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Molecular and Cellular Biology, May 1999, p. 3515-3528, Vol. 19, No. 5
Institute of General Microbiology, University
of Bern, 3012 Bern, Switzerland1; Peter
MacCallum Cancer Institute, East Melbourne 3002, Australia2; and Department of Cell
Biology and Genetics,
Received 20 July 1998/Returned for modification 8 September
1998/Accepted 29 January 1999
Our work and that of others defined mitosis-specific (Rad21
subfamily) and meiosis-specific (Rec8 subfamily) proteins involved in
sister chromatid cohesion in several eukaryotes, including humans.
Mutation of the fission yeast Schizosaccharomyces pombe rec8 gene was previously shown to confer a number of meiotic
phenotypes, including strong reduction of recombination frequencies in
the central region of chromosome III, absence of linear element
polymerization, reduced pairing of homologous chromosomes, reduced
sister chromatid cohesion, aberrant chromosome segregation, defects in
spore formation, and reduced spore viability. Here we extend the
description of recombination reduction to the central regions of
chromosomes I and II. We show at the protein level that expression of
rec8 is meiosis specific and that Rec8p localizes to
approximately 100 foci per prophase nucleus. Rec8p was present in an
unphosphorylated form early in meiotic prophase but was phosphorylated
prior to meiosis I, as demonstrated by analysis of the mei4
mutant blocked before meiosis I. Evidence for the persistence of Rec8p
beyond meiosis I was obtained by analysis of the mutant
mes1 blocked before meiosis II. A human gene, which we
designate hrec8, showed significant primary sequence
similarity to rec8 and was mapped to chromosome 14. High
mRNA expression of mouse and human rec8 genes was found
only in germ line cells, specifically in testes and, interestingly, in
spermatids. hrec8 was also expressed at a low level in the
thymus. Sequence similarity and testis-specific expression indicate
evolutionarily conserved functions of Rec8p in meiosis. Possible roles
of Rec8p in the integration of different meiotic events are discussed.
Meiosis is an essential step in the
sexual reproduction of eukaryotes. It serves to reduce the chromosome
number from diploidy in the germ line to haploidy in the gametes. This
is accomplished by two rounds of chromosome segregation, meiosis I and
II, after a single round of DNA replication. During meiotic prophase,
the replicated homologous chromosomes pair, recombination occurs
between nonsister chromatids, and the resulting crossovers lead to
chiasma formation in all bivalents. Chiasmata and sister chromatid
cohesion are required for correct segregation of homologous chromosomes at meiosis I (reductional division). Throughout meiotic prophase, numerous events occur at the chromosomes in highly ordered fashion (for
reviews, see references 37 and
68).
The events occurring at the DNA level are coordinated with many other
cellular processes, and all are integrated into the meiotic cell cycle.
Much additional research is required for the elucidation of the
regulatory mechanisms governing meiosis. However, it is known that
checkpoint proteins involved in the regulation of the mitotic cell
cycle are also needed for checkpoint controls in meiosis
(47). A number of protein kinases have important functions
in meiosis. An example is the kinase encoded by the Drosophila
melanogaster mei-41 gene, which is homologous to the human
ATM gene (30). Mutation of this gene affects the
number and morphology of recombination nodules (12). The
CDC28, MEK1/MRE4, ESR2, and
IME2 genes, coding for identified or putative protein kinases, have meiosis-specific functions in Saccharomyces
cerevisiae: (19, 34, 44, 67, 74). A protein phosphatase
was shown to interact with Red1, which is a component of the lateral
elements of the S. cerevisiae synaptonemal complex (77,
83). A component of the lateral elements of the rat synaptonemal
complex, SCP3, is multiply phosphorylated (43). SCP3 is
identical to the Cor1 protein, which relocates from lateral elements to
centromeres after anaphase I (20).
An important structural feature of chromosomes during mitotic and
meiotic prophase is sister chromatid cohesion. It contributes to
meiotic pairing and recombination of the homologous chromosomes. After
crossover formation and degradation of the synaptonemal complex, the
homologs are kept in alignment by the microscopically visible
chiasmata, which do not resolve until anaphase I. If sister chromatid
cohesion is resolved prematurely, chromosomes do not segregate
properly, resulting in daughter cells with unbalanced genomes (for a
review, see reference 52).
In several organisms, genes involved in meiotic sister chromatid
cohesion have been identified. The spo76 mutant of
Sordaria shows precocious separation of sister chromatids
and reduced meiotic recombination levels (55). Mutation of
the ord gene of D. melanogaster also leads to
premature separation of sister chromatids (7, 51). In
mei-S332 mutants of D. melanogaster, meiotic
recombination and segregation of the homologs at meiosis I is normal.
However, sister chromatids separate precociously during anaphase I,
leading to random segregation at meiosis II (35). Mei-S332
protein associates with centromeres in late meiotic prophase and
disappears at anaphase II, when sister chromatids separate completely
(36). A function similar to Mei-S332 was attributed to mouse
Cor1 on the basis of its specific localization to centromeric regions
from anaphase I to anaphase II (20, 53).
