![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
Vol. 16, Issue 3, 1449-1455, March 2005
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
* Department of Genetics, The Hebrew University of Jerusalem, Jerusalem 91904, Israel;
Division of Molecular Genetics, Netherlands Cancer Institute, 1066CX Amsterdam, The Netherlands
Submitted July 15, 2004;
Revised November 29, 2004;
Accepted December 21, 2004
Monitoring Editor: Douglas Koshland
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
|---|
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
|---|
) as well as in fission yeast (Cervantes et al., 2000; Zenvirth and Simchen, 2000) and in mouse (Mahadevaiah et al., 2001; Zenvirth et al., 2003). Meiotic DSBs occur preferentially in regions that have a high tendency to undergo recombination, the so-called recombination hot spots, at least in budding yeast. A DSB in one of the four chromatids initiates the process of meiotic recombination; resection of the DSBs, to form 3' single-strand overhangs, was found both in yeast (Sun et al., 1991) and in mouse spermatocytes (Zenvirth et al., 2003). Meiotic DSBs are repaired by interaction with homologous nonsister chromatids, thus forming recombinant molecules and chiasmata, which hold together the two homologous chromosomes and facilitate their proper segregation.
The universality of meiotic DSBs and their repair by homologous recombination is further enhanced by the findings that yeast artificial chromosomes (YACs), consisting largely of DNA from human cells, also undergo breakage during meiosis in yeast (Klein et al., 1996a, 1996b), as well as meiotic recombination (Sears et al., 1992, 1994) at rates characteristic to yeast rather than to meiosis in humans. Moreover, YACs containing DNA from recombination hot spots in the human genome undergo 2–3 times more double-strand breakage in yeast meiosis than YACs containing DNA from cold spots (Klein et al., 1996b), indicating that important features of the recombination machinery are conserved and shared between humans and yeast.
Here we report on mouse-DNA YACs that, unlike all other YACs previously examined, do not undergo meiotic DSBs during yeast meiosis. We also find that recombination on these YACs is severely compromised and that their segregation in meiosis is very distorted. We show that silencing of meiotic DSBs on these YACs is relieved by mutations in two SIR genes, suggesting that the YACs assume a chromatin configuration that is inaccessible to the meiotic-DSB forming proteins. As the two refractory YACs contain DNA from a recombination hot spot in the MHC class III region of mouse chromosome 17 (Snoek et al., 1998), we try to speculate on the behavior of this region during meiosis in the mouse.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
|---|
, 1998). YAC109 (150 kb), YAC35 (370 kb), and YAC328 (500 kb) contain DNA from mouse chromosome 2. The five YACs were obtained by M. Snoek by selection from the YACs at Genethon (Paris), using microsatellite markers located in the desired region. The YACs were derived from plasmid pYAC4 (Burke and Olson, 1991) and carry the yeast markers URA3 (at the end of the long arm) and TRP1 (on the short arm, near the centromere), unless otherwise indicated in the text. The YACs were originally generated in yeast strains derivatives of AB1380 MATa, ura3, trp1, ade2-l, his5, lys2, ile-, thr-, can1 (Burke and Olson, 1991) and were transferred by Kar1- matings (Hugerat et al., 1994; Spencer et al., 1994) to haploids of SK1 genetic background. For examination of meiotic DSBs, the final recipients of the YACs were of the following genotypes: Strain 2850: MATa, ho::hisG, ura3, lys2, trp1, rad50S::ura3, can-1; Strain 2851: MAT
, ho::hisG, ura3, lys2, trp1, rad50S::ura3, cyh2; and Strain 2860: MAT, ho:LYS2, ura3
, lys2, leu2, rad50S::ura3, cyh2. The YAC-carrying haploids were mated with SK1 haploids of the opposite mating type, carrying the rad50S mutation, either with the same YAC or without a YAC.
To test the effect of sir mutations on meiotic DSBs, sir gene disruptions were introduced by one-step transformation with the following plasmid-derived fragments: HindIII fragment of plasmid pJH540 for disrupting SIR2 (Ivy et al., 1986), SalI-XhoII fragment of plasmid pKL12 for disrupting SIR3 (Stone et al., 1991) and SmaI-PvuII fragment of pBe200 for disrupting SIR4 (Ivy et al., 1986). The sir deletions were verified by PCR analysis and by their phenotypic expression (these haploid strains did not mate and started sporulation.)
