Loss of a Histone Deacetylase Dramatically Alters the Genomic Distribution of Spo11p-Catalyzed DNA Breaks in Saccharomyces Cerevisiae

Loss of a histone deacetylase dramatically alters the genomic distribution of Spo11p-catalyzed DNA breaks in Saccharomyces cerevisiae

Piotr A. Mieczkowski*, Margaret Dominska*, Michael J. Buck†, Jason D. Lieb†, and Thomas D. Petes*‡

*Department of Molecular Genetics and Microbiology, Box 3054, Duke University School of Medicine, Durham, NC 27710; and †Department of Biology, and Carolina Center for Genome Sciences, University of North Carolina, Chapel Hill, NC 27599-3280

Contributed by Thomas D. Petes, January 17, 2007 (sent for review November 29, 2006) In eukaryotes, meiotic recombination events are distributed nonran- HML and HMR, and genes inserted within the rRNA gene cluster domly in the genome, with certain regions having high levels of (14). Although no global analysis of the effects of the sir2 mutation recombination (hotspots) and others having low levels (coldspots). on recombination have been reported previously, sir2 strains have Species with similar DNA sequences (for example, chimpanzees and elevated rates of intrachromosomal meiotic recombination in the humans) can have strikingly different patterns of hotspots and rRNA genes (15) and elevated DSB formation in yeast artificial coldspots. Below, by using a microarray analysis that allows us to chromosomes containing mouse DNA (16), but reduced crossovers measure the frequency of the meiosis-speciﬁc double-strand DNA in one genetic interval near the end of chromosome I (17). As breaks (DSBs) of all 6,000 yeast genes, we show that mutation of a described below, we find that the sir2 mutation has a very pro- single gene (SIR2), which encodes a histone deacetylase, signiﬁcantly nounced effect on the distribution of meiosis-specific DSBs in S. changes DSB frequencies of 12% of yeast genes, elevating DSBs of cerevisiae, significantly elevating DSBs for 5% of the genes and 5%, and reducing DSBs of 7%. Many of the genes with repressed significantly reducing DSBs for 7% of the genes. recombination are located in large (50–100 kb) regions located near, but not at, the telomeres. Some of the genes with altered frequencies Results of DSBs (including the ribosomal RNA gene cluster) are known targets Genome-Wide Mapping of DSB Activity in sir2 Strains by DNA Mi- of Sir2p deacetylation in the wild-type strain. croarrays. To map the frequency of meiosis-specific DSBs throughout the genome in the sir2 strain MD311 [strain construction chromatin ͉ meiosis ͉ Sir2p ͉ yeast described in supporting information (SI) Table 1], we used the same method that we used previously to map DSBs in the wild-type strain n the yeast Saccharomyces cerevisiae and in most other eukaryotes JG169 (3, 5). In brief, we used chromatin immunoprecipitation to Ithat have been examined, meiotic recombination is initiated by purify DNA that is covalently associated with Spo11p. These double-strand DNA breaks (DSBs) catalyzed by the topoisomer- samples were labeled with a fluorescent probe and hybridized to a ase-related Spo11p (1). The frequencies of DSBs vary widely at DNA microarray (containing all yeast genes and intergenic regions) different sites in the yeast genome and are affected by both global along with total genomic DNA that was labeled with a different and local factors (2). Examples of global effects are the suppression fluorescent probe. We will refer to the log2 of the hybridization ratio of recombination in large (50–100 kb) regions near the telomeres of (Spo11p-enriched DNA/total genomic DNA) as the DSB activity of the gene. Because the DNA fragments used for the microarray and centromeres (3–5). Examples of local effects are the preference Ϸ for DSBs to occur within nuclease-sensitive regions of chromatin analysis were sheared to a size of 2 kb, the DSB activity is related (6) at the 5Ј ends of genes (3). Some hotspots (␣ hotspots) require to the frequency of local DSBs.