Loss of mitotic sister chromatid cohesion in S. cerevisiae
requires degradation of Pds1p at the metaphase-to-anaphase transition (14). No homologs of Pds1p were identified in other
organisms, but Cut2p of the fission yeast S. pombe may have
a similar function (24). Recently, additional proteins
involved in mitotic sister chromatid cohesion in S. cerevisiae were described (49). One of them, Scc1p, is
related to Rad21p of the fission yeast S. pombe (8,
9). Scc1p binds to chromosomes during S phase, dissociates at the
metaphase-to-anaphase transition, and is needed for sister chromatid
cohesion near centromeres and in the chromosome arms. Binding of Scc1p
to chromosomes is Smc1p dependent. The same protein, but named Mcd1p,
was also identified in a screen for structural proteins of chromosomes
and for proteins interacting with Smc1p, suggesting an additional role
for Scc1p/Mcd1p in chromosome condensation (28). In addition
to Rad21p, homologs of Scc1p/Mcd1p appear to exist in all eukaryotes,
including Homo sapiens (reference 48 and
this study).
The rec8 gene of S. pombe was identified by
screening for mutants with reduced meiotic recombination in the
ade6 gene (63). Point mutations in
rec8 reduce meiotic but not mitotic recombination and result
in mutants with normal resistance to the DNA-damaging agents UV
radiation and methyl methanesulfonate (17). Strong reduction
of recombination was reported to be specific for a 2-Mb region around
the centromere of chromosome III (18; but see below). Expression of rec8 RNA was concluded to be meiosis
specific based on meiosis induction in haploid pat1 mutant
cells (46). In mutants with a rec8-110 point
mutation, linear-element formation is defective and only amorphous
aggregates of linear-element proteins were observed (54).
The linear elements appearing in S. pombe prophase resemble
axial cores (precursors of the lateral elements in the synaptonemal
complex) of other eukaryotes (2). Bouquet formation was
normal in rec8-110 mutants, but pairing of chromosomes, studied by fluorescent in situ hybridization (FISH), was reduced. The
strongly reduced spore viability was assumed to be a consequence of
precocious separation of sister chromatids, which was observed in
genetic assays and by FISH. It was concluded that linear-element formation contributes both to sister chromatid cohesion and to high
meiotic recombination frequency (54).
Here we expand on the characterization of fission yeast Rec8p by
performing an analysis of a new rec8 allele, a gene
deletion/disruption likely to create a null phenotype, and using an
anti-Rec8p antibody in the study of Rec8p localization and Rec8p
phosphorylation. We report the cloning and characterization of a novel
human gene, hrec8, and present the results of experiments on
complementation of the fission yeast rec8 disruption strain
by the human homolog. We describe in detail the rad21/rec8
gene family. Finally, the results are discussed with respect to
possible roles of Rec8p in the regulation of meiotic events.
Strains, plasmids, media, and general methods.
The genotypes
of S. pombe strains used in this study are listed in Table
1. All rec8::ura4
mutant strains were constructed from PA32 by genetic crosses. Plasmid
pYL3 (46) contains a 3.9-kb rec8 fragment
subcloned in pSP2 (16). Plasmid pREP41-hrec8 was prepared by
insertion of the full-length human rec8 (hrec8)
cDNA into the NdeI site of plasmid pREP41 (5).
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Rec8p, a Meiotic Recombination and Sister Chromatid Cohesion
Phosphoprotein of the Rad21p Family Conserved from Fission Yeast
to Humans
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
TABLE 1.
Description of S. pombe strains used in
this study
N (PM without NH4Cl)
(86) were used.
rec8 gene disruption.
To construct a
rec8 gene disruption mutant, a 3.9-kb SacI
fragment from pYL3 (46) containing the rec8 gene
and flanking sequences was subcloned into pBluescript KS (Stratagene).
From the resulting plasmid, pBSrec8S-1, a 1.5-kb
NsiI-NheI fragment was replaced by a 1.8-kb
PstI-XbaI fragment from pB4-3 containing the
ura4 marker gene (25) to yield the gene
disruption plasmid pBSrec8S-1::ura4. From this plasmid, a
4.2-kb SacI fragment was used to transform the strain
h
leu1-32 ura4-D18 by the lithium acetate
method (33). Proper integration of the fragment into the
genome was verified by Southern blot analysis.
Anti-Rec8p antibody. A 1.2-kb rec8 DNA fragment covering the originally published open reading frame (ORF) from positions 742 to 1923 (46) was amplified by PCR to introduce a BglII and an EcoRI restriction site at the 5' and 3' ends of the fragment, respectively. This PCR product was ligated in frame into the BamHI and EcoRI sites of pGEX-2T (Pharmacia) to obtain a rec8-glutathione S-transferase (rec8-GST) fusion. The construct was verified by DNA sequence analysis.
Fusion protein was produced as described previously (78). Briefly, Escherichia coli DH5
cells were transformed with
pGEX-2T-rec8 and selected for the presence of plasmid with ampicillin
(50 µg/ml). Synthesis of fusion protein was induced by the addition
of isopropyl-
-D-thiogalactoside (IPTG) to a final
concentration of 0.1 mM. After further incubation for 3 to 4 h at
37°C, the cells were harvested and stored at 70°C. For
preparation of E. coli extracts, approximately 4 g of
cells was resuspended in 40 ml of phosphate-buffered saline (PBS)
containing 2 mM EDTA, 100 mM benzamidine, 1 mM phenylmethylsulfonyl
fluoride, and 1% Triton X-100. The GST fusion protein was isolated
from the cleared lysate by the addition of 2 ml of 50% slurry of
glutathione-Sepharose 4B (Pharmacia). The fusion protein was eluted in
50 mM Tris-HCl (pH 8.0)-10 mM reduced glutathione, and eluted
fractions were concentrated and analyzed by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The 72-kDa
Rec8-GST fusion protein was isolated from the polyacrylamide gel and
used for immunization.
