosomes on Cdc28-focus formation. Red1, an AE component of the SC (Rockmill & Roeder 1988), is required for normal Cdc28 localization. The red1 mutant shows a significant defect in Cdc28-focus formation (Fig. 2A,B), although the mutant showed some residual Cdc28 foci on chromosomes at late prophase. Hop1, which is also a component of the chromosome axis, binds to and recruits Red1 to chromosomes (Hollingsworth et al. 1990). The hop1 mutant almost abolished Cdc28-focus formation (Fig. 2A,B). These results suggest that Red1 and Hop1, particularly Hop1, play a critical role in the recruitment of CDK to meiotic chromosomes. Interestingly, a null mutation in the MEK1 ⁄ MRE4 gene does not affect the formation of Cdc28 foci (Fig. 2A,B). Mre4 ⁄ Mek1 kinase is thought to collaborate with and requires Red1 and Hop1 for its binding to chromosomes (Wan et al. 2004).


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Figure 2 Meiotic chromosome components promotes Cdc28 binding to chromosomes. (A, B) Kinetics of Cdc28-focus formation were analyzed in various strains: wild-type, spo11-Y135F, red1, hop1, mek ⁄ mre4, rec8, ndt80 single mutants and clb5 clb6 double mutant. Typical staining images of Cdc28 foci at 4-h meiosis are shown for each mutant and wild type (A). Cdc28, green; DNA, blue. Percentage of Cdc28-focus-positive cells was counted and plotted at each time point (B). Wild type (CDC28-3HA), open circles with gray lines; each mutant with CDC28-3HA, closed circles. White bars, one micrometer. (C) Meiotic cell lysates from various strains with the CDC28-3HA allele at various times were analyzed by Western blotting using anti-HA antibody (upper panel) and antitubulin (bottom panel).

Rec8 is a component of the meiosis-specific cohesin complex and plays a fundamental role in chromosome morphogenesis during meiosis (Klein et al. 1999). The rec8 mutant greatly decreases Cdc28-focus formation (Fig. 2A,B). It is likely that the effect of the rec8 on Cdc28 localization may be indirect through Red1-Hop1, whose proper localization requires Rec8. We also confirm that the deletion of the HOP1, RED1 and REC8 genes does not affect a level of Cdc28 protein in a cell (Fig. 2C). Taken together, these results suggest that meiotic chromosome axis plays a critical role in efficient recruitment of CDK on chromosomes. 1040

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CDK localization to meiotic chromosomes requires S-phase cyclins

During early meiotic prophase I, Cdc28 works with two B-type cyclins, Clb5 and ⁄ or Clb6 (Dirick et al. 1998; Stuart & Wittenberg 1998; Benjamin et al. 2003). Clb5 and Clb6 are expressed earlier in the meiotic pro-phase, with redundant functions in premeiotic S phase and DSB formation. To determine the role of B-type cyclins in Cdc28-localization, a clb5 clb6 double mutant was analyzed for Cdc28focus formation. Interestingly, Cdc28-focus formation was severely compromised in the clb5 clb6 double



mutant, suggesting that Clb5 and ⁄ or Clb6 is necessary for efficient Cdc28 loading (Fig. 2B). The clb5 clb6 double mutant does not form SC, but does form axial elements, showed by Red1 staining (Smith et al. 2001) and does not form meiotic DSBs (Smith et al. 2001; Henderson et al. 2006). As the spo11 mutant is proficient in Cdc28-focus formation, it is unlikely that the effect of clb5 clb6 mutations on Cdc28 localization is attributed to inability of the mutant to form either SCs or DSBs. Rather, we postulate that Cdc28 binds to the meiotic chromosome axis when the kinase forms a complex with partner B-type cyclins, Clb5 or Clb6. Alternatively, phosphorylation of target(s) by Cdc28-Clb5 or -Clb6 might promote binding of Cdc28 to the chromosomes. We also examined the effect of the mutation of the NDT80 gene that encodes a transcriptional activator for expression of middle sporulation genes such as B-type cyclins such as Clb1, Clb3 and Clb4 (Chu & Herskowitz 1998). The ndt80 mutant can form Cdc28 foci (Fig. 2A; OS unpublished results), suggesting that the mitotic B-type cyclins are not required for CDK localization on chromosomes. CDK activity is necessary for the formation of the SC

