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Originally published as Genetics Published Articles Ahead of Print on March 2, 2005.

Genetics, Vol. 170, 95-106, May 2005, Copyright © 2005
doi:10.1534/genetics.104.036301

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Differential Activation of M26-Containing Meiotic Recombination Hot Spots in Schizosaccharomyces pombe

David W. Pryce*, Alexander Lorenz{dagger}, Julia B. Smirnova*,1, Josef Loidl{dagger} and Ramsay J. McFarlane*,2

* North West Cancer Research Fund Institute, University of Wales Bangor, Bangor LL57 2UW, United Kingdom
{dagger} Department of Chromosome Biology, University of Vienna, A-1030 Vienna, Austria

2 Corresponding author: North West Cancer Research Fund Institute, Memorial Bldg., University of Wales Bangor, Deiniol Rd., Bangor, Gwynedd LL57 2UW, United Kingdom.
E-mail: ramsay{at}sbs.bangor.ac.uk

Manuscript received September 14, 2004. Accepted for publication February 4, 2005.


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 LITERATURE CITED
 
Certain genomic loci, termed hot spots, are predisposed to undergo genetic recombination during meiosis at higher levels relative to the rest of the genome. The factors that specify hot-spot potential are not well understood. The M26 hot spot of Schizosaccharomyces pombe is dependent on certain trans activators and a specific nucleotide sequence, which can function as a hot spot in a position- and orientation-independent fashion within ade6. In this report we demonstrate that a linear element (LE) component, Rec10, has a function that is required for activation of some, but not all, M26-containing hot spots and from this we propose that, with respect to hot-spot activity, there are three classes of M26-containing sequences. We demonstrate that the localized sequence context in which the M26 heptamer is embedded is a major factor governing whether or not this Rec10 function is required for full hot-spot activation. Furthermore, we show that the rec10-144 mutant, which is defective in full activation of ade6-M26, but proficient for activation of other M26-containing hot spots, is also defective in the formation of LEs, suggesting an intimate link between higher-order chromatin structure and local influences on hot-spot activation.

PROGRAMMED genetic recombination is central to the formation of structures required to ensure a high fidelity of meiotic chromosome segregation, as well as serving as a major evolutionary driving force (for review see SMITH and NICOLAS 1998; ZICKLER and KLECKNER 1999; PAGE and HAWLEY 2003). Meiotic recombination events do not occur with an even distribution along chromosomes, with some loci that have levels suppressed (cold spots) or elevated (hot spots) relative to the genome-wide mean (for review see LICHTEN and GOLDMAN 1995; WAHLS 1998; PETES 2001; NACHMAN 2002).

In the fungi Saccharomyces cerevisiae and Schizosaccharomyces pombe, meiotic recombination hot spots are closely associated with sites of elevated levels of double-strand DNA breaks (DSBs), which are required for the initiation of meiotic recombination (SUN et al. 1989; STEINER et al. 2002). It is widely accepted that a critical first step in hot-spot activation is to make chromatin more accessible to the recombination factors that mediate DSB formation, although this alone may not be sufficient to confer hot-spot activity to a locus (WU and LICHTEN 1995). Heightened sensitivity of chromatin to micrococcal nuclease (MNase) is found at meiosis-specific hot spots in both S. cerevisiae and S. pombe, indicating a conserved and intimate link between a more open chromatin configuration and recombination initiation (OHTA et al. 1994; WU and LICHTEN 1994; MIZUNO et al. 1997). This has been termed the chromatin transition and is dependent on premeiotic DNA replication in S. cerevisiae (MURAKAMI et al. 2003).

The M26 hot spot of S. pombe has been well characterized (for review see SMITH 1994; DAVIS and SMITH 2001; PETES 2001). It is defined by the heptanucleotide sequence 5'-ATGACGT-3' (PONTICELLI et al. 1988; SZANKASI et al. 1988; SCHUCHERT et al. 1991) and was first identified as a G-to-T transversion in the ade6-M26 mutant (GUTZ 1971). The heptamer acts as a binding site for the Atf1/Pcr1 (Mts1/Mts2; Gad7/Pcr1) heterodimeric transcription factor and both atf1+ and pcr1+ genes are required for hot-spot activation (WAHLS and SMITH 1994; KON et al. 1997). Atf1/Pcr1 also binds to other M26-related sequences, collectively termed cAMP-response elements (CREs), which share the consensus sequence 5'-NTGACGT(C/A)-3' and also function as Atf1/Pcr1-dependent meiotic recombination hot spots (FOX et al. 2000). Furthermore, the mitogen-activated protein kinase pathway that is responsible for the regulation of the Atf1-mediated transcriptional response to stress is also required for full M26 hot-spot activation (KON et al. 1998; FOX et al. 2000; MIZUNO et al. 2001).

