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...