The CDK activity is required for various meiotic processes, including premeiotic DNA replication, the formation and resection of DSBs, the exit from pachytene as well as meiosis I and II (Benjamin et al. 2003; Henderson et al. 2006; Manfrini et al. 2010). As described previously, cytological analysis of Cdc28 strongly suggests the role of the kinase in meiotic chromosome morphogenesis such as SC formation. However, it is very difficult to evaluate an actual role of the CDK in SC formation using the mutant, because the CDK activity may affect indirectly through the other events, particularly ones before SC formation. More complicated, in the budding yeast, SC formation is tightly coupled with meiotic recombination such as DSB formation. Mutants defective in DSB formation and processing, which depends on CDK, also show a defect in SC formation (e.g. the spo11 and dmc1 mutants; Bishop et al. 1992). To know the role of CDK in SC formation, we used an inhibitors-sensitive allele of the CDC28, which encodes a catalytic subunit of the kinase; the cdc28-as1 mutant (Bishop et al. 2000; Benjamin et al. 2003). We added an inhibitor, 1NM-PP1, at different times after the induction of meiosis and monitored Rad51-focus formation as a marker for DSB formation.

Without the inhibitor, the cdc28-as1 mutant shows almost normal kinetics of various meiotic events (Fig. S1A in Supporting Information, Fig. 3). At 4 h, up to 70% of the cells are positive for Rad51 foci. When the cdc28-as1 cells were incubated with 0.5 lM of 1NM-PP1 from 0 h, there is no focus formation of Rad51 on chromosomes after 4-h incubation, consistent with the idea that the CDK activity is essential for DSB formation and ⁄ or resection (Henderson et al. 2006; Huertas et al. 2008). As reported previously (Benjamin et al. 2003), FACS and DAPI staining analyses show that there is normal meiotic DNA replication under the condition but arrest at MI (by the addition of 0.5 lM of inhibitor; data not shown). When the inhibitor was added at either 2 or 2.5 h and chromosome spreads were prepared at 4 h, both spreads show Rad51-focus formation (Fig. S1A in Supporting Information). Forty-two percent and 79% of cells treated at 2 and 2.5 h are positive for the Rad51 foci, respectively. Similar staining was seen for foci containing a meiosis-specific MutS homologue, Msh5 (Fig. S1A in Supporting Information), which also binds to recombination intermediates. This indicates that the addition at 2 h reduced DSB formation and ⁄ or resection slightly but 2.5 h addition did not. Based on these results, we concluded that the addition of the inhibitor at 2.5- h incubation allows us to analyze the role of CDK in events after the formation DSBs. Addition of the 1NM-PP1 at 2.5 h slightly delays the progression of meiotic S phase (Fig. S1B in Supporting Information), suggesting a role of the kinase in progression of S phase or firing late origins. When the kinetics of Rad51 foci were analyzed (Fig. 3A,D), the addition of the inhibitor at 2.5 h delays appearance of Rad51 slightly, possibly attributed to delay in S-phase progression. In the presence of the inhibitor, Rad51 persists on chromosomes with slow disassembly at late times, suggesting the role of CDK in late events of meiotic recombination after the formation of Rad51 ensemble on DSBs. Because the addition of 1NM-PP1 to the cdc28-as1 ensures the formation of DSBs, we next examined the SC formation by immunostaining of SC components, Zip1 and Red1. The inactivation of CDK after DSB formation does not affect the loading of Red1 (Fig. 3B). However, Zip1 assembly is abnormal under the condition (Fig. 3A,C). In the presence of the CDK inhibitor, dots (Class I) and partial lines (Class II) of Zip1 are predominantly observed, but a fraction containing fully elongated Zip1 lines (Class III) is severely reduced compared