MIZUNO et al. (1997) have demonstrated that the local chromatin in which the ade6-M26 heptamer is embedded becomes more sensitive to MNase early on during meiotic entry, indicating that an active chromatin transition is occurring at M26. This transition is repressed by the Tup11/Tup12 repressors and requires Gcn5 and Snf22 for a normal transition profile (HIROTA et al. 2003; YAMADA et al. 2004). Gcn5 is a histone acetyl transferase, while Snf22 is a bromodomain Swi2/Snf2-like potential chromatin remodeller; deletion of gcn5+ results in a partial loss of hot-spot activation, while loss of snf22+ function results in an almost total loss of ade6-M26 hot-spot activation, indicating that the chromatin transition is essential for activation of the ade6-M26 hot spot (YAMADA et al. 2004).

Here we demonstrate that M26-containing hot spots at different positions within a single open reading frame have different requirements for their activation and that this is influenced by minimal changes to the local context in which each M26 heptamer is embedded. We discuss the possibility that this is a highly conserved phenomenon involving the chromosomal linear element (LE) protein Rec10, which has similarities to the S. cerevisiae axial element (AE) protein Red1 (LORENZ et al. 2004).


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 LITERATURE CITED
 
S. pombe strains and plasmids:
A list of the strains employed in this study and their genotypes are shown in Table 1. The ade6-52 and ade6-M375 alleles were used as marker alleles during two factor crosses against hot-spot and non-hot-spot control alleles of ade6. ura4-294 was used as the test allele during two factor crosses against hot-spot and non-hot-spot alleles of ura4. All ade6 and rec10(20)-144 alleles were subjected to DNA sequencing to ensure that alleles were correct.


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  		TABLE 1 Strains used in this study

 
Plasmid pJS3 was constructed by cloning a 3.25-kbp fragment of DNA generated by PCR using genomic DNA as a template (the GenBank accession number for rec10+ is U09871) (LIN and SMITH 1995). The PCR primers had EcoRI restriction sites at their 5'-ends enabling the PCR products to be cloned directly into the S. pombe vector, pFY20 (LI et al. 1997). The PCR primers were rec10 JS-F EcoRI (5'-GCGCGAATTCCTGCGGTGTGTTCAGTTTC-3'; rec10+ nucleotides are nucleotides 12908–12926 of S. pombe cosmid c25G10; http://www.sanger.ac.uk/Projects/S_pombe/) and rec10 JS-R EcoRI (5'-CGCGGAATTCCCTGTCTTCTGCATTGATATG-3'; rec10+ nucleotides are nucleotides 9688–9709 of S. pombe cosmid c25G10; http://www.sanger.ac.uk/Projects/S_pombe/).

Meiotic crosses:
Cultures were grown in yeast extract liquid (YE; MORENO et al. 1991) supplemented with adenine (200 mg/liter) to a density of ~2 x 107 cells/ml. Equal volumes (600 µl) of each culture were mixed in microfuge tubes, pulse centrifuged, and aspirated. Cell pellets were washed with 1 ml of dH2O and finally resuspended in 20 µl dH2O. Suspensions were spotted onto fully supplemented synthetic sporulation media (SPA) plates and incubated at the required temperature for 3–4 days (4–5 days for 20° crosses). After incubation, sporulating cells were scraped into a microfuge tube containing 1 ml of a 0.6% ß-glucuronidase (Sigma, St. Louis)/dH2O solution and incubated for 16 hr at 25°. After incubation, spores were harvested and resuspended in 30% ethanol and incubated at room temperature for not longer than 5 min. Suspensions were then centrifuged and aspirated dry and cell pellets were resuspended in 1 ml dH2O.

Determination of meiotic recombinant frequencies:
Recombination frequencies were determined as either Ura+ or Ade+ prototrophs/106 viable spores. To determine total viable spore numbers, aliquots from serial dilutions of spore suspensions were plated onto yeast extract agar (YEA) plates. After 3 days incubation at 34°, at least two plates, each with >50 colonies, where counted and their average used to determine viable spore totals. To measure Ade+ recombinant totals, we took advantage of the fact that S. pombe cells exhibit purine antagonism (POURQUIE 1970); high guanine concentrations inhibit the uptake of exogenous adenine, rendering adenine auxotrophs incapable of growth on YEA, which contains a limited amount of adenine (CUMMINS and MITCHISON 1967). Aliquots of spore suspensions were plated onto YEA + 20 mg/ml guanine, pH 6.5. After 3 days incubation at 34°, two plates, each with >50 colonies, were used to determine each Ade+ total. If <10 Ade+ colonies were present on individual plates derived from neat spore suspensions, neat suspensions were pelleted, resuspended into ~100 µl volume, and then plated onto a single YEA + guanine plate. To measure Ura+ recombinant totals, we plated spore suspensions on EMM2 (MORENO et al. 1991) agar plates with and without uracil (200 µg/ml).