For the genetic analysis of meiotic recombination, end-markers on the YACs had to be replaced by other markers. Thus on one YAC, TRP1 was replaced by ADE2 by one-step transformation with a NotI-SacI fragment of plasmid pPHH2 (Hugerat and Simchen, 1993), and on another, homologous YAC, URA3 was replaced by LEU2, by transformation with the HindIII-PvuII fragment of plasmid pPH3. The latter was obtained by inserting the LEU2 SalI-XhoI fragment (2.3 kb) from YEp13, into the SalI site of pBR322. These two replacements provided us with two homologous YACs, between which recombination could easily be examined by tetrad analysis. One YAC was marked ADE2-URA3 and the other TRP1-LEU2. The two YACs were transferred sequentially by Kar1- matings (Hugerat and Simchen, 1993) to strain NE29 (MATa, ura3, lys2, leu2, trp1, ade2, his4, can1), which was later mated to NE30 (MAT, ura3, lys2, leu2, trp1, ade2, HIS4), to form the diploids that were sporulated and of which tetrads were dissected.
Media, Growth, and Sporulation Conditions
Yeast cultures were grown on rich (YEPD), on synthetic defined (SD) medium (Sherman, 1991), or on SPO plates (Kassir and Simchen, 1991). To keep the YACs from getting lost, cells were grown on SD plates that lacked the nutrients for which the markers on the YACs provided (e.g., for a strain containing a YAC marked LEU2-URA3 the medium lacked leucine and uracil).
For determination and mapping of meiotic DSBs (Zenvirth et al., 1992), cells were grown overnight in liquid YEPD resuspended in YEPA at dilution 1:400, and grown with vigorous agitation to cell density of 1–2 x 107/ml and then washed in water and resuspended at the same cell density in liquid SPM.
For tetrad analysis of YAC segregation and recombination, cells were sporulated on plates; first they were grown for 2–3 d on YEPD plates and then the culture was spread on SPO plates (Kassir and Simchen, 1991).
Identification of Meiotic DSBs in Sporulating Cultures
Chromosome-length DNA was prepared from samples withdrawn immediately (time 0) and 6 h after transfer to SPM sporulation medium and analyzed by pulsed-field gel electrophoresis (PFGE) in a CHEFDR apparatus (Bio-Rad, Richmond, CA), as described earlier (Zenvirth et al., 1992). Southern blots were hybridized to a series of 32P-labeled probes and the autoradiograms compared.
Probes used in this work were prepared from digested DNA fragments or PCR products: pBR322: marks all YACs by hybridizing to YAC ends; B2: repetitive sequences specific to the mouse genome, used to identify mouse DNA in YACs. Fragment of gene YCR48w was used to test meiotic DSBs on yeast chromosome III. The probes were labeled by random priming protocol (Roche, Mannheim, Germany) with [-32P]dCTP.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
|---|
, 1996b; and unpublished results), yeast strains carrying mouse DNA YAC99 (
200 kb) or YAC100 (450 kb) did not show any signs of DSBs during meiosis. The experimental procedure by which these YACs were examined was as follows. Each of the YACs was transferred from the strain in which the YAC library was generated, AB1380 (Burke and Olson, 1991), to a rad50S MATa strain (2580), by two-stage Kar1- mating (Hugerat et al., 1994; Spencer et al., 1994). The rad50S MATa strains with the YACs were mated to a rad50S MAT strain, to form diploids, in which meiotic DSBs could be easily examined. The diploid strains were grown in YEPA medium and sporulated in SPM, as described in Materials and Methods. Cell samples were collected at times 0 and 6 h after the initiation of meiosis (transfer from YEPA to SPM), from which intact, chromosome-size DNA molecules were prepared (Zenvirth et al., 1992). These DNA samples were run on a PFGE apparatus, the gels were blotted onto membranes, and these were hybridized to mouse-DNA specific 32P-labeled probe, B2 (Figure 1A); similar results were obtained when pBR322 DNA was used as a probe (unpublished data). Three "trivial" explanations for the absence of meiotic DSBs on the MHC class III YACs were considered: 1) The strains carrying YAC99 or YAC100 did not undergo meiosis and therefore did not initiate the recombination process. We know that due to homozygosity of the rad50S mutation, these cells cannot complete meiosis and only aberrant asci are formed. Such cells were observed in the cultures undergoing sporulation, but it was not clear whether meiotic DSBs were formed in these cells (on the "native" yeast chromosome). To check whether early events of meiosis and recombination had taken place, we stripped off the blotted membranes from the B2 radioactive label and rehybridized the blots with another 32P-labeled probe, specific for yeast chromosome III. As seen in Figure 1B, the native yeast chromosome did undergo meiotic breakage in the same DNA samples in which the YACs remained intact. Therefore it appears that silencing of double-strand breakage on these mouse YACs is specific to the alien chromosomes. 