Regulation Mechanisms of Meiotic Recombination Revealed from the Analysis of a Fission Yeast Recombination Hotspot ade6-M26

Abstract

Meiotic recombination is a pivotal event that ensures faithful chromosome segregation and creates genetic diversity in gametes. Meiotic recombination is initiated by programmed double-strand breaks (DSBs), which are catalyzed by the conserved Spo11 protein. Spo11 is an enzyme with structural similarity to topoisomerase II and induces DSBs through the nucleophilic attack of the phosphodiester bond by the hydroxy group of its tyrosine (Tyr) catalytic residue. DSBs caused by Spo11 are repaired by homologous recombination using homologous chromosomes as donors, resulting in crossovers/chiasmata, which ensure physical contact between homologous chromosomes. Thus, the site of meiotic recombination is determined by the site of the induced DSB on the chromosome. Meiotic recombination is not uniformly induced, and sites showing high recombination rates are referred to as recombination hotspots. In fission yeast, ade6-M26, a nonsense point mutation of ade6 is a well-characterized meiotic recombination hotspot caused by the heptanucleotide sequence 5′-ATGACGT-3′ at the M26 mutation point. In this review, we summarize the meiotic recombination mechanisms revealed by the analysis of the fission ade6-M26 gene as a model system.

1. Introduction

Meiosis is a special cell division process that creates gametes by reducing the number of chromosomes by half to maintain the original chromosome number during sexual reproduction. Meiosis consists of DNA replication followed by two sequential rounds of nuclear division: MI and II. Homologous chromosomes segregate away from each other in the first nuclear division (MI), whereas the sister chromatids separate in the second meiotic division (MII) [1]. Before MI, the two homologous chromosomes are in close contact to form bivalent chromosomes, which is pivotal for the faithful segregation of homologous chromosomes [2]. This contact is mediated by crossover (CO) (cytologically seen as chiasma) formed between two homologous chromosomes due to homologous recombination [3,4,5].

Meiotic homologous recombination contributes to the proper segregation of homologous chromosomes and augmentation of genetic diversity. This is a highly regulated process initiated by DNA double-strand breaks (DSBs), which are induced by the conserved Spo11 protein and other accessory DSB proteins including Rec114 and Mer2, which assemble on chromosomes [6,7,8,9,10,11]. Spo11 is an enzyme similar to topoisomerase II and induces DSB via nucleophilic attack of the hydroxy group in its catalytic residue tyrosine (Tyr) on the phosphodiester bond [5,12] (Figure 1A,B, DSB formation). The programmed DSBs are repaired by homologous recombination using the homologous chromosome donor sequence (Figure 1B). DSB ends are processed into 3′ single-stranded overhangs via end resection by the CtIP, Exo1, and Mre11-Rad50-Nbs1 (MRN) complex [13,14,15,16] (Figure 1B, End resection). The resultant single stranded DNA (ssDNA) can invade homologous chromosomes via homologous recombination [17,18] (Figure 1B). From bacteria to humans, most types of homologous recombination rely on the RecA family of recombination proteins (eukaryotic homologs, Rad51, and Dmc1) to promote strand pairing and invasion [17,19]. This view is supported by the facts that mutations in DMC1 gene cause infertility in human and mouse [20,21]. Each of these recombination proteins forms a nucleoprotein filament by binding to 3 nucleotides of the ssDNA [22]. The resultant nucleoprotein filament helps identify a homologous genome sequence within the donor double-stranded DNA (dsDNA) sequence and then promotes an exchange of base pairs, leading to the formation of a displacement loop (D-loop) [23]. After invasion and exchange of the base pairs, a DNA polymerase extends the 3′ end of the invading strand using a homologous template and eventually repairs the DSB. If the synthesized DNA end returns and rehybridizes to the original strand, a portion of the sequence in the homologous chromosome is copied. This process is referred to as synthesis-dependent strand annealing (SDSA) and results in gene conversion (non-crossover [NCO]) (Figure 1B, SDSA). Alternatively, annealing between the displaced strand and complementary ssDNA (second-end capture) results in the formation of a double Holliday junction (dHJ), leading to CO [24,25] (Figure 1B, dHJ). This dHJ model was proposed from studies in Saccharomyces cerevisiae (budding yeast), while other model in which COs are induced from single Holliday junction (sHJ) was proposed in the highly divergent yeast Schizosaccharomyces pombe (fission yeast) [26]. In this model, the second-end capture results in the formation of sHJ, which is resolved by Mus81 resulting in CO or NCO [26] (Figure 1B, sHJ).