Determination of hot-spot values:
Individual experiments consisting of a number of independent repeats were performed under identical mating conditions. Recombination frequencies for both hot-spot and control alleles were then derived as above. Hot-spot activities were calculated as the ratio of the recombination frequency of a hot-spot allele divided by that of its control allele. Mean hot-spot values and 95% confidence limits were generated by calculating all the possible hot-spot values from the recombination frequencies obtained during a single experiment.

Microscopical preparation and staining:
Meiotic time courses for cytology were performed as described previously (BäHLER et al. 1993; LORENZ et al. 2004). Ten-microliter samples were taken from sporulating cultures and centrifuged for 4 min at 2000 rpm. The pellet was resuspended in 2 ml 0.65 M KCl supplemented with 0.01 M dithiothreitol (Sigma), 5 mg/ml Novozyme 234 (Sigma), and 50 µg/ml Zymolyase 100T (Seikagaku, Tokyo) and incubated with shaking for 30 min at 30° (see BäHLER et al. 1993; LORENZ et al. 2004). The reaction was stopped by adding 5 volumes of ice-cold 1 M sorbitol solution, pH 6.4, containing 0.1 M 2-morpholinoethanesulfonic acid, 1 mM EDTA, and 0.5 mM MgCl2. The spheroplasts were pelleted and resuspended in an appropriate volume (normally 150–200 µl) of the sorbitol solution. Twenty microliters of the spheroplasted cells were mixed with 40 µl fixative (4% paraformaldehyde + 3.4% sucrose) and 80 µl detergent ("Lipsol," LIP, Shipley, United Kingdom) and spread on a microscopical slide. After ~30 sec another 80-µl fixative was added. The slides were dried in a chemical hood and kept in the freezer until further use.

For electron microscopy, the slides were washed with distilled water and air dried, and the material was contrasted by coating slides with 50% aqueous solution of AgNO3 under a nylon gauze for 40 min at 60°. Slides were washed with distilled water, dried, and coated with 1% formvar in chloroform. By applying drops of 1% hydrofluoric acid, the formvar film with the adherent material was detached from the glass slides and transferred to electron microscopic carrier grids (LOIDL et al. 1998).

For immunostaining, slides were washed 3 x 15 min in 1 x PBS containing 0.05% Triton X-100. Excess liquid was removed and the primary antibody was applied under a coverslip. The slides were incubated for several hours at room temperature or at +4° overnight. Concentrations for the primary antibodies were 1:2000 for rabbit {alpha}-Rec10 and 1:50 for guinea pig {alpha}-S.p.Hop1 (for detailed information on the antibodies see LORENZ et al. 2004). After incubation the slides were washed as indicated above. Incubation with appropriate secondary fluorescence-tagged (FITC, Cy3) antibodies was performed for at least 4 hr at room temperature. After another round of washing in 1x PBS containing 0.05% Triton X-100, slides were mounted in antifade solution (Vectashield, Vector Labs, Burlingame, CA) supplemented with 1 µg/ml DAPI for the staining of DNA.

Fluorescent signals were captured on a Zeiss Axioskop epifluorescence microscope equipped with a cooled CCD camera and with single band pass filters for excitation of DAPI, FITC, and Cy3. Images were recorded and analyzed using the IPLab Spectrum software (Scanalytics, Fairfax, VA).


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 ACKNOWLEDGEMENTS
 LITERATURE CITED
 
rec20-144 is a novel allele of the linear element regulator rec10+:
rec20-144 was previously identified as a mutant exhibiting minor defects in intragenic meiotic recombination at the ade6 locus (DE VEAUX et al. 1992). We have demonstrated that rec20-144 is defective in activation of the ade6-M26 meiotic recombination hot spot (see below), and so we attempted to clone the rec20+ gene. During strain construction we noted a weak linkage to the leu2+ locus on chromosome I (data not shown). Using the published S. pombe genome sequence database (http://www.sanger.ac.uk/Projects/S_pombe/), we scanned the region flanking the leu2+ gene for possible candidate genes. The rec10+ gene maps to this region and was an attractive candidate for rec20+, and so we extended previous complementation analyses by testing rec20-144 with an insertion inactivation mutant, rec10-155 (LIN and SMITH 1995). rec20-144 failed to complement rec10-155, and a rec10+ plasmid suppressed the recombination defect observed in a rec20-144 homozygous cross (Table 2). Previously, rec20-144 was placed in a complementation group separate from the more extensively studied rec10-109 allele; our data demonstrate that rec10-109 and rec20-144 show a partial complementation at 30°, consistent with a previous report (DE VEAUX et al. 1992). The rec20-144/rec10+ and rec10-155/rec10+ heterozygote crosses resulted in recombination frequencies approximately half those observed in the rec10+ homozygous crosses (Table 2); this observation is highly reproducible, indicating semidominance for rec10-144 and rec10-155 mutants (J. WELLS, D. PRYCE and R. MCFARLANE, unpublished data).