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Figure 3 Cyclin-dependent kinase activity is necessary for synaptonemal complex (SC) formation. (A, B) The cdc28-as1 mutant treated with 0.5 lM of 1 NM-PP1 at 2.5 h was chromosome spread at different times and stained with different combination of antibodies. (A) Rad51 (green) and Zip1 (red); Zip3 (green) and Zip1 (red). (B) Mer3 (green) and Zip1 (red); small ubiquitin-like modifier (green) and Red1 (red). White bars, one micrometer. (C) Kinetics of SC formation in wild type and the cdc28-as1 mutant in the presence and absence of 1 NM-PP1 were analyzed by Zip1 staining. Staining patterns were classified as described in main text. Class I, blue, Zip1 dotted foci; Class II, green, short linear Zip1 stretches; Class III, red, nuclei containing long linear Zip1 staining. (D) Kinetics of Rad51-focus and Zip1-PC formation were analyzed for wild-type and the cdc28-as1 mutant by immunostaining of Rad51 (blue) and Zip1–PC (red).

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to that in the absence of the inhibitor. At 4 h, only 3.5% shows Class III in the presence of the inhibitor, whereas in its absence 32% of the cdc28-as1 is class III. Furthermore, 54% of cells at 4 h treated with 1NM-PP1 accumulate aggregates of Zip1, PCs, compared to 26% in its absence (Fig. 3D). We also examined the other CE marker, SUMO. SUMO binds to Zip1 in meiosis and shows line-like staining (Cheng et al. 2006; Hooker & Roeder 2006). Under CDK inactivation, like Zip1, SUMO shows dotty staining and accumulates abnormal aggregates, which is not seen in the absence of the inhibitor (Fig. 3B). These findings suggest that CDK activity is necessary for proper SC formation, probably the elongation of CEs. In addition, under CDK inactivation condition, the disassembly of SC is delayed by more than 3 h compared to the absence of the inhibitor. This is consistent with the delay of disassembly of Rad51. Because a recent study shows that Cdc28 is not necessary for SC disassembly (Sourirajan & Lichten 2008), this delay of SC disassembly may be caused by defects in earlier events such as recombination or SC assembly. We also analyzed the effect of the addition of 1 NM-PP1 at 3.5 h on Zip1 elongation. The addition of the inhibitor does not affect drastically a fraction of Class III, thus Zip1 elongation (Fig. S2 in Supporting Information). However, there is a significant increase in Zip1-PC at 4 h, an half hour after the addition, suggesting a weak defect in SC assembly even under the condition. The ZMM protein promotes SC assembly. We also studied the effect of CDK depletion on the localization of ZMM proteins such as Zip3 and Mer3. In wild type, Zip3 and Mer3 show dotty staining on chromosomes (Agarwal & Roeder 2000; Shinohara et al. 2008). Under CDK inactivation condition, wild-type levels of Mer3 and Zip3 foci were seen, although the CDK inactivation induces accumulation of PCs containing Mer3 and Zip3 with Zip1, which is possibly caused by Zip1 elongation defects (Fig. 3A,B). These results suggest that CDK activity is not necessary for initial loading of ZMM components such as Zip3 and Mer3 as a focus. CDK may phosphorylate several chromosomal proteins in meiosis

