Mer2 is chromatin-associated and is phosphorylated during meiosis
was isolated in two screens for genes involved in meiotic recombination (Engebrecht et al., 1990
; Malone et al., 1991
). Null mer2
mutants do not make DSBs, leading to spore inviability. The predicted 35.5 kDa protein has no obvious motifs aside from a region of heptad repeats (Rockmill et al., 1995
homologs are present in database sequences from six more Saccharomyces
species and two other ascomycetes, with 25–90% amino acid sequence identity (Supplemental Figure 1A and 1B
An intriguing feature of MER2
is its meiosis-specific regulation: its transcript has an intron with a non-canonical 5′ splice site that is efficiently spliced only during meiosis under the control of Mer1, a meiosis-specific RNA-binding protein (Engebrecht et al., 1991
; Nandabalan and Roeder, 1995
). Each putative MER2
ortholog has an intron within the same poorly conserved region (Supplemental Figure 1A
). Splicing regulation may be conserved, because the five homologs most closely related to S. cerevisiae
have the same non-canonical splice site (Supplemental Figure 1C
) and because Mer1 homologs are also present (data not shown). Conservation of sequence and (apparently) regulation suggests that Mer2 has a similar role in meiosis in these organisms.
To characterize S. cerevisiae
Mer2, we epitope-tagged the protein at the C-terminus with multiple copies of the myc epitope. MER2myc
complemented a mer2
null mutant and supported nearly normal levels of meiotic recombination (see Experimental Procedures). Given its role in DSB formation, Mer2 is expected to localize to meiotic chromosomes. Nuclear spreads were double-stained for Mer2myc and Zip1, a component of the synaptonemal complex (SC) (Sym et al., 1993
). The Zip1 pattern indicates the stage in meiosis (). In leptonema (prior to SC formation), Mer2myc was on chromatin in many foci (), which places Mer2 on chromosomes prior to or at the time DSBs are formed. Staining was brighter and patchy in zygonema (during SC formation) () and remained on chromosomes but became weaker in pachynema (full-length SC), i.e., past the period when DSBs form (). In contrast to sudden loss of Rec102 and Rec104 from chromosomes at mid-pachynema (Kee et al., 2004
), Mer2myc persisted, similar to Ski8 and Spo11 (Arora et al., 2004
; Prieler et al., 2005
). Mer2myc staining only partially overlapped with Zip1 (), suggesting that much of Mer2myc localized to chromatin loops rather than chromosome axes, similar to Rec102, Rec104, and Ski8 (Arora et al., 2004
; Kee et al., 2004
). Bulk differential extraction confirmed association of Mer2 with meiotic chromatin (described below in ) and revealed that chromatin association did not require any of the other DSB proteins tested (Spo11, Ski8, Mei4, Rec114, Rec102, Rec104, Xrs2, Mre11, data not shown).
Localization of Mer2 to premeiotic and meiotic chromosomes
Physical and functional characterization of Mer2 phosphorylation
Because Mer2 was already abundant on leptotene chromosomes, we also examined earlier times. Mer2myc was present in extracts from premeiotic cells immediately upon transfer to sporulation medium and from cycling haploid and diploid cells (), consistent with small amounts of spliced message detected in non-meiotic cells (Engebrecht et al., 1991
). Mer2myc formed a few distinct foci (7.3 ± 5.1, mean ± SD, n=107) on chromosomes from premeiotic cells (). Foci were also found on nuclei from haploid cells cultured identically to the premeiotic cells, and on nuclei from vegetatively growing haploid and diploid cells (data not shown). No foci were detected on spreads from an untagged diploid control (). Thus, Mer2 forms chromatin-associated complexes in non-meiotic cells. The function of these complexes is not known.
