MCD1 Was Isolated by Two Screens Designed to Identify Genes Encoding Chromosomal Structural Proteins
eterminant) mutant was identified in a screen to isolate mutants defective in sister chromatid cohesion (Guacci et al., 1993
). To enrich for mutants defective in mitotic functions, mutants temperature-sensitive for growth (Ts−
) were screened for enhanced inviability after arrest in M phase as compared to G1 phase. mcd1–1
was one mutant with these phenotypes (). Similar mitotic lethality has been observed for cells defective in the mitotic checkpoint (Hoyt et al., 1991
; Li and Murray, 1991
). However, mcd1
cells have a functional mitotic checkpoint since nocodazole-treated mcd1
cells do not undergo new rounds of DNA replication or new bud formation (data not shown). Therefore, the mitotic lethality of mcd1–1
cells is not due to a defect in cell cycle regulation but reflects a potential role for Mcd1p in chromosome cohesion. The mcd1
mutant was subsequently shown to exhibit precocious dissociation of sister chromatids (see below). The MCD1
gene was cloned by complementation of the Ts−
and mitotic lethal phenotypes of mcd1
Characterization of the mcd1–1 Mutant and the MCD1 Gene
MCD1 was also identified in a screen for proteins that interact with the Smc1 protein (Smc1p), a member of the SMC family. To this end, high copy suppressors of the Ts− phenotype of an smc1 mutant (smc1–2) were isolated. One plasmid suppressed the Ts− phenotype and associated morphological defects (). The suppressor gene was found to be MCD1 (Experimental Procedures). While multiple copies of MCD1 were required to suppress the smc1–2 defects, they did not suppress the Ts− phenotype of either an smc1 deletion mutant or an smc2 mutant (data not shown). These results indicate that MCD1 suppression is specific and occurs by augmenting smc1–2 function rather than by replacing it.
The genetic interaction between MCD1 and SMC1 suggested an in vivo physical interaction between the Mcd1 protein (Mcd1p) and Smc1p. To test this possibility, a functional full-length Mcd1p tagged with the T7 epitope (Novagen) was overexpressed in an otherwise wild-type strain. Overexpression of Mcd1p recapitulates the conditions under which MCD1 and SMC1 genetically interact (see above). Mcd1p and Smc1p were found to coimmunoprecipitate in a chromatin-independent manner since large chromatin fragments were removed by high-speed centrifugation () and coimmunoprecipitation was unaffected by DNase treatment. Polyclonal anti-Mcd1p antibodies failed to immunoprecipitate Mcd1p when expressed under its own promoter, but in preliminary experiments Mcd1p and Smc1p co-fractionate through several steps of a biochemical purification (data not shown). No immunoprecipitation of Mcd1p or Smc1p was observed in strains lacking a T7 epitope tag. Taken together, these data suggest that Mcd1p and Smc1p are in a common complex.
MCD1 Is Essential and Encodes a Member of a Conserved Protein Family
MCD1 was shown to be essential by two approaches. First, a diploid strain heterozygous for a complete deletion of MCD1 was constructed and sporulated. All tetrads contained two viable and two inviable spores. None of the viable spores contained the deleted MCD1 gene. Second, strains were constructed in which the sole source of Mcd1p was from an MCD1 gene under control of an inducible GAL1 promoter. These strains were inviable in the absence of inducing agent (galactose) for MCD1 (data not shown). Thus, loss of MCD1 is a lethal event.
encodes a protein with a predicted molecular mass of 63 kDa. Its sequence was compared to other proteins in the database. Mcd1p shares homology with the S. pombe Rad21 protein and with putative proteins encoded by ORFs from human, mouse, and C. elegans. These proteins are 25% identical over their entire length except for the human and mouse sequences, which are 97% identical. Three blocks of >50% similarity are shared, and their spacing and relative position is conserved (). The rest of the predicted protein sequence is not conserved, but in the central region there are numerous potential PEST sequences, which have been proposed to target polypeptides for rapid degradation by the 26S proteasome (Coux et al., 1996
; Rechsteiner and Rogers, 1996
Mcd1p Activity Is Essential for Proper Chromosome Segregation
The mitotic lethality of mcd1–1 cells suggested a mitotic function for Mcd1p. To determine the role of MCD1 in cell cycle progression, wild-type and mcd1–1 haploid cells growing at 23°C were shifted to 37°C and cell, spindle, and DNA morphologies as well as DNA content were scored. At 23°C, mcd1 cells were indistinguishable from wild-type cells at 23°C or 37°C (). However, at 37°C, the mcd1 culture was enriched for cells with 2C DNA content () and showed a 3-fold increase in the frequency of large budded cells with short or partially elongated spindles.