To look for a target of CDK phosphorylation in SC formation, we searched a CDK phosphorylation site containing S ⁄ TP, more strictly S ⁄ TPXK ⁄ R sequence, among proteins involved in SC formation and ⁄ or

meiotic recombination. For comparison, we also listed the frequency of S ⁄ TQ sites in these proteins, which are the target sites for Tel1 ⁄ Mec1 (ATM ⁄ ATR) kinases (Table 1). Mer3, Red1, Zip3 and Zip1 contain 10, 6, 6, 4 S ⁄ TP sites, respectively, which are over-representative compared to other proteins. Previous systematic biochemical analysis to identify yeast CDK substrates in vitro showed that CDK (Cdc28-Clb2) can phosphorylate Zip1, Zip2, Hop1 and Tid1 in vitro (Ubersax et al. 2003). It is shown that Red1 protein is phosphorylated during meiosis (Wan et al. 2004; Cheng et al. 2006). Table 1 Numbers of CDK and ATM ⁄ ATR consensus sites in various proteins involved in meiotic recombination and SC formation Proteins

Amino acid length

TP or SP

SQ or TQ

Zip3 Zip1 Zip2 Mer3 Spo22 ⁄ Zip4 Spo16 Msh4 Msh5 Rec8 Hop1 Red1 Mek1 ⁄ Mre4 Ndj1 Csm4 Dmc1 Mei5 Sae3 Tid1 ⁄ Rdh54 Mnd1 Hop2 Rad51 Rad52 Rad55 Rad57 Rad54 Rfa1 (RPA) Rfa2 (RPA) Rfa3 (RPA) Mlh1 Mlh3 Psy3 Csm2 Shu1 Shu2

482 875 704 1187 975 198 878 901 680 605 827 497 352 156 334 222 91 958 219 218 400 471 406 460 898 621 273 122 769 715 242 213 150 223

6 4 1 10 1 2 1 0 1 2 6 3 0 0 1 0 1 5 1 0 1 3 1 4 3 1 2 2 3 1 1 2 1 0

4 9 4 8 8 2 3 8 4 8 1 3 3 2 2 1 1 12 0 2 4 0 5 4 9 2 2 2 6 5 1 3 1 0

CDK, cyclin-dependent kinase; RPA, replication protein-A; SC, synaptonemal complex.


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Meiotic yeast cell lysates contained fuzzy bands of Red1 (Fig. 4A,B), as previously described (Wan et al. 2004). The fuzzy band species of Red1 appear to be phosphorylated forms, as the treatment with a phosphatase increased the mobility as a sharp band (Fig. 4B), consistent with the previous report (Wan et al. 2004). To examine the involvement of CDK in Red1 phosphorylation, the cdc28-as1 allele was used (Bishop et al. 2000). In the cdc28-as1 mutant without the inhibitor, fuzzy Red1 bands are observed on a Western blot as in wild type, but with a reduced intensity (Fig. 4A). (A)

This might be attributed to a reduced activity of Cdc28-as1 kinase. In the presence of 0.5 lM of 1 NMPP1, the upper bands migrate faster than those in its absence. In contrast, the inhibitor does not affect mobility of Red1 in wild-type cells. These findings indicate that Red1 modification is dependent on the CDK activity. In meiosis, Zip1 shows a band shift (Fig. 4C,D). This putative phosphorylation of Zip1 occurs 2- to 6-h incubation with SPM (Fig. 4C). The appearance of fuzzy Zip1 is compromised in the clb5 clb6 mutant, indicating that Clb5 and Clb6 are required for