The COs/chiasmata and meiotic DSBs are non-randomly distributed along the chromosome and concentrated in short DNA intervals called DSB hotspots [27,28,29,30]. The global distribution of DSB has been extensively studied in budding yeast and fission yeast [31,32,33,34]. Nucleotide resolution analysis of DSB sites using next-generation sequencing in budding yeast has provided insights into DSB sites and hierarchical contexts, such as chromosome structures, chromatin, transcription factors, and local sequence composition [35]. Many of the DSB hotspots are located in promoter regions for mRNA or noncoding RNA (ncRNA) [36,37], which are characterized by transcription factor binding, histone modifications, and low nucleosome density, suggesting a close relationship between meiotic recombination and transcription. Chromatin consists of arrays of nucleosomes, which are one of the critical determinants of all bioreactions on chromosomes, such as transcription, replication, and recombination, with the following correlations: open chromatin configuration correlates with active reactions and closed (condensed) chromatin configuration correlates with inactivation [38,39]. Moreover, the involvement of the state of chromatin figurations and histone variant in the control of CO/NCO has been shown in the genome wide researches in arabidopsis (reviewed in [40]). In addition to chromatin status, the status of mRNA and ncRNA transcription may also be critical determinants of meiotic recombination [36,37]. For several decades, researchers have explored the multiple layers of meiotic recombination modulators by analyzing the fission yeast gene ade6-M26 as a model meiotic recombination hotspot. Herein, we discuss how analyses of the fission yeast gene ade6-M26 have provided knowledge on the multiple regulatory mechanisms involved in the regulation of meiotic recombination.

2. ade6-M26, a Meiotic Hotspot Created by a Mono-Nucleotide Substitution Mutation

The ade6 gene encodes phosphoribosylaminoimidazole carboxylase, which is required for purine biosynthesis. The adenine auxotrophic strains with a nonsense mutation in the ade6 gene form red (or pink) colonies on yeast extract medium with a limiting adenine concentration [41], providing a visual distinction between wild-type and mutant alleles (Figure 2A). Several hundred ade6 mutations induced by ultraviolet or X-ray irradiation or nitrous acid treatment were assessed for recombination to map the mutation point, and the mutation ade6-M26 was shown to create a meiotic recombination hotspot [42]. The ade6-M26 hotspot contains a G-to-T substitution in the ade6 open reading frame (ORF) [43,44] (Figure 2B, Red letter). During the early stage of genetic analysis in meiotic recombination, the rec genes required for meiotic recombination were screened using this system and several rec genes were identified [45,46]. Originally, ade6-M26 hotspot was identified as a gene conversion (NCO) hotspot [42] (Figure 1B, left SASD). Later, it was showed that this mutation hotspot also augments the meiotic CO recombination [47] (Figure 1B, left Crossover).