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  		TABLE 2 rec20-144 is an allele of rec10+

 
Taken together, these data indicate that rec20-144 is a novel rec10– allele. To confirm this, we sequenced the rec10 gene in a number of rec20-144 strains. In all cases the rec10 gene carried a single G-to-A base substitution at position 2180 relative to translational start. This changes glycine to glutamate at codon 727 within the C-terminal Red1 domain (LORENZ et al. 2004). In the interests of clarity, we will rename the rec20-144 allele and refer to it as rec10-144.

Rec10 is a novel temperature-dependent activator of the ade6-M26 hot spot:
In an attempt to identify novel M26 trans activators, we employed two factor crosses followed by random spore analysis to measure heteroallelic intragenic recombination at the ade6+ locus in S. pombe mutants previously reported to have only mild defects in intragenic meiotic recombination at ade6+ (<10-fold) (PONTICELLI and SMITH 1989; DE VEAUX et al. 1992). The rec10-144 mutant exhibited a greater reduction in intragenic meiotic recombination when meiosis was carried out at elevated temperatures (25°–33°; Table 3). The temperature-dependent reduction was noted for both hot-spot (ade6-M26) and non-hot-spot (ade6-M375) alleles (both crossed with the same test allele, ade6-52). However, the reduction in recombination frequency was significantly greater for the hot-spot allele (ade6-M26; maximal reduction of 90-fold) (Table 3; supplementary Table 1 at http://www.genetics.org/supplemental/) than for the non-hot-spot control (ade6-M375; maximal reduction of 23-fold) (Table 3; supplementary Table 1 at http://www.genetics.org/supplemental/).


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  		TABLE 3 Reduced activation of the ade6-M26 meiotic recombination hot spot in the rec10-144 mutant

 
This differential loss of recombination function between the two ade6– alleles indicates a requirement for some function of rec10+ for the full activation of the ade6-M26 hot spot at elevated temperatures (Table 3).

Differential trans activation of M26 heptamers:
The M26 heptamer can function within the ade6+ gene in a position- and orientation-independent fashion (FOX et al. 1997). This was achieved by introducing the M26 heptamer (and appropriate control sequences) at different positions and in opposing orientations within the ade6 open reading frame, with minimal change to the higher-order structure of the region (FOX et al. 1997). Since these hot spots have been reported to have similar activities (FOX et al. 1997), we hypothesized that other M26 heptamers at different positions, and in different orientations, within ade6+ would show a similar requirement for the Rec10 function lost in the rec10-144 mutant. To test this, we measured the intragenic recombination frequency of another M26-containing allele, ade6-3005, whose heptamer is introduced at the same position as that of ade6-M26, but in the opposite orientation (Figure 1A). As with ade6-M26 and ade6-M375, both ade6-3005 (inverted M26 heptamer) and ade6-3006 (non-hot-spot control for ade6-3005) exhibit a temperature-dependent reduction in intragenic recombination in the rec10-144 background (Table 4; supplementary Table 2 at http://www.genetics.org/supplemental/). Surprisingly, both alleles exhibit a largely uniform reduction in recombination frequency with increased temperature (Table 4). Hot-spot values derived from these data show that there is generally no significant loss of hot-spot activity for the ade6-3005 allele in the rec10-144 mutant (Table 4). However, if cells are mated in more restrictive concentrations of supplementary adenine (10 µg/ml), then a small, but statistically significant, reduction in hot-spot value is observed between rec10+ and rec10-144 for ade6-3005 at 30° (supplementary Table 2 at http://www.genetics.org/supplemental/, 30°, experiment 2). The adenine concentration-dependent change in hot-spot activation for ade6-3005 in the rec10-144 mutant at 30° is highly reproducible. None of the other hot spots tested during this study (see below) exhibited any change in hot-spot activation under the altered mating medium adenine concentration and 30° was the only temperature at which an altered adenine concentration had a significant effect on ade6-3005 activity (data not shown). The molecular basis of this is not understood, although the adenine status of a cell has previously been reported to have effects on meiotic gene conversion frequencies in S. cerevisiae (ABDULLAH and BORTS 2001).