Polyclonal antiserum was obtained by injecting a rabbit and three rats
three times with approximately 200 µg of Rec8 fusion protein at
2-week intervals. Antisera were affinity purified by three rounds of
adsorption to nitrocellulose Western blot strips (65)
containing three different proteins (GST, E. coli heat shock
protein GroEL, and Rec8 fusion protein). GroEL was a contamination in
the preparation of Rec8 fusion protein as identified by protein sequencing.
Meiotic time courses and Rec8p immunofluorescence on nuclear
spreads.
The induction of meiosis, the preparation of nuclear
spreads, and 4',6-diamino-2-phenylindole (DAPI) staining of DNA were as
described previously (2). Briefly, S. pombe PA39
diploid cells were induced to undergo meiosis by being shifted to
nitrogen-free medium. Aliquots collected immediately after the shift (0 h) and at 2-h intervals thereafter were used to prepare spreads of
nuclei. For flow-cytometric analysis, 1-ml aliquots of cells were fixed in 70% ethanol and stained with propidium iodide (Sigma) as described elsewhere (6). For immunofluorescence experiments, the
wild-type diploid strain PA39 and the control diploid strain PA40
homozygous for the rec8::ura4 deletion were used
(Table 1). Slides with nuclear spreads stored at 70°C were soaked
in PBS containing 0.1% Photo-Flo (Kodak) to remove the sucrose layer
and then washed for 15 min in PBS containing 0.05% Triton X-100 and
for 15 min in twofold-diluted blocking buffer (100 mM lysine, 3%
nonfat dry milk, 0.05% Triton X-100, and 0.02% NaN3 in
PBS [pH 7.3]). The nuclear spreads were then blocked overnight in
blocking buffer and subsequently probed with the first antibody (1:10
dilution of rabbit anti-Rec8p in blocking buffer) for 24 h. The
slides were sequentially washed in PBS containing 0.1% Photo-Flo and PBS containing 0.05% Triton X-100 for 15 min each prior to incubation with the second antibody for 24 h (1:80 dilution of goat
anti-rabbit immunoglobulin G-fluorescein isothiocyanate conjugate in
PBS [Sigma]). Before being mounted, the slides were washed once more
for 15 min in PBS containing 0.1% Photo-Flo and for 15 min in PBS
containing 0.05% Triton X-100, with two additional washes in
H2O for 5 min each. The chromatin was counterstained with
DAPI in a Vectashield antifade solution (Vecta Laboratories Inc.). The
slides were analyzed with an epifluorescence (Zeiss Axiovert)
microscope. Foci were quantified with the UTHSCSA Image Tool software
(version 1.27).
RNA extraction, Northern blot hybridization, and reverse transcription-PCR. Total RNA from S. pombe cells was prepared as described previously (26). Hybridization with a 32P-labelled rec8 or byr1 (58) DNA probe was performed as specified by the manufacturer (Bio-Rad) in its standard hybridization protocol. The rec8 probe was the same 1.2-kb DNA fragment used to construct pGEX-2T-rec8. The byr1 probe (a gift from A. M. Schweingruber) was the 0.4-kb PCR fragment from positions 976 to 1387 in the ORF.
A 300-µg portion of total RNA from a meiotic time course (8 h after the shift) was used to prepare meiotic mRNA with the Oligotex poly(A)+ mRNA isolation kit (Qiagen). First-strand rec8 cDNA was synthesized from 1 µg of meiotic mRNA with an oligo(dT)18 primer and Superscript reverse transcriptase (GIBCO). Reverse transcription-PCR was performed with the primers 5'-GGAAAAGGGAGGAATGGGAGTAATTTGG-3' (positions 186 to 213 of rec8) and 5'-GTGAAAAGTTTCAAATGGCATCGGTGC-3' (positions 2013 to 2039 of rec8). The resulting PCR fragment was subcloned into pGEM-T (Promega) and subjected to DNA sequencing. Northern blot analysis of the human rec8 mRNA was performed as described previously (48). A multiple human tissue blot was obtained from Clontech (no. 7754-1); hybridization and wash conditions were as specified by the manufacturer. The blots were hybridized with 32P-labelled cDNA probes.Immunoblotting, immunoprecipitation, and phosphatase
treatment.
For the preparation of S. pombe extracts,
approximately 1 g of cells from different time points of a meiotic
time course was suspended in disruption buffer (25 mM Tris-HCl [pH
7.5], 150 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol, 2 mM
phenylmethylsulfonyl fluoride, 5 mM benzamidine, 13 mM
-mercaptoethanol), mixed with an equal volume of glass beads, and
disrupted in a mini bead-beater. Following SDS-PAGE, the proteins were
transferred to nitrocellulose and the filter was blocked overnight in
TBST (150 mM NaCl, 10 mM Tris-HCl [pH 8.0], 0.05% Tween 20, 0.01%
NaN3) containing 5% nonfat dry milk. The filter was then
incubated with the primary rat anti-Rec8 antibody (1:20 in TBST
containing 5% nonfat dry milk) for 2 h. After three washes in
TBST, the second antibody (1:500 dilution of rabbit anti-rat IgG
peroxidase conjugate [DAKO] in TBST) was added for 2 h, and the
mixture was washed three times in TBST and developed with the ECL
chemiluminescent detection kit (Amersham).