the binding of transcription factors (2), whereas others (␥ hotspots) The DSB activities for each ORF and intergenic region derived GENETICS are associated with local regions of high G ϩ C base composition from 13 sir2 microarrays and 11 isogenic wild-type microarrays (5) (3). Although the mechanistic explanations of these local effects are are shown in SI Table 2. Based on a two-tailed t test, comparing the wild-type and sir2 microarrays, we found 784 genes (12%) had not clear, it has been suggested (3, 7, 8) that the preference for DSB Ͻ formation to occur in regions of high G ϩ C content is a conse- significantly (P 0.005) altered DSB activity (346 increased and quence of cohesins blocking DSB formation at their AT-rich 438 decreased). In all, we found that 3,441 ORFs had elevated DSBs binding regions (3, 7–9). in the sir2 strain and 2,946 had decreased DSBs, although many of No single well defined DNA sequence motif defines preferred these changes were not statistically significant. The summary of the sites for DSB formation in S. cerevisiae. This observation and the median DSB activities for all ORFs is shown in SI Table 3. other observations described above indicate that the frequency of We also calculated the difference in DSB activities between sir2 and wild-type strains by subtracting the wild-type DSB activity from DSB formation is determined, at least in part, by chromatin ⌬ structure rather than primary DNA sequence. In support of this the activity observed in the sir2 strain. These values [termed R, conclusion, Yamada et al. (10) showed that deletion of a histone

acetylase or an ATP-dependent chromatin remodeling factor re- Author contributions: P.A.M., M.D., and T.D.P. designed research; P.A.M. and M.D. per- sulted in a partial reduction in recombination activity at one hotspot formed research; M.J.B. contributed new reagents/analytic tools; P.A.M., M.J.B., J.D.L., and in Schizosaccharomyces pombe. In this report, using DNA microar- T.D.P. analyzed data; and P.A.M. and T.D.P. wrote the paper. rays, we show that the DSB frequencies throughout the yeast The authors declare no conﬂict of interest. genome are dramatically altered by a mutation in a single gene Freely available online through the PNAS open access option. (SIR2) encoding a chromatin-modifying enzyme. This result dem- Abbreviations: DSB, double-strand DNA break; TPSR, telomere-proximate suppressed onstrates that one aspect of recombination is extremely malleable region. and is responsive to small changes in the global chromatin state. Data deposition: The microarray data have been deposited in the Gene Expression Omnibus Sir2p is a histone deacetylase, one of whose substrates is H4K16 (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. GSE6245). (11). Sir2p binds near the telomeres and within the rRNA gene ‡To whom correspondence should be addressed. E-mail: [email protected]. cluster (12), and Robyr et al. (13) showed that Sir2p-mediated This article contains supporting information online at www.pnas.org/cgi/content/full/ modifications are concentrated near the telomeres. Sir2p is involved 0700412104/DC1. in silencing expression of telomeric genes, the silent mating type loci © 2007 by The National Academy of Sciences of the USA

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0700412104 PNAS ͉ March 6, 2007 ͉ vol. 104 ͉ no. 10 ͉ 3955–3960 Downloaded by guest on September 25, 2021 A I B II 1.5 III IV 1 V VI 0.5

VII 2 VIII (log Ra tio) tio) Ra (log 0

IX R X ∆ XI -0.5 XII 4 XIII -1 XIV 1 0kb 375 kb 750 kb

Rat io XV Chromosome coordinate XVI 0.4

Fig. 1. Depictions of the differences in DSB activities [log2(Spo11p-enriched DNA/total genomic DNA)] in a sir2 strain compared with a wild-type strain. For this analysis, the DSB activity in the wild-type strain was subtracted from the recombination activity in the sir2 strain, generating the ⌬R values. (A) GeneSpring software was used to depict ⌬R for all genes. Blue indicates a ⌬RofϽ0 (fewer DSBs in the sir2 mutant than in wild-type), yellow indicates a ⌬RofϷ0 (DSBs similar in sir2 and wild-type), and red indicates ⌬RofϾ0 (DSBs higher in sir2 than in wild-type). Roman numerals specify each chromosome, black arrows show the position of the centromere, green arrows indicate the positions of genes in which the DSB frequencies were measured by using standard Southern blot analysis, and the red arrow indicates the position of the rRNA gene array. (B) The ChIPOTle software (18) was used to measure ⌬R on chromosome X in a window of 20 kb, moved along the chromosome in intervals of 10 kb.

because the DSBs are the recombination (R)-initiating lesion] are Genes and Genomic Regions with Suppressed DSBs in the sir2 Strain. shown for all elements on the microarray and for genes only are in One salient feature of the sir2 strain is the increased suppression of SI Tables 3 and 4, respectively. In Fig. 1A, we depict ⌬R for all genes DSBs in regions beginning Ϸ10–20 kb from the telomere and on all chromosomes, showing DSB activities that are reduced extending Ϸ100 kb toward the centromere (Figs. 1–4, SI Tables 5 (blue), increased (red), or unchanged (yellow) in the sir2 strain. and 6). One method of visualizing this suppression is to calculate These results, described in detail below, show that the Sir2p can act ⌬R along the chromosome in a moving window of 20 kb. For to suppress or promote DSB formation in different genomic example, on chromosome X, the genes located within 20 kb of the regions, and can act either locally or regionally on the chromosome.