3. Transcription Factor Required for Meiotic Recombination at M26

The M26-mutation is a nonsense mutation that creates a cyclic adenosine monophosphate (cAMP)-responsive element (CRE)-like heptanucleotide sequence 5′-ATGACGT-3′ (Figure 2B, Red square). Later, biding factors to this CRE-like heptanucleotide sequence was identified and named as Mst1 and Mst2 and it was revealed that these bind factors activate meiotic recombination [48,49,50] (Figure 2C). After this identification, three groups almost simultaneously identified Atf1, a basic leucine zipper (bZIP) transcription factor that is activated by the mitogen-activated protein kinase (MAPK) pathway [51,52,53]. Moreover, a similar bZIP protein, Pcr1, was also identified [54], and Mts1 and Mts2 were found to be identical to Atf1 and Pcr1, respectively [48]. In fission yeast, meiosis is induced by the depletion of the nitrogen source, and the signal is mediated by PKA and stress-activated MAPK signal pathway, resulting in the activation of the genes required for meiosis [55]. In addition, during nitrogen starvation, the phosphorylation of Atf1 through a stress-activated MAPK, Spc1, is required for conjugation and meiosis [52]. Atf1 phosphorylation is also required for the activation of the fbp1 gene upon glucose starvation stress via the modulation of chromatin configuration in fbp1 upstream sequence [56,57,58,59,60,61,62,63]. Thus, Atf1 phosphorylation is involved in ade6-M26 hotspot activation, gene induction during mating and meiosis, and fbp1 transcription. Interestingly, the canonical Spc1-phospholylation sites in Atf1 are required for fbp1 transcription but not for the activation of the ade6-M26 hotspot or induction of meiotic genes, indicating a distinctly different phosphorylation mechanism required for ade6-M26 hotspot activation, gene transcriptional induction in mating and meiosis, and fbp1 transcription [64]. Similarly, a meiotic recombination hotspot, ade6-3049 allele possesses a point mutation, which concurrently creates a nonsense mutation and a CRE-like sequence at the 3′ region of ade6 gene [37]. Both ade6-M26 and ade6-3049 strains express shorter versions of mRNA initiated from M26 and 3049 point mutations, respectively [37,65] (Figure 2C), suggesting a link between transcriptional initiation and meiotic recombination. Involvement of transcriptions in the meiotic recombination activation at ade6-M26 is also highlighted by the following facts. First, hotspot activity of ade6-M26 requires ade6 promoter [66,67]. Second, insertions of other transcription factor binding sequences at the M26 mutation site in ade6 gene also created meiotic recombination hotspot [68,69]. This indicates that meiotic recombination hotspots can be created by transcription factors other than the Atf1-Pcr1 transcription factor required for the ade6-M26 hotspot [48,49,50]. This was proved to be the case when the loss of each transcription factor bound to the inserted binding site inactivated hotspot activity [70]. Moreover, the subset of naturally occurring M26 sequence sites (M26 heptanucleotide sequence occurring in wild-type cells) that facilitate meiotic recombination is implicated in approximately 20% of all natural meiotic recombination events in the fission yeast genome [71]. This might also be the case in another highly diverged yeast, Saccharomyces cerevisiae (budding yeast), since binding sites for the Atf1 ortholog Sko1 tend to be highly enriched for meiotic DSBs [35]. Furthermore, other sequence-specific binding proteins proven to activate hotspots in the ade6 gene in fission yeast (Php2, Php3, Php5, Rst2) [68,69] also have orthologs in budding yeast (Hap2, Hap3, Hap5, Adr1) in which DSBs are preferentially directed to their binding sites [35]. Such sequence-specific transcription factor-dependent hotspots created in the fission yeast ade6 gene are mediated through a common chromatin modulation system [72] (see Section 4). Taken together, these results suggest that some shared regulatory systems between recombination and transcription may play a role in a subset of meiotic recombination sites created by the binding sequence of transcription factors.

4. Chromatin Modulation Mechanisms Involved in the Regulation of ade6-M26 Meiotic Recombination Hotspot

The chromatin configurations play pivotal role in the transcriptional control (reviewed in [38,39]). This can be attributed to the fact that tightly packed chromatin limits the accessibility of trans-acting proteins such as transcription factors. Regulation of chromatin structure involves two major classes of chromatin modulation machinery. The first comprises histone modifications, such as acetylation, that are involved in chromatin modulation. For example, histone acetyltransferases add acetyl groups that are usually associated with active chromatin [73,74]. The second class comprises ATP-dependent chromatin remodelers. ATP-dependent chromatin remodelers alter chromatin configuration by sliding and disassembling nucleosomes in an ATP-dependent manner [75]. The yeast Swi2/Snf2 family ATP-dependent chromatin remodeler contains a helicase/ATPase motif as a catalytic domain and a bromodomain that recognizes acetylated histones [76,77]. Proteins carrying bromodomains, including Swi2/Snf2 family ATP-dependent chromatin remodelers, can serve as decoders for histone acetylation.

The direct involvement of histone acetylation and ATP-dependent chromatin remodeler-mediated chromatin remodeling in the regulation of meiotic recombination was first demonstrated in ade6-M26 [78]. The chromatin configurations around the M26 mutation site were assessed based on the sensitivity of the chromatin DNA to micrococcal nuclease (MNase). The chromatin configuration at the M26 mutation point was shown to convert into an open state during meiosis, which requires the involvement of Atf1, Pcr1, and CRE-like heptanucleotide sequences [79] (Figure 3A). Acetylation of the histones H3 and H4 around the M26 mutation site is augmented in an M26- and Atf1/Pcr1-dependent manner early in meiosis. Gcn5 histone acetyl transferase is involved in the histone H3 acetylation around M26 [78]. Moreover, deletion of the gcn5 gene results in a significant delay in chromatin remodeling and partial reduction in the M26 meiotic recombination frequency [78]. Deletion of the snf22 gene (which encodes Swi2/Snf2-chromatin remodeler) abolishes chromatin remodeling and results in the critical reduction of meiotic recombination at ade6-M26 site [78]. These results suggest that Gcn5 histone acetyl transferase and Snf22 Swi2/Snf2-chromatin remodeler cooperatively alter the local chromatin structure to activate meiotic recombination at M26 in a site-specific manner (Figure 3B). Later, two CHD-1 family chromatin remodelers (Hrp1 and Hrp3) involved in chromatin regulation, a Gcn5 histone acetyltransferase component (Ada2), a member of the Moz-Ybf2/Sas3-Sas2-Tip60 family histone acetyltransferase (Mst2), and a histone variant H2A.Z have been reported in the regulation of the of ade6-M26 hotspot activity [80,81]. Moreover, proteomic analysis of the ade6-M26 recombination hotspot revealed that chromatin-mediated regulators, including histone chaperones (Nap1, Hip1/Hir1), subunits of the Ino80 complex (Arp5, Arp8), a DNA helicase/E3 ubiquitin ligase (Rrp2), components of a Swi2/Snf2 family remodeling complex (Swr1, Swc2), and a nucleosome evictor (Fft3/Fun30), interact with the ade6-M26 recombination hotspot during meiosis [82]. Taken together, these studies revealed that remarkably diverse chromatin modulators and histone post-translational modifications participate in the regulation of M26 meiotic recombination.