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Figure 4 Cyclin-dependent kinase-dependent phosphorylation of Red1 and Zip1. (A) Meiotic cell lysates from various strains (wild type, red1 and cdc28-as1) at 4 h were analyzed by Western blotting using anti-Red1 antibody. The cdc28-as1 and wild-type cells were treated with 0.5 lM of 1 NM-PP1 after 2-h incubation with sporulation medium (SPM). (B) Immunoprecipitates of Red1-3HA by anti-HA antibody were incubated with calf intestine phosphatase as described in Experimental Procedures and analyzed by Western blotting using anti-HA antibody. (C) Meiotic cells of different strains with Zip1-3HA were analyzed for Zip13HA expression by Western blotting using anti-HA antibody. (D) Meiotic cell lysates from various strains with the ZIP1-3HA (wild type, zip1 and cdc28-as1) at 4 h were analyzed by Western blotting using anti-HA antibody. The cdc28-as1 and wild-type cells were treated with 0.5 lM of 1 NM-PP1 after 2-h incubation with SPM. (E) Meiotic cell lysates from various strains with the MER3-3HA allele (wild-type and cdc28-as1) at various times were analyzed by Western blotting using anti-HA antibody (upper panel) and antitubulin (bottom panel). The cdc28-as1 and wild-type cells were treated with 0.5 lM of 1 NM-PP1 after 2-h incubation with SPM. (F) Meiotic cell lysates from various strains with the ZIP3-3Flag allele (wild-type and cdc28-as1) at various times were analyzed by Western blotting using anti-Flag antibody (upper panel) and antitubulin (bottom panel). The cdc28-as1 and wildtype cells were treated with 0.5 lM of 1 NM-PP1 after 2-h incubation with SPM.

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efficient phosphorylation of Zip1 (Fig. 4C). Consistent with this, in the presence of the inhibitor, the cdc28-as1 mutant shows reduced mobility of Zip1 protein (Fig. 4D). Interestingly, the hop1, but not spo11 mutants, shows a slight delay in the appearance of Zip1 phosphorylation (Fig. 4C), suggesting that Hop1, maybe through the loading of CDK, might be required for efficient Zip1 phosphorylation. Because Zip1 is very unstable protein in cell lysates, only fixation of cells with trichloroacetic acid (TCA) gave clear bands of a full-length Zip1 protein on Western blots. For this technical difficulty, it was very hard to characterize the phosphorylation status of Zip1 in cell lysates [e.g. by calf intestine phosphatase (CIP) treatment]. We also checked whether CDK inactivation affects the mobility of Zip3 and Mer3, which contains 6 and 10 putative CDK sites, respectively (Fig. 4E,F). Both proteins exhibit fuzzy bands on Western blot, suggesting possible post-translational modification of the proteins, possibly phosphorylation. However, CDK inactivation does not affect the mobility of Zip3 and Mer3. These findings suggest that most of modification of Zip3 and Mer3 in lysates is independent on CDK. Recently, it was reported that knocking out putative phosphorylation sites (including CDK sites) of Red1 does not show any apparent meiotic defects (Wang personal communication; Lin et al. 2009). We then focused on four Zip1 CDK sites, three of which are located in the C-terminal region of Zip1 (S89, S763, S783 and S828) and constructed two mutants of the ZIP1 gene; the zip1-3SA (S763A, S783A and S828A) and zip1-4SA mutants (S89A, S763A, S783A and S828A). The zip1-3SA and zip1-4SA mutants show 93% and 94% spore viability, respectively. The zip1-3SA and zip1-4SA mutants show 1-h delay in the onset of MI, suggesting a weak defect in prophase I (Fig. 5A,B). Indeed, the zip1-4SA mutant is proficient in both assembly of Rad51 and the formation of full-length SCs but is partially defective in disassembly of Rad51 and SC (Fig. 5C–E). These results suggest that the putative CDK phosphorylation of Zip1 plays little role in SC formation. By tagging of Zip1-4SA protein with HA, we confirmed that band shifts of Zip1-4SA-HA are less intense than wild-type Zip1HA (Fig. S3 in Supporting Information).

