Roles of histone acetylation and chromatin remodeling factor in a meiotic recombination hotspot
2004; Springer Nature; Volume: 23; Issue: 8 Linguagem: Inglês
10.1038/sj.emboj.7600138
ISSN1460-2075
AutoresTakatomi Yamada, Ken‐ichi Mizuno, Kouji Hirota, Ning Kon, Wayne P. Wahls, Edgar Hartsuiker, Hiromu Murofushi, Takehiko Shibata, Kunihiro Ohta,
Tópico(s)Fungal and yeast genetics research
ResumoArticle26 February 2004free access Roles of histone acetylation and chromatin remodeling factor in a meiotic recombination hotspot Takatomi Yamada Takatomi Yamada Genetic Dynamics Research Unit-Laboratory, RIKEN, Wako, Saitama, Japan Cellular & Molecular Biology Laboratory, RIKEN/CREST of the JST, Wako, Saitama, Japan Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, Bunkyo-ku, Tokyo, Japan Search for more papers by this author Ken-ichi Mizuno Ken-ichi Mizuno Genetic Dynamics Research Unit-Laboratory, RIKEN, Wako, Saitama, Japan Cellular & Molecular Biology Laboratory, RIKEN/CREST of the JST, Wako, Saitama, Japan Search for more papers by this author Kouji Hirota Kouji Hirota Genetic Dynamics Research Unit-Laboratory, RIKEN, Wako, Saitama, Japan Cellular & Molecular Biology Laboratory, RIKEN/CREST of the JST, Wako, Saitama, Japan Search for more papers by this author Ning Kon Ning Kon Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, AR, USA Search for more papers by this author Wayne P Wahls Wayne P Wahls Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, AR, USA Search for more papers by this author Edgar Hartsuiker Edgar Hartsuiker Genome Damage and Stability Centre, University of Sussex, Falmer Brighton, UK Search for more papers by this author Hiromu Murofushi Hiromu Murofushi Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, Bunkyo-ku, Tokyo, Japan Search for more papers by this author Takehiko Shibata Takehiko Shibata Cellular & Molecular Biology Laboratory, RIKEN/CREST of the JST, Wako, Saitama, Japan Search for more papers by this author Kunihiro Ohta Corresponding Author Kunihiro Ohta Genetic Dynamics Research Unit-Laboratory, RIKEN, Wako, Saitama, Japan Cellular & Molecular Biology Laboratory, RIKEN/CREST of the JST, Wako, Saitama, Japan Search for more papers by this author Takatomi Yamada Takatomi Yamada Genetic Dynamics Research Unit-Laboratory, RIKEN, Wako, Saitama, Japan Cellular & Molecular Biology Laboratory, RIKEN/CREST of the JST, Wako, Saitama, Japan Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, Bunkyo-ku, Tokyo, Japan Search for more papers by this author Ken-ichi Mizuno Ken-ichi Mizuno Genetic Dynamics Research Unit-Laboratory, RIKEN, Wako, Saitama, Japan Cellular & Molecular Biology Laboratory, RIKEN/CREST of the JST, Wako, Saitama, Japan Search for more papers by this author Kouji Hirota Kouji Hirota Genetic Dynamics Research Unit-Laboratory, RIKEN, Wako, Saitama, Japan Cellular & Molecular Biology Laboratory, RIKEN/CREST of the JST, Wako, Saitama, Japan Search for more papers by this author Ning Kon Ning Kon Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, AR, USA Search for more papers by this author Wayne P Wahls Wayne P Wahls Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, AR, USA Search for more papers by this author Edgar Hartsuiker Edgar Hartsuiker Genome Damage and Stability Centre, University of Sussex, Falmer Brighton, UK Search for more papers by this author Hiromu Murofushi Hiromu Murofushi Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, Bunkyo-ku, Tokyo, Japan Search for more papers by this author Takehiko Shibata Takehiko Shibata Cellular & Molecular Biology Laboratory, RIKEN/CREST of the JST, Wako, Saitama, Japan Search for more papers by this author Kunihiro Ohta Corresponding Author Kunihiro Ohta Genetic Dynamics Research Unit-Laboratory, RIKEN, Wako, Saitama, Japan Cellular & Molecular Biology Laboratory, RIKEN/CREST of the JST, Wako, Saitama, Japan Search for more papers by this author Author Information Takatomi Yamada1,2,3,‡, Ken-ichi Mizuno1,2,‡, Kouji Hirota1,2, Ning Kon4, Wayne P Wahls4, Edgar Hartsuiker5, Hiromu Murofushi3, Takehiko Shibata2 and Kunihiro Ohta 1,2 1Genetic Dynamics Research Unit-Laboratory, RIKEN, Wako, Saitama, Japan 2Cellular & Molecular Biology Laboratory, RIKEN/CREST