Genes and Genomic Regions with Elevated DSBs in the sir2 Strain. We Chromosome XII A 2

observed increased DSBs at the telomeres, within the rRNA wt tio

gene cluster and its flanking sequences, and in genes scattered a 0

R 2 throughout the genome. Genes very close (within 10 kb) to the telomeres generally had elevated rates of DSBs in the sir2 strain log (Fig. 1A). Fig. 1B shows the elevation of ⌬R at the telomeres of -3 chromosome X, as visualized by calculating ⌬R along the 2 B sir2

chromosome in a moving window of 20 kb. Throughout the tio a 0 genome, the regions within 10 kb of the telomeres had an 2

overrepresentation of genes with positive ⌬R values (SI Table 5). R log Because Sir2p deacetylates H4K16 near the telomeres (13), we -2 suggest that increased levels of acetylation in the sir2 strain C 2 wt, sir2

results in a more open chromatin structure, allowing Spo11p and o ti

associated proteins increased access to the DNA. a 0

R g

In wild-type strains, recombination is suppressed within the lo rRNA genes (19) and flanking regions (4, 5), and loss of sir2 -3 elevates the rate of intrachromosomal recombination in the 2

rRNA gene cluster (15). Our analysis showed elevated DSBs D o sir2-wt ti

within the rRNA gene cluster in the sir2 strain (Fig. 2), although a 2 R 0

these regions are still colder than the average gene. In addition, g lo we found that the suppression of DSBs in genes flanking the -2 cluster is partially relieved by the sir2 mutation (Fig. 2). Of the Chromosome coordinate 146 genes in the region flanking the rRNA gene cluster (SGD 0 kb 1078 kb ~ 900 kb coordinates 300–600 kb), 139 have elevated DSB activity and 7 100-150 repeats of rDNA have reduced DSB activity in the sir2 strain, a very significant (P Ͻ 0.0001) departure from the frequency of genes with Fig. 2. DSB activities in wild-type and sir2 strains on chromosome XII. This elevated DSBs in the sir2 strain in the genome as a whole. figure depicts the effect of the sir2 mutation on the rRNA gene cluster and its The final class of genes with elevated DSBs in the sir2 strain had flanking sequences. Because only two of the Ϸ150 repeats are present on the no obvious common characteristic. One example is CYS3 on microarray, the rRNA gene cluster is much larger than it appears in this A representation. For all four graphs, DSB activities were measured in a window chromosome I (Fig. 3 ). It is possible that the elevated recombi- of 20 kb moved in intervals of 10 kb. (A) DSB activity in the wild-type strain nation in these genes reflects a redistribution of recombination- JG169. (B) DSB activity in the sir2 mutant strain MD311. (C) Depiction of the promoting proteins from the regions proximate to the telomeres overlap in the wild-type (red) and sir2 (blue) patterns of DSBs. (D) ⌬R calcu- (which have suppressed DSBs in sir2 strains) to other regions in the lated along chromosome XII. The elevation in DSBs in the sir2 strain within the genome. rRNA genes and flanking sequences is evident.

3956 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0700412104 Mieczkowski et al. Downloaded by guest on September 25, 2021 A B