2) Another possibility that we considered was that YACs carrying mouse DNA did not show meiotic DSBs because they differ in a fundamental way from native yeast chromosomes. Such a difference was not found with human DNA YACs, however. Nevertheless, we checked the behavior, during yeast meiosis, of several additional YACs that contain DNA from chromosome 2 of the mouse (Figure 2 and unpublished data). Figure 2 represents an experiment, in which meiotic DSBs were examined in four yeast strains, three with YACs containing DNA from chromosome 2 and a fourth strain with YAC100, from the MHC class III region. The former three YACs underwent meiotic breakage, like the yeast chromosomes, whereas the latter did not. We conclude that silencing of meiotic DSBs occurs only on the YACs containing DNA from this region, YAC99 and YAC100, and not on other mouse DNA YACs. 3) Another possible reason why the two MHC YACs were silenced with respect to meiotic DSBs could be that they lacked the DNA sequence that are normally associated with the main meiotic DSBs in yeast, the CoHR motif (Blumental-Perry et al., 2000). (However, a recent study by Haring et al. (2004) has questioned the association of the defined CoHR with some DSBs). We checked for the presence of CoHR in the sequence containing the genes G7e, G7a, and G7c of the MHC class III region, which are found in YAC99 and YAC100 (van Kooij et al., 2001), both forward and reverse DNA strands, using the program ProfileGap (Blumental-Perry et al., 2000). Five high-quality matches to CoHR were found (Figure 3); thus, the absence of DSBs on the MHC YACs could not be explained by lack of the CoHR motif. We conclude that these, mouse MHC class III YACs, are refractory to meiotic DSBs despite containing the sequence motifs that are often found near or at the breaks.
|
|
|
We have also considered the possibility that the MHC class III YACs undergo DSBs only very late in meiosis, because it is known that late-occurring DSBs might not be detected in rad50S strains (Borde et al., 2000). We therefore examined DSBs during meiosis in a Rad+ strain that contain a pair of YAC100. No obvious breaks were observed on the YACs (using pBR322 DNA as a probe), whereas transient meiotic DSBs were clearly seen on chromosome III (unpublished data).
Meiotic Recombination of the MHC Class III YACs Is Severely Compromised
As the two YACs that contained DNA of the mouse MHC class III region were refractory to meiotic double-strand breakage in yeast cells, we wanted also to see whether these chromosomes undergo recombination during yeast meiosis. Human-DNA YACs have been shown to recombine readily during yeast meiosis, at high frequencies, comparable to recombination along native yeast chromosomes (Sears et al., 1992). To study meiotic recombination on YACs, one needs to construct diploid strains with pairs of homologous YACs that differ from each other at markers in at least two loci. The original YACs carry the marker TRP1 on the short arm, near the centromere, and URA3 at the end of the long arm. For each YAC in the original strains, TRP1 was replaced by ADE2 by transformation, as described in Materials and Methods, thus resulting in a YAC with the markers ADE2–URA3. In parallel to this transformation, the original strain (with YAC TRP1–URA3) was also subject to replacement of URA3 by the marker LEU2, thus resulting in a YAC with the markers TRP1–LEU2. The two YACs were each transferred by two-stage Kar1- mating (Hugerat et al., 1994) to the same haploid strain, NE29, from which MATa/MAT diploids were derived by mating to strain NE30; each diploid was homozygous for the mutations ade2, ura3, trp1, leu2, lys2, and carried the two homologous YACs, marked ADE2–URA3 and TRP1–LEU2. The diploids did not carry any rad mutation and underwent normal meiosis with high efficiency. On tetrad dissection, good spore germination was observed (>90%). These YAC manipulations and strain constructions were done separately for YAC99, YAC100, and YAC109, the latter being a 150-kb YAC with DNA from mouse chromosome 2. No significant rearrangements or aberrations of these YACs were observed during these manipulations or during vegetative growth of the yeast cells (chromosomes were examined by pulsed-field gel electrophoresis at each stage of the genetic manipulation). Similar strains were previously constructed in our lab and analyzed (Table 1), with YAC pairs from the pseudoautosomal region (PAR) of human chromosome X, YAC18ED5 (190 kb) carrying DNA from the recombination hot spot in PAR, and YACy-WXD4932 (100 kb) from a recombination colder spot in the same region (Klein et al., 1996b).