of the JST, Wako, Saitama, Japan 3Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, Bunkyo-ku, Tokyo, Japan 4Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, AR, USA 5Genome Damage and Stability Centre, University of Sussex, Falmer Brighton, UK ‡These authors contributed equally to this work *Corresponding author. Genetics Dynamics Research Unit-Laboratory, RIKEN, Hirosawa 2-1, Wako-shi, Saitama 351-0198, Japan. Tel.: +81 48 467 9538; +81 48 462 4671; E-mail: [email protected] The EMBO Journal (2004)23:1792-1803https://doi.org/10.1038/sj.emboj.7600138 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Histone acetyltransferases (HATs) and ATP-dependent chromatin remodeling factors (ADCRs) are involved in selective gene regulation via modulation of local chromatin configuration. Activation of the recombination hotspot ade6-M26 of Schizosaccharomyces pombe is mediated by a cAMP responsive element (CRE)-like sequence, M26, and a heterodimeric ATF/CREB transcription factor, Atf1·Pcr1. Chromatin remodeling occurs meiotically around M26. We examined the roles of HATs and ADCRs in chromatin remodeling around M26. Histones H3 and H4 around M26 were hyperacetylated in an M26- and Atf1-dependent manner early in meiosis. SpGcn5, the S. pombe homolog of Gcn5p, was required for the majority of histone H3 acetylation around M26 in vivo. Deletion of gcn5+ caused a significant delay in chromatin remodeling but only partial reduction of M26 meiotic recombination frequency. The snf22+ (a Swi2/Snf2-ADCR homologue) deletion and snf22+gcn5+ double deletion abolished chromatin remodeling and significant reduction of meiotic recombination around M26. These results suggest that HATs and ADCRs cooperatively alter local chromatin structure, as in selective transcription activation, to activate meiotic recombination at M26 in a site-specific manner. Introduction Eucaryotic chromosomal DNA is packaged into a highly condensed chromatin structure, which inhibits various DNA-associated processes, such as replication, transcription, and recombination, presumably by preventing the loading of trans-acting factors onto target DNA sites. Thus, the local chromatin structure around cis-acting DNA sites should be converted into an open configuration before the initiation of DNA-associated processes (Wolffe, 1997). Chromatin-modifying machineries, such as histone acetyltransferase (HAT) complexes and ATP-dependent chromatin remodeling factors (ADCRs), are often recruited to promoters via sequence-specific DNA binding proteins, bound to their target sites, to activate transcription (Cosma et al, 1999; Krebs et al, 1999; Agalioti et al, 2000). Chromatin-modifying machineries may also activate other DNA-associated reactions. For example, histone acetylation is involved in DNA repair and site-specific V(D)J recombination (McBlane et al, 1995; McMurry and Krangel, 2000; Bird et al, 2002). Homologous recombination is elevated markedly in meiosis and contributes to the genetic diversity of the next generation and the proper segregation of meiotic chromosomes. Most meiotic recombination in Saccharomyces cerevisiae is initiated by transient, meiosis-specific DNA-double strand breaks (DSBs) that map to hotspots for meiotic gene conversion. Such DSB sites are often found in transcription promoters that show hypersensitivity to nucleases (Ohta et al, 1994; Wu and Lichten, 1994). Meiotic DSBs also initiate recombination in fission yeast (Cervantes et al, 2000; Zenvirth and Simchen 2000). These observations suggest that the chromatin structure and its modification, possibly mediated by some sequence-specific transcriptional activators, are important to regulate the initiation of meiotic homologous recombination. In support of this idea, certain transcription factors influence DSB formation at 'α-hotspots' in S. cerevisiae (Petes, 2001). The ade6-M26 (M26) locus in the fission yeast Schizosaccharomyces pombe is a well-characterized meiotic recombination hotspot that provides a good model system for studies of recombination regulation. The ade6-M26 allele is a single G/T transversion in the 5′ end of the ade6 coding region (Ponticelli et al, 1988; Szankasi et al, 1988). This mutation creates