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  		FIGURE 1.— M26-containing hot spots within the ade6 open reading frame. (A) The position and orientation of M26 heptamers introduced within the ade6 open reading frame (FOX et al. 1997). Arrows show 5'–3' orientation (the gene is transcribed from left to right). The numbers in parentheses are the nucleotide positions of the heptamers (nucleotide 1 is the A of the ATG start codon of the ade6 open reading frame). (B) Creation of heptamers in the ade6-3005 and ade6-3049 alleles generated novel CRE sequences in the opposite orientation to the heptamer (see text). The arrows indicate the position and the orientation of the M26 heptamer and the boxes delineate the CRE sequence, which is in opposition to the M26 heptamer. The CRE consensus is given at the bottom (FOX et al. 2000).

 

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  		TABLE 4 ade6-3005 meiotic recombination hot-spot activation in the rec10-144 mutant

 
To dismiss the possibility that ade6-3005 is activated via a distinct Atf1/Pcr1-independent pathway, which does not require Rec10, we tested ade6-3005 activity in an atf1{Delta} mutant. The mean atf1+ hot-spot value at 30° was 14.2 (95% confidence interval is 12.2–16.3; n = 9), whereas the atf1{Delta} mutant had a hot-spot value of 1.1 at 30° (95% confidence interval is 0.7–1.5; n = 9). This demonstrates a total loss of ade6-3005 hot-spot activation in the aft1{Delta} mutant, dismissing the possibility that ade6-3005 is activated by an Atf1-independent pathway (the atf1{Delta} mutant exhibits no reduction in recombination frequency for the non-hot-spot control allele, ade6-3006; data not shown).

The ade6-M26 and ade6-3005 alleles differ in the M26 heptamer being in the opposite orientation. However, during this study we noted that the creation of the inverted M26 heptamer in the ade6-3005 allele resulted in creation of an additional CRE sequence, which is in the same orientation as the M26 heptamer in the ade6-M26 allele (FOX et al. 1997; Figure 1B). This CRE site could be masking the true result for the inverted M26 heptamer, although at least one of these two potential hot spots must be Rec10 function independent (Table 4). To further explore the possibility of an orientation-dependent requirement for Rec10, we tested the activation of other M26-containing hot-spot alleles of ade6 (Figure 1A; Table 5; supplementary Tables 3 and 4 at http://www.genetics.org/supplemental/).


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  		TABLE 5 Activation of other M26 heptamer-containing meiotic recombination hot spots in a rec10-144 mutant

 
The first point to note is that ade6-3049 gives a hot-spot activity far exceeding the others in a rec+-proficient background. The elevated hot-spot ratio in the rec10+ background is due, in part, to an elevated recombination frequency for the hot-spot allele, ade6-3049, relative to other hot-spot alleles (see Table 5). This is consistent with the high levels of DSBs observed for an ade6– allele with a similar hot-spot configuration, ade6-3011 (STEINER et al. 2002). However, there is also a low recombination frequency for the non-hot-spot control allele, ade6-3009, relative to other control alleles that have a shorter distance between markers. The high recombination frequencies observed for ade6-3049 indicate that there are inherent differences in M26 heptamer behavior, which are dependent on unknown factors. The ade6-3049 hot spot is similar to ade6-3005: both create an additional CRE sequence (FOX et al. 1997) (Figure 1B). In this case, as with the ade6-3005 allele, the other CRE sequence is also in the opposite orientation to the M26 heptamer, and the major elevation in hot-spot activity could be a result of the combined effect of these two potential hot-spot-activating sequences. Why ade6-3049 recombination frequency should be significantly higher than that of the ade6-3005 hot spot, which also has an M26 heptamer and CRE sequences in opposition, is not clear, but this disparity is consistent with an inherent difference in activation of M26 hot spots in different local contexts.

Of the other ade6– hot spots tested, none exhibited any notable dependency on the Rec10 function lost in the rec10-144 mutant, including ade6-3049 (Table 5), indicating that orientation is not the factor dictating dependency on some function of Rec10. We expanded this analysis to explore whether an M26 heptamer located at another locus was dependent on Rec10. FOX et al. (1997) had created an M26 heptamer in the ura4+ gene, ura4-167, in the same orientation as the ade6-M26 heptamer, relative to transcription. Despite relatively low hot-spot values for the ura4-167 M26-containing hot spot (which indicates inherent differences in M26-containing hot spots), there is a clear dependency on some function of Rec10 for full ura4-167 hot-spot activation (Table 5).

rec10-144 mutants are defective in linear element formation:
Rec10 is required for the formation of the proteinaceous LEs, which are thought to be analogous to the lateral elements of the synaptonemal complex found in most other organisms, but not in the fission yeast (BäHLER et al. 1993; MOLNAR et al. 2003). Immunocytochemistry shows that Rec10 is a structural component of LEs (LORENZ et al. 2004). A mutant of rec10, rec10-155, generated by insertional inactivation of the rec10+ open reading frame, results in total loss of LE formation and a significant reduction in meiotic recombination throughout the genome (LIN and SMITH 1995; MOLNAR et al. 2003; LORENZ et al. 2004; our unpublished observations).