Phosphorylation of Mer2 in meiotic prophase, independent of DSB formation
Nuclear spreads were also prepared from cells early in meiosis (2 hr after transfer to sporulation medium) and double-stained for Mer2myc and Red1. Red1 is a meiosis-specific protein associated with chromosomes prior to SC formation and can be used to define early meiotic stages (Smith and Roeder, 1997
). In nuclei with a few Red1 foci, the number of Mer2myc foci had increased substantially from premeiotic levels (; 75 ± 54 foci per spread, n=7). Nuclei with more Red1 immunostaining structures showed even more foci, reaching 150 or more per nucleus (; 166 ± 53 foci per spread, n=8). Thus, progression into premeiotic S phase is associated with formation of numerous Mer2 foci. Aside from the Rad50-Mre11-Xrs2 complex, none of the other DSB proteins can be detected on chromosomes so abundantly this early (Arora et al., 2004
; Kee et al., 2004
; Prieler et al., 2005
, and our unpublished results).
In pre- and non-meiotic cells, Mer2myc migrated on SDS-PAGE as a single band at ~54 kDa, larger than its predicted size of ~43 kDa. Similar aberrant mobilities of tagged proteins were observed previously (Kee et al., 2004
). After transfer to sporulation medium, Mer2myc levels increased and the mobility changed so that multiple forms were seen (). Slower migrating species first appeared between ~2–3 hr, at or before the beginning of SC formation, and the protein persisted past the first meiotic division (). Phosphatase treatment converted slow-migrating to rapidly migrating forms, revealing that Mer2 is a phosphoprotein (). Mer2myc phosphorylation was similar to wild type in DSB-defective mutants (). Hence, phosphorylation is not a consequence of DSB formation.
Mer2 phosphorylation requires CDK activity in vivo
Cdc28-Clb5/Clb6 activity accumulates during premeiotic S phase, increases through prophase and peaks at about MI (Stuart and Wittenberg, 1998
). We tested whether this kinase is required for Mer2 phosphorylation. First, we used a mutant version of Cdc28 (Cdc28-as1) that can be inactivated in vivo by the inhibitor 1-NM-PP1 (Bishop et al., 2000
). Addition of 0.5 μM 1-NM-PP1 to the sporulation medium allows DNA replication but arrests cells prior to the first meiotic division, whereas more complete inhibition with 5 μM 1-NM-PP1 prevents both S phase and the first division (Benjamin et al., 2003
). Mer2myc was phosphorylated in a cdc28-as1
mutant in the absence of 1-NM-PP1 (). In the presence of 0.5 μM 1-NM-PP1, meiotic induction of Mer2myc occurred, but phosphorylation was delayed and reduced (). Phosphorylation was reduced even further when the culture was exposed to 5 μM 1-NM-PP1 (). We also reduced CDK activity by deleting CLB5
. Mer2myc levels increased as cells entered meiosis but the protein mobility did not change, indicating that the protein was not phosphorylated normally ().
Direct phosphorylation of Mer2 by Cdc28-Clb5/Clb6
Steady-state Mer2myc levels were elevated when Cdc28as1 activity was decreased (). The reason for this effect is not known, but analysis of nonphosphorylatable mer2 mutants suggests that this is a consequence of blocking Mer2 phosphorylation (see below). In addition, a faster migrating form of Mer2myc accumulated when CDK activity was reduced (asterisks in ). This species is presumably an N-terminally truncated proteolytic product (the epitope tag is on the C terminus), although it is not known whether proteolysis occurred in vivo or during extraction. Based on analysis of mer2 mutants, susceptibility to cleavage also appears to be a specific consequence of blocking Mer2 phosphorylation (see below).