5. Meiotic Double-Strand Break Formation around M26 Mutation Site

Meiotic recombination is thought to be initiated by either single-strand breaks or double-strand breaks in the DNA [83,84]; however, which of the two is specifically responsible has been a long-standing question. This controversy was resolved by physical detection of double-strand breaks (DSBs) in budding yeast during meiosis [85]. The formation of DSBs in budding yeast requires multiple gene products that are also required for meiotic recombination and proper homologous chromosome segregation [86]. One such gene product, Spo11 is required for the formation of meiotic DSBs [87]. The fission yeast Spo11 homolog, Rec12 protein, is also essential for meiotic recombination [45], and thus this protein is hereafter referred to as Spo11/Rec12. The catalytic tyrosine residue of Spo11/Rec12 is pivotal for its action, suggesting that the DNA–Spo11/Rec12 covalent bond is mediated by this tyrosine residue [88,89] (Figure 1A). These results suggest a conserved mechanism for meiotic DSB formation and recombination. However, the initial attempt to assess meiotic DSBs in the fission yeast genome suffered due to a lack of a detectable level of DSBs at or near the ade6-M26 hotspot [89]. The unresolved question whether DSBs are associated with the ade6-M26 hotspot was resolved by using the rad50s mutant. This mutant, originally obtained from budding yeast, was found to be defective in end resection [90,91], and covalent Spo11/Rec12-DNA complexes were detected in this mutant [87,92] (DSB formation and end resection step in Figure 1B). In fission yeast, the rad50s mutant also shows accumulation of DSBs and no detectable repair [93]. Multiple DSBs were detected on both sides near the M26 mutation site (5′-ATGACGT-3′ heptanucleotide sequence) with closely spaced M26 sites [94]. Interestingly, the M26 site itself lies in a region of approximately 70 bp that is free of visible breaks (Figure 4), suggesting that Spo11/Rec12 is recruited to the genomic DNA where the chromatin configuration has been meiotically opened by the Atf1-Pcr1 and stochastic digest the surrounding sequences.

6. Induction of Meiotic DSB via the Expression of Long ncRNA

DSBs induced by Spo11/Rec12 are not uniformly distributed in the genome [87,92,93], and a significant overlap between the DSB sites and the regions of ncRNA expression was identified by bioinformatics analysis in fission yeast [36]. These facts suggest that the accessibility of Spo11/Rec12 to the genome could be controlled by ncRNAs. In this section, the role of ncRNA transcriptions in the induction of meiotic DSB is discussed.

The ncRNAs can be classified into two classes, small RNAs and long ncRNAs (lncRNAs), according to their length. The first class of ncRNAs, small RNAs are ncRNAs comprised of less than 200 nucleotides. siRNAs and miRNAs is well characterized examples of small RNAs. Small RNAs regulate gene expression via degradation of mRNA and inhibition of translation through their interaction with RNA-induced silencing complexes [95]. The second class of ncRNAs is long ncRNA (lncRNA) carrying over 200 nucleotides length. They are involved in various biological processes, including gene regulation, development, nuclear organization, and cancer development [96,97]. Deregulation of several lncRNAs in cancer cells has been identified and such lncRNAs can be used as a cancer marker in the diagnosis [98,99]. The expressed lncRNAs may serve as enhancers, scaffolds, or decoys by interacting with other RNAs or proteins, resulting in the aberrant activation of genes required for cancer proliferations [100]. In addition to the roles played by transcribed lncRNA molecules, several studies have suggested the importance of cis-acting RNA polymerase II-transcribing nascent lncRNAs in the regulation of neighboring genes through their effects on histone modifications [101,102]. The lnc RNAs expressed from gene promoter region are referred to as promoter-associated lncRNA, which are categorized into most abundant ncRNA, and play pivotal roles in the regulation of downstream genes [103].