To establish the nature of the context-dependent influence of Rec10 on M26 hot-spot activation, we assessed the integrity of LE formation in the rec10-144 mutant. Homozygous rec10-144 diploids were induced to traverse azygotic meioses and immuno- and silver staining of nuclear spreads showed that LEs were severely compromised in the rec10-144 mutant (Figure 2). LEs have been subclassified according to the morphological features observed by silver and immunostaining (BäHLER et al. 1993; LORENZ et al. 2004) (Figure 2C). The Rec10-positive structures present in meiotic rec10-144 nuclei, as identified by immunostaining, were counted following increasing times in sporulation medium. Of the nuclei containing Rec10-positive structures, only two classes of structure could be observed at all time points. One is comparable to the class Ia structures observed in rec10+ cells (LORENZ et al. 2004) (Figure 2C), comprising only small Rec10-positive foci. The second class contained a number of small foci plus one to four short, fat linear structures; such nuclei were not observed in rec10+ cells (LORENZ et al. 2004; this study). At all time points taken (4–8 hr after transfer to sporulating medium, at 1-hr intervals), these two classes were present at slightly changing proportions with a mean of 39.5% class Ia nuclei and 60.5% aberrant nuclei. Among 1290 Rec10-positive nuclei scored, none contained long or reticulated or bundled LEs, corresponding to classes Ib, IIa, and IIb or to the rec10 cells (LORENZ et al. 2004) (Figure 2C).



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  		FIGURE 2.— Linear elements are defective in the rec10-144 mutant. (A and B) Electron micrographs of silver-stained meiotic prophase nuclei demonstrate that LEs do not fully form in the rec10-144 mutant. The wild-type cells show the classical thread-like structures of mature LEs (A). These threads do not form in the rec10-144 mutant and only a number of short dense structures can be observed (examples shown by black arrows), indicating severely impaired LEs (B). Bar in 2B, 5 µm. (C) Immunostaining of Rec10 in spread meiotic prophase nuclei. (Left) Immunostained Rec10; (right) immunostained Rec10 (red) merged with DAPI-stained DNA (blue). Wild-type nuclei show a range of classes of staining patterns for Rec10, including spots (class Ia), short lines (class Ib), thin extended lines (class IIa), and linear bundles (class IIb) (individuals classes are marked) (also see LORENZ et al. 2004). In rec10-144 nuclei, dot-like structures are apparent and remain present in all nuclei, along with a small number of larger aggregated structures (examples shown by arrows). Thread-like structures and extensive linear bundles do not appear to form, indicating a severe defect in LE formation and progression. Bar, 2 µm. (D) Hop1 protein loads onto defective LEs. Examples of spread rec10-144 prophase nuclei stained for both Rec10 (red) and Hop1 (green) show a significant amount of colocalization (yellow), indicating that Hop1 protein is capable of loading onto the defective LE structures that form. Blue is DNA stained with DAPI.

 
Hop1 protein is localized to LEs after the formation of Rec10-containing structures (LORENZ et al. 2004; our unpublished data). Figure 2D shows that Hop1 is capable of loading onto the limited Rec10-containing structures in the rec10-144 mutant, demonstrating that the defect in ade6-M26 activation is not due to a failure to recruit Hop1 to the LE structures present in the rec10-144 mutant. Moreover, a hop1{Delta} null mutant is proficient in activation of the ade6-M26 hot spot (our unpublished data).