a nonsense codon and also confers an up to 15-fold, meiosis-specific elevation of recombination as compared to control alleles such as ade6-M375 (M375) (Gutz, 1971; Ponticelli et al, 1988; Schuchert et al, 1991). The ade6-M375 allele is also a G/T base substitution that creates a nonsense mutation in the codon adjacent to that altered by the M26 mutation, but M375 does not show the hotspot activity. Thus, the ade-M375 allele provides an excellent negative control for studies of hotspot recombination at ade6-M26. Recent studies have revealed a molecular basis for hotspot recombination at ade6-M26. Atf1 and Pcr1, which both belong to the ATF/CREB transcription factor family, bind as a heterodimer specifically to a heptameric DNA sequence, ATGACGT, which is created by the M26 mutation and is similar to the CRE (cAMP Response Element, TGACGT) sequence (Wahls and Smith, 1994; Kon et al, 1997). Binding of the Atf1·Pcr1 complex to this 'M26 DNA site' is required for the recombination hotspot activity (Wahls and Smith, 1994; Kon et al, 1997). Interestingly, the Atf1·Pcr1 heterodimer is a transcription factor involved in meiotic induction, but it is dispensable for the steady-state levels of ade6 transcription (Wahls and Smith, 1994; Kon et al, 1997). Activation of meiotic recombination at M26 is at least partly due to meiosis-specific formation of DSBs around M26, which are dependent upon Rec12 (the S. pombe homologue of Spo11) and Pcr1 (Steiner et al, 2002). We previously reported that the chromatin structure around ade6-M26 is remodeled meiotically (Mizuno et al, 1997). The nucleosome phasing along the ade6 open reading frame is lost, and in turn a micrococcal nuclease (MNase)-sensitive site appears de novo. This process, referred to as M26 chromatin remodeling, is regulated by meiosis-inducing signaling pathways (Mizuno et al, 2001). In addition, similar chromatin remodeling occurs around naturally occurring CRE-related sequences, such as ctt1+ and fbp1+ promoter sequences (Mizuno et al, 2001; Hirota et al, 2003), controlled by cAMP-dependent kinase (PKA) and stress-activated kinase (SAPK) pathways and fission yeast Tup1-like corepressors (Hirota et al, 2003). This suggests that M26 chromatin remodeling might reflect an intrinsic physiological response occurring at S. pombe natural CRE-related sequences. These findings, together with the dual roles of the Atf1·Pcr1 heterodimer in meiotic development and hotspot activation (Kon et al, 1998), suggest that the induction of recombination in meiosis may be partly regulated at the chromatin or DNA accessibility level, and may be coupled in some way to transcriptional regulation in a developmental stage-specific manner. To further understand the mechanisms underlying the induction of meiotic recombination, we have studied the roles of histone acetylation in M26 chromatin remodeling. Here we report roles of the Atf1·Pcr1-, SpGcn5 HAT-mediated histone acetylation, and SpSnf22 ADCR-like factor in chromatin remodeling and meiotic recombination at the M26 hotspot. These results provide important insights into molecular mechanisms on site-specific chromatin regulation at CRE-like sequences in the activation of meiotic recombination. Results Atf1 and Pcr1 are required for chromatin remodeling at ade6-M26 Activation of the M26 recombination hotspot requires Atf1·Pcr1 (Kon et al, 1997), which is constitutively expressed and binds to the M26 DNA site, as revealed by a methylation interference assay (Kon et al, 1998) and a chromatin immunoprecipitation (Ch-IP) assay (W. Wahls, unpublished results). To study the molecular basis of M26 chromatin remodeling, we first analyzed the effects of atf1Δ and pcr1Δ (null) mutations on chromatin structure. Wild-type (atf1+ pcr1+), atf1Δ, pcr1Δ, and atf1Δ pcr1Δ mutant cells were cultured in a pre-sporulation medium and were then cultured in a sporulation medium prior to analysis of chromatin structure. In atf1+ pcr1+ cells, the chromatin structure around ade6-M26 changed by 3 h of meiosis, while little or no change was observed around M375 (Figure 1A). However, in atf1Δ or pcr1Δ cells, the M26 DNA site-dependent chromatin remodeling was not observed at 3 h and even at 6 h (data not shown) after