Cloning and sequence analysis of hrec8. General molecular biology procedures were essentially as described previously (69). Two 20-mer primers were designed based on the nucleotide sequence of EST T33286, whose translated product was homologous to the C-terminal end of hHR21sp (48) (see Results). The primers had their 5' ends at 102 nucleotides before and 140 nucleotides after the terminal nucleotide of the hrec8 ORF and were used to amplify a 241-bp fragment of the hrec8 gene from a human T-cell leukemia cell line cDNA (cDNA kindly supplied by Karin van Gool). This PCR fragment was used as a probe to isolate hrec8 cDNA clones from a normalized, gridded human infant-brain plasmid library (79), kindly supplied by P. Heutink. These hrec8 clones were sequenced on both strands. Amino acid sequences were aligned with the ESEE program (version 1.09e; supplied by Eric Cabot). An unrooted phylogenic tree (see Fig. 5C) was constructed based on the protein sequence alignments of the rec8/rad21 homologs shown in Fig. 5B; a tree based on alignment of all full-length rec8/rad21 proteins with the DNA Man program (version 3.2; Lynnon Biosoft, Quebec, Canada) gave very similar results (not shown). Trees were drawn with the PHYLIP software package (22); pairwise distances were calculated with the Dayhoff PAM 001 matrix. PEST sequences (66) were identified by using PESTfind at IMB Jena.
Complementation of rec8::ura4 by the human
hrec8 gene.
S. pombe PA43 was transformed with
either pREP41 or pREP41-hrec8 by the lithium acetate method
(33). Transformants were selected on EMM plates lacking
leucine. Transformants were plated onto MEA and allowed to sporulate
for 72 h at 25°C. Cell-free spore suspensions were prepared by
incubation of cell material with -glucuronidase (Sigma) at 22°C
for 16 h. The spore titer was determined by counting. The
percentage of viable spores was determined by plating on YEA and
counting of the forming colonies.
Chromosomal localization. FISH experiments were performed with biotinylated hrec8 cDNA probes, hybridized to metaphase spreads of normal human lymphocytes, as described previously (62, 84). Chromosomes were banded with DAPI and actinomycin D and counterstained with propidium iodide in antifade solution.
Nucleotide sequence accession numbers. The GenBank accession numbers for the fission yeast and human rec8 DNA sequences are AJ223299 and AF006264, respectively.
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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The phenotypes of a rec8 gene disruption and reevaluation of the region specific reduction of recombination. A renewed analysis of the rec8 gene led to extension of the ORF at both ends (see the legend to Fig. 1). This result led to the discovery of homology to other genes in the data banks (see below). A gene disruption strain was constructed by replacement of a large part of the rec8 ORF by the ura4 marker gene (Fig. 1) for comparison of its phenotypes with those of the rec8-110 point mutation studied previously (54). Spore viabilities measured by random spore analysis were 12 and 20% in the disruption and point mutants, respectively. This difference is statistically significant but of doubtful relevance. The frequencies of intragenic and intergenic recombination were clearly reduced in comparison to those of the wild type, but no significant differences between disruption and point mutation crosses were found. At lys7 (lys7-1 × lys7-2) and at ade6 (ade6-M26 × ade6-52), the differences were at most threefold. In the three intervals pro2-arg3, leu2-lys7, and ade6-arg1, the values were two- to threefold lower in the rec8::ura4 than in the point mutation crosses (data not shown). The cytological analysis revealed an absence of linear elements and a shortened meiotic prophase in the rec8-110 strain (54). Linear-element formation in rec8::ura4 was impaired to a similar degree: no filaments and amorphous complexes of linear-element material were found. In contrast to the point mutation, the rec8::ura4 strain showed no shortening of prophase in three independent time courses (data not shown). Shortening of the prophase in the point mutation strain may indicate a role of Rec8p in meiosis regulation. Alternative explanations, like shortening of prophase by an additional mutation, were not excluded.
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Rec8p is localized in foci in the nucleus. When intact wild-type cells from vegetative or meiotic cultures were fixed and treated with affinity-purified anti-Rec8p antibody, no signal was detected unless Rec8p was overexpressed from a plasmid, in which case the signal was confined to the nucleus (data not shown).
Gentle lysis of cells and spreading of nuclei on surfaces were used previously for the analysis of linear-element formation and chromosome pairing (2, 54, 70). Thus, spread meiotic nuclei from wild-type diploids were treated with affinity-purified anti-Rec8p antibody (see Materials and Methods). Fluorescence microscopy revealed that Rec8p is localized in foci throughout the spread nuclei. Meiotic time course experiments were then performed with the wild-type diploid strain PA39 and with the rec8::ura4 disruption diploid strain PA40. An example of the analysis of meiotic landmarks, the presence of rec8 mRNA, and the subnuclear localization of Rec8p (quantitation of Rec8p foci) are presented in Fig. 3.
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Rec8p is phosphorylated during prophase and persists beyond meiosis I. To further examine Rec8p expression during meiosis, time course Western analysis and immunoprecipitation experiments were performed with both wild-type diploid PA39 cells and diploid cells homozygous for mutations that block meiosis at specific stages. The particular PA39 culture used for the experiment in Fig. 4A was somewhat delayed compared to the cultures used for the study of Rec8p foci (Fig. 3). Rec8p first appeared 6 h after induction of meiosis, when the first horse-tail nuclei also became visible (data not shown), and grew more abundant at the later time points. At 6 h, Rec8p was visible as a single band of 87 kDa. At 8 h, an additional band migrating at 95 kDa became apparent and its intensity increased (10 and 12 h). After 12 h, protein extraction became difficult due to the presence of high percentages of spores. At 12 h, 40% of cells had performed the first meiotic division and fewer than 10% of the cells were still in meiotic prophase (horse-tail nuclei [data not shown]). The apparently large amount of Rec8p in the extract from cells harvested at 12 h indicated to us that Rec8p may persist beyond meiosis I (see below).