CDK-cyclin complexes phosphorylate Mer2 directly
These findings demonstrate that phosphorylation of Mer2 requires CDK activity. To determine whether this requirement is direct, we tested for Mer2 phosphorylation by CDK in vitro. Whole-cell extracts were prepared from CDC28
strains at 3 hr in meiosis and incubated with [γ32
-(benzyl)ATP with or without purified recombinant Mer2 protein (). The source of radioactive label is a bulky ATP analog that is used by Cdc28-as1 protein 130-fold more efficiently than ATP but is used very inefficiently by wild-type Cdc28 (Ubersax et al., 2003
). Under these conditions, proteins in the extract (, lane 2) as well as added histone H1 (, lane 3) were labeled by Cdc28-as1 but not by wild-type Cdc28 (, lane 1). The cdc28-as1
extract also labeled Mer2 (, lane 4). Because Cdc28-as1 is the only kinase in the extract capable of using N6
-(benzyl)ATP efficiently, we conclude that Mer2 can be directly phosphorylated by CDK-cyclin complexes.
Identification of CDK target sites on Mer2
The consensus CDK target is the sequence T/S-P-x-K/R where x is any amino acid, although CDK targets can also be phosphorylated at a minimal site, S/T-P (Nigg, 1993
; Ubersax et al., 2003
). Mer2 contains an optimal consensus site (SPFR) at Ser-30 and a minimal site (SP) at Ser-271 (). Both sites are well conserved despite low overall conservation ().
Mutating Ser-30 to alanine decreased the electrophoretic mobility shift and delayed the appearance of shifted species until after the normal time of DSB formation (). Mutating Ser-271 also altered the mobility pattern, but less drastically, and with kinetics similar to wild type (). Mutating both residues led to greater loss and delay of shifted species (). Phosphatase treatment increased the mobility of both Mer2(S30A)myc and Mer2(S30,271A)myc (), indicating that both are still phosphorylated. Similar to global inhibition of CDK, mutation of Ser-30 increased steady-state Mer2 levels, particularly at late time points () and increased the proteolytic product (asterisks in ). Thus, Ser-30 and Ser-271 are both required for normal Mer2 phosphorylation in vivo, with mutation of Ser-30 having a more profound effect. A simple interpretation is that these residues are both phosphorylated by Cdc28-Clb5/Clb6, although these are not the only sites that can be phosphorylated.
To confirm that Ser-30 and Ser-271 are direct targets of CDK, recombinant mutant proteins were tested as substrates in cdc28-as1 extracts with [γ32P] N6-(benzyl)ATP. Mer2(S271A) was efficiently phosphorylated, but phosphorylation of Mer2(S30A) and Mer2(S30,271A) was reduced ~5-fold and ~20-fold, respectively, compared to wild-type Mer2 (). We conclude that Cdc28 directly phosphorylates Mer2 on Ser-30. Because Mer2(S30,271A) phosphorylation was decreased even more, it appears that Ser-271 is also phosphorylated.
CDK targets on Mer2 are required for normal DSB formation
To determine the significance of Mer2 phosphorylation, phenotypes of phosphorylation mutants were analyzed. In mer2(S30A), spore viability was greatly reduced and intragenic recombination and DSB formation were not detectable over background (). These phenotypes are indistinguishable from a mer2 null, revealing that Ser-30 is essential for meiotic DSB formation. The mer2(S30,271A) mutant was similar ().
In contrast, Ser-271 phosphorylation is less important for recombination, because spore viability in untagged mer2(S271A)
was only slightly reduced compared to wild type (94% vs. 98%; ), DSB formation was also slightly reduced (96% of wild type at 6 hr in meiosis; ), and intragenic recombination was similar to wild type (). Interestingly, combining the mer2(S271A)
mutation with the myc tag caused a more severe defect than either change alone. Addition of the epitope tag to otherwise wild-type Mer2 gave a small but significant reduction in spore viability from 98% to 91% (p<0.01, chi-square test), indicating that the tag slightly reduces Mer2 activity in vivo (). However, when mer2(S271A)
was tagged, spore viability was further reduced (83%, ), suggesting that mer2(S271A)
is not fully functional and that its mild defect is exacerbated by the tag. Similar effects from combining point mutations with epitope tags were observed for SPO11
(Diaz et al., 2002
Phosphorylation is not required for association of Mer2 with meiotic chromatin
To determine if Mer2 phosphorylation is required for proper localization of the protein, we used a previously described differential extraction assay to follow the subcellular distribution of wild-type and mutant Mer2myc () (Kee et al., 2004
). Meiotic cells were hypotonically lysed to generate whole-cell extract (W) which was separated by centrifugation into a soluble cytoplasmic fraction (S1) and a pellet (P1) containing nuclei and other insoluble material. The pellet was extracted with nonionic detergent to release nucleoplasmic proteins, then separated by centrifugation again into a soluble fraction (S2) and a pellet containing chromosomes and other insoluble material (P2). Mer2myc was insoluble after detergent extraction (P2) and was quantitatively solubilized by DNase I (S3), indicating that Mer2myc is chromatin-associated (), consistent with the immunocytological analysis above.