meiotic induction. Similarly, atf1Δ pcr1Δ double mutant cells did not exhibit chromatin remodeling at M26 (Figure 1A). We conclude that both Atf1 and Pcr1 are strictly required for the meiotic chromatin remodeling around ade6-M26. Figure 1.Atf1·Pcr1 is required for meiotic chromatin remodeling around ade6-M26. (A) Disruption of atf1+ or pcr1+ abolishes meiotic chromatin remodeling around ade6-M26. Diploid strains ELD205 (ade6-M26), WSP779 (ade6-M26, atf1Δ), WSP857 (ade6-M26, pcr1Δ), WSP859 (ade6-M26, atf1Δ, pcr1Δ), ELD203 (ade6-M375), and WSP780 (ade6-M375, atf1Δ) were cultured in presporulation medium (lanes 0 h). Cells were then transferred to sporulation medium and cultured further for 3 h (lanes 3 h). Chromatin isolated from cells was digested with 0 (lanes −) or 20 (lanes +) units/ml of MNase and analyzed as described (Mizuno et al, 1997). Probe 1 was used for indirect end labeling. The vertical and the horizontal arrows indicate the ade6 ORF and the position of the M26 mutation, respectively. Numbers by the right vertical arrow show the positions in nucleotides of the Xho I (−485), Bam HI (26), and Hind III (842) sites relative to the first A of the ade6 coding region. Open and hatched ovals represent phased and randomly positioned nucleosomes, respectively. The broken line by lane 4 indicates the region where the chromatin structure is remodeled. (B) Ectopically induced meiosis does not induce chromatin remodeling in atf1Δ and pcr1Δ cells. (Lanes 1–12) Chromatin structures of WSP857 (ade6-M26, atf1+, pcr1Δ) diploids harboring the empty vector (lanes vector(pDB)), the Pcr1 expressing plasmid (lanes pDB(Pcr1)), and the Ste11 expressing plasmid (lanes pDB(Ste11)) were analyzed. Experiments were performed as in Figure 1A. (Lanes 14–23) The K213 (ade6-M26, atf1Δ, pat1-114), K214 (ade6-M375, atf1Δ, pat1- 114), and GP1725x (ade6-M26, pat1-114) cells were cultured for 24 h in presporulation medium (lanes 0 h). Cells were transferred to sporulation medium and cultured at 34°C for 3 h (lanes 3 h). Chromatin analyses were performed as in Figure 1A. Overexpression of Pcr1 caused chromatin remodeling in premeiotic cells (lane 6). This may be due to artificial activation of chromatin remodeling by enhanced binding of Atf1·Pcr1 to the M26 site. Download figure Download PowerPoint As Atf1 and Pcr1 are transcription factors that induce some genes during meiotic differentiation (Takeda et al, 1995; Kanoh et al, 1996; Watanabe and Yamamoto, 1996), it was possible that the deletion of atf1+ or pcr1+ indirectly affected the chromatin remodeling around ade6-M26. Kon et al. previously ruled out the possibility that the loss of atf1+ or pcr1+ decreased ade6 transcription and consequently abolished the chromatin remodeling (Kon et al, 1997). The entire loss of the M26 chromatin remodeling in the atf1Δ and the pcr1Δ strains is also unlikely to be due to their deficiencies in the meiotic proficiency, since nearly half of the mutant cells underwent sporulation. However, it remained possible that Atf1·Pcr1 regulated the expression of other meiotic factors that mediated the chromatin remodeling. To test this hypothesis more rigorously, we used two approaches to bypass the requirement for the Atf1·Pcr1 heterodimer in meiotic induction. First, we induced meiosis ectopically by expressing Ste11 (Sugimoto et al, 1991) in pcr1Δ cells. Second, we induced meiosis ectopically in atf1Δ cells by inactivation of the Pat1 kinase (Iino and Yamamoto, 1985). Each approach alleviated the early meiotic defects of pcr1Δ and atf1Δ cells (Watanabe and Yamamoto, 1996), but did not compensate for the loss of the M26 chromatin remodeling, even though meiosis was almost fully induced in these mutant cells (Figure 1B). These results, together with the fact that Atf1·Pcr1 occupies the M26 site in cells, led us to conclude that the M26 chromatin remodeling is mediated directly in cis by the Atf1·Pcr protein bound to the M26 DNA site. Histones around ade6-M26 are hyperacetylated HATs are often recruited by sequence-specific transcriptional activators, and thereafter convert the local chromatin configuration into a state suitable for active transcription (Brown et al, 2000). This led us to speculate that