C D E

Fig. 3. Comparison of DSB activities by Southern blot analysis and microarray analysis. (A) The DSB activities along chromosome I (based on microarray data) were examined in a sliding window of 1 kb, moved in intervals of 250 bp. DSB formation is suppressed in the sir2 strain on the left (coordinates, 20–110 kb) and right (coordinates, 160–210 kb) arms of the chromosome; CEN1 is located at coordinate 151 kb. DSB formation at CYS3 (shown by an arrow) is elevated in the sir2 strain compared with wild-type. (B) Intact chromosomal DNA was isolated from wild-type (PG169) and sir2 (MD311) strains immediately after the cells were incubated in sporulation medium (0 h) or after 24 h in sporulation medium. Chromosomes were separated by pulse–field gel electrophoresis, and the separated chromosomes were examined by Southern blot analysis by using a chromosome I-specific probe (further details in SI Materials and Methods). DSBs in the left (arrow 4) and right (arrow 1) arms were reduced in the sir2 strain relative to DSBs near the middle of the chromosome (arrows 2 and 3). (C–E) We show standard Southern blots of DNA samples isolated from wild-type and sir2 strains hybridized to three different probes (enzymes used for DNA treatment shown in parentheses): HIS4 (BglII) (C), YGR177 (BglII) (D), and CYS3 (HindIII) (E); the specific probes used are described in SI Materials and Methods.

telomere have slightly elevated DSB formation in the sir2 strain chromosome end (SI Table 6). We term these areas of suppressed relative to wild-type, followed by the region of suppressed DSBs DSB formation ‘‘telomere-proximate suppressed regions’’ (Fig. 1B). We estimate that 21 of the 32 telomeres have large (Ն50 (TPSRs). The average size of TPSRs is 135 kb and the region kb) regions of suppressed DSBs that begin Ϸ20 kb from the telomere-distal to the TPSRs that escapes suppression averages Ϸ20 kb. In SI Table 5, we show the number of ORFs with increased and 0.6 decreased recombination activity in the sir2 strain in 10-kb ‘‘bins’’ GENETICS from the telomeres toward the centromere. There is a significant Յ 0.4 (P 0.0001) excess of ORFs with elevated DSBs in the 20 kb closest s) to the telomere and a significant (P Ͻ 0.0001) excess of ORFs with

D S B S D

decreased DSBs for all ORFs between 30 and 90 kb from the e 0.2 telomere. Of the 438 ORFs with significantly reduced DSBs in the sir2 strain, 297 are in TPSRs. The elevation of DSBs very close to

ld -t yp -t ld i the telomeres and the suppression of DSBs in the regions between w 0

s 20 and 150 kb from the telomeres averaged over all of the

S chromosomes are shown in Fig. 4. In addition to the TPSRs, there

-0.2 were two other large regions of reduced DSBs in the sir2 strain on

sir2 D

( chromosomes II and IV (Fig. 1A). Although many of the genes with suppressed DSBs in the sir2 R -0.4 ∆ strain were located in TPSRs, there were also some isolated genes with reduced DSB formation, presumably reflecting a local alteration in chromatin structure. We looked for correlations between -0.6 0-10 10-20 20-30 30-40 40-50 50-60 60-70 70-80 80-90 90- 100- 110- 120- 130- 140- our ⌬R data set and other global studies done in yeast, and found 100 110 120 130 140 150 a correlation between genes that have reduced DSBs and genes that Distance from telomere (kb) have reduced expression in sir2 haploids (SI Table 7). The interpretation of this correlation is discussed below. Fig. 4. Differences in DSB formation between sir2 and wild-type strains (⌬R) as a function of distance from the telomere. For this analysis, we averaged ⌬R in bins Confirmation of the Microarray Results by Southern and Tetrad of 10 kb for all elements on the microarray for all chromosomes. Standard error bars are shown. The elevation of DSB formation within Ϸ10 kb of the chromo- Analysis. Our conclusions based on microarray studies were sup- some end is evident, as is the suppression of DSBs that extends to Ϸ120 kb from ported by Southern analysis and tetrad dissection. Fig. 3B shows a the telomere. Southern analysis of chromosomal DNA samples (untreated with