Cell Cycle Progression in Wild-Type and mcd1–1 Cells
mcd1–1 cells were further characterized using synchronous populations obtained after release at 37°C from S phase arrest. While arrested in S phase, wild-type and mcd1 cells had a 1C DNA content, large bud, undivided DNA mass, and short spindle (). After release (1 hr), most wild-type cells completed both DNA replication and chromosome segregation since cells had a 2C DNA content, elongated spindle, and segregated DNA masses (). In contrast, most mcd1 cells had a stretched nuclear DNA mass and partially elongated spindle indicative of a chromosome segregation defect (). By 2 hr, 50% of wild-type cells exited mitosis (unbudded and small budded cells), compared to only 20% for mcd1 cells (). The majority of mcd1 cells remained in mitosis with cell and DNA morphologies similar to that at 1 hr (). Similar results were obtained when Mcd1p was depleted in strains in which the sole source of MCD1 was under control of an inducible promoter (data not shown). These data suggest that mcd1 inactivation causes a mitotic defect that disrupts chromosome segregation and delays, but does not prevent, exit from mitosis. Finally, mcd1 cells exhibited a 10 min delay in early S phase, indicating a requirement for Mcd1p at this time (data not shown).
Mcd1p Is Required for Sister Chromatid Cohesion
To establish that mcd1–1 cells are defective in sister chromatid cohesion, synchronized populations of wild-type and mcd1 haploid cells were arrested in mid-M phase by either of two regimens and subjected to FISH. For regimen 1, cells were at the nonpermissive temperature for the mcd1–1 mutation through S and mid-M phases, which is the time sister chromatid cohesion is both established and maintained (Experimental Procedures). Cells were processed for FISH using either a chromosome XVI centromere-proximal (CEN-proximal) or distal probe to assay cohesion at different sites along chromosome XVI.
Consistent with our previous studies, in mid-M phase most wild-type cells had one FISH signal per nuclear DNA mass, demonstrating that sister chromatids were paired (). In a small number of DNA masses, two FISH signals were detected due to either a low level of precocious sister chromatid dissociation or spurious background (). In contrast, most mcd1 cells in mid-M phase at 37°C had two FISH signals per DNA mass, indicating that sister chromatids had precociously dissociated (). The cohesion defect is not restricted to nocodazole arrested cells since inactivation of Mcd1p also caused precocious sister chromatid dissociation in cells arrested in G2/M by a mutation in an anaphase promoting complex (APC) subunit (Cdc16p). For example, 60% of mcd1 cdc16 cells exhibited precocious sister separation at CEN-proximal and distal loci compared to only 15% in cdc16 cells. The second FISH signal in mid-M phase mcd1 cells was not due to preexisting aneuploidy since in G1 phase, most cells had only one FISH signal (). The few cells that had two FISH signals in G1 in wild-type and mcd1 cells were likely due to spurious background. Similar results were obtained using probes from CEN-proximal regions of chromosomes I and IV and a more CEN-distal chromosome XVI region (data not shown). Thus, Mcd1p is required for sister chromatid cohesion at CEN-proximal and distal chromosomal regions in yeast cells.
Analysis of Sister Chromatid Cohesion in Mid-M Phase Wild-Type and mcd1–1 Cells
The previous regimen can not distinguish between defects in establishment or maintenance of cohesion. To assay for maintenance of cohesion, mcd1–1 and wild-type cells were allowed to establish cohesion at permissive temperature before mcd1–1 function was inactivated (Experimental Procedures, regimen 2). As expected, mid-M wild-type cells at 23°C and 37°C had one FISH signal per DNA mass (). Most mid-M mcd1 cells had one FISH signal at 23°C but two signals upon shift to 37°C. Similar results were obtained using probes from chromosomes I, IV, and XVI (data not shown). These results demonstrate that Mcd1p is required for maintenance of sister chromatid cohesion.
Mcd1p Is Required for Chromosome Condensation
The stretched DNA mass in mcd1
mutants at 37°C is reminiscent of yeast mutants defective in condensation (Strunnikov et al., 1995
). To test the role of Mcd1p in condensation, we used FISH to examine chromosome condensation at the rDNA locus, a 500 kb block of repetitive DNA (Guacci et al., 1994
). We had shown that in G1 phase cells, an amorphous rDNA FISH signal characteristic of a decondensed chromosome is detected, while in mid-M phase haploid cells a single line-like FISH signal characteristic of condensed and paired sister chromatids is seen (Guacci et al., 1994
). As expected, wild-type cells arrested in mid-M using regimen 2 had a single line-like FISH signal in 85% of the DNA masses (). In contrast, 74% of DNA masses from similarly treated mcd1
cells had an amorphous FISH signal (). An amorphous FISH signal is not expected for separation of sister chromatid, since in wild-type cells precociously separated sisters are seen as two line-like signals (, inset). Furthermore, the rDNA is line- like after sister chromatids have separated and segregated in anaphase nuclei cycling wild-type cells (Guacci et al., 1994
). Thus, mcd1–1
cells in mid-M phase at 37°C have an aberrant rDNA morphology indicative of a defect in rDNA condensation.