the Atf1·Pcr1 heterodimer might recruit HATs to the M26 site and mediate the chromatin remodeling through histone acetylation. To test this idea, we used Ch-IP for the analysis on the state of histone acetylation around M26. Cells were induced to enter meiosis and cell extracts were subjected to Ch-IP using antibodies specific for acetylated isoforms of histones. The DNA from the immunoprecipitated chromatin and the input material was placed on slot blots and was hybridized with either total genomic DNA (serving as a reference for global histone acetylation levels) or with DNA sequences around the M26 hotspot. We reproducibly detected stronger signals on the blots of immunoprecipitated chromatin from the M26 cells as compared to those from the M375 cells (Figure 2A). Quantitative analysis demonstrated that the histones H3 and H4 around ade6-M26 were highly acetylated at 0.5 h after the meiotic induction. The histone H3 and H4 acetylation levels at M26 (about 20% of the total input fraction) were 2- and 1.5-fold higher, respectively, than those at ade6-M375, (Figure 2B and C), and were ∼20-fold higher than the global (genome average) levels (Figure 2B and C, about 1% of the total input fraction). The relatively high levels of histone acetylation around ade6-M375 (5–10% of the total input fraction), compared to the global levels, may be due to the active transcription of the ade6 gene, which is an essential housekeeping gene. The histone acetylation levels gradually decreased to 50% of their maximal values by 2 h after the meiotic induction, a time point when premeiotic DNA synthesis was almost completed and the M26 chromatin remodeling was initiated (Mizuno et al, 1997). Separate experiments revealed that the global levels of histone acetylation in cells harboring ade6-M26 were similar to those in ade6-M375 cells (Figure 2B-C, broken lines). These results demonstrate that histones at the ade6 locus are hyperacetylated in an M26 DNA site-dependent manner. Figure 2.Histones H3 and H4 around ade6-M26 are highly acetylated in an Atf1-dependent manner during meiosis. Chromatin obtained from DTY2 (ade6-M26), DTY4 (ade6-M375), and WSP779 (atf1Δ, ade6-M26) diploid cells was immunoprecipitated with antiacetylated histone H3 and H4 antibodies. DNA obtained from input and immunoprecipitated material was applied to slot blots. (A) Example of primary data showing autoradiograms of slot blots probed for the ade6-M26 or ade6-M375 region. (B–G) Quantitative data are expressed as the ratio of the bound to the input material (immunoprecipitation efficiency: IE). All data were averages of at least three independent experiments. Labels inset into each figure panel indicate the relevant cell genotype and the probe used for each experiment. The data for WT in panels D–G are the same as those in panels B, C (solid line) and B, C (broken line). (B, C) Effects of ade6 alleles on acetylation of histone H3 and H4, respectively. (D, E) Requirement for Atf1 in histone H3 and H4 acetylation at ade6-M26, respectively. (F, G) Role of Atf1 in global (genome average) acetylation of histone H3 and H4, respectively. Download figure Download PowerPoint We next applied the Ch-IP analysis to atf1Δ cells, in which no meiotic chromatin remodeling was observed around M26 (see Figure 1). As shown in Figure 2D and E, the acetylation of histones H3 and H4 around ade6-M26 was greatly reduced in the atf1Δ cells, indicating that histone acetylation around M26 requires Atf1. Interestingly, the global levels of histone acetylation were also reduced significantly in atf1Δ cells (Figure 2F and G). S. pombe Gcn5p is a HAT We next sought to identify the HAT that may be involved in histone acetylation around M26. Referring to the amino-acid sequences of the conserved HAT domains and bromodomains of budding yeast and human Gcn5p, we searched for a fission yeast homologue of Gcn5p by a BLAST search. One gene, SPAC1952.05, showed the highest similarity to yeast and human Gcn5p (Figure 3A). SPAC1952.05 was previously identified as a gene encoding a putative S. pombe homologue of Gcn5p, and the gene product was found in a SAGA-like complex (Mitsuzawa et al, 2001), although it was unknown whether the protein had HAT