Discussion In this study, we showed meiosis-specific localization of CDK on chromosomes. The CDK binds to

chromosomes during meiotic prophase I when recombination and SC formation occur, suggesting the role of CDK in these processes. Indeed, compromised CDK activity leads to the abnormal SC formation and a defect in late recombination events. As described here, CDK in the budding yeast binds to meiotic chromosomes like in mammalian cells (Ashley et al. 2001). The binding of CDK to the chromosomes depends on meiosis-specific chromosomal proteins, Rec8, Red1 and Hop1. As Rec8 facilitates normal Red1 assembly (Klein et al. 1999), the role of a meiosis-specific cohesin containing Rec8 might be indirect. Red1 and Hop1 form a complex both in vivo and in vitro (Hollingsworth & Ponte 1997). It is proposed that Hop1, which can bind to DNAs, recruits Red1 on chromosomes. This suggests that Red1-Hop1 complex, or possibly Red1 alone, might be a platform for recruitment of the CDK complex. This is consistent with the finding that Red1 is a phosphoprotein, with phosphorylation dependent on CDK. Recently, Diffley and colleagues elegantly showed the interaction of a cyclin, Clb2, with phosphorylated sequences of target protein(s), such as Cdc6 by CDK (Mimura et al. 2004). The same might be true for Clb5 (or Clb6). For example, a phosphorylated region of Red1 by CDK or other kinase might create a binding site for Clb5 or Clb6, which may recruit Cdc28 to chromosomes as a complex. However, further analysis is necessary to determine the interaction of the CDK with Red1. Chromosome-bound CDK, which is a small fraction of total kinase in a cell, seems to promote local phosphorylation of target proteins, which in turn plays a critical function on meiotic chromosomes such as SC formation. Although recruitment of CDK on chromosomes depends on chromosome axis proteins, Cdc28 forms a focus on chromosomes axes covered with Red1, suggesting that there is the other regulatory mechanism to target CDK on a specific region of chromosomes as foci. Local events such as recombination, which coupled with chromosome morphogenesis, might trigger local concentration of CDK. Because Cdc28-focus formation is independent on SPO11 function, the involvement of the recombination is less likely. The other chromosomal event(s) such as DNA replication may play a role in the recruitment of CDK on chromosome axis. CDKs are involved in various cellular processes including cell cycle regulation. In meiosis, the CDK controls various events. Pre-DNA replication and cell cycle progression of MI and MII depend on CDK


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(B)

(C)

(D)

(E)

Figure 5 Meiotic phenotypes of the zip1 mutants in putative cyclin-dependent kinase sites. (A, B) Meiotic cell cycle progression of wild-type (A, B; open circlers) and zip1-3SA (A; closed circles), zip1-4SA (B; closed circles) cells were examined by DAPI staining. (C) Kinetics of Rad51-focus appearance and disappearance were analyzed in wild-type (open circles) and the zip1-4SA mutant (closed circles). (D) Kinetics of Zip1-poly-complex were analyzed in wild-type (open circles) and the zip1-4SA mutant (closed circles). (E) Nuclear spreads from wild type and zip1-4SA were stained with anti-Zip1. Zip1 staining was classified in Fig. 3C. Percentage of each class was calculated for mutants at each time point.

activity as in mitotic cell cycle (Benjamin et al. 2003; Carlile & Amon 2008). In addition, the CDK is involved in meiosis-specific events such as recombination. In meiotic recombination, direct CDKdependent phosphorylation of Mer2 promotes the formation of meiotic DSBs, which triggers the initiation of the recombination (Henderson et al. 2006). Recent studies showed that CDK is involved in downstream of DSB formation in mitosis and meiosis. CDK-dependent phosphorylation of Sae2 ⁄ Com1 is necessary for the activation of end processing activity of the protein for ssDNA formation (Huertas et al. 1046

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2008). It is suggested that the phosphorylation of Sae2 ⁄ Com1 promotes the removal of Spo11 from DSB end for the processing (Manfrini et al. 2010). In S. cerevisiae, SC formation as well as later steps of meiotic recombination depends on early events such as DNA replication and DSB formation. To know the role of CDK in SC formation and late recombination events, we have to inactivate the CDK activity specifically after the DSB formation. Indeed, using the chemical-sensitive allele of the cdc28, cdc28-as1 (Bishop et al. 2000; Benjamin et al. 2003), we could inactivate the activity after the DSB