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Cloning and sequence analysis of a human rec8 gene. Since the conservation of the rad21 class of genes extended from yeasts to mammals and rec8 genes existed in both budding and fission yeast species (see below), it was likely that rec8 was also conserved in mammals. A human expressed sequence tag, T33286, was identified by its homology to the human rad21 homolog, hHR21sp (for human homolog of rad21, S. pombe [48]) on BLAST (1) database searching. A cDNA fragment corresponding to the C-terminal region of this new human gene, which we denote hrec8, was amplified from a human T-cell leukemia cDNA pool and used to screen a cDNA library from which full-length hrec8 cDNA clones were obtained.
hrec8 corresponded to an ORF of 1,641 nucleotides. Consistent with the generally low mRNA expression of this gene (see below), the sequence around the initiation codon did not conform strongly to the Kozak consensus (41); however, its translation start site was likely to be the correct one because of its highly conserved nature (below) and the presence of stop codons in all three reading frames in the 5' untranslated region (5' UTR) (data not shown). The 5' UTR was at least 477 nucleotides long, while the 3' UTR was 160 nucleotides (data not shown). The translation of hrec8 is shown in Fig. 5A, aligned with the full-length fission yeast Rec8p, a putative Rec8p homolog in budding yeast (Rec8sc; see below), and the founding member of the rad21/rec8 gene family, Rad21p.
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Phylogenetic analysis of rad21/rec8 gene family. A phylogenetic comparison of the Rec8 and Rad21 proteins from the different eukaryotes was performed. Overall, the estimated phylogenetic relationships between the Rec8/Rad21 proteins from the different species (indicated by the lengths of the lines in Fig. 5C) are consistent with those observed for other groups of orthologous and paralogous genes; i.e., they are congruent with the evolutionary relationship of the various species (32, 76, 82). There is no clear distinction between the rad21 and rec8 members of the gene family based on phylogeny. The common root of the two nematode proteins and their proximity to human rad21 indicates that unlike in the other species examined to date, there are two rad21 genes in Caenorhabditis elegans. However, no genes with sequence homology closer to rec8 than to rad21 have yet been identified in that species, although its sequence analysis has been completed (82).
Chromosomal localization and mRNA expression of hrec8. By using FISH, hrec8 was mapped to chromosome 14q11.2-12 (Fig. 6A). This locus is not apparently implicated in human disease syndromes. The mRNA expression of hrec8 was examined in different mammalian tissues. On blots prepared with total RNA, a 2.4-kb mouse rec8 mRNA species, consistent with the size of the hrec8 ORF, was detected only in testis cells (Fig. 6B, top left) and not in other tissues. Mouse testis tissues were fractionated into meiotic and postmeiotic compartments (spermatocytes and spermatids, respectively [27]). In contrast to the pattern seen for the related mHR21sp gene (48), weak mrec8 expression was detected in meiotic cells and greater expression was detected in postmeiotic spermatids (Fig. 6B, top middle). In addition, a multiple-human-tissue poly(A)+ RNA blot was hybridized with an hrec8 probe. Although not detected on the total-RNA blot, the hrec8 mRNA level was increased in thymus tissue and, unlike in testis tissue, there was an additional 3.2-kb mRNA species (Fig. 6B, top right). Expression was not detectable in other tissues, including some with high cellular proliferation. This indicates that increased thymic and testicular mrec8 expression was unlikely to simply reflect cellular proliferation in these tissues. We conclude that hrec8 mRNA expression is increased in meiotic as well as postmeiotic testis cells and is also detectable in thymus cells.
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Experiments on complementation of fission yeast rec8::ura4 by human hrec8 cDNA. Sequence similarity was demonstrated for fission yeast and human Rec8p, and both genes were expressed in meiosis. If the two proteins have common functions, hRec8p might be able to substitute for Rec8p function in S. pombe. Two sets of experiments were performed. The homothallic strain PA43 with the rec8::ura4 disruption was transformed with pREP41 (control) and pREP41-hrec8. Individual transformants were brought to conjugation and sporulation, and the resulting spores were checked for viability. Untransformed PA43 showed a low spore viability of 9% compared to that of a rec8 wild-type strain. The vector alone had no effect after transformation into PA43, but pREP41-hrec8 increased spore viability to 48% (normalized to that of the rec8 wild-type strain [data not shown]).