|
The results of tetrad analysis of asci dissected from all five strains are shown in Table 1. The first part of the table contains data on the segregation patterns of the YACs (Table IA). Meiotic segregation of YAC99 and YAC100 is very distorted. Table IB contain the different types of aberrant segregation as describe earlier (Sears et al., 1992). About half the cells undergoing meiosis had lost one or both YACs or have shown to contain three YACs instead of two. This YAC instability could be at least partly premeiotic. For YAC109, only 20% of meioses showed YAC loss or duplication and similar levels of YAC instability were found with the human DNA YACs, 18ED5 and WXD4932. In the genetic analysis, the three mouse YACs and the cold-spot human YAC also showed high frequencies of meiotic nondisjunction, especially in meiosis I. Recombination analysis of the YACs is shown in Table IC, which only contains data from tetrads that showed normal segregation of the YACs (i.e., the four spore colonies containing one YAC each, with markers on both YAC ends segregating 2:2). Here one can see that in very few cases YAC99 and YAC100 underwent recombination events that lead to tetratype segregation (TT), presumably one event per meiosis. The given proportion of these events is obviously an overestimate for the frequency of recombination, as only one fifth of the meioses showed normal segregation of the YACs, out of which this proportion was calculated. None of the other tetrads (80%, showing abnormal segregation and instability of YACs) contained evidence of recombinant YACs. Unexpectedly, the diploid with YAC99 had six tetrads that showed nonparental ditype segregation (NPD) of the YACs, the same frequency as TT. NPD usually indicates two independent exchange events in meiosis and its frequency is considerably lower than TT, which results from a single exchange, as seen for YAC109 and yWXD4932. Even when numerous exchanges occur between the two markers, and almost all segregation patterns (PD, TT, NPD) involve more than one exchange, like in YAC18ED5, the ratio NPD:TT only reaches 1:4. A plausible explanation for the six NPD tetrads of YAC99 is that they represent cases of reciprocal recombination between the two YACs in the mitotic division that preceded the meiosis at question, or in G1 of meiosis, before DNA replication.
We conclude that in parallel to the absence of meiotic DSBs on the MHC class III YACs, these YACs recombine very rarely in meiosis and their segregation is also seriously compromised. Meiotic segregation of a pair of homologous chromosomes clearly depends on the occurrence of at least one recombination event (and chiasma) between them. Therefore it is not surprising that these YACs show aberrant segregations. However, the genetic analysis (tetrad analysis, in Table 1) shows that these YACs are also very unstable, both in meiosis and in the preceding divisions. To evaluate instability of these YACs during mitotic divisions and under conditions leading to meiosis/sporulation, we tested the loss of YAC markers in vegetatively grown cells, on YEPD plates, and in the mitotic cell divisions before meiosis, on SPO plates. Diploid cells that contained two homologues YACs (with different end markers) were spread on these two types of plates and then transferred to YEPD plates at times 0 and 8 h to form single colonies. The latter were replica plated onto differential media, to determine the presence of the YAC markers. A colony that did not grow on the two media indicative of markers at the ends of a given YAC was considered as having arisen from a cell that had lost that particular YAC. The percentage of YAC loss was calculated by dividing the number of such colonies by the total number of colonies (Table 2). From these data it appears that the two mouse YACs that carry MHC III DNA are somewhat unstable during mitotic divisions, compared with the other YACs, especially during the few divisions before meiosis (on SPO plates). Thus the instability of YAC99 and YAC100 observed in the analysis of tetrads (Table 1) might be an extension of the (milder) instability observed already during mitotic divisions (Table 2).