Analysis of Chromosome Condensation in Wild-Type and mcd1–1 Cells in Mid-M Phase
To monitor chromosome condensation at unique chromosomal regions, synchronized populations of wild-type and mcd1
haploid cells arrested in mid-M phase at 37°C (regimen 1) were subjected to FISH using a mixture of four chromosome XVI probes or six chromosome VIII probes. The number and spacing of FISH signals from the mixture of chromosome XVI or VIII probes provides a qualitative measure of chromosome condensation. For example, when haploid cells are arrested in G1 phase and hybridized with chromosome XVI probes, up to four, often widely spaced, FISH signals are detected per DNA mass whereas in mid-M phase, one or two closely associated FISH signals are detected (Guacci et al., 1994
). This change from dispersed in G1 phase to clustered in mid-M phase is characteristic of the change from a decondensed chromosome to a condensed chromosome with paired sisters.
As expected, in wild-type cells arrested in mid-M phase at 37°C and hybridized with either the chromosome XVI or VIII probe mixtures, a few closely associated FISH signals were detected in most DNA masses as expected for condensed and paired sister chromatids (). In contrast, in mcd1
cells hybridized with either the chromosome XVI or VIII probe mixtures, many, often widely spaced FISH signals were detected in many DNA masses (). Some of the increased numbers of FISH signals are expected due to sister chromatid dissociation. However, if sister chromatids remained condensed, there should be two tight clusters of FISH signals, one from each separated and condensed sister chromatid. Instead, the dispersed FISH signals are reminiscent of decondensed chromosomes (Guacci et al., 1994
). Taken together, the results from FISH using rDNA, chromosome VIII, and chromosome XVI probes indicate that the mcd1–1
mutant exhibits defects in chromosome condensation as well as sister chromatid cohesion.
Mcd1p Is Nuclear and Its Levels Are Cell Cycle Regulated
To determine if the in vivo localization of Mcd1p was consistent with its proposed role as a chromosomal structural protein, cells were processed for both Western blot analysis and indirect immunofluorescence using anti-Mcd1p antibodies. Initially, we examined a strain in which Mcd1p was overexpressed (). Many cells had prominent punctate nuclear staining, with the exception of cells undergoing anaphase, where Mcd1p was dispersed evenly throughout the cell, suggesting possible redistribution (). When expressed from its endogenous promoter in wild-type cells, Mcd1p staining was highly variable but nuclear when detected (data not shown). To examine whether this variability was cell–cycle dependent, wild-type cells were arrested in G1, S, or mid-M phase and processed for Western blotting and indirect immunofluorescence. Mcd1p was barely detectable in G1 phase, at high levels in S phase, and at lower levels in mid-M phase with a punctate nuclear localization in S and mid-M phases (). Similarly, the S. pombe Rad21 protein also localized to the nucleus (Birkenbihl and Subramani, 1995
). The nuclear localization of Mcd1p during S phase and mitosis is consistent with its proposed role in chromosome structure.
To monitor the dynamics of cell-cycle dependent changes in Mcd1p levels, a synchronous population of wild-type cells was examined. Aliquots of cells were taken in G1 phase, as well as every 20 min after release from G1, then processed to monitor Mcd1p levels and scored for DNA content to assess cell cycle position. Mcd1p levels were barely detectable in G1 phase cells (T = 0), reached a peak in S phase (T = 40) for many cells, and decreased in G2/M phases (T = 60) (). To more precisely determine the time when Mcd1p levels decline, the relative levels of Mcd1p and Pds1p were compared. The decrease in Mcd1p levels occurred 20 min prior to the decrease in Pds1p levels (, arrows). Since Pds1p degradation is required for the metaphase-to-anaphase transition, the decrease in Mcd1p levels occurred before this cell cycle progression landmark (Cohen-Fix et al., 1996
). Finally, Mcd1p levels were examined by Western at discrete cell cycle stages using cdc
mutants or wild-type cells treated with α
factor, HU, or Nz. Mcd1p was barely detectable in G1 (cdc28–4
factor), peaked in early S (HU), and decreased to a lower but steady level in late S (cdc9
), G2 (cdc28–1N
), G2/M (cdc13
), mid-M (cdc20
, and Nz), and telophase (cdc14
) cells (data not shown). Taken together, these data show that Mcd1p levels peak in early S phase, are reduced by late S to a lower level that remains constant through telophase and decreases to nearly undetectable levels by G1.
Cell Cycle–Dependent Expression of Mcd1p
Insights into the cell cycle–dependent changes in Mcd1p levels came from examination of MCD1
mRNA in synchronized cycling cells and Mcd1p levels in cdc
mRNA levels peak in early S phase then decrease 9-fold 20 min prior to the Mcd1p decrease (). This pattern of mRNA regulation is likely due to two MluI boxes in the MCD1
promoter region (data not shown; McIntosh et al., 1991
). The decrease in mRNA coupled with possible PEST-mediated degradation is sufficient to account for the decrease in Mcd1p levels in late S. Mcd1p levels dropped 20 min prior to Pds1p degradation, a known landmark of anaphase initiation and the earliest known target of the anaphase promoting complex, APC (). Moreover, the decrease in Mcd1p levels observed in late S/G2 was not affected by mutations in APC components (cdc16
) (data not shown). Thus, the change in Mcd1p levels in late S/G2 is independent of APC, although it may play a role in the subsequent decrease in Mcd1p levels in G1.