Complementation of spore viability is expected to be due to restoration of high recombination frequency. Therefore, the heterothallic strains PA21 and PA22, suitable for recombination analysis, were transformed by pREP41 (control) and pREP41-hrec8. In this experiment, crossing the two untransformed strains resulted in low (18%) spore viability while transformation with the vector yielded 24% spore viability. Transformation with pREP41-hrec8 increased the spore viability to 38%. These results are qualitatively similar to those described above and may indicate partial complementation of the reduced spore viability phenotype by Hrec8p (data not shown). Neither intragenic recombination measured between the ade6-M26 and ade6-52 alleles nor intergenic recombination examined in the pro2-arg3 interval was significantly different from that in crosses of the untransformed strains (data not shown). These results show that the partial complementation of spore viability may be of doubtful significance.| |
DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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In this study, we have further elaborated the function of the fission yeast rec8 gene, whose product is a phosphoprotein critical for meiotic sister chromatid cohesion and correct chromosome segregation. We describe the sequence conservation of rec8 in humans and present evidence that hrec8 is the human homolog of fission yeast rec8. We also demonstrate that the rec8 class of genes shows sequence homology to the rad21 class of mitotic, cell cycle-regulated phosphoproteins involved in sister chromatid cohesion, chromosome condensation, and DNA double-strand break repair. The findings suggest that rad21 and rec8 are the mitotic and meiotic members, respectively, of a new gene family involved in multiple aspects of DNA metabolism.
Here we present a unifying hypothesis to explain the observations on Rec8p. In this context, some special features of fission yeast meiosis need to be kept in mind. S. pombe maintains the bouquet structure of chromosomes throughout meiotic prophase, with concomitant absence of a fully tripartite synaptonemal complex and of crossover interference (for reviews, see references 37, 39, and 68). The retention of linear elements resembling the axial cores of synaptonemal complex indicates that these structures are of fundamental importance for meiotic recombination and chromosome segregation. During prophase of fission yeast meiosis the horse-tail nuclei move continuously from end to end of the cylindrical cells. At the leading end of the elongated nuclei, all telomeres are clustered to form and maintain the bouquet structure of chromosomes (13, 81). Disruption of the integrity of the spindle-pole body and/or telomere clustering by mutation leads to reduction of meiotic intra- and intergenic recombination frequencies (15, 59, 73). When nuclear movement was abolished by mutation of a motor protein required exclusively for horse-tail nucleus movement, recombination was also reduced (31). In all cases, about a fivefold reduction of recombination frequencies resulted.
The primary functions of Rec8p: a working hypothesis. We propose that freshly synthesized Rec8p binds to the sites of initiation of recombination that are a subset of the sites of early pairing between related DNA sequences (re)established immediately after premeiotic DNA replication. It is likely that at this early stage, ectopic interactions between repeated sequences on different chromosomes occur as well as truly homologous DNA contacts. Little is known about the mechanisms involved in the formation of the first contacts between homologous chromosomes and the resolution of ectopic interactions. In S. cerevisiae, early contacts between homologous chromosomes occur before double-strand breaks initiate recombination, and it was proposed that assembly of the recombination initiation complex occurs in a succession of events at sites of sister chromatid cohesion (for reviews, see references 37 and 68).
Rec8p binding to the developing recombination initiation complex is proposed to be required for the start of linear-element polymerization. Linear elements, in turn, are proposed to contribute to extension and stabilization of homologous chromosome pairing and to maintenance of recombination intermediates once initiation has occurred. In particular, we suggest that linear-element formation is important in chromosome regions far from the telomeres. Rec8p binding to early homologous contacts is also proposed to enhance sister chromatid cohesion directly, or indirectly by promotion of linear-element polymerization. The phosphorylation of Rec8p may play a structural role in organizing the chromosomes during prophase (e.g., repulsion of DNA backbones). Alternatively, or in addition, phosphate groups may be introduced in response to completion of specific steps of the pairing and recombination pathway, in order to signal their occurrence to other components of the nucleus. Another regulatory function of the kinase(s) phosphorylating Rec8p may be the promotion of events at the chromosomes after completion of processes elsewhere in the cell, in particular, resolution of recombination intermediates and relaxation of sister chromatid cohesion in the chromosome arms at meiosis I. The persistence of phosphorylated Rec8p after meiosis I may be required for cohesion of the centromeres, as proposed for the phosphoprotein Cor1/SCP3 of rats. Cor1 relocates from the dissolving lateral elements to the centromeric regions of chromosomes and persists there until anaphase II (20, 53). A role in centromere cohesion before anaphase II has been established for the Mei-S332 protein of Drosophila (36).Evaluation of the hypothesis on primary Rec8p functions. First, we consider early Rec8p functions. An alternative to the model presented above may propose binding of Rec8p to specific sites on replicated chromosomes and subsequent formation of homologous contacts, leading to linear-element formation and recombination initiation. If this equivalent model applies, it may also be proposed that Rec8p binds to sites of sister chromatid cohesion formed during premeiotic DNA replication by the same mechanism as in mitotic DNA replication and thus involving mitotic cohesins. Scc1p/Mcd1p, the S. cerevisiae homolog of Rad21p, was reported to bind to chromosomes during mitotic S phase (28, 49).