formation and possibly the end processing, which is supported by focus formation of Rad51 that binds to ssDNA. Under this condition, the elongation of Zip1 lines, thus SC assembly, is severely hampered, whereas the formation of chromosome axis structure such as AE looks normal, judging from normal localization of axis-associated proteins such as Red1. This suggests that the CDK is necessary for the elongation of CE of SC, rather than formation of AE. In addition, we showed normal loading of two ZMM components Mer3 and Zip3, which promote Zip1 elongation, as foci on chromosomes. These findings suggest that the CDK activity, thus the phosphorylation of a target protein(s) by CDK, controls SC elongation per se, rather blocking of the binding of the SC assembly factors of ZMM to the chromosomes. Our results strongly suggest that the CDK phosphorylates proteins involved in SC formation. Zip1 is a strong candidate for the CDK target, because in vitro biochemical studies showed that CDK phosphorylates Zip1 (Ubersax et al. 2003). Consistent with this, we found that not only Red1 but also Zip1 protein shows CDK-dependent band shift on Western blots. However, the mutants in three and four candidate CDKphosphorylation sites of Zip1; zip1-3SA, zip1-4SA look like wild type in SC assembly with a subtle defect in SC disassembly (Fig. 5). Therefore, we conclude that phosphorylation of Zip1 by CDK plays little role in SC assembly. One possibility to explain this is there are multiple targets by CDK for SC formation. Inactivation of a single target is not sufficient to see clear defects in SC formation. Alternatively, it is possible to think that the effect of CDK inactivation is indirect through defective recombination and ⁄ or chromosome movement processes. CDK inactivation after ssDNA formation indeed delays disassembly of Rad51 and Msh5 foci (Fig. 3 and SM unpublished), suggesting that CDK may play a role in processing in recombination intermediates. Previously, it is shown that CDK is not required for the resolution of dHJs for crossover formation (Sourirajan & Lichten 2008). Taken together, these results suggest that CDKdependent phosphorylation may control the activity of proteins working in steps from ssDNA to dHJ formation. Because the ZMM proteins are involved not only in this process but also in SC assembly, the ZMM protein might be a candidate for CDK-dependent phosphorylation. Interestingly, some ZMM proteins including Mer3 and Zip3 contain many CDK sites (Table 1). Because CDK inhibition does not affect the loading of Mer3 and Zip3 (Fig. 3), CDK plays a role after the loading of these ZMM proteins.

However, we did not observe CDK-dependent phosphorylation for both of the proteins in cell lysates (Fig. 4). As discussed earlier, this does not necessarily mean that Mer3 or Zip3 is not a CDK target. Because only proteins on chromosomes might be phosphorylated by chromosome-bound CDK. More complicated, it is known that, during meiosis of the budding yeast, SC formation is tightly coupled with chromosome movement and rearrangement (Koszul & Kleckner 2009). Therefore, the deficiencies in chromosome movements may also induce abnormal SC formation. It is possible that a protein involved in meiotic chromosome dynamics is also a CDK target (H.B.D.P. Rao and A.S., unpublished results). Further studies are necessary to evaluate a direct CDK target for SC formation.

Experimental procedures Strains and plasmids All yeast strains used in this study were derivatives of SK1 (Table S1 in Supporting Information). The C-terminus of Cdc28, Zip1, Zip3, Mer3 and Red1 was tagged with three HA (or Flag) sequences using PCR-mediated epitope tagging methods (De Antoni & Gallwitz 2000). The red1, mek1 ⁄ mre4, rec8, ndt80 and spo11-Y135F single mutants as well as clb5 clb6 double mutant are described (Xu et al. 1995; Bergerat et al. 1997; Hollingsworth & Ponte 1997; Stuart & Wittenberg 1998; Klein et al. 1999). A hop1 deletion was made using plasmid, pNH46-1, a kind gift from Nancy Hollingsworth. cdc28as1 strains were made from SK1 background strains, KBY437 and -438, kind gifts from Kirsten Benjamin (Benjamin et al. 2003). The zip1-3SA and zip1-4SA mutants were constructed by PCR-mediated site-directed mutagenesis. Detailed methods including oligonucleotides are available on request.