Requirement for Rec10 is suppressed by changing the nucleotide context of M26:
Activation of M26 is dependent on some feature of the macromolecular structure in which it is located, most likely the chromatin context (PONTICELLI and SMITH 1992; VIRGIN et al. 1995; YAMADA et al. 2004). Given that Rec10 is required for the formation of normal meiotic chromosomal architecture (MOLNAR et al. 2003; LORENZ et al. 2004), we explored the possibility that Rec10/LE function is related to the generation of a favorable chromatin configuration for full hot-spot activation. We tested whether full Rec10 function is required for activation of a variant of ade6-M26, ade6-M26-16C, in which the 3' nucleotide following the heptamer has been changed from a G to a C, resulting in a more restricted chromatin transition, relative to ade6-M26 (Figure 3) (MIZUNO et al. 1997). We found that changing the nucleotide at the 3' flank of the ade6-M26 heptamer from a G to a C resulted in restoration of full hot-spot activity in the rec10-144 mutant at 33° and in partially restored activity at 30° (Figure 3; Table 6; supplementary Table 5 at http://www.genetics.org/supplemental/); alterations to the adenine concentration in the mating medium did not alter these observations (the 30° experiment in Table 6 was carried out using restricted adenine concentrations). Previously, others have reported that the ade6-M26-16C sequence results in an elevation of hot-spot activity and a tighter binding of Atf1/Pcr1 (SCHUCHERT et al. 1991; WAHLS and SMITH 1994); however, the mean hot-spot activity for ade6-M26-16C at 30° and 33° was not significantly different from that of ade6-M26 (Table 6). This dismisses the possibility that an intrinsically higher activity of ade6-M26-16C is responsible for an apparent suppression of the rec10-144 defect.



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  		FIGURE 3.— Chromatin structure of the ade6-M26 and ade6-M26-16C hot spots on meiotic entry. (A) The wild-type ade6 sequence gives a regular nucleosome pattern on meiotic entry. The ade6+ open reading frame is represented by an open box, nucleosome positions by shaded circles, and the sequence at the position of the ade6-M26 heptamer is shown (the 3' flanking nucleotide is underlined). (B) Changing a G to a T in ade6 generates the M26 heptamer, to which Atf1/Pcr1 binds (boldface type; arrow indicates orientation of the heptamer) in the ade6-M26 allele; this is flanked at the 3' boundary with a G (underlined). On meiotic entry this results in an open chromatin configuration at the heptamer and up to three nucleosomes downstream (MIZUNO et al. 1997). This hot spot exhibits a dependence on some function of Rec10 for full activation (Table 2). (C) Changing the 3' flanking nucleotide from a G to a C (underlined) in the ade6-M26-16C allele results in a more restricted opening of chromatin. MIZUNO et al. (1997) propose that this is due to a chromatin barrier of unknown function (solid oval adjacent to the Atf1/Pcr1 heterodimer). Activation of this hot spot is largely independent of the Rec10 function required for ade6-M26 activation (Table 6).

 

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  		TABLE 6 A change in the nucleotide at the 3' flank of the M26 heptamer of the ade6-M26 hot spot from a G to a C suppresses the requirement for the function of Rec10, which is defective in the rec10-144 mutant

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
ACKNOWLEDGEMENTS
 LITERATURE CITED
 
Although ade6-M26 can function as a meiotic recombination hot spot at some loci, when it is located on a plasmid or when it is positioned in a locus that has suffered a major alteration to the local chromosomal architecture, it is inactive (PONTICELLI and SMITH 1989; VIRGIN et al. 1995). This dependence on the context in which the M26 heptamer is embedded suggests that broader features of DNA sequence or chromatin configuration have a strong influence on hot-spot behavior. Moreover, a chromatin transition occurs at M26 under conditions that result in meiotic entry and becomes more pronounced early in meiosis (MIZUNO et al. 1997, 2001). This transition, and hot-spot activity, is dependent not only on the Atf1/Pcr1 heterodimer, but also on the chromatin modulators Gcn5 and Snf22 (YAMADA et al. 2004). Taken together, these facts indicate that regulators of local chromatin structure influence the activation of the M26 heptamer. To date, no regulators of higher-order chromosomal architecture have been implicated in M26 heptamer activation. In this report we demonstrate that a function of the chromosomal protein Rec10, which is a component of LEs and is most likely essential for LE function, is important for full activation of the M26 heptamer in a context-dependent fashion.

To some degree, the differential requirement for some function of Rec10 for full hot-spot activation is paralleled in the distantly related S. cerevisiae. Red1 of S. cerevisiae has features in common with Rec10 at a number of levels (LORENZ et al. 2004). RED1 null mutants are defective in intragenic recombination for some, but not all, cis activator elements (ROCKMILL and ROEDER 1990), while Rec10 activates recombination in a region-specific fashion (DE VEAUX and SMITH 1994; KRAWCHUK et al. 1999). Moreover, similar cis activators of DSB formation exhibit differential requirements for Red1 function in a context-dependent fashion (PECINA et al. 2002). Red1 forms part of the AEs in S. cerevisiae (SMITH and ROEDER 1997), structures analogous to the LEs of S. pombe, of which Rec10 is a major component (LORENZ et al. 2004). Taken together these observations suggest that the context-dependent influences on whether or not LEs/AEs are required for hot-spot activation is a common feature of meiosis. However, the studies in S. cerevisiae employed a null mutant of RED1, whereas this study has used a hypomorphic mutant of rec10, indicating that although they have common features, Red1 and Rec10 do differ in some important respects.