Mieczkowski et al. PNAS ͉ March 6, 2007 ͉ vol. 104 ͉ no. 10 ͉ 3957 Downloaded by guest on September 25, 2021 restriction enzymes), demonstrating meiosis-specific DSBs on chro- analysis, not discussed below, are provided in SI Materials and mosome I in the wild-type (JG169) and sir2 (MD311) strains. It is Methods. evident that DSBs near the ends of the chromosome are suppressed relative to DSBs near the middle in the sir2 strain. In the same pair Genes with Elevated DSBs in the sir2 Strain. We can divide the genes of strains, we examined DSBs at the HIS4, YGR177C, and CYS3 loci with elevated DSBs into four classes: class 1, the genes located near by standard Southern analysis (Fig. 3 C–E). These loci represent (within 10 kb) of the telomeres; class 2, the rRNA genes; class 3, the ORFs that, by microarray analysis, had lower (HIS4) or higher genes flanking the rRNA gene cluster (within 150 kb of the cluster); (CYS3) DSB activities in sir2 strains compared with wild-type class 4 all other genes with elevated DSBs. Telomeres bind Sir2p strains. YGR177C had very high levels of DSBs in both wild-type (12) and Sir2p represses gene expression at the telomeres (14), and sir2 strains, although the activity was slightly higher in the sir2 presumably as a consequence of its H4K16 deacetylation activity. It strain. For all loci examined, the Southern analysis was consistent is likely that class 1 genes represent a direct effect of the enzymatic with our conclusions from the microarrays. activity of Sir2p on DSB formation: elevated levels of H4K16 In fungi, meiotic recombination activity can be monitored by acetylation leading to a more open chromatin structure that allows the measuring the rate of aberrant segregation (gene conversion Spo11p increased access to the telomeric DNA. and related events) of a heterozygous marker (20, 21). Because The DSB frequencies of the class 2 (rRNA gene cluster) and class the HIS4 gene is in a region of suppressed DSB formation in the 3 (genes flanking the cluster) were also elevated by the sir2 sir2 mutant, we constructed wild-type (MD251) and sir2 mutation (Fig. 2). In wild-type strains, recombination is suppressed (MD258) strains that were heterozygous for a mutation in HIS4, within the rRNA genes (19) and flanking regions (4, 5). San- as well as a mutation in ARG4, a gene with a DSB that is not Segundo and Roeder (25) suggested that the increased level of affected by the sir2 mutation. These two strains were sporulated recombination observed in sir2 strains reflects increased levels of and tetrads were dissected. The HIS4 aberrant segregation rate Hop1p (a positive regulator of recombination) in the nucleolus, was significantly (P Յ 0.001) reduced by the sir2 mutation, from because Pch2p excludes Hop1p from the nucleolus, and Pch2p 23% in MD251 to 8% in MD258 (total of 1,127 and 242 tetrads requires Sir2p for its nucleolar localization. Similarly, the elevated for MD251 and MD258, respectively). The aberrant segregation rates of DSBs in genes that flank the rRNA gene cluster in the sir2 rate for ARG4 was not significantly affected (10% in MD251 and strain could be the result of elevated levels of Hop1p extending 8% in MD258). Thus, the tetrad analysis confirms the microarray from the rRNA gene cluster. analysis and demonstrates that the suppression of DSBs in Class 4 genes had no common characteristic that we have been TPSRs leads to an absolute reduction in recombination activity. able to discern. These genes are not associated with any obvious The strains MD251 and MD258 were heterozygous for markers structural element of the chromosome (centromeres, replication allowing us to map crossovers in three intervals on chromosome III origins or repetitive DNA elements). No class of genes is very (CHA1-HIS4, HIS4-LEU2, and LEU2-CEN3) and between ARG4 significantly (P Յ 0.001) overrepresented among these genes (de- and CEN8. The map lengths were significantly reduced by the sir2 termined by using the Stanford Genome Database Gene Ontology mutation in the CHA1-HIS4 interval (from 41 cM in MD251 to 18 Term Finder). Because introduction of a strong DSB site can cM in MD258) and the HIS4-LEU2 interval (from 23 cM in MD251 