Both single mutant proteins were quantitatively chromatin associated, with no detectable increase in the non-chromatin bound fractions (S1 and S2) compared to wild type (). However, both proteins were more susceptible to degradation by endogenous proteases during extraction, showing increases of the N-terminally truncated species (). Mer2(S30A)myc was more sensitive, because the truncated form was apparent at the first step (P1), whereas the truncated form did not become abundant for Mer2(S271A)myc until the DNase digestion (S3). The final yield of Mer2(S30A)myc was also reduced. Both the increased truncation product and decreased overall yield were even more pronounced for the double mutant Mer2(S30,271A)myc (). Thus, protease sensitivity correlated with the extent of the phosphorylation defect. Even so, this protein was also chromatin-associated as judged by the quantitative recovery in the first pellet (P1). Immunocytological analysis confirmed that the mutant proteins still associated with meiotic chromosomes (data not shown). These results reveal that CDK-dependent phosphorylation is not required to target Mer2 to chromatin, consistent with the finding that Mer2 was associated with chromosomes of premeiotic cells, i.e., when it was not yet detectably phosphorylated (). Moreover, the DSB defect of mer2(S30A) cannot be attributed to improper localization.
Protein degradation in this assay probably reflects activation of cellular proteases during extraction and is thus likely to be non-physiological (Kee et al., 2004
). This result may indicate that the degradation described above () also occurred ex vivo. More importantly, however, the increased degradation suggests that the mutations alter Mer2 conformation and/or association with other factors such that the mutant proteins are more accessible to proteases.
CDK target site mutations perturb Mer2 interaction with other DSB proteins
Mer2 interacts physically with itself and with other DSB proteins (Arora et al., 2004
). To determine if the DSB defect of the phosphorylation site mutants can be attributed to defects in Mer2 association with its known partners, we analyzed a subset of two-hybrid interactions that produced the highest β-galactosidase signals with wild-type proteins: Mer2 with itself and Mer2 with Rec114, Mei4 and Xrs2 () (Arora et al., 2004
). These interactions can be detected when the two-hybrid reporter strain is grown vegetatively, with the exception of the Mer2-Rec114 interaction, which is only detected when the reporter strain is induced to enter meiosis (Arora et al., 2004
). Expression levels for two-hybrid fusion proteins were indistinguishable for wild type and mutant Mer2 constructs (data not shown). All of the mutant Mer2 proteins retained the capacity to interact with wild-type Mer2 and with Mei4, albeit with reduced signal for Mer2(S30A) and Mer2(S30,271A) (). In contrast, when each mutant was assayed for interaction with itself, all three showed severe defects, although they continued to yield β-galactosidase expression significantly above background (). Interactions with Rec114 and Xrs2 were even more substantially compromised for some or all of the mutants. For both Mer2(S30A) and Mer2(S30,271A), signals were similar to background controls (). For Mer2(S271A), the interaction with Rec114 was unaffected, but the interaction with Xrs2 was decreased to nearly the same extent as for the other two mutants, although still above background ().
Effects of phosphorylation site mutations on Mer2 interactions with other DSB proteins