Immunostaining Chromosome spreads were prepared and immuno stained as described previously (Shinohara et al. 2000). Stained samples were observed using an epifluorescent microscope (BX51; Olympus) with a 100 X objective (NA; 1.3). Images were captured by CCD camera (Cool Snap; Roper) and then processed using IP lab (Sillicon) and Photoshop (Adobe) software. For focus- and class-counting, more than 100 nuclei were counted at each time point. Anti-HA antibody (16B12; Babco), anti-Flag M2 (Sigma), anti-SUMO (Biomol) and anti-Rad51 (Shinohara et al. 1992) were used for staining. Antisera against Red1, Zip1 and Zip3 were raised using recombinant proteins purified from E. coli (Shinohara et al. 2008). Anti-Mer3 serum was made against a recombinant Mer3 fragment protein produced in bacteria by the manufacture (MBL. Co. Ltd.).


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Immunoprecipitation and Western blotting Yeast cell lysates were prepared by glass-bead disruption method using the glass beater (Yasui-Kiki, Co. Ltd). Lysates were incubated with magnetic beads (Dybnal M280, GE Health-care) coated with anti-HA for 12 h and washed extensively. In the case of the treatment of alkaline phosphatase, a buffer was exchanged with CIP butter (20 mM Tris–HCl [pH8.8], 1 mM MgCl2). Ten units of CIP (TAKARA Co. Ltd) were added to the reaction mixture and incubated for 1 h. Bound fractions were analyzed on a SDS-PAGE gel, transferred to a nylon membrane and probed with antibodies.

Western blotting using TCA fixation To visualize Zip1-HA, Zip3-Flag and Mer3-HA without degradations efficiently, cell lysates were prepared from cells fixed with TCA. Briefly, 1.5 mL of meiotic culture was harvested. After washing with water, cell suspensions were mixed with 100 lL of 1.85 N NaOH and then placed on ice for 15 min. TCA (150 lL of 55%) was then added, and the mixture incubated for a further 15 min on ice. Pellets were recovered, suspended in 100 lL of 1· cracking buffer, then dissolved completely at 65 C for 10 min. Samples were analyzed by SDS-PAGE using standard methods.

Acknowledgements We thank Drs N. Hollingsworth, H. Araki, K. Benjamin and K. Ohta for materials used for this study. We are grateful to members of this laboratory, particularly Dr H. Sasanuma and T. Usui for critical reading of the manuscript. This study is supported by a Grant-in-Aid from the Ministry of Education, Science, Sports and Culture, Japan (SM, MS and AS) and as well as a grant from Asahi-Glass Science Foundation, Uehara Science Foundation, Mochida Medical Science Foundation and Takeda Science Foundation to AS. ZZ was supported by fellowships from JASSO and Osaka University. KM is a JSPS fellow (DC2).

Author contribution ZZ, SM and HO contributed equally to the works described in the paper. SM, HO and ZZ characterized cdc28 mutants and HO and ZZ carried out Cdc28 cytology. ZZ, HO and KM checked in vivo phosphorylation. HO and MS constructed strains and prepared antisera. AS wrote the paper and SM, ZZ, HO, KM and MS checked the manuscript.

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Figure S1 Characterization of the cdc28-as1 mutant.

Received: 1 April 2010 Accepted: 24 June 2010

Additional Supporting Information may be found in the online version of this article.

Supporting Information/Supplementary material

Please note: Wiley-Blackwell are not responsible for the content or functionality of any supporting materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article.

Figure S2 CDK activity inactivation lead abnormal SC formation. Figure S3 Western analysis of Zip1-4SA-3HA protein. Table S1 Strain list

The following Supporting Information can be found in the online version of the article:

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