activity. Figure 3.HAT activity of SpGcn5 in vitro and in vivo around M26 (A) Sequence alignments of ScGcn5p and SpGcn5 showing identical (dark gray) and conserved (light gray) amino acids in motif 'A' of the HAT domain. The asterisk indicates the glycine residue replaced with alanine in SpGcn5(G205A). (B) SpGcn5 has histone (H3>H4) acetyltransferase activity in vitro. Recombinant SpGcn5 and SpGcn5(G205A) proteins were purified and assayed for HAT activity by incubation with human histone octamers and [14C] acetyl-CoA. Labeled proteins were separated by SDS–PAGE, stained with Coomassie brilliant blue (left panel), and subjected to autoradiography (right panel) to reveal acetylated histones (control, no enzyme). (C–H) Chromatin obtained from DTY9 (gcn5Δ, ade6-M26) and DTY11 (gcn5Δ, ade6-M375) cells was analyzed as in Figure 2. All data are averages of at least three independent experiments. Labels inset into each figure panel indicate the relevant cell genotype and the probe used for each experiment. The data for WT in panels C–H are same as those in Figures 2B, C, F and G. Requirement for Gcn5 in histone H3 (C, E) and H4 (D, F) acetylation at ade6-M26 (C, D) and at ade6-M375 (E, F). (G, H) Role of Gcn5 in global (genome average) acetylation of histone H3 and H4, respectively. Download figure Download PowerPoint To test the hypothesis that SPAC1952.05 encoded a HAT enzyme, we produced and purified bacterially expressed, wild-type protein and a protein with an alanine substitution at the glycine205. The Gly205 residue is in the putative acetyl CoA-binding site and corresponds to an amino acid required for the HAT activity of budding yeast Gcn5p (Kuo et al, 1998) (Figure 3A). We then assayed the potential HAT activities of these proteins, using human histone octamers as substrates (Figure 3B). The wild-type protein had HAT activity, with preference toward histone H3 compared to H4 (∼30-fold, Figure 3B, lane WT), which is similar to the specificity of the budding yeast Gcn5p (Kuo et al, 1996). The protein with the G205A substitution exhibited very low HAT activity (Figure 3B, lane G205A). We concluded that SPAC1952.05 encodes a functional HAT enzyme homologous to human and budding yeast Gcn5, and therefore use the names 'gcn5+' and 'SpGcn5' to refer to the S. pombe gene and protein, respectively. To determine whether SpGcn5 was responsible for the acetylation of histones at ade6 in vivo, we deleted the gcn5+ gene. Haploid gcn5Δ (null) mutants were viable and grew slightly slower than the gcn5+ cells. Diploid gcn5Δ mutants underwent meiosis I normally, except that the meiosis I process seems slightly quick and a small fraction of the cells (less than 10%) entered meiosis prematurely when cultured in a presporulation medium (Supplemental data 1). In addition, the gcn5Δ cells produced asci with four viable spores (78% spore viability). Moreover, northern analyses revealed that loss of gcn5+ did not affect the transcriptional induction of the meiosis-specific recombination genes, rec6+ and rec12+ (data not shown). These phenotypes differ markedly from those of budding yeast, where the deletion of GCN5 completely inhibits meiotic events (Burgess et al, 1999). The Ch-IP experiments revealed that histone H3 acetylation levels around ade6-M26 were much lower in gcn5Δ cells than in gcn5+ cells (∼75% reduction, Figure 3C). A less severe decrease in histone H4 acetylation was observed, consistent with the substrate preference of SpGcn5 toward histone H3 (Figure 3D), while such in vivo effects might include an indirect influence on acetylation by other HATs in the absence of SpGcn5. We also observed a significant reduction of histone H3 acetylation around ade6-M375 (Figure 3E) and in the entire genome (Figure 3G) in gcn5Δ cells. Notably, in gcn5Δ cells the absolute levels of histone H3 acetylation around ade6-M26 were very similar to those around ade6-M375. These results demonstrate that SpGcn5 is required for the acetylation of histone H3 around ade6 and in the whole genome (see discussion), and more importantly it is required for the M26-specific hyperacetylation. Effects of gcn5+ deletion on genome-wide DSB formation and meiotic recombination We next examined the effects