repress recombination at neighboring sites (26–28), it is likely that to 14 cM in MD258). The map lengths in the other intervals were loss of strong DSB sites in some sites will elevate DSB formation at unaffected (SI Table 8). The microarray analysis indicates that the other sites. Consequently, it is possible that the elevated DSBs in CHA1-HIS4 and the HIS4-LEU2 intervals are within a single TPSR class 4 genes are a consequence of a redistribution of recombina- on chromosome III, whereas the LEU2-CEN3 and ARG4-CEN8 tion-promoting proteins from the TPSRs to class 4 genes. intervals do not include large numbers of genes with altered DSB activities. Thus, these results support the conclusion that the Genes with Suppressed DSBs in the sir2 Strain. There are also several alterations in DSB frequency detected by the microarrays in the sir2 classes of genes that have reduced DSBs in the sir2 strains: class strain affect the crossover frequencies and, therefore, the genetic 1, ORFs located in TPSRs; class 2, ORFs that are associated with map of S. cerevisiae. reduced gene expression in sir2 strains; class 3, all other ORFs. Of the 438 ORFs with significantly reduced recombination, Discussion approximately two-thirds are class 1 genes as defined in SI Table We show that the pattern of meiosis-specific DSBs in S. cerevisiae 6. In wild-type strains, we have previously shown that DSB is very substantially altered by a mutation that results in loss of one formation is suppressed in regions extending Ϸ80 kb from the of the yeast histone deacetylases. Winckler et al. (22) recently telomeres (5). In the sir2 strains, the suppression of DSBs is more demonstrated that, despite Ϸ99% sequence conservation between pronounced and extends to Ϸ130 kb from the telomeres (Fig. 4). human and chimpanzees, the positions of recombination hotspots Although it is possible that the increased extent of suppression are not conserved between these two genomes. Although there is represents a mechanism qualitatively different from that ob- evidence that sequence polymorphisms can influence the strength served in the wild-type strain, we prefer the simpler hypothesis of local recombination events (a cis effect) in humans (23, 24), our that the increased suppression observed in sir2 strains in an results demonstrate that trans effects mediated by altering a chro- enhancement of the suppression that occurs in wild-type strains. matin-modifying protein can substantially change the genomic The region of DSB suppression near the telomeres in the distribution of DSBs in budding yeast and the altered frequencies wild-type strain is similar to the location of the regions in which the of DSBs associated with some genes affect the rates of crossing over. level of H3K18 acetylation is regulated by Hda1p (13). Although It is likely that similar types of alterations influence the differences Hda1p acts at many positions scattered throughout the genome, in the human and chimpanzee genetic maps. contiguous regions located between 20 and 100 kb from the Of the 12% of the genes with significantly altered DSBs in the sir2 telomeres (termed HAST domains) are modified by Hda1p (13). strains, Ϸ5% had elevated DSBs (positive ⌬R) and 7% had reduced One explanation of our results is that Hda1p-mediated deacetyla- DSBs (negative ⌬R). As described below, by correlating these ⌬R tion in HAST domain suppresses DSBs in the wild-type strain. If values with various genomic data sets (studies of gene expression, Sir2p and Hda1p compete in their ability to act in these regions, protein binding, chromatin modifications, timing of replication, and deletion of Sir2p might allow Hda1p to act in a more extended other properties), we were able to distinguish a number of different region. Because both Sir2p and Hda1p are histone deacetylases, we classes within the positive and negative ⌬R groups that presumably also need to postulate that Hda1p-mediated deacetylation (target- reflect different mechanisms. Most of our conclusions are based on ing H3K18) more effectively suppresses DSB formation than the correlations shown in SI Table 7. Some of the details of the Sir2p-mediated deacetylation (targeting H4K16).