We propose that {alpha} hot spots (hot spots dependent on the binding of a trans activator) (PETES 2001) with a common cis-activating primary DNA sequence can be subgrouped into three classes dependent on context: first, those that are inactive, due to a restrictive context, such as certain ade6-M26 transplacements (VIRGIN et al. 1995); second, those that are active but require a higher-order chromosomal structure to maintain a favorable context, such as ade6-M26 (this study); third, those that are active and are located in a context that does not require higher-order chromosomal structures to maintain a favorable, active configuration, such as the M26 heptamer-containing hot spots reported in this study, which do not require the Rec10 function lost in the rec10-144 mutant for full activity. Such a subclassification is further supported by the fact that changes to the DNA sequence context of S. cerevisiae hot spots can result in changes to the activity of that hot spot (PETES and MERKER 2002).

It has been proposed that Red1 status is dependent on chromosomal isochore configuration (BLAT et al. 2002), which suggests that the differential requirements for AE/LEs in recombination activation are governed by global genome configuration. However, our data also support a model in which there is a highly localized influence of context and suggest that although more global features may play an important role, the fundamental determinant for the LE (AE) requirement in hot-spot activation is primarily influenced by highly localized chromosomal features at or near the DSB site. BLAT et al. (2002) propose that DSBs occur in chromatin loops, emanating from the axis, and that recombination at the DNA level is mediated by an axis-associated recombinosome, which draws the chromatin loop containing the DSB to the axis for processing. Our data are not inconsistent with such a model as global and local requirements for LE/AE formation are unlikely to be mutually exclusive.

The compromised LEs observed in the rec10-144 mutant are capable of interacting with Hop1 (Figure 2D). hop1{Delta} null mutants do not exhibit a loss of ade6-M26 activity, although, like the rec10-144 mutant, they do exhibit a limited defect in intragenic recombination at the ade6+ locus (our unpublished data). This suggests that the differential requirements for LEs for M26-containing hot-spot activation are Hop1 independent.

MIZUNO et al. (1997) suggest that the restriction in chromatin transition in the ade6-M26-16C allele is a result of the nucleotide change forming a chromatin boundary element, the nature of which remains unknown (Figure 3). It may be that, in the absence of this unknown restrictive boundary element, Rec10 is required for a specific localized function to mediate a more extensive opening of chromatin, which in turn is required for full hot-spot activation in the absence of the boundary element. Alternatively, Rec10 may function to limit the more extensive chromatin opening in the absence of the boundary; if further opening results in an inhibition of hot-spot activity, loss of Rec10 function would lift a control of further chromatin opening. Both scenarios are possible and both may place Rec10 function in intimate local proximity with chromatin regulators, such as Gcn5 and Snf22. Recently, the Atf1/Pcr1 heterodimer has been implicated in heterochromatin nucleation at the mating-type locus in S. pombe (JIA et al. 2004). This demonstrates that Atf1/Pcr1 can function differentially in different chromosomal contexts, functioning to regulate both "opening" and "closing" of chromatin during hot-spot activation and mating-type heterochromatin formation, respectively. Rec10 may be required in some chromosomal contexts to facilitate the decision of which Atf1/Pcr1 pathway is utilized.

Our data demonstrate that a single nucleotide polymorphism (SNP) can have a profound effect on the requirements for trans activation of a meiotic recombination hot spot. In human males the FG11 SNP located within 5 bp of the cumulative crossover center of the DNA2 hot spot influences hot-spot activity (JEFFREYS and NEUMANN 2002). A simple interpretation of this is that a trans-activator binding site is created by the SNP, analogous to the way in which the Atf1/Pcr1-binding site was created in the ade6-M26 allele. However, it might be the case that SNPs can result in subtle, but significant, changes to chromatin structure, making a specific site more amenable to becoming a recombination hot spot.


   ACKNOWLEDGEMENTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
LITERATURE CITED
 
We thank Jürg Kohli and Gerry Smith for strains. We are grateful to Barry Wells and Chris Gliddon for advice on statistical analysis of data. We thank Jürg Bähler and Gerry Smith for helpful comments on the manuscript. This work was supported by Biotechnology and Biological Sciences Research Council (BBSRC), studentship no. 99/B1/G05482; Wellcome Trust Project grant no. 057317; and the Austrian Science Fund, grant no. P16282. D.W.P. is funded by a North West Cancer Research Fund Fellowship.


   FOOTNOTES
 
1 Present address: Department of Biomolecular Science, UMIST, Manchester M60 1QD, United Kingdom. Back


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