|
Mutations in SIR Genes Release Meiotic DSB Silencing
The best-documented case of chromosomal regions in yeast that are refractory to double-strand breakage are the silent mating-type cassettes, HML and HMR (Haber, 1998). Although these cassettes contain DNA sequences that are also present in the mating-type locus, MAT, in which the Ho endonuclease efficiently generates DSBs, the silent cassettes are normally devoid of such breaks, remain inaccessible to Ho, and may participate in mating-type interconversion only as donors of genetic information. HMR and HML are protected from HO-induced breaks by their unique chromatin structure, consisting of the Sir proteins. Mutants with mutations in SIR genes show mating-type switching in the silent cassettes, which may (in these mutants) also serve as recipients of genetic information, following the occurrence of DSBs in the cassettes (Klar et al., 1981; Nasmyth, 1982). There is also a case of Sir2-mediated silencing of meiotic recombination in the array of ribosomal DNA genes of S. cerevisiae (Gottlieb and Esposito, 1989). We asked whether the mouse MHC class III YACs were also protected from meiotic DSBs by assuming a Sir-based chromatin structure. To examine this possibility, we introduced the mutations sir2, sir3, or sir4 into the rad50S haploid strains carrying YAC99 or YAC100. Such strains enter meiosis as haploids because a sir mutation allows the expression of the silent cassettes HML and HMRa. Figure 4 presents experiments in which meiosis was induced in Sir- rad50S haploids, carrying YAC100. DNA was gently extracted at times 0 and 6 h after the induction of meiosis, DNA samples were run on PFGE, blotted onto membranes, and probed with 32P-labeled YAC-specific probe pBR322. Meiotic DSBs can be seen at 6 h in the sir2 and sir4 strains but not in the sir3 strain The DSBs on the YACs, however, could not be accurately mapped because their DNA was not sequenced. Stripping the membranes of the YAC-specific 32P label and rehybridization with yeast chromosome III–specific probe has shown that in the sir3 strain, as well as in the others, meiosis was initiated and native yeast chromosomes underwent DNA breakage (unpublished data). Interestingly, the pattern of breakage of the YACs in the sir2 strain was different from that found in sir4 (Figure 4). This could be due to the different roles of the two proteins in chromatin structure (Gasser and Cockell, 2001; Luo et al., 2002): binding of Sir2, a histone deacetylase, to the chromatin depends on the presence of Sir4, whereas the latter can be present even without Sir2. We conclude that Sir 2 and Sir4 proteins, but not Sir3, take part in making the MHC class III YACs inaccessible to DSBs during yeast meiosis.
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
|---|
), in fission yeast (Cervantes et al., 2000; Zenvirth and Simchen, 2000), and in mouse spermatocytes (Mahadevaiah et al., 2001; Zenvirth et al., 2003), we believe that they are common to all eukaryotes and meiotic recombination assumes similar features in different organisms. Moreover, YACs containing human DNA undergo DSBs during budding yeast meiosis (Klein et al., 1996a, 1996b), as do YACs with DNA of the plant Arabidopsis (D. Zenvirth, unpublished results) and several mouse YACs (see above, Figure 2), supporting the universality of meiotic DSBs. YACs with DNA from recombination hot spots in the human genome show considerably more breakage during yeast meiosis than cold-spot YACs, implying similarities between the meiotic recombination machinery in humans and in yeast (Klein et al., 1996b). These differences in DSBs between hot-spot and cold-spot YACs are also reflected by the frequencies of recombination on the YACs (Table 1B). YAC18ED5 has undergone considerably more recombination events than yWXD4932—in most meioses of the former two or more exchanges have occurred, and therefore the distribution of PD:TT:NPD is as expected if the two end-markers segregated freely from each other (1:4:1). YAC109 and yWXD4932 have undergone only one or two exchanges (or none at all), reflecting their shorter length, as well as their genomic origin: YAC109 can be expected to represent an average recombination potential in the mouse genome; yWXD4932 comes from pseudoautosomal region (PAR) on the human chromosome X, next to the hot spot covered by 18ED5 (Ried et al., 1995)—this is a relatively cold region (compared with 18ED5), but even such "cold" regions in PAR may show at least 10-fold higher frequency than the genome average (Lien et al., 2000).
In contrast to the behavior in meiosis of most YACs, the two containing DNA from the mouse MHC class III region did not undergo double-strand breakage during meiosis in rad50S yeast strains (Figure 1A) and showed very few recombination events (Table 1B). This peculiar behavior was unique to the YACs, because the native yeast chromosomes in the same cells behaved as expected during meiosis, showing meiotic DSBs and undergoing normal segregation. Absence of meiotic DSBs on YAC99 and YAC100 was observed in spite of the presence of CoHR DNA sequences (Figure 3), which are commonly associated with most preferred meiotic DSBs in S. cerevisiae (Blumental-Perry et al., 2000). We presume that the MHC class III inserts on these YACs are responsible for their unusual behavior in meiosis, perhaps reflecting their origin and role in the mouse genome. These two YACs also showed serious mal-segregation in meiosis: high rates of nondisjunction in meiosis I and II, and chromatid loss (together 12–14% of tetrads), and of premeiotic loss of one or both YACs, or duplication of one of the YACs (40–50% of tetrads). During vegetative growth on YEPD plates, and especially on SPO plates (before sporulation), these YACs also showed marked instability (Table 2), although to a lesser extent. We therefore speculate that these premeiotic changes could be induced by the poor nutritional conditions just at the onset of meiosis and sporulation. The meiotic NDJ events could of course be related to the absence of recombination, as was previously found for a pair of nonhomologous YACs in meiosis (Sears et al., 1994). Moreover, a number of premeiotic gene-conversion events, with or without YAC loss, were also observed (Table 1B); these could also occur during meiotic DNA replication (also called premeiotic S). We conclude that the behavior of the MHC class III YACs might have also been abnormal in the mitotic divisions just preceding meiosis or during meiotic DNA replication. Some of these defects could reflect difficulties in DNA replication or centromere functioning on the YACs in these divisions, possibly resulting from a unique chromatin confirmation (see below).