3958 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0700412104 Mieczkowski et al. Downloaded by guest on September 25, 2021 A second class of ORFs that have reduced DSBs are those that surprising, because no single factor correlates with the pattern of have reduced expression in sir2 haploids. Because sir2 haploids recombination hotspots and coldspots in the wild-type strain, and express both mating types, many genes involved in mating (pro- because DSB frequencies are elevated for some genes and reduction or response to pheromones) and retrotransposition are pressed for others in the sir2 strain. The elevated levels of DSBs at repressed, directly or indirectly by the a1.␣2 repressor (29) that is known targets of Sir2p-directed deacetylation (telomeres and the encoded by genes from both mating type loci. Of the 100 genes that rRNA gene cluster) are likely to be fairly direct effects of loss of have the greatest decrease in expression in the sir2 haploid strain, Sir2p. The very striking reduction in DSBs in the TPSRs may reflect 36 are related to conjugation or retrotransposition, as expected an increased level of Hda1p activity, although many other possi- because both conjugation and retrotransposition are repressed in bilities exist. Whatever the mechanism(s) responsible for the al- a/␣ diploids or sir2 haploids (29, 30). Of these 36 genes, 26 have tered patterns of recombination observed in the sir2 strain, it is clear reduced DSBs in the sir2 strain and 10 have elevated DSBs, a that alteration of a single gene can dramatically change the distri- significant (P ϭ 0.003) enrichment in genes with reduced DSBs bution of DSBs of an organism without a substantial effect on compared with the genome as a whole (2,946 ORFs reduced and fertility (spore viability). 3,441 elevated). Why is there an association between the gene expression levels Materials and Methods in a sir2 haploid and DSB formation in a sir2 diploid? Previously, Strains. All diploid strains used in this study were constructed by we showed that the binding of transcription factors stimulates local crossing derivatives of the haploid strains AS4 and AS13, which we meiotic DSB formation, but transcription per se is not required for have used in many previous studies (5). Alterations in AS4 and hotspot activity (2). Our interpretation of this result was that AS13 were made by transformation as described in detail in SI chromatin modifications related to transcription factor binding Materials and Methods and SI Table 1. The isogenic wild-type strain facilitate the association of both the transcription and recombina- JG169 (5) and the sir2 diploid MD311 were used in the microarray tion machinery. Because much of the repression of haploid-specific experiments. Both strains had an epitope-tagged version of Spo11p ␣ ␣ genes in the a/ diploid is a consequence of the a1- 2 repressor, one and the rad50S mutation, allowing the purification of DNA se- would not necessarily expect that the repression levels would differ quences located near the site of Spo11p-associated DSBs by im- in a wild-type diploid and a sir2 diploid. One possibility is that, as munoprecipitation (5). a consequence of expression of the silent mating type loci in the sir2 ␣ diploid, more a1- 2 repressor is produced in a sir2 diploid than in Genetic Methods and Southern Analysis. Methods for transforma- a wild-type diploid and, consequently, the genes normally regulated tion and tetrad analysis were standard. For Southern blot by the repressor are turned off more completely. Although this analysis, DNA was isolated by using the procedure described by hypothesis requires additional experimental support, we suggest Nag and Petes (31). The hybridization probes were prepared by that class 2 genes may represent genes with increased levels of PCR and the primers used for their preparation are described in binding of a repressor that reduces both gene expression and DSB SI Materials and Methods. formation. The class 3 genes with reduced DSBs do not have a common feature that we have been able to identify. Microarray Analysis of DSB Activities. The methods that we used A final correlation is between the level of DSBs in intergenic were the same as used previously (5). In brief, we isolated Spo11p- regions and the promoter activity of these regions. Of the 6,357 associated DNA from sporulated cultures of JG169 or MD311 by intergenic regions represented on our microarrays, 1,666 are bidi- using IgG Sepharose beads. The Spo11p in JG169 and MD311 is rectional promoters (located between divergently transcribed tagged with a epitope (ZZ) that binds the beads. The experimental genes), 3,112 are unidirectional promoters, and 1,579 are nonpro- DNA samples were labeled with Cy3 and hybridized to microarrays moters (located between convergently transcribed genes). The along with total genomic DNA that had been labeled with Cy5; in average ⌬Rs for these regions (95% confidence limits in parenthe- Ϫ some experiments, we labeled the experimental DNA with Cy5 and ses) are: 0.05 (0.01–0.08) for the bidirectional promoters, 0.06 genomic DNA with Cy3. The hybridization images were acquired (Ϫ0.03 to Ϫ0.08) for unidirectional promoters, and Ϫ0.14 (Ϫ0.11 GENETICS Ϫ with a GenePix 4000B scanner and analyzed with GenePix Pro 5.0 to 0.17) for nonpromoters. Thus, the nonpromoter regions have software. Subsequent steps in the data analysis are described in SI substantially reduced ⌬Rs. We noted previously (3, 5) that DSBs Materials and Methods. Excel spreadsheets summarizing the analysis associated with recombination hotspots were preferentially associ- are in SI Tables 2–4 and 7, and the raw data for these experiments ated with bidirectional promoters and had a negative correlation are available at the University of North Carolina Microarray with nonpromoters. There is also a strong correlation between the Database (https://genome.unc.edu) and through GEO accession binding of cohesin and intergenic regions that are nonpromoters no. GSE6245. (7). One explanation of our data, therefore, is that the sir2 mutation, directly or indirectly, increases the binding of cohesins in the We thank M. Grunstein, J. Rine, A. Johnson, J. Gerton, J. Kohli, and nonpromoter regions, reducing the level of DSBs associated with members of the T.D.P. and J.D.L. laboratories for useful discussions. We these already-cold regions. thank J. Gerton (Stowers Institute for Medical Research, Kansas City, MO) for supplying the wild-type strain JG169 used in the microarray Summary. No single known factor explains the difference in DSB experiments. This research was supported by National Institutes of formation between the wild-type and sir2 strains. This result is not Health Grants GM24110 (to T.D.P.) and GM72518 (to J.D.L.).

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