A number of proteins involved in recombination were shown to form foci on the chromosomes during meiotic prophase (see, e.g., references 10 and 72). It remains to be demonstrated whether Rec8p foci colocalize with recombination enzymes loading onto the initiation sites of recombination. We propose loading of Rec8p onto emerging recombination initiation complexes as a parsimonious way to ensure that linear-element formation occurs at the sites where stabilization of recombination intermediates is required. Ultimately, sister chromatid cohesion in the chromosome arms is required after crossover formation and recombination complex (nodule) dissolution, and it must then be maintained up to anaphase I. However, sister chromatid cohesion may also have a function in early prophase of fission yeast meiosis. Its presence in wild-type early prophase and its absence in rec8 mutant prophase have been demonstrated (54, 70). With Rec8p antisera, the protein was localized to about 100 foci in prophase nuclei (Fig. 3). The average number of crossovers per fission yeast meiosis is 45 (56). Thus, sufficient Rec8p foci form during prophase to account also for conversion events not associated with crossing over. A roughly 1:2 relation between crossover and conversion events was proposed early in the analysis of intragenic recombination of S. cerevisiae (for a review, see reference 61) and is consistent with the existing data on S. pombe (88). Smith and Roeder (77) proposed that Red1 protein nucleates the formation of axial elements in S. cerevisiae and plays a role in meiotic sister chromatid cohesion. Red1 also interacts with a protein phosphatase (83). The respective roles of Red1 and the S. cerevisiae Rec8p homolog (see below) remain to be investigated. As reviewed by Kleckner (37), the lateral elements may stabilize the initial homologous contacts by firmly organizing the two sister chromatids attached to them. DNA regions close to the sites of attachment of chromatin loops to the lateral elements may then be protected for maintenance of their interactions with one of the homologous chromatids throughout prophase, until the recombination intermediates are resolved. Genetic and cytological data collected on rec8 mutants clearly demonstrated the involvement of Rec8p in sister chromatid cohesion and consequently in correct chromosome segregation in meiosis I (54). Rec8p may also be involved in late sister chromatid cohesion that is required for maintenance of chiasmata until transition from metaphase to anaphase I (for a review, see reference 52). Late sister chromatid cohesion must be independent of linear elements, since degradation of linear elements clearly occurs before meiosis I (2). Concurrently, nuclei approaching meiosis I also stop nuclear movement (13, 81). Rec8p is phosphorylated during meiotic prophase. Its mitotic homolog, Rad21p, is multiply phosphorylated on serine and threonine residues, and its phosphorylation status varied through the cell cycle (9). The transition from unphosphorylated to phosphorylated Rec8p occurs early in prophase (Fig. 4), perhaps in connection with linear-element polymerization. Additional (de)phosphorylation steps may occur at the transitions to completion of recombination and linear-element degradation. The presence of Rec8p even after meiosis I suggests a further role for the protein that may be similar to the one proposed for the rat SCP3/Cor1 lateral-element component. SCP3/Cor1 is a phosphoprotein whose phosphorylation state changes during pachytene (43). Relocation of Rec8p to the centromeres of chromosomes, as demonstrated for Cor1 in rat meiosis (20, 53), is consistent with our results in Fig. 4. Recently, Watanabe and Nurse (87) obtained evidence for binding of Rec8p to the centromeric regions of chromosomes before meiosis I and persistence of this binding up to meiosis II. Their work emphasizes the function of Rec8p at the centromeric regions of the chromosomes, but they also observed localization of Rec8p throughout the nucleus during meiotic prophase.Region specificity of recombination reduction in rec8 mutants. De Veaux and Smith (18) showed that rec8, rec10, and rec11 point mutations reduce the recombination frequency more than 100-fold in the central part of chromosome III but not more than 10-fold in chromosomes I and II. They proposed that the respective wild-type proteins activate recombination in some chromosome regions but not in others. Our additional data showing strong reduction also in the central regions of chromosomes II and I (Fig. 2), as well as similar results obtained independently (42), do not exclude this hypothesis. However, the results are at odds with our hypothesis proposing that Rec8p binds to all early homologous contacts that will be resolved as recombination events. We suggest an alternative explanation, assuming that several factors contribute to the high level of meiotic recombination and that they are of variable importance in different regions of the genome.
Meiotic recombination frequencies are 2 to 3 orders of magnitude above the mitotic recombination frequencies in fission yeast (50), as in other organisms (3). Disruption of spindle pole integrity and telomere clustering leads to about a fivefold reduction of recombination frequencies (15, 59, 73). A similar result was obtained when nuclear movement was abolished (31). Thus, formation and maintenance of the bouquet and nuclear movement are two factors contributing to high levels of meiotic recombination. However, this contribution is minor compared to other factors. We suggest that the vigorous nuclear movement may also act negatively, leading to disruption of homologous pairing before recombination intermediates are resolved. This would reduce recombination frequencies most strongly in regions that are under greater mechanical stress. Our model for primary Rec8p function proposes that linear elements contribute to the stabilization of recombination intermediates. The small reduction of recombination in the vicinity of the telomeres (Fig. 2) may then result from maintenance of pairing due to telomere clustering and little disruption by chromosome movement. In contrast, in the regions far from the telomeres, the loss of stabilization by linear elements may frequently lead to disruption of recombination intermediates due to mechanical stress. While recombination reduction is larger than 2 orders of magnitude in the centromeric regions of chromosomes II and III, it is less pronounced in the centromeric region of chromosome I. Interestingly, both arms of chromosome III and one arm of chromosome II are short, while both arms of chromosome I are longer. Long arms may experience less mechanical stress than short arms. An alternative explanation for the strong region-specific reduction of recombination frequency in rec8 mutant strains may be the involvement of another protein, fulfilling similar functions to Rec8p only at the ends of chromosomes. However, the slight reduction of recombination frequencies at the telomere regions in rec8 mutant strains indicates that Rec8p also has a function at the ends of chromosomes.The rad21/rec8 gene family and cloning of a human rec8 homolog. On the basis of amino acid similarity to Rec8p, we identified a human homolog of fission yeast rec8. hrec8 was homologous both to rec8 and rad21 of fission yeast and to the previously described human rad21 homolog (48) (see above). The degree of homology is well within the range previously found for orthologous yeast-mammalian DNA repair and metabolism genes (23).