We found that mutations in SIR2 or SIR4, but not in SIR3, remove the inaccessibility of the MHC class III YACs to meiotic DSBs (Figure 4). This may suggest that the proteins Sir2 and Sir4 take part in a unique chromatin structure that makes the YACs refractory to the meiotic DNA breakage and recombination enzymes. This is a new role for Sir proteins, previously regarded mostly as transcriptional silencers (Gasser and Cockell, 2001). Meiotic DSB silencing may, for instance, be important near telomeres and centromeres. Meiotic DSBs are indeed silenced near the Telomeres of S. cerevisiae (Blumental-Perry et al., 2000) and preliminary experiments suggest that such silencing may be removed by the sir4 mutation (Raizman, Zenvirth and Simchen, unpublished results). A similar role was documented for Sir proteins in protecting the silent mating-type cassettes HML and HMRa from DNA cleavage by the HO endonuclease, despite these loci having the HO recognition sequences (Klar et al., 1981; Nasmyth, 1982). The unique chromatin structure of the MHC class III YACs could also account for their unusual segregation behavior in the premeiotic divisions, although at present we cannot offer a precise mechanism for the latter phenomenon.
What does meiotic DSB silencing of MHC class III YACs tell us about gametogenesis and meiosis in the mouse? We have previously suggested that the frequency of meiotic DSBs on human-DNA YACs may reflect their recombination potential in the human genome (Klein et al., 1996b). Recombination in yeast meiosis of two YACs from the human pseudoautosomal region also reflects their recombination potential (Table 1C, above). By extrapolation to the MHC class III region in the mouse genome, we speculate that its refraction to DSBs and recombination in yeast meiosis may reflect similar characteristics during mouse spermatogenesis. Thus the recombination hot spot identified in this region (Snoek et al., 1996, 1998) may not represent events that occur during meiosis in the mouse, but rather during premeiotic divisions. From the behavior of the MHC class III YACs in yeast, we speculate that the region may be protected from the recombination machinery during meiosis in the mouse, probably by a unique chromatin structure that is associated with unknown sequences in this region. These DNA sequences may be recognized by Sir2 or Sir4 in yeast or by proteins that are associated with the Sirs and by similar proteins in mouse. (No homologues were found in mammalian genomes to Sir3 and Sir4, although homologues of the histone deacetylase Sir2 were found in humans, mice, and other eukaryotic organisms [Sherman et al., 1999; Smith et al., 2000; Yang et al., 2000].) Support for the proposition that recombination in MHC class III occurs before meiosis comes from a PCR analysis of gene-conversion events in the MHC class II region at different stages of mouse spermatogenesis (Hogstrand and Bohme, 1997). On the other hand, recombination at the Psmb9 (Lmp2) recombination hot spot in the mouse MHC class II region has been recently shown to occur during spermatogenesis in the pachytene stage of meiosis (Guillon and de Massy, 2002). Thus it seems that on chromosome 17 of the mouse, in the 4500-kb region containing the MHC genes, recombination in subregions occurs at different stages during spermatogenesis. We suggest that these differences in recombination timing may reflect temporary changes in accessibility of the subregions. Accessibility may at least partly be determined by elements of the chromatin, namely proteins that are homologous or similar to the yeast Sir proteins.
|
ACKNOWLEDGMENTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
|---|
|
Footnotes |
|---|
Address correspondence to: Giora Simchen (simchen{at}vms.huji.ac.il).
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
|---|
Borde, V., Goldman, A., and Lichten, M. ((2000). ). Direct coupling between meiotic DNA replication and recombination initiation. Science 290, , 806-809.
