The Double-Strand Break Landscape of Meiotic Chromosomes Is Shaped by the Paf1 Transcription Elongation Complex in Saccharomyces cerevisiae

Abstract

Histone modification is a critical determinant of the frequency and location of meiotic double-strand breaks (DSBs), and thus recombination. Set1-dependent histone H3K4 methylation and Dot1-dependent H3K79 methylation play important roles in this process in budding yeast. Given that the RNA polymerase II associated factor 1 complex, Paf1C, promotes both types of methylation, we addressed the role of the Paf1C component, Rtf1, in the regulation of meiotic DSB formation. Similar to a set1 mutation, disruption of RTF1 decreased the occurrence of DSBs in the genome. However, the rtf1 set1 double mutant exhibited a larger reduction in the levels of DSBs than either of the single mutants, indicating independent contributions of Rtf1 and Set1 to DSB formation. Importantly, the distribution of DSBs along chromosomes in the rtf1 mutant changed in a manner that was different from the distributions observed in both set1 and set1 dot1 mutants, including enhanced DSB formation at some DSB-cold regions that are occupied by nucleosomes in wild-type cells. These observations suggest that Rtf1, and by extension the Paf1C, modulate the genomic DSB landscape independently of H3K4 methylation. KEYWORDS meiosis; recombination; H3K4 methylation; PAF; Rtf1; double-strand breaks D NA double-strand breaks (DSBs) are a kind of DNA damage that is deleterious to cells unless it is repaired. Unrepaired DSBs lead to genome instability by forming broken chromosomes. DSBs are repaired by either homologous recombination or nonhomologous end joining (Krogh and Symington 2004;Symington and Gautier 2011). During vegetative growth, cells have to repair DSBs, which are accidentally created by exogenous impacts, such as ionizing radiation, as well as by internal sources, e.g., due to oxidation. Meiotic cells are equipped with a program to introduce DSBs along the genome for the induction of homologous recombination. Recombination generates a crossover, a reciprocal exchange of parental DNAs, which is essential for the correct segregation of homologous chromosomes during meiosis I (Petronczki et al. 2003;Lichten and de Massy 2011). Diversity in the gamete genome is also produced by meiotic recombination.Programmed formation of DSBs on meiotic chromosomes is catalyzed by the topoisomerase II-like protein Spo11 and its binding partner Ski8 (Keeney et al. 1997;Lam and Keeney 2015). The activity of Spo11 is also regulated by essential accessory proteins or protein complexes: the Mre11-Rad50-Xrs2 (MRX) complex, the Rec114-Mer2-Mei4 (RMM) complex, Rec102, and Rec104 (de Massy 2013). In addition, DSB formation during meiosis is controlled by the chromosomal structure. Three meiosis-specific chromosomal proteins, Hop1, Red1, and Mek1/Mre4, are necessary for efficient DSB formation (Xu et al. 1997). Furthermore, a meiosis-specific cohesin complex containing Rec8 kleisin regulates the distribution of DSBs in the genome (Klein et al. 1999;Kugou et al. 2009;Sun et al. 2015). Interestingly, the majority of proteins required for DSB formation, including co...

Genetic Interactions of Histone Modification Machinery Set1 and PAF1C with the Recombination Complex Rec114-Mer2-Mei4 in the Formation of Meiotic DNA Double-Strand Breaks

“…As reported [21], REC114-9myc (hereafter REC114-myc) cells showed similar spore viability to the wild type ( Table 1). The rtf1∆ decreased the viability to 75.3%, consistent with the previous results [13]. When rtf1∆ was combined with REC114-myc, the viability was synergistically reduced to 7.83%, indicating a strong synthetic defect between the rtf1∆ and REC114-myc alleles.…”

“…; Figure 3A) per Rad51-foci positive nuclei [27,28]. As reported [13], the rtf1∆ mutant reduced the Rad51 foci number to 23.1 ± 3.8 at 4 h (p value < 0.0001 against wild type, Mann-Whitney U test; Table S2). Although the REC114-myc cells showed normal spore viability (Table 1), REC114-myc nuclei also showed a decreased number of the foci of 16.7 ± 4.5 at 4 h (p value < 0.0001 against wild type, Mann-Whitney U test).…”

“…Importantly, Spp1 binds to Mer2 for DSB formation on the axis [6,8]. Although the Spp1 seems to be a key regulator for meiotic DSB formation by Spo11, the spp1 deletion shows significantly higher levels of DSBs compared to histone modification-defective mutants [6,8,13]. This may imply an additional role of the histone H3K4me for meiotic DSB formation.…”

“…In this study, we describe unique synthetic interactions between mutations in the histone modification machinery genes and a wild-type-like tagged-allele of RMM genes in DSB formation during meiosis. Yeast cells with the deletion of the RTF1 gene, only when combined with either a myc-tagged REC114 or MER2 alleles, produce few meiotic DSBs with a big reduction in spore Although the Spp1 seems to be a key regulator for meiotic DSB formation by Spo11, the spp1 deletion shows significantly higher levels of DSBs compared to histone modification-defective mutants [6,8,13]. This may imply an additional role of the histone H3K4me for meiotic DSB formation.…”

“…Set1-mediated H3K4 methylation depends on the other epigenetic marker, histone H2B K123 ubiquitination, which is catalyzed by two ubiquitination enzymes, Rad6 (E2) and Bre1 (E3), with the aid of RNApolymerase II-associated factor I complex (PAF1C) [11]. Indeed, mutations in the SET1, RAD6, BRE1, and RTF1 (PAF1C component) showed marked reduction of meiotic DSB formation in the genome [7,[12][13][14]. Interestingly, these mutants still form significant levels of DSBs, which account for the high spore viability.…”