We also identified a rec8 homolog, rec8sc, within the published budding-yeast genome. Interestingly, disruption of the S. cerevisiae rec8sc gene resulted in no detectable phenotype under vegetative growth (spore germination, growth on different media, and resistance to UV radiation and methyl methanesulfonate). However, homozygous deletion of rec8sc resulted in loss of sporulation (4). Given the sequence homology between rad21 and rec8 family members, which genes are true homologs in the different species? For a number of reasons, it is likely that the designation we use here is correct. First, in the regions of conserved sequence, there are key residues conserved between the rec8 family members which are not conserved in the rad21 group, and vice versa. For example, with reference to human Hrec8p (Fig. 5A and B), the residues at position 17 are T or V in rec8 genes but mostly K in rad21 genes; at position 25, rec8 genes have all G residues whereas the others have different amino acids; at position 94, H and S are present in rec8 genes and mostly E is present in rad21 genes. Likewise, T23 and S26 are conserved between Rec8p and Hrec8p, but the rad21 genes carry a chemically different amino acid. Second, deletion of the rad21 and SCC1/MCD1 genes in fission and budding yeasts revealed that they are essential for mitotic growth. Hypomorphic alleles show similar phenotypes in mitotic cells (8, 28, 49). In contrast, deletion of the rec8 genes in both yeast species resulted in nonvital phenotypes restricted to meiosis and sporulation (reference 4 and our results). Third, Hrec8p may partially complement the low spore viability of a fission yeast rec8 deletion strain but fails to complement recombination deficiency. We did not find a clear separation of rad21 from rec8 classes on phylogenetic analysis of the rad21/rec8 gene family. For some duplicated genes involved in DNA metabolism, e.g., RAD23 and RAD6 of S. cerevisiae (40, 84), gene duplication apparently occurred during the one billion or more years of evolution from yeasts to mammals (38). For the rad21/rec8 family, however, it appears that gene duplication occurred earlier, before differentiation of yeasts and other eukaryotes, probably with the evolution of meiosis itself. Further, as evidenced by the lengths of the lines in Fig. 5C, the rec8 genes appear to have evolved at a higher rate than the corresponding rad21 genes in the respective species. The common root between the two C. elegans proteins and hHR21sp (Fig. 5C) also suggests that these sequences are derived from the same ancestral gene. Since rad21 and rec8 genes exist in budding yeast, fission yeast, Drosophila (85), and mammals, they are likely to be present in intermediate species as well. Thus, a rec8 homolog may eventually be identified in C. elegans. hrec8 was mapped by FISH to chromosome 14q11.2-12 (Fig. 6A). This locus does not correspond to any known human disease. Previously, sequence-tagged sites corresponding to hrec8 were mapped to chromosomes 1, 5, 14, and X (Genethon). The mapping to X may represent cross-hybridization to the hHR21sp intronless pseudogene located on the X chromosome (GenBank accession no. HSU85A3). Apparently this pseudogene originated recently in evolution, given the high degree of identity to hHR21sp (data not shown). As with the mouse rad21 homolog, mHR21sp, mRNA formation from mrec8 was high in testis tissue and detectable above baseline in thymus tissue. Recombination occurs in both these tissues. However, unlike mHR21sp, mrec8 was highly expressed in postmeiotic testis tissue (Fig. 6B). The transcript seen in postmeiotic cells did not represent cross-hybridization of mrec8 with the mHR21sp gene, since mHR21sp mRNAs differ in size and since nucleotide sequence similarity between these genes is poor in the region of the probe used for hybridization (data not shown). The significance of mrec8 expression in postmeiotic testis cells is unknown. However, in view of the role for a rad21/rec8 family member in chromatin condensation (28), mrec8 may be involved in the marked chromatin repackaging accompanying spermiogenesis.| |
ACKNOWLEDGMENTS |
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The first two authors contributed equally to this work.
We thank the following persons for communication of unpublished results: Y. Lin, D. Evans, and G. Smith; W.-D. Heyer and V. Bashkirov; Y. Hiraoka; M. D. Krawchuk and W. P. Wahls; Y. Watanabe and P. Nurse; K. Nasmyth and F. Klein; and W. Warren. We thank H. Scherthan for technical advice, S. Verschoor for technical assistance, J. Hoogerbrugge and A. Grootegoed for fractionated mouse testis cells, and F. Fabre for supplying basic budding yeast strains. M.J.M. thanks colleagues in the Bootsma/Hoeijmakers laboratory for general advice and discussion.
R.K. is a Fellow of the Royal Netherlands Academy of Arts and Sciences. This work was supported by the Swiss National Science Foundation, a grant of the Human Frontier Science Program, the Royal Australasian College of Radiologists, the Netherlands Organization for Scientific Research (NWO; 901-01-097), and International Human Frontier Science Program postdoctoral fellowship LT-506/94 to M.J.M.
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FOOTNOTES |
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* Corresponding author. Mailing address for Jürg Kohli: Institute of General Microbiology, University of Bern, Baltzer-Strasse 4, CH-3012 Bern, Switzerland. Phone: 41-31-631-4654. Fax: 41-31-631-4684. E-mail: juerg.kohli{at}imb.unibe.ch. Mailing address for Michael J. McKay: Peter MacCallum Cancer Institute, St. Andrew's Place, East Melbourne 3002, Australia. Phone: 61-3-9656-1246. Fax: 61-3-8656-1411. E-mail: mmckay{at}petermac.unimelb.edu.au.
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REFERENCES |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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