Burke, D., and Olson, M. ((1991). ). Preparation of clone libraries in yeast artificial-chromosome vectors. Methods Enzymol. 194, , 251-270.[Medline]
Cervantes, M., Farah, J., and Smith, G. ((2000). ). Meiotic DNA breaks associated with recombination in S. pombe. Mol. Cell 5, , 883-888.[CrossRef][Medline]
Gasser, S., and Cockell, M. ((2001). ). The molecular biology of the SIR proteins. Gene 279, , 1-16.[CrossRef][Medline]
Gottlieb, S., and Esposito, R. ((1989). ). A new role for a yeast transcriptional silencer gene, SIR2, in regulation of recombination in ribosomal DNA. Cell 10, , 771-776.
Guillon, H., and de Massy, B. ((2002). ). An initiation site for meiotic crossing-over and gene conversion in the mouse. Nat. Genet. 32, , 296-299.[CrossRef][Medline]
Haber, J. ((1998). ). Mating-type gene switching in Saccharomyces cerevisiae. Annu. Rev. Genet. 32, , 561-599.[CrossRef][Medline]
Haring, S., Lautner, L., Comeron, J., and Malone, R. ((2004). ). A test of the CoHR motif associated with meiotic double-strand breaks in Saccharomyces cerevisiae. EMBO Rep. 5, , 41-46.[CrossRef][Medline]
Hogstrand, K., and Bohme, J. ((1997). ). Gene conversion of major histocompatibility complex genes in the mouse spermatogenesis is a premeiotic event. Mol. Biol. Cell 8, , 2511-2517.
Hugerat, Y., and Simchen, G. ((1993). ). Mixed segregation and recombination of chromosomes and YACs during single-division meiosis in spo13 strains of Saccharomyces cerevisiae. Genetics 135, , 297-308.[Abstract]
Hugerat, Y., Spencer, F., Zenvirth, D., and Simchen, G. ((1994). ). A versatile method for efficient YAC transfer between any two strains. Genomics 22, , 108-117.[CrossRef][Medline]
Ivy, J., Klar, A., and Hicks, J. ((1986). ). Cloning and characterization of four SIR genes of Saccharomyces cerevisiae. Mol. Cell. Biol. 6, , 688-702.
Kassir, Y., and Simchen, G. ((1991). ). Monitoring meiosis and sporulation in Saccharomyces cerevisiae. Methods Enzymol. 194, , 94-110.[Medline]
Keeney, S. ((2001). ). The mechanism and control of meiotic recombination initiation. Curr. Topics Dev. Biol. 52, , 1-53.[Medline]
Klar, A., Strathern, J., and Hicks, J. ((1981). ). A position-effect control for gene transposition: state of expression of yeast mating-type genes affects their ability to switch. Cell 25, , 517-524.[CrossRef][Medline]
Klein, S., Zenvirth, D., Dror, V., Barton, A. B., Kaback, D. B., and Simchen, G. ((1996a). ). Patterns of meiotic double-strand breakage on native and artificial yeast chromosomes. Chromosoma 105, , 276-284.[Medline]
Klein, S., Zenvirth, D., Sherman, A., Ried, K., Rappold, G., and Simchen, G. ((1996b). ). Double-strand breaks on YACs during yeast meiosis may reflect meiotic recombination in the human genome. Nat. Genet. 13, , 481-484.[CrossRef][Medline]
Lien, S., Szyda, J., Schechinger, B., Rappold, G., and Arnheim, N. ((2000). ). Evidence for heterogeneity in recombination in the human pseudoautosomal region: high resolution analysis by sperm typing and radiation-hybrid mapping. Am. J. Hum. Genet. 66, , 557-566.[CrossRef][Medline]
Luo, K., Vega-Palas, M. S., and Grunstein, M. ((2002). ). Rap1-Sir4 binding independent of other Sir, y Nospacekuqq, or histone interactions initiates the assembly of telomeric heterochromatin in yeast. Genes Dev. 16, , 1528-1539.