of gcn5+ deletion on the meiotic DSB formation on whole chromosomes (Figure 4A) using haploid strains with the pat1-114 rad50S background, which enabled us to detect the accumulation of discrete meiotic DSB bands in highly synchronized meiosis. In gcn5+ cells, we detected the accumulation of smeared chromosomal bands, reflecting meiotic DSBs, after 4 h in sporulation culture (Figure 4A lanes 4 and 5). The abundance of these subchromosomal fragments was markedly reduced at 4 h in gcn5Δ cells (Figure 4A lane 9), while gcn5Δ cells undergo slightly quicker meiosis I (see above). However, the final extent of chromosomal breakage in the gcn5+ and gcn5Δ cells was similar at a later time point (Figure 4A, lane 10). These results indicate that SpGcn5 is required for the proper timing of the DSB formation, but has only a nominal effect on the final level of the genome-wide DSB formation. Figure 4.Effects of gcn5+ disruption on the genome-wide DSB formation and recombination. (A) Effects of gcn5Δ mutation upon meiotic DSBs in whole chromosomes. Genomic DNA was prepared from K341 (ade6-M26, rad50S, lanes WT) and TY24 (ade6-M26, rad50S, gcn5Δ, lanes gcn5Δ) cells cultured for the indicated times in sporulation medium, and was analyzed by pulsed-field gel electrophoresis. The electrophoresis image was obtained by an FM BIO II image analyzer (HITACHI) after Syber Green staining. The smeared DNA shows the broken DNA generated during meiosis. (B) Effects of the gcn5Δ mutation upon intergenic recombination. Standard genetic crosses between haploids containing appropriate markers were performed, and spores were plated on complete medium. Then the colonies arising from the spores were replicated onto appropriate test media to check the prototrophy and to measure the recombination frequencies. Each recombination frequency represents average of five independent crosses. Download figure Download PowerPoint To determine whether the gcn5Δ mutation influences global recombination, we measured the frequency of intergenic recombination (crossing-over) between lys7 and leu2 on chromosome I and between lys4 and ade1 on chromosome II (Figure 4B). We found that the gcn5+ disruption did not significantly affect the recombinant frequencies. A possibility of overestimating recombinants in gcn5Δ mutants was eliminated, since there is no significant effect of the gcn5Δ mutation on viable or diploid spore formation. Thus, we concluded that although SpGcn5 is required for proper timing of meiotic DSB formation, it is dispensable for intergenic, nonhotspot recombination, at least for the two intervals tested. Effects of gcn5+ disruption on chromatin remodeling, DSB formation, and recombination at the ade6-M26 hotspot We studied the effects of the gcn5Δ mutation on chromatin remodeling, meiotic DSB formation, and recombination at the ade6-M26 hotspot. In wild-type diploid cells, the M26 chromatin remodeling occurred by 2–3 h after meiotic induction (Figures 1 and 5A). However, in gcn5Δ diploid cells, chromatin remodeling was not observed after 3 h, but partial remodeling occurred at 4.5 h (data not shown) and at 6 h (Figure 5A). Similar results were obtained with highly synchronized meioses in pat1-114 cells (data not shown). These results demonstrate that SpGcn5 is involved in the M26-dependent chromatin remodeling during meiosis. Figure 5.Effects of gcn5+ disruption on the chromatin remodeling, DSB formation, and recombination at M26. (A) Requirement for SpGcn5 in meiotic chromatin remodeling. Meiotic chromatin remodeling at the ade6 locus in DTY2 (ade6-M26, lanes WT) and DTY9 (ade6-M26, gcn5Δ, lanes gcn5Δ) diploids was analyzed as in Figure 1, using 0, 20, and 30 units/ml of MNase. Three independent experiments gave the same results. The broken line by lane 6 indicates the region where the chromatin structure is remodeled. (B) Requirement for SpGcn5 in meiotic DSB formation around M26. Genomic DNA was prepared as in Figure 4A, was digested with Afl II, and analyzed by Southern analysis. Horizontal and vertical arrows indicate the positions of the M26 (or M375) sites and the ade6 ORF, respectively. (C) Meiotic DSBs at the M26-independent site
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