The fission yeast fbp1 gene has been extensively analyzed for understanding gene regulation systems (reviewed in [104]), and several promoter-associated lncRNAs are identified in its promoter region [59,105]. These lncRNAs are referred to as metabolic stress-induced long-ncRNA (mlonRNA) and are expressed from specific cis-element, mlon-BOX, before induction of fbp1 mRNA upon glucose starvation stress [106,107]. Transcriptional initiation of mlonRNAs modulates chromatin structure around the fbp1-promoter and converts it into an open configuration, thereby promoting the binding of transcription factors to the promoter [59]. A significant overlap between the mlon-BOX and meiotic DSB sites in the fission yeast genome suggests interesting hypothesis that mlonRNA transcriptional initiations determine the DSB site via the modulation of chromatin architecture [65,108]. This hypothesis was assessed by inserting the mlon-BOX sequence into the ade6-M26 hotspot. To mimic fbp1 regulation, in which the mlon-BOX sequence is located 200 bp downstream from the Atf1 binding site in fbp1, the mlon-BOX sequence was placed 200 bp downstream from the M26 mutation site in ade6-M26 (Figure 5A,B). This insertion caused additional activation of transcription at the insertion site (Figure 5B) and stimulated meiotic recombination via local chromatin remodeling [65] (Figure 5C). These results suggest that mlonRNA transcription plays a universal role in chromatin remodeling and the regulation of transcription and recombination. Taken together with the regulatory mechanisms discussed in earlier sections, following model can be illustrated. The accessibility of Spo11/Rec12 to the genome could be one of the critical determinants of DSB sites and that Spo11/Rec12 might preferentially make DSBs at genomic regions showing open chromatin configurations induced by cis-acting RNA polymerase II transcribing nascent mlonRNAs.

7. Summary and Perspective

In this review, we summarized the multiple layers of regulation involved in meiotic recombination revealed from the analysis of the fission yeast ade6-M26 hotspot as a model system. Analyzed regulatory layers include transcription factors, chromatin modulators (histone modifiers and ADCRs), and lncRNA transcriptions. Although intensive analyses have been conducted using this model system, several important questions remained unsolved. The first question is what the proportion of meiotic recombination sites is dependent on transcription factors. The subset of naturally occurring M26 (binding sequence for the Atf1 transcription factor) sites that facilitate meiotic recombination supposedly mediate approximately 20% of all meiotic recombination events in the fission yeast genome [71]. The chromatin structure at prominent natural hotspots, such as major break site 1 (mbs1) in fission yeast, shows a constitutive open configuration independent of Atf1 [109,110]. These constitutive hotspots might be important for ensuring the basal recombination level required for CO, which is essential for the progression of meiosis. In contrast, recombination sites linked to transcription factor-binding sequences, such as M26 sites, might be important for the plasticity of meiotic recombination hotspots. Environmental conditions and stresses also affect the frequency and distribution of meiotic recombination [111]. The binding patterns of the transcription factors associated with meiotic recombination could possibly be affected by environmental conditions and thereby affecting the frequency and distribution of recombination. This was proved to be the case when hotspots created in the ade6 gene carrying a binding sequence for different transcription factors such as Atf1-Pcr1, Rst2, or Php2-Php3-Php5 were affected differently by distinct types of stresses [70]. The second unsolved question involves the factor(s) and/or conditions required for creating M26 sequence hotspots. The ade6 gene has a CRE sequence (5′-TGACGTCA-3′) at +1131 to +1136 bp starting from the first A in the ade6 ORF. However, Atf1-Pcr1 could not bind to this CRE sequence and could not facilitate recombination. Consistently, little meiotic DSB is introduced at CRE sequence in the regulatory region of fbp1 gene (our unpublished observation), indicating that not the all CRE sequences in fission yeast genome are meiotic recombination hotspot. Similarly, the HIS4 locus in budding yeast was initially shown to be linked to the activity of the transcription factors Bas1 and Bas2 [112], but subsequent genome-wide analyses failed to detect a clear correlation between Bas1/2 activity and DSB formation [113]. Thus, other factor(s) and/or condition(s) may be involved in making these transcription factors-binding sites meiotic recombination hotspot. Further research is required to clarify these questions.