Mahadevaiah, S., Turner, J., Baudat, F., Rogakou, E., de Boer, P., Blanco-Rodriguez, J., Jasin, M., Keeney, S., Bonner, W., and Burgoyne, P. ((2001). ). Recombinational DNA double-strand breaks in mice precede synapsis. Nat. Genet. 27, , 271-276.[CrossRef][Medline]
Nasmyth, K. ((1982). ). The regulation of yeast mating-type chromatin structure by SIR: an action at a distance affecting both transcription and transposition. Cell 30, , 567-578.[CrossRef][Medline]
Ried, K., Mertz, A., Nagaraja, R., Trusgnich, M., Riley, J. H., Anand, R., Lehrach, H., Page, D., Ellison, J. W., and Rappold, G. ((1995). ). Characterization of a YAC contig spanning the pseudoautosomal region. Genomics 29, , 787-792.[CrossRef][Medline]
Sears, D. D., Hegemann, J. H., and Hieter, P. ((1992). ). Meiotic recombination and segregation of human
derived artificial chromosomes in Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA 89, , 5296-5300.
Sears, D. D., Hieter, P., and Simchen, G. ((1994). ). An implanted recombination hot spot stimulates recombination and enhances sister chromatid cohesion of heterologous YACs during yeast meiosis. Genetics 138, , 1055-1065.[Abstract]
Sherman, F. ((1991). ). Getting started with yeast. Methods Enzymol. 194, , 3-21.[CrossRef][Medline]
Sherman, J., Stone, E., Freeman-Cook, L., Brachmann, C., Boeke, J., and Pillus, L. ((1999). ). The conserved core of a human SIR2 homologue functions in yeast silencing. Mol. Biol. Cell 10, , 3045-3059.
Smith, J. et al. ((2000). ). A phylogenetically conserved NAD+-dependent protein deacetylase activity in the Sir2 protein family. Proc. Natl. Acad. Sci. USA 97, , 6658-6663.
Snoek, M., Teuscher, C., and Van Vugt, H. ((1998). ). Molecular analysis of the major MHC recombinational hot spot located within the GFc gene of the murine class III region that is involved in diseases susceptibility. J. Immunol. 160, , 266-272.
Snoek, M., van Dinten, L., and van Vugt, H. ((1996). ). A novel gene, G7e, resembling a viral envelope gene, is located at the recombinational hot spot in the class III region of the mouse MHC. Genomics 15, , 5-12.
Spencer, F., Hugerat, Y., Simchen, G., Hurko, O., Connelly, C., and Hieter, P. ((1994). ). Yeast kar1 mutants provide an effective method for YAC transfer to new hosts. Genomics 22, , 118-126.[CrossRef][Medline]
Stone, E., Swanson, M., Romeo, A., Hicks, J., and Sternglanz, R. ((1991). ). The SIR1 gene of Saccharomyces cerevisiae and its role as an extragenic suppressor of several mating-defective mutants. Mol. Cell. Biol. 11, , 2253-2262.
Sun, H., Douglas, T., and Jack, W., Szostak. ((1991). ). Extensive 3'-overhanging, single-stranded DNA associated with the meiosis-specific double-strand breaks at the ARG4 recombination initiation site. Cell 64, , 1155-1161.[CrossRef][Medline]
van Kooij, M., de Groot, K., van Vugt, H., Aten, J., and Snoek, M. ((2001). ). Genotype versus phenotype: conflicting results in mapping a lung tumor susceptibility locus to the G7c recombination interval in the mouse MHC class III region. Immunogenetics 53, , 656-661.[CrossRef][Medline]
Yang, Y., Chen, Y., Zhang, C., Nimmakayalu, M., Ward, D., and Weissman, S. ((2000). ). Cloning and characterization of two mouse genes with homology to the yeast Sir2 gene. Genomics 69, , 355-369.[CrossRef][Medline]
Zenvirth, D., Arbel, T., Sherman, A., Goldway, M., Klein, S., and Simchen, G. ((1992). ). Multiple sites for double-strand breaks in whole meiotic chromosomes of Saccharomyces cerevisiae. EMBO J. 11, , 3441-3447.[Medline]
Zenvirth, D., Richler, C., Bardhan, A., Baudat, F., Barzilai, A., Wahrman, J., and Simchen, G. ((2003). ). Mammalian meiosis involves DNA double-strand breaks with 3' overhangs. Chromosoma 111, , 369-376.[Medline]
Zenvirth, D., and Simchen, G. ((2000). ). Meiotic double-strand breaks in Schizosaccharomyces pombe. Curr. Genet. 38, , 33-38.[CrossRef][Medline]
This article has been cited by other articles:
|
|
 |
|
  P. A. Mieczkowski, M. Dominska, M. J. Buck, J. D. Lieb, and T. D. Petes Loss of a histone deacetylase dramatically alters the genomic distribution of Spo11p-catalyzed DNA breaks in Saccharomyces cerevisiae PNAS, March 6, 2007